WO2015084999A1

WO2015084999A1 - Complement component biomarker assays

Info

Publication number: WO2015084999A1
Application number: PCT/US2014/068435
Authority: WO
Inventors: Graham Parry; Sandip PANICKER
Original assignee: True North Therapeutics, Inc.
Priority date: 2013-12-06
Filing date: 2014-12-03
Publication date: 2015-06-11

Abstract

The present disclosure provides methods for monitoring treatment of a complement-mediated disease or disorder. The present disclosure provides assays for monitoring response to a treatment regimen for a complement-mediated disease or disorder.

Description

COMPLEMENT COMPONENT BIOMARKER ASSAYS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

61/913,171, filed December 6, 2013, which application is incorporated herein by reference in its entirety.

INTRODUCTION

[0002] The complement system is a well-known effector mechanism of the immune

response, providing not only protection against pathogens and other harmful agents but also recovery from injury. The complement pathway comprises a number of proteins that typically exist in the body in inactive form. The classical complement pathway is triggered by activation of the first component of complement, referred to as the CI complex, which consists of Clq, Clr, and Cls proteins. Upon binding of CI to an immune complex or other activator, the Cls component, a diisopropyl fluorophosphate (DFP)-sensitive serine protease, cleaves complement components C4 and C2 to initiate activation of the classical complement pathway. The classical complement pathway appears to play a role in many diseases and disorders. C4 is involved in both the classical complement pathway and lectin complement pathway.

[0003] There is a need in the art for assays for monitoring treatment of a complement- mediated disease or disorder; and for assays for monitoring response to a treatment regimen for a complement-mediated disease or disorder. SUMMARY

[0004] The present disclosure provides methods for monitoring treatment of a complement- mediated disease or disorder. The present disclosure provides assays for monitoring response to a treatment regimen for a complement-mediated disease or disorder. FEATURES

[0005] The present disclosure features a method of monitoring C4a levels in an individual, the method comprising assaying C4a levels in a sample obtained from the individual following administration of a therapeutic agent. The therapeutic agent can be an inhibitor of the classical complement pathway or the lectin complement pathway. The present disclosure features a method of monitoring treatment in an individual, the method comprising: assaying, in a sample obtained from the individual following a treatment regimen for a complement-mediated disease or disorder, a level of complement component 4a (C4a), wherein a level of C4a that is lower than a pre- treatment level of C4a indicates treatment efficacy. The treatment regimen can comprise administration of a therapeutic agent that is an inhibitor of the classical complement pathway or the lectin complement pathway. The present disclosure features a method of monitoring treatment in an individual for a complement- mediated disease or disorder in an individual, the method comprising: assaying, in a sample obtained from the individual following a treatment regimen for the complement-mediated disease or disorder, a level of complement component 4a

(C4a), wherein a level of C4a that differs significantly from a pre-treatment level of C4a indicates treatment efficacy. The treatment regimen can comprise administration of a therapeutic agent that is an inhibitor of the classical complement pathway or the lectin complement pathway. In some cases, the individual has a complement- mediated disease or disorder. In some cases, the individual does not have a complement-mediated disease or disorder (e.g., the individual is a healthy individual, e.g., a healthy volunteer). In some cases, the sample is blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, a solid tissue sample, a tissue culture sample, or a cellular sample. A method of the present disclosure can further comprise adjusting the treatment regimen based on the assayed level of C4a. For example, in some cases, the adjusting comprises adjusting dose and/or dosage intervals, and/or duration of the treatment regimen. In some cases, the treatment regimen comprises administering an agent that inhibits interaction between Cls and C4. For example, in some cases, the agent is an antibody that inhibits interaction between Cls and C4. In some instances, the antibody comprises a complementarity-determining region (CDR) having an amino acid sequence selected from the group consisting of: a) SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and b) SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:3, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO:13. In some cases, the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO:7 or SEQ ID NO: 15 and a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO:8 or SEQ ID NO: 15. In some cases, the antibody comprises: i) a light chain variable region comprising amino acid sequence SEQ ID NO:7 and a heavy chain variable region comprising amino acid sequence SEQ ID NO:8; or ii) a light chain variable region comprising amino acid sequence SEQ ID NO: 14 and a heavy chain variable region comprising amino acid sequence SEQ ID NO: 15. In some cases, the assaying step comprises: contacting the biological sample with an antibody that specifically binds C4a; and quantitating binding of the antibody to complement C4a protein present in the sample. In some cases, a method of the present disclosure further comprises generating a report indicating efficacy of treatment. In some cases, the method further comprises assaying the level of the therapeutic agent in a biological sample obtained from the individual. In some cases, where the treatment regimen involves administering an antibody, the method can further comprise assaying the level of the antibody in the biological sample. In some cases, a level of C4a that is lower than a pre-treatment level indicates that the agent is present in the individual in an effective amount.

The present disclosure features a method of monitoring response in an individual to a treatment regimen fora complement-mediated disease or disorder in an individual, the method comprising: (a) determining a first amount of a complement C4a protein in a biological sample obtained from the individual at a first time point; (b) determining a second amount of the complement C4a protein in a biological sample obtained from the individual at a second time point; and (c) comparing the second amount of complement C4a protein with the first amount of complement C4a protein. In some cases, the first time point is a time point before initiation of a treatment regimen, and the second time point is a time point after initiation of the treatment regimen. In some cases, the method further comprises adjusting the treatment regimen based on the amount of complement a C4a protein in a biological sample obtained from the individual at the second time point. In some cases, the method further comprises generating a report indicating the status of the disease or disorder. For example, the report can comprise guidance to a clinician for adjusting the treatment regimen. In some cases, the adjusting comprises adjusting dose and/or dosage intervals, and/or duration of the treatment regimen. In some embodiments of a method of the present disclosure, the treatment regimen comprises administering an agent that inhibits interaction between Cls and C4. In some cases, the agent is an antibody that inhibits interaction between Cls and C4. In some cases, the antibody comprises a complementarity-determining region (CDR) having an amino acid sequence selected from the group consisting of: a) SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; or b) SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:3, SEQ ID NO: l l, SEQ ID NO: 12, and SEQ ID NO: 13. In some cases, the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO:7 or SEQ ID NO: 14 and a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO: 8 or SEQ ID NO: 15. In some cases, the antibody comprises: i) a light chain variable region comprising amino acid sequence SEQ ID NO:7 and a heavy chain variable region comprising amino acid sequence SEQ ID NO: 8; or ii) a light chain variable region comprising amino acid sequence SEQ ID NO: 14 and a heavy chain variable region comprising amino acid sequence SEQ ID NO: 15. In some cases, assaying step comprises: contacting the biological sample with an antibody that specifically binds C4a; and quantitating binding of the antibody to complement C4a protein present in the sample. In some cases, the method further comprises generating a report indicating efficacy of treatment. In some cases, where the treatment regimen comprises administering an antibody, the method can comprise assaying the level of the antibody in the biological sample. In some cases, a level of C4a that is lower than a pre-treatment level indicates that the agent is present in the individual in an effective amount.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 provides the amino acid sequence of Homo sapiens complement Cls protein (SEQ ID NO: 16).

[0008] Figure 2 depicts the in vivo effect of TNT003 on C4a generation following

administration to non-human primates.

[0009] Figure 3 provides the amino acid sequence of Homo sapiens complement C4a protein

(SEQ ID NO: 17).

[0010] Figure 4 provides the amino acid sequence of Homo sapiens complement C4 protein (SEQ ID NO: 18).

[0011] Figure 5 provides a general schematic of a computerized system for use in the

methods of the present disclosure.

[0012] Figures 6A-C depict the effect of TNT009 on C4a serum levels in cynomolgus

monkeys dosed with 5 mg/kg, 10 mg/kg, or 30 mg/kg. [0013] Figures 7A-C depict the effect of TNT009 on C4a plasma levels in cynomolgus monkeys dosed with 30 mg/kg, 45 mg/kg, or 60 mg/kg TNT009.

DEFINITIONS

[0014] The terms "individual," "subject," and "patient," used interchangeably herein, refer to a mammal, e.g., a non-human primate, a rodent (e.g., a mouse; a rat), a lagomorph (e.g., a rabbit), a human, etc..

[0015] A "biological sample" encompasses a variety of sample types obtained from an

individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term "biological sample" encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions such as plasma and serum, and the like. The term "biological sample" also includes solid tissue samples, tissue culture samples, and cellular samples.

[0016] As used herein, the terms "determining", "assessing", "assaying", "measuring" and "detecting" refer to both quantitative and semi-quantitative determinations and as such, the term "determining" is used interchangeably herein with "assaying," "measuring," and the like. Where a quantitative determination is intended, the phrase "determining an amount" of an analyte and the like is used. Where either a quantitative and semi-quantitative determination is intended, the phrase "determining a level" of an analyte or "detecting" an analyte is used.

[0017] "Quantitative" assays in general provide information on the amount of an analyte in a sample relative to a reference (control), and are usually reported numerically, where a "zero" value can be assigned where the analyte is below the limit of detection.

"Semi-quantitative" assays involve presentation of a numeric representation of the amount of the analyte in the specimen that is relative to a reference (e.g., a threshold, e.g., normal threshold or an abnormal threshold), where a "zero" value can be assigned where the analyte is below the limit of detection. In general, semi- quantitative results are compared against an accompanying reference interval to provide a qualitative interpretation of the result.

[0018] The terms "antibodies" and "immunoglobulin" include antibodies or

immunoglobulins of any isotype, fragments of antibodies that retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAb), single domain antibodies (dAb), single domain heavy chain antibodies, a single domain light chain antibodies, bi-specific antibodies, multi-specific antibodies, and fusion proteins comprising an antigen-binding (also referred to herein as antigen binding) portion of an antibody and a non-antibody protein. The antibodies can be detectably labeled, e.g., with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, and the like. The antibodies can be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. The antibodies can also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the term are Fab' , Fv, F(ab')₂, and or other antibody fragments that retain specific binding to antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which were produced from a single cell by repetitive cellular replication. That is, the clone of cells only produces a single antibody species. While a monoclonal antibody can be produced using hybridoma production technology, other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). An antibody can be monovalent or bivalent. An antibody can be an Ig monomer, which is a "Y-shaped" molecule that consists of four polypeptide chains: two heavy chains and two light chains connected by disulfide bonds.

[0019] The terms "polypeptide," "peptide," and "protein", used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. [0020] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0021] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0023] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a biological sample" includes a plurality of such samples and reference to "the C4a polypeptide" includes reference to one or more C4a polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0024] It is appreciated that certain features of the invention, which are, for clarity,

described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0025] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0026] The present disclosure provides methods for monitoring treatment of a complement- mediated disease or disorder. The present disclosure provides assays for monitoring response to a treatment regimen for a complement-mediated disease or disorder. MONITORING TREATMENT

[0027] The present disclosure provides a method of monitoring C4a levels in an individual, the method comprising assaying C4a levels in a sample obtained from the individual following administration of a therapeutic agent. The therapeutic agent can be an inhibitor of the classical complement pathway or the lectin complement pathway. The present disclosure provides a method of monitoring treatment in an individual, the method comprising: assaying, in a sample obtained from the individual following a treatment regimen for a complement-mediated disease or disorder, a level of complement component 4a (C4a), wherein a level of C4a that is lower than a pre- treatment level of C4a indicates treatment efficacy. The treatment regimen can comprise administration of a therapeutic agent that inhibits the classical complement pathway. The treatment regimen can comprise administration of a therapeutic agent that inhibits the lectin complement pathway.

[0028] The present disclosure provides methods for monitoring treatment of a complement- mediated disease or disorder. The methods generally involve assaying, in a sample obtained from the individual following a treatment regimen for the complement- mediated disease or disorder, a level of complement component 4a (C4a), wherein a level of C4a that differs significantly from a pre-treatment level of C4a indicates treatment efficacy. The present disclosure provides methods for monitoring treatment of a complement-mediated disease or disorder. The methods generally involve assaying, in a sample obtained from the individual following a treatment regimen for the complement-mediated disease or disorder, a level of complement component 4a (C4a), wherein a level of C4a that is significantly lower than a pre-treatment level of C4a indicates treatment efficacy. The individual can be an individual who has a complement-mediated disease or disorder (e.g., an individual who has been diagnosed as having a complement-mediated disease or disorder). The individual can be an individual who does not have a complement- mediated disease or disorder (e.g., an individual who has not been diagnosed as having a complement-mediated disease or disorder; e.g., a healthy individual; e.g., a healthy control individual).

[0029] In some cases, the post-treatment level of C4a is lower than a pre-treatment C4a level. In such cases, treatment efficacy is indicated where the treatment results in an at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, or more than 75%, lower level of C4a, compared to the pre-treatment level of C4a.

[0030] A monitoring method of the present disclosure can be used to monitor response to therapy of a complement-mediated disease or disorder in an individual. A monitoring method of the present disclosure can be used to determine an individual's response to a treatment regimen for treating a complement-mediated disease or disorder.

[0031] The present disclosure provides a method of response to therapy for a

complement-mediated disease or disorder in an individual. The method generally involves comparing the amount of a complement C4a protein in a biological sample obtained from the individual at a first time point with the amount of a complement C4a protein in a biological sample obtained from the individual at a second time point. A difference in the amount of complement C4a protein in a biological sample obtained from the individual at a second time point (e.g., a second time point during treatment), compared to the amount of complement C4a protein in a biological sample obtained from the individual at a first time point (e.g., a first time point during treatment), can provide an indication as to: i) whether the treatment regimen is effective to treat the complement-mediated disease or disorder; and/or ii) whether the individual is exhibiting a beneficial clinical response to treatment with a drug or other treatment regimen for treating the complement-mediated disease or disorder. In some cases, the steps of determining comprise contacting the biological sample with an anti-C4a antibody and quantitating binding of the antibody to complement C4a protein present in the sample

[0032] The present disclosure provides a method of monitoring response to treatment of a complement-mediated disease or disorder in an individual. The method generally involves comparing the amount of a complement C4a protein in a biological sample obtained from the individual at a first time point (e.g., a pre-treatment time point) with the amount of a complement C4a protein in a biological sample obtained from the individual at a second time point (e.g., a time point after treatment has begun). A difference in the amount of complement C4a protein in a biological sample obtained from the individual at a second time point, compared to the amount of complement C4a protein in a biological sample obtained from the individual at a first time point, can provide an indication as to whether the individual is exhibiting a beneficial clinical response to treatment with a drug or other treatment regimen for treating the complement-mediated disease or disorder. In some embodiments, the steps of determining comprise contacting the biological sample with an anti- C4a antibody and quantitating binding of the antibody to complement C4a protein present in the sample. In some embodiments, the step of comparing indicates if treatment has been effective.

[0033] Any biological sample obtained from the individual in whom treatment is being monitored is suitable. Examples of suitable biological samples include blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, a solid tissue sample, a tissue culture sample, and a cellular sample.

[0034] In some cases, a subject method of monitoring treatment can comprise adjusting the treatment regimen based on the assayed level of C4a. For example, one or more of the dose, dosage intervals, and duration of the treatment regimen can be adjusted.

[0035] In some cases, the treatment regimen comprises administering an agent that inhibits interaction between Cls and C4. Examples of such agents include an antibody that inhibits interaction between Cls and C4. For example, an antibody that inhibits interaction between Cls and C4 can comprise a complementarity-determining region (CDR) having an amino acid sequence selected from the group consisting of:

[0036] a) SEQ ID NO: l , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; or [0037] b) SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:3, SEQ ID NO:l l, SEQ ID NO: 12, and SEQ ID NO: 13.

[0038] For example, an antibody that inhibits interaction between Cls and C4 can comprise: a) a light chain region comprising one, two, or three CDRs selected from SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3; and b) a heavy chain region comprising one, two, or three CDRs selected from SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some of these embodiments, the anti-Cls antibody includes a humanized V_H and/or V_L framework region.

[0039] SEQ ID NO:l : SSVSSSYLHWYQ;

[0040] SEQ ID NO:2: STSNLASGVP;

[0041] SEQ ID NO:3: HQYYRLPPIT;

[0042] SEQ ID NO:4: GFTFSNYAMSWV;

[0043] SEQ ID NO:5: ISSGGSHTYY;

[0044] SEQ ID NO:6: ARLFTGYAMDY.

[0045] In some of these embodiments, the anti-Cls antibody includes a humanized V_H

and/or V_L framework region.

[0046] For example, an antibody that inhibits interaction between Cls and C4 can comprise a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:7. In some of these embodiments, the anti-Cls antibody includes a humanized V_H and/or V_L framework region.

[0047] SEQ ID NO:7:

DIVMTQTTAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYST SNLASGVPARFSGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTK LELK.

[0048] For example, an antibody that inhibits interaction between Cls and C4 can comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:8. In some of these embodiments, the anti-Cls antibody includes a humanized V_H and/or V_L framework region.

[0049] SEQ ID NO:8:

QVKLEESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQIPEKRLEWVATIS SGGSHTYYLDSVKGRFTISRDNARDTLYLQMSSLRSEDTALYYCARLFTGYA MDYWGQGTSVT.

[0050] In some cases, an antibody that inhibits interaction between Cls and C4 can

comprise: a) a light chain region comprising one, two, or three CDRs selected from SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO:3; and b) a heavy chain region comprising one, two, or three CDRs selected from SEQ ID NO: l l , SEQ ID NO: 12, and SEQ ID NO: 13. In some of these embodiments, the anti-Cls antibody includes a humanized VH and/or VL framework region.

[0051] SEQ ID NO:9: TASSSVSSSYLH;

[0052] SEQ ID NO: 10: STSNLAS;

[0053] SEQ ID NO:3: HQYYRLPPIT;

[0054] SEQ ID NO: l l : NYAMS;

[0055] SEQ ID NO: 12: TIS S GGSHT Y YLDS VKG ;

[0056] SEQ ID NO: 13: LFTGYAMDY.

[0057] In some cases, an antibody that inhibits interaction between Cls and C4 comprises a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 14.

[0058] SEQ ID NO: 14:

QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTS NL AS G VP ARFS GS GS GTFYS LTIS SME AEDD ATY YCHQ Y YRLPPITFG AGTKL ELK.

[0059] In some cases, an antibody that inhibits interaction between Cls and C4 comprises a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15.

[0060] SEQ ID NO: 15:

EVMLVESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQIPEKRLEWVATI SSGGSHTYYLDSVKGRFTISRDNARDTLYLQMSSLRSEDTALYYCARLFTGY AMD YWGQGTS VT VS S .

[0061] The amount of a complement C4a protein in a biological sample can be assessed by any suitable method known in the art. Suitable methods include, but are not limited to, a protein ("Western") blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectroscopy (MS), matrix-assisted laser desorption/ionization-time of flight-MS (MALDI-TOF), surface-enhanced laser desorption ionization- time of flight (SELDI-TOF), high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS), and laser densitometry.

[0062] In some embodiments, an immunological assay involves use of an antibody specific for a C4a polypeptide. The anti-C4a antibody can be immobilized on an insoluble support (e.g., a test strip, a well of a multi-well plate, a bead (e.g., a magnetic bead), etc.). Suitable supports are well known in the art and comprise, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, wells of reaction trays (e.g., multi-well plates), plastic tubes, etc. A solid support can comprise any of a variety of substances, including, e.g., glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses,

polyacrylamides, agaroses, and magnetite. Suitable methods for immobilizing a subject antibody onto a solid support are well known and include, but are not limited to ionic, hydrophobic, covalent interactions and the like. Solid supports can be soluble or insoluble, e.g., in aqueous solution. In some embodiments, a suitable solid support is generally insoluble in an aqueous solution.

[0063] In some embodiments, an immunological assay involves use of an antibody specific for a C4a polypeptide. The antibody can include a detectable label. The antibody will in some embodiments be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, a chromogenic protein, and the like. An antibody can be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. An antibody can also be bound to a solid support, including, but not limited to, polystyrene plates or beads, magnetic beads, test strips, membranes, and the like.

[0064] In some embodiments, the antibody is detectably labeled, either directly or indirectly.

Direct labels include radioisotopes (e.g., ¹²⁵I; ³⁵S, and the like); enzymes whose products are detectable (e.g., lucif erase, β-galactosidase, horse radish peroxidase, alkaline phosphatase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu, or others of the lanthanide series, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds, e.g., luciferin; fluorescent proteins; and the like. Indirect labels include second antibodies specific for a gene product-specific antibody, wherein the second antibody is labeled as described above; and members of specific binding pairs, e.g., biotin-avidin, and the like.

[0065] In some embodiments, an antibody comprises, covalently linked to the antibody, a protein that provides for a detectable signal. Suitable proteins include, but are not limited to, fluorescent proteins and enzymes (e.g., β-galactosidase, lucif erase, horse radish peroxidase, alkaline phosphatase, etc.). Suitable fluorescent proteins include, but are not limited to, a green fluorescent protein (GFP), including, but not limited to, a GFP derived from Aequoria victoria or a derivative thereof, a number of which are commercially available; a GFP from a species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle et al. (2001) /. Protein Chem. 20:507-519; a yellow fluorescent protein; a blue fluorescent protein; a red fluorescent protein; any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, U.S. Patent Publication No. 2002/0197676, or U.S. Patent Publication No. 2005/0032085; and the like.

Subjects

[0066] Individuals who are to be tested using a method of the present disclosure can include individuals who have never previously been diagnosed as having a complement- mediated disease or disorder. Individuals who are to be tested using a method of the present disclosure can include individuals who have been diagnosed as having a complement-mediated disease or disorder. Individuals who are to be tested using a method of the present disclosure can include individuals who are undergoing treatment for a complement-mediated disease or disorder. Individuals who are to be tested using a method of the present disclosure can include individuals who do not have a complement- mediated disease or disorder, e.g., individuals who have not been diagnosed as having a complement-mediated disease or disorder complement- mediated disease or disorder; e.g., healthy individuals. For example, a healthy individual can include an individual who is participating as a healthy control in a clinical trial.

Examples of a complement-mediated disease or disorder include, but are not limited to, age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, argyrophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behcet's disease, British type amyloid angiopathy, bullous pemphigoid, Buerger's disease, Clq nephropathy, cancer, catastrophic antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt- Jakob disease, Crohn's disease, cryoglobulinemic vasculitis, dementia pugilistica, dementia with Lewy Bodies (DLB), diffuse neurofibrillary tangles with calcification, Discoid lupus erythematosus, Down's syndrome, focal segmental glomerulosclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration,

Gerstmann-Straussler-Scheinker disease, Guillain-Barre syndrome, Hallervorden- Spatz disease, hemolytic -uremic syndrome, hereditary angioedema,

hypophosphastasis, idiopathic pneumonia syndrome, immune complex diseases, inclusion body myositis, infectious disease (e.g., disease caused by bacterial (e.g., Neisseria meningitidis or Streptococcus) viral (e.g., human immunodeficiency virus (HIV)), or other infectious agents), inflammatory disease, ischemia / reperfusion injury, mild cognitive impairment, immunothrombocytopenic purpura (ITP), molybdenum cofactor deficiency (MoCD) type A, membranoproliferative glomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis (MPGN) II (dense deposit disease), membranous nephritis, multi-infarct dementia, lupus (e.g., systemic lupus erythematosus (SLE)), glomerulonephritis, Kawasaki disease, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann- Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, Pemphigus vulgaris, Pick's disease, postencephalitic parkinsonism, polymyositis, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga-toxin E coli (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, Tangle only dementia, transplant rejection, vasculitis (e.g., ANCA associated vasculitis), Wegner's granulomatosis, sickle cell disease,

cryoglobulinemia, mixed cryoglobulinemia, essential mixed cryoglobulinemia, Type II mixed cryoglobulinemia, Type III mixed cryoglobulinemia, nephritis, drug- induced thrombocytopenia, lupus nephritis, bullous pemphigoid, Epidermolysis bullosa acquisita, delayed hemolytic transfusion reaction, and platelet refractoriness. Generating a report

[0068] In some embodiments, a subject method involves generating a report. A subject method can include generating a report that provides an indication regarding one or more of: a) the status of a complement-mediated disease or disorder; b) response of an individual to treatment for a complement-mediated disease or disorder; and c) a level of C4a in a biological sample. A report can also include information such as a recommendation regarding further evaluation; a recommendation regarding therapeutic drug intervention (including, e.g., therapeutic antibody treatment, such as anti-Cls treatment); a recommendation regarding adjusting a current treatment regimen (e.g., adjusting a dose, a dosing interval, a treatment duration, etc.), and the like.

[0069] For example, the methods disclosed herein can further include a step of generating or outputting a report providing the results of a subject method, which report can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium). A person or entity that prepares a report ("report generator") may also perform steps such as sample gathering, sample processing, and the like. Alternatively, an entity other than the report generator can perform steps such as sample gathering, sample processing, and the like. A report can be provided to a user. A "user" can be a health professional (e.g., a clinician, a laboratory technician, a physician, etc.).

[0070] A subject report can further include one or more of: 1) service provider information;

2) patient data; 3) data regarding the level of C4a; 4) follow-up evaluation recommendations; 5) therapeutic intervention recommendations; and 6) other features. Further evaluation

[0071] Based on detection of a level of C4a, as described above, and/or based on a report (as described above), a physician or other qualified medical personnel can determine whether further evaluation of the test subject (the patient) is required.

Therapeutic intervention

[0072] Based on detection of a level of C4a, and/or based on a report (as described above), a physician or other qualified medical personnel can determine whether appropriate therapeutic intervention is advised, e.g., in order to treat the complement-mediated disease or disorder. Based on detection of a level of C4a, and/or based on a report (as described above), a physician or other qualified medical personnel can determine whether a current treatment regimen should be adjusted, where adjusting a treatment regimen can include on one or more of adjusting a dose, adjusting a dosing interval, and adjusting a treatment duration.

COMPUTER-IMPLEMENTED METHODS, SYSTEMS AND DEVICES

[0073] The methods of the present disclosure can be computer-implemented, such that

method steps (e.g., assaying, comparing, calculating, and/or the like) are automated in whole or in part. Accordingly, the present disclosure provides methods, computer systems, devices and the like in connection with computer-implemented methods of facilitating carrying out a subject method.

[0074] For example, the method steps, including obtaining values for biomarker levels (e.g., C4a levels; C4 levels), comparing biomarker levels to a control level and/or comparing biomarker levels to a pre-treatment level, generating a report, and the like, can be completely or partially performed by a computer program product. Values obtained can be stored electronically, e.g., in a database, and can be subjected to an algorithm executed by a programmed computer.

[0075] For example, the methods of the present disclosure can involve inputting a biomarker level (e.g., a C4a level; a C4 level) into a computer programmed to execute an algorithm to perform the comparing and calculating step(s) described herein, and generate a report as described herein, e.g., by displaying or printing a report to an output device at a location local or remote to the computer.

[0076] The present invention thus provides a computer program product including a

computer readable storage medium having a computer program stored on it. In certain aspects, the storage medium is non-transitory (e.g., a storage medium that is not a transitory wave or signal). The program can, when read by a computer, execute relevant calculations based on values obtained from analysis of one or more biological sample from an individual. The computer program product has stored therein a computer program for performing the calculation(s).

[0077] The present disclosure provides systems for executing the program described above, which system generally includes: a) a central computing environment; b) an input device, operatively connected to the computing environment, to receive patient data, wherein the patient data can include, for example, biomarker level or other value obtained from an assay using a biological sample from the patient, as described above; c) an output device, connected to the computing environment, to provide information to a user (e.g., medical personnel); and d) an algorithm executed by the central computing environment (e.g., a processor), where the algorithm is executed based on the data received by the input device, and wherein the algorithm calculates a value, which value is indicative of the likelihood the subject has a complement- mediated disease or disorder.

Computer Systems

[0078] A generalized example of a computerized embodiment in which programs to

facilitate execution of the methods of the present disclosure can be implemented is depicted in Figure 5, which illustrates a processing system 100 which generally comprises at least one processor 102, or processing unit or plurality of processors, memory 104, at least one input device 106 and at least one output device 108, coupled together via a bus or group of buses 110. In certain embodiments, input device 106 and output device 108 can be the same device. An interface 112 can also be provided for coupling the processing system 100 to one or more peripheral devices, for example interface 112 can be a PCI card or PC card. At least one storage device 114 which houses at least one database 116 can also be provided.

[0079] The memory 104 can be any form of memory device, for example, volatile or nonvolatile memory, solid state storage devices, magnetic devices, etc. In certain aspects, the memory includes a non-transitory storage medium (e.g., a storage medium that is not a transitory wave or signal). The processor 102 can comprise more than one distinct processing device, for example to handle different functions within the processing system 100. Input device 106 receives input data 118 and can comprise, for example, a keyboard, a pointer device such as a pen-like device or a mouse, audio receiving device for voice controlled activation such as a microphone, data receiver or antenna such as a modem or wireless data adaptor, data acquisition card, etc. Input data 118 can come from different sources, for example keyboard instructions in conjunction with data received via a network.

[0080] Output device 108 produces or generates output data 120 and can comprise, for

example, a display device or monitor in which case output data 120 is visual, a printer in which case output data 120 is printed, a port for example a USB port, a peripheral component adaptor, a data transmitter or antenna such as a modem or wireless network adaptor, etc. Output data 120 can be distinct and derived from different output devices, for example a visual display on a monitor in conjunction with data transmitted to a network. A user can view data output, or an interpretation of the data output, on, for example, a monitor or using a printer. The storage device 114 can be any form of data or information storage means, for example, volatile or non-volatile memory, solid state storage devices, magnetic devices, etc.

[0081] In use, the processing system 100 is adapted to allow data or information to be stored in and/or retrieved from, via wired or wireless communication means, at least one database 116. The interface 112 may allow wired and/or wireless communication between the processing unit 102 and peripheral components that may serve a specialized purpose. In general, the processor 102 can receive instructions as input data 118 via input device 106 and can display processed results or other output to a user by utilizing output device 108. More than one input device 106 and/or output device 108 can be provided. The processing system 100 may be any suitable form of terminal, server, specialized hardware, or the like.

[0082] The processing system 100 may be a part of a networked communications system.

Processing system 100 can connect to a network, for example the Internet or a WAN. Input data 118 and output data 120 can be communicated to other devices via the network. The transfer of information and/or data over the network can be achieved using wired communications means or wireless communications means. A server can facilitate the transfer of data between the network and one or more databases. A server and one or more databases provide an example of an information source.

[0083] Thus, the processing computing system environment 100 illustrated in Fig. 5 may operate in a networked environment using logical connections to one or more remote computers. The remote computer may be a personal computer, a server, a router, a network PC, a peer device, or other common network node, and typically includes many or all of the elements described above. [0084] The logical connections depicted in Fig. 5 may include a local area network (LAN) and a wide area network (WAN), but may also include other networks such as a personal area network (PAN). Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets, and the Internet. For instance, when used in a LAN networking environment, the computing system environment 100 is connected to the LAN through a network interface or adapter. When used in a WAN networking environment, the computing system environment typically includes a modem or other means for establishing communications over the WAN, such as the Internet. The modem, which may be internal or external, may be connected to a system bus via a user input interface, or via another appropriate mechanism. In a networked environment, program modules depicted relative to the computing system environment 100, or portions thereof, may be stored in a remote memory storage device. It is to be appreciated that the illustrated network connections of Fig. 5 are examples and other means of establishing a

communications link between multiple computers may be used.

[0085] Fig. 5 is intended to provide a brief, general description of an illustrative and/or suitable example of a computing environment in which embodiments of the methods disclosed herein may be implemented. Fig. 5 is an example of a suitable

environment and is not intended to suggest any limitation as to the structure, scope of use, or functionality of an embodiment of the present invention. A particular environment should not be interpreted as having any dependency or requirement relating to any one or combination of components illustrated in an exemplary operating environment. For example, in certain instances, one or more elements of an environment may be deemed not necessary and omitted. In other instances, one or more other elements may be deemed necessary and added.

[0086] Certain embodiments may be described with reference to acts and symbolic

representations of operations that are performed by one or more computing devices, such as the computing system environment 100 of Fig. 5. As such, it will be understood that such acts and operations, which are at times referred to as being computer-executed, include the manipulation by the processor of the computer of electrical signals representing data in a structured form. This manipulation transforms the data or maintains them at locations in the memory system of the computer, which reconfigures or otherwise alters the operation of the computer in a manner understood by those skilled in the art. The data structures in which data is maintained are physical locations of the memory that have particular properties defined by the format of the data. However, while an embodiment is being described in the foregoing context, it is not meant to be limiting as those of skill in the art will appreciate that the acts and operations described hereinafter may also be

implemented in hardware.

[0087] Embodiments may be implemented with numerous other general-purpose or special- purpose computing devices and computing system environments or configurations. Examples of well-known computing systems, environments, and configurations that may be suitable for use with an embodiment include, but are not limited to, personal computers, handheld or laptop devices, personal digital assistants, multiprocessor systems, microprocessor-based systems, programmable consumer electronics, network, minicomputers, server computers, web server computers, mainframe computers, and distributed computing environments that include any of the above systems or devices.

[0088] Embodiments may be described in a general context of computer-executable

instructions, such as program modules, being executed by a computer. Generally, program modules include routines, programs, objects, components, data structures, etc., that perform particular tasks or implement particular abstract data types. An embodiment may also be practiced in a distributed computing environment where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote computer storage media (e.g., non-transitory storage media, where the medium is not a transitory wave or signal) including memory storage devices.

Computer program products

[0089] The present disclosure provides computer program products that, when executed on a programmable computer such as that described above with reference to Fig. 5, can carry out the methods of the present disclosure. As discussed above, the subject matter described herein may be embodied in systems, apparatus, methods, and/or articles depending on the desired configuration. These various implementations may include implementation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device (e.g. video camera, microphone, joystick, keyboard, and/or mouse), and at least one output device (e.g. display monitor, printer, etc.).

[0090] Computer programs (also known as programs, software, software applications,

applications, components, or code) include instructions for a programmable processor, and may be implemented in a high-level procedural and/or object-oriented programming language, and/or in assembly /machine language. As used herein, the term "machine-readable medium" (e.g., "computer-readable medium") refers to any computer program product, apparatus and/or device (e.g., magnetic discs, optical disks, memory, etc.) used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. According to certain

embodiments, the machine-readable medium is non-transitory (e.g., a machine readable medium that is not a transitory wave or signal).

[0091] It will be apparent from this description that aspects of the present invention may be embodied, at least in part, in software, hardware, firmware, or any combination thereof. Thus, the techniques described herein are not limited to any specific combination of hardware circuitry and/or software, or to any particular source for the instructions executed by a computer or other data processing system. Rather, these techniques may be carried out in a computer system or other data processing system in response to one or more processors, such as a microprocessor, executing sequences of instructions stored in memory or other computer-readable medium (e.g., a non-transitory computer-readable medium) including any type of ROM, RAM, cache memory, network memory, floppy disks, hard drive disk (HDD), solid-state devices (SSD), optical disk, CD-ROM, and magnetic-optical disk, EPROMs, EEPROMs, flash memory, or any other type of media suitable for storing instructions in electronic format.

[0092] In addition, the processor(s) may be, or may include, one or more programmable general-purpose or special-purpose microprocessors, digital signal processors (DSPs), programmable controllers, application specific integrated circuits (ASICs), programmable logic devices (PLDs), trusted platform modules (TPMs), or the like, or a combination of such devices. In alternative embodiments, special-purpose hardware such as logic circuits or other hardwired circuitry may be used in combination with software instructions to implement the techniques described herein. KITS

[0093] Kits for use in a method of the present disclosure can include a binding reagent(s) for a biomarker (e.g., C4a; C4). The binding reagent can be, for example, and antibody that specifically binds a biomarker (e.g., an anti-C4a antibody; an anti-C4 antibody).. "Binding reagent" as used here encompasses both capture reagents and detection reagents. A "capture reagent" refers to a binding partner for a biomarker that is suitable for use in, for example, enriching a sample for its respective biomarker, e.g., an anti-biomarker antibody. Where the capture reagent comprises an antibody, the antibody may be a polyclonal or monoclonal antibody. "Detection reagent" refers to a binding partner for a biomarker that is suitable for use in detection of an immobilized biomarker, e.g., an anti-biomarker antibody, and is optionally detectably labeled. Where the detection reagent comprises an antibody, the antibody may be a polyclonal or monoclonal antibody. Kits may further include one or more reagents for detection of binding of a detection reagent, e.g., detection of an anti-biomarker antibody, e.g., as when bound to an anti-biomarker binding reagent/biomarker complex.

[0094] Binding reagents for a control analyte (e.g., a control biomarker) can also be

included.

[0095] Capture reagents provided in a kit suitable for use in a subject method can be

immobilized on an insoluble support, e.g., an assay substrate, such as an array, bead, and the like as described herein. Detection reagents can include a detectable label. Where the detection reagents are not detectably labeled, the kit can include reagents for detecting the detection reagents, such as an antibody. The various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container, as desired.

[0096] Kits can include instructions for using the components of the kit to practice a method of the present disclosure. The instructions are generally recorded on a suitable recording medium, such as paper, plastic, electronic storage medium, and the like. For example, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. compact disc-read only memory (CD-ROM), digital versatile disk (DVD), diskette, etc. In other examples, the instructions provided do not contain many or all assay details, but rather provide direction as to a remote source for obtaining detailed instructions, e.g. via the internet.

EXAMPLES

[0097] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal (ly); s.c, subcutaneous(ly); and the like.

Example 1 : C4a levels indicate efficacy of treatment

[0098] In vivo effects of anti-Cls antibody TNT003 were tested in a non-human primate.

The data are shown in Figure 2.

[0099] Cynomolgus monkeys were given a single i.v. dose of TNT003 (30 mg/kg). As

shown in Figure 2, TNT003 inhibits in vivo C4a generation in cynomolgus monkeys given a single i.v. dose of TNT003. C4a concentrations in serum samples taken from TNT003 -dosed monkeys were determined using commercially available ELISA kits (plotted on the right Y-axis) The data show that C4a levels drop by approximately 90% immediately following TNT003 administration and are depressed in all samples containing detectable TNT003 (up to and including 72 hours). At 96 hours, TNT003 fell below detectable levels (as determined by an ELISA capture assay; plotted on the left Y-axis) at which point serum C4a were restored to pre-bleed levels.

[00100] Cynomolgus monkeys were given a single i.v. dose (5 mg/kg, 10 mg/kg, or 30 mg/kg) of TNT009 (humanized TNT003); and C4a serum levels were measured. The data are shown in Figures 6A-C. C4a measured in serum samples taken from cynomolgous monkeys dosed with TNT009 at A) 5 mg/kg, B) 10 mg/kg, and C) 30 mg/kg show a reduction in C4a levels in all dose groups following TNT009 administration (n = 3 animals/dose group).

[00101] Cynomolgus monkeys were given a single i.v. dose (30 mg/kg, 45 mg/kg, or

60 mg/kg) of TNT009 (humanized TNT003); and C4a plasma levels were measured. The data are shown in Figures 7A-C. C4a measured in plasma samples taken from cynomolgus monkeys dosed with TNT009 at A) 30 mg/kg, B) 45 mg/kg, and C) 60 mg/kg show a reduction in C4a levels in all dose groups following TNT009 administration (n = 2 animals/dose group). [00102] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

What is claimed is: 1. A method of monitoring C4a levels in an individual, the method comprising assaying C4a levels in a sample obtained from the individual following administration of a therapeutic agent.

2. A method of monitoring treatment in an individual, the method comprising: assaying, in a sample obtained from the individual following a treatment regimen for a complement-mediated disease or disorder, a level of complement component 4a (C4a), wherein a level of C4a that is lower than a pre-treatment level of C4a indicates treatment efficacy.

3. The method of claim 1 or claim 2, wherein the individual has a complement- mediated disease or disorder.

4. The method of claim 1 or claim 2, wherein the individual does not have a complement-mediated disease or disorder.

5. The method of any one of claims 1-4, wherein the sample is blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, a solid tissue sample, a tissue culture sample, or a cellular sample.

6. The method of any one of claims 1-5, comprising adjusting the treatment regimen based on the assayed level of C4a.

7. The method of claim 6, wherein said adjusting comprises adjusting dose and/or dosage intervals, and/or duration of the treatment regimen.

8. The method of any one of claims 1-7, wherein the treatment regimen comprises administering a therapeutic agent that inhibits interaction between Cls and C4.

9. The method of claim 8, wherein the therapeutic agent is an antibody that inhibits interaction between Cls and C4.

10. The method of claim 9, wherein the antibody comprises a complementarity- determining region (CDR) having an amino acid sequence selected from the group consisting of:

a) SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and

SEQ ID NO:6; or

b) SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:3, SEQ ID NO:l l, SEQ ID NO: 12, and SEQ ID NO: 13.

11. The method of claim 9, wherein the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO:7 or SEQ ID NO: 14 and a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO:8 or SEQ ID NO: 15.

12. The method of claim 9, wherein the antibody comprises:

i) a light chain variable region comprising amino acid sequence SEQ ID NO:7 and a heavy chain variable region comprising amino acid sequence SEQ ID NO: 8; or

ii) a light chain variable region comprising amino acid sequence SEQ ID NO: 14 and a heavy chain variable region comprising amino acid sequence SEQ ID NO: 15.

13. The method of any one of claims 1-12, wherein said assaying step comprises

(i) contacting the biological sample with an antibody that specifically binds C4a; and

(ii) quantitating binding of the antibody to complement C4a protein present in the sample.

14. The method of any one of claims 1-13, comprising generating a report indicating efficacy of treatment.

15. The method of any one of claims 1-14, comprising assaying the level of the therapeutic agent in the biological sample.

16. The method of any one of claims 9-12, comprising assaying the level of the antibody in the biological sample.

17. The method of any one of claims 2-16, wherein a level of C4a that is lower than a pre-treatment level indicates that the therapeutic agent is present in the individual in an effective amount.

18. A method of monitoring response in an individual to a treatment regimen for a complement-mediated disease or disorder in an individual, the method comprising:

(a) determining a first amount of a complement C4a protein in a biological sample obtained from the individual at a first time point;

(b) determining a second amount of the complement C4a protein in a biological sample obtained from the individual at a second time point; and

(c) comparing the second amount of complement C4a protein with the first amount of complement C4a protein.

19. The method of claim 18, wherein the first time point is a time point before initiation of a treatment regimen, and wherein the second time point is a time point after initiation of the treatment regimen.

20. The method of claim 18, comprising adjusting the treatment regimen based on the amount of complement a C4a protein in a biological sample obtained from the individual at the second time point.

21. The method of claim 18, comprising generating a report indicating efficacy of treatment.

22. The method of claim 21, wherein the report comprises guidance to a clinician for adjusting the treatment regimen.

23. The method of claim 22, wherein said adjusting comprises adjusting dose and/or dosage intervals, and/or duration of the treatment regimen.

24. The method of claim 18, wherein the treatment regimen comprises administering an agent that inhibits interaction between Cls and C4.

25. The method of claim 24, wherein the agent is an antibody that inhibits interaction between Cls and C4.

26. The method of claim 25, wherein the antibody comprises a complementarity- determining region (CDR) having an amino acid sequence selected from the group consisting of:

a) SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and

SEQ ID NO:6; or

27. The method of claim 25, wherein the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO:7 or SEQ ID NO: 14 and a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO:8 or SEQ ID NO: 15.

28. The method of claim 25, wherein the antibody comprises:

29. The method of claim 18, wherein said assaying step comprises

30. The method of claims 25-28, comprising assaying the level of the antibody in the biological sample.

31. The method of claims 25-28, wherein a level of C4a that is lower than a pre- treatment level indicates that the agent is present in the individual in an effective amount.