Immunoassay of intermediate region pro-adrenomedullin using monoclonal antibodies

Abstract

The present invention relates to a method for measuring intermediate region proadrenomedullin (MR-proADM) in a sample, comprising using two labeled monoclonal antibodies that bind to (preferably different) epitopes of MR-proADM, and detecting a signal when both antibodies bind to MR-proADM. The method further relates to monoclonal antibodies or antibody fragments that bind to intermediate region proadrenomedullin (MR-proADM). The present invention further relates to diagnostic applications and methods, such as in vitro methods, for measuring MR-proADM in a sample using antibodies or antibody fragments.

Description

Technical Field

[0001] The present invention relates to the fields of diagnostic reagents, immunoassay methods, and antibodies for measuring or quantifying biomarkers in a sample.

[0002] The present invention relates to a method for quantifying mid-regional proadrenomedullin (MR-proADM) in a sample, comprising two types of labeled monoclonal antibodies that bind to (preferably different) epitopes of MR-proADM, and detecting a signal to MR-proADM by the binding of both antibodies.

[0003] In one aspect, it relates to an in vitro method for measuring mid-regional proadrenomedullin (MR-proADM) in a sample, the method comprising subjecting the sample to the following steps: (i) contacting with a first monoclonal antibody specific for a first epitope of MR-proADM, or a fragment thereof, and (ii) a second monoclonal antibody specific for a second epitope of MR-proADM or a fragment thereof, detecting the binding of the first and second monoclonal antibodies or fragments thereof to the MR-proADM, the first and second antibodies being labeled with a detectable label, and when these antibodies bind within the MR-proADM complex, the signal generated by these labels is modulated, said method.

[0004] In a preferred embodiment, the proximity of the donor and acceptor labels within the antibody-MR-proADM complex and the spectral overlap between the donor emission spectrum and the acceptor absorption spectrum of these labels enhance the signal and / or extend the lifetime of the signal, resulting in the ability to measure delayed fluorescence over time. In other preferred embodiments, the donor label contains a terbium cryptate or chelate, and the acceptor label contains: It is a fluorescent dye that has excitation and fluorescence spectra compatible with those of terbium (in time-resolved fluorescence resonance energy transfer (TR-FRET) reactions and / or time-resolved amplified cryptotate emission (TRACE) reactions).

[0005] The present invention further relates to a monoclonal antibody or antibody fragment that binds to the intermediate region proadrenomedullin (MR-proADM).

[0006] The present invention further relates to diagnostic applications and methods, such as in vitro diagnostic methods for measuring MR-proADM in a sample using the antibody or antibody fragment of the present invention, as well as corresponding methods for administering the antibody or antibody fragment of the present invention in medical applications and the treatment and / or prevention of medical conditions.

[0007] In a further embodiment, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antibody fragment of the present invention, a host cell comprising the nucleic acid molecule, a host cell capable of producing the antibody or antibody fragment of the present invention, and a composition such as a pharmaceutical composition comprising the antibody or antibody fragment of the present invention. [Background technology]

[0008] (Background of the invention) Adrenomedullin (ADM) is a peptide composed of 52 amino acids that is believed to have various physiological functions. Its potent vasodilatory effect has been reported in several studies. Measuring ADM has been shown to be useful in various clinical diagnostic and prognostic assessment situations. For example, elevated plasma ADM concentrations have been reported in the diagnosis, monitoring, and prognosis of various cardiovascular diseases and sepsis.

[0009] However, accurate measurement of ADM in the bloodstream is difficult. In addition to the immediate binding of ADM to receptors near its production site, peripheral measurement is also hindered by the presence of protein conjugates via autocrine and / or paracrine reactions, the short half-life of ADM, and technical difficulties. ADM is produced by post-translational processing from a larger precursor peptide (preproADM; 185 amino acids). Cleavage of pre-processed ADM (pre-pro-ADM) produces various smaller peptides, such as PAMP, mature (biologically active) ADM, or intermediate region proADM (MR-proADM).

[0010] Methods for detecting pro-ADM, particularly MR-proADM, have been previously reported, but these methods rely on polyclonal antibodies obtained, for example, by immunizing sheep. Morgenthaler (Clinical Chemistry 51:10; 1823-1829, 2005) reported on the technical characteristics of a sandwich immunoassay for measuring MR-proADM in human plasma, its reference interval in healthy individuals, and findings on elevated plasma concentrations in patients with cardiovascular disease or sepsis.

[0011] Despite the effectiveness of these assays, polyclonal antibodies may have potential problems such as cross-reactivity and limited stock. The "B·R·A·H·M·S MR-proADM KRYPTOR" system enables MR-proADM concentration measurement, providing a more accurate assessment of disease severity and patient risk management, while enhancing clinical investigation and treatment decision-making. In the intensive care unit, it enables immediate assessment of disease severity to maximize patient safety, guide optimal treatment, and provide early warning of complications.

[0012] The B·R·A·H·M·S KRYPTOR analyzer is a fully automated, closed-system testing system that can perform numerous tests using a random-access method based on its unique measurement principle called TRACE® (Time Resolved Amplified Cryptate Emission).

[0013] Nevertheless, KRYPTOR technology is based on the concept of immunoassay, and accurate MR-proADM measurement in clinical diagnosis requires sensitive, specific, and highly affinity antibodies. Caruhel et al. (Clinical Biochemistry 42 (2009) 725-728) describe the B·R·A·H·M·S KRYPTOR system and analysis of EDTA plasma samples from community-acquired pneumonia patients. The system uses TRACE technology and is based on non-radioactive energy transfer between the donor, the long-lived phosphor europium cryptote (EuC), and the acceptor, cyanine 5 (Cy5). However, the method described by Caruhel et al. has a common drawback to these systems because it relies on a polyclonal MR-proADM antibody.

[0014] U.S. Patent 2019 / 178897 discloses a method for diagnosing organ dysfunction, including the measurement of pro-ADM and histones in patient samples. The use of a KRYPTOR device is disclosed within this method.

[0015] Although immunoassays for MR-proADM are available on the market, there is a need for alternative or improved immunoassays and antibodies that overcome the shortcomings of conventional techniques, such as those associated with polyclonal antibodies (cross-reactivity and limited stock), and that offer higher sensitivity and specificity than those previously described, thereby improving the detection of MR-proADM biomarkers. [Overview of the project]

[0016] Considering the prior art, the technical problem inherent in the present invention was to provide alternative or improved means for determining and / or measuring MR-proADM and / or diagnosing and / or treating conditions associated with MR-proADM.

[0017] Another objective of the present invention was to provide an immunoassay method that overcomes the shortcomings of the prior art.

[0018] One of the objectives of the present invention was to provide an immunoassay method with higher sensitivity than conventional methods. Another objective of the present invention is to provide a highly sensitive immunoassay method, preferably an even more sensitive immunoassay method, or an assay that has at least the same sensitivity as conventional methods and improved or equivalent accuracy in the quantification of MR-proADM in patient samples.

[0019] One of the objectives of the present invention is to provide a higher level of signal RFU than conventional methods, thereby improving or maintaining equivalent accuracy in the quantification of MR-proADM in patient samples.

[0020] Another object of the present invention is to provide an antibody or antibody fragment with improved properties. These problems are resolved by the features described in the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0021] Therefore, one aspect of the present invention relates to an in vitro method for measuring intermediate region pro-adrenomedullin (MR-proADM) in a sample, comprising the following steps: - The sample, i. A first monoclonal antibody, or a fragment thereof, that is specific to the first epitope of MR-proADM. ii. Contacting with a second monoclonal antibody or fragment thereof that is specific to the second epitope of MR-proADM, and - Detecting the binding of the first and second monoclonal antibodies or fragments thereof to MR-proADM. - The first and second antibodies are labeled with a detectable label, and when the first and second antibodies bind within an antibody-MR-proADM complex, the signal generated by these labels is modulated.

Mode for Carrying Out the Invention

[0022] As will be described in more detail below, the method and immunoassay of the present invention improve the existing B·R·A·H·M·S MR-proADM system by replacing the polyclonal antibodies used in the conventional system with monoclonal antibodies having improved characteristics, thereby leading to a system with improved sensitivity and signal intensity compared to the conventional system.

[0023] As described by Morgenthaler (Clinical Chemistry 51:10; 1823 - 1829, 2005), polyclonal antibodies were generated and effectively used to realize the current B·R·A·H·M·S MR-proADM system. Three peptides related to precursor proadrenomedullin (preproADM) were chemically synthesized and purified, and after immunizing sheep with these peptides, the resulting antibodies were purified. The corresponding peptide immobilized on a solid phase.

[0024] In the present invention, mouse antibodies were produced using different antigens and epitopes, isolated and developed as monoclonal antibodies, and tested in an assay format similar to the existing B·R·A·H·M·S MR-proADM system.

[0025] Surprisingly, by using two types of monoclonal antibodies as in the present invention, the results were significantly improved compared to conventional polyclonal techniques. The accuracy of MR-proADM quantification in patient samples is maintained equivalent to the prior art, but unexpectedly, a significantly improved RFU signal is obtained by using the monoclonal antibody approach.

[0026] The epitope targeted by the monoclonal antibody of the present invention is described in more detail below.

[0027] In one embodiment, the first antibody and the second antibody are dispersed in a liquid reaction mixture (homogeneous immunoassay).

[0028] In one embodiment, the first antibody is labeled with a donor label and the second antibody is labeled with an acceptor label.

[0029] In one embodiment, due to the proximity of the donor label and the acceptor label within the antibody-MR-proADM complex, and the overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, the signal is enhanced, or the lifetime of the signal is extended, enabling the measurement of time-delayed fluorescence.

[0030] In embodiments of the present invention, a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction or a time-resolved amplified cryptate luminescence (TRACE) reaction is utilized.

[0031] In this embodiment, the donor label contains a rare earth cryptate or chelate, preferably containing lanthanide ions, and the acceptor label contains a fluorescent dye or a chemiluminescent dye.

[0032] In a preferred embodiment, the first antibody is labeled with a donor label and the second antibody is labeled with an acceptor label. The donor and acceptor labels are in close proximity within the antibody-MR-proADM complex, and the donor emission spectrum and acceptor absorption spectrum overlap, modulating the signal (for example, the signal may be enhanced, the signal lifetime extended, or time-delayed fluorescence measurement may be possible).

[0033] Here, the donor label contains a rare earth cryptotate or chelate, and the acceptor label contains a fluorescent dye or chemiluminescent dye.

[0034] In a preferred embodiment, the method of the present invention utilizes the TRACE technology of a KRYPTOR system using lanthanide ions, and the acceptor label includes the following:

[0035] It is a fluorescent dye or chemiluminescent dye that is bound to the target monoclonal antibody. A unique combination of MR-proADM epitopes. Lanthanide emission is well-established in this field and offers several advantages for fluorescence-based biological assays, including a large Stokes shift (>150 nm) and multiple narrow emission bands (typically <10 nm with half-maximum), thus enabling efficient spectroscopy.

[0036] Lanthanide emission is typically characterized by the following properties:

[0037] The long luminescence lifetime, ranging from microseconds to milliseconds, enables time-resolved detection. Lanthanide ions (especially terbium and europium) offer significant advantages over organic fluorescent dyes. Compared to conventional organic fluorescent materials, lanthanide coordination compounds exhibit relatively long lifetimes, large Stokes shifts, and sharp luminescence characteristics.

[0038] Fluorescence Peak Profile: Due to these excellent properties, time-resolved reagents are based on the lanthanide fluorescence produced.

[0039] Terbium and europium probes typically incorporate metal ions into organic chelate ligands containing sensitizing chromophores. When excited by near-ultraviolet light in the absorption band, intramolecular energy transfer occurs via system crossovers to a triplet excited state and to the emission level of the chelated metal.

[0040] By directly binding lanthanum-based probes to antibodies, it becomes possible to develop highly sensitive time-resolved fluorescence resonance energy transfer (TR-FRET) assays and time-resolved amplification cryptotate luminescence (TRACE) assays for various practical applications. The B·R·A·H·M·S MR-proADM KRYPTOR system utilizes this TRACE technology, which is also used in preferred embodiments of the present invention.

[0041] Surprisingly, the signal intensity obtained with the method and system of the present invention was improved compared to similar assays based on conventional polyclonal antibodies, based on labeled monoclonal antibodies. Furthermore, the use of terbium labeling allowed for further enhancement of signal intensity in appropriate assay formats.

[0042] In one embodiment, the donor label contains terbium cryptotate or chelate.

[0043] In some embodiments, the donor label may include terbium, a terbium derivative, or terbium N-hydroxysuccinimide ester, such as C65H78N14O17Tb.

[0044] In one embodiment, the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye having an excitation and emission spectrum compatible with the following: the excitation and emission spectrum of terbium (in time-resolved fluorescence resonance energy transfer (TR-FRET) reactions and / or time-resolved amplified cryptotate emission (TRACE) reactions).

[0045] In this specification, the terms “compatible with the excitation and emission spectra of terbium” or “time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or time-resolved amplified cryptotate emission (TRACE) reaction” refer to selecting a suitable fluorescent dye molecule with complementary (compatible) excitation and emission spectra that enables effective energy transfer and subsequent detection in a time-resolved method (FRET or TRACE). For example, using terbium as the donor label, the acceptor label is selected to satisfy the following conditions: a matching excitation spectrum that is excited upon binding to the donor, and an emission spectrum detectable at an appropriate wavelength.

[0046] For example, this method is an advanced version of conventional methods that utilize radioactive energy transfer between a donor, the long-lived phosphor europium cryptote, and an acceptor, such as a phosphor that emits red wavelengths, like cyanine 5 (Cy5).

[0047] Energy transfer depends on the proximity between molecules and the overlap of the spectra of the two fluorescent dyes. In sandwich immunoassays, europium and acceptors are bound to specific antibodies. In conventional assays, europium is excited at 337 nm by a nitrogen laser, its energy is transferred to Cy5, and specifically re-emitted at 665 nm. This homogeneity assay uses time-resolved measurements and spectral selection to remove background signals from the medium and select specific signals.

[0048] Therefore, the present invention enables remarkable progress compared to the prior art by allowing the use of terbium instead of europium, and / or a novel monoclonal antibody, thereby enabling unexpected improvements over the prior art.

[0049] In one embodiment, the donor label comprises a terbium cryptotate or chelate having the following luminescence.

[0050] That is, the acceptor label is composed of a fluorescent dye having a spectrum in the range of 485-495, 540-550, 585-595 and / or 615-625 nm, and the acceptor label has an excitation spectrum corresponding to the emission spectrum of the donor label, and an emission spectrum different from the emission spectrum of the donor label, preferably in the wavelength range of 500-570 nm, more preferably 510-560 nm or 515-555 nm (green), or 580-770 nm, more preferably 600-780 nm, or 670-690 nm (red).

[0051] Appropriate acceptor luminescent dye molecules can be selected based on the compatibility of their excitation and emission spectra, details of which are described below.

[0052] In a preferred embodiment, the method comprises a fully automated homogeneous assay, preferably a homogeneous sandwich fluorescence immunoassay, employing time-resolved amplified cryptotate emission (TRACE) technology with non-radioactive energy transfer.

[0053] This process takes place between a donor and an acceptor, and the energy transfer depends on their proximity.

[0054] The overlap of the spectra of two fluorescent dyes.

[0055] In embodiments, the first and / or second antibody or fragment thereof is a murine antibody, preferably a mouse antibody, and for example, the antibody of the fragment may contain or be composed of an amino acid sequence isolated after immunization of a mouse subject.

[0056] In this embodiment, the first and second epitopes are distinct from each other and selected from the MR-proADM epitopes shown in SEQ ID NOs. 5 to 18.

[0057] As shown below, various monoclonal antibodies have been generated and validated in this system. These antibodies may be defined by their sequence, the associated epitope of MR-proADM to which they bind, or one or more of the structural and / or functional properties described herein.

[0058] The antibodies were named Ab1-7 and are described in detail below. In one embodiment, the antibody "Ab1" binds to an epitope according to SEQ ID NO: 5 or 6. In one embodiment, the antibody "Ab2" binds to an epitope according to SEQ ID NO: 7 or 8. In one embodiment, the antibody "Ab3" binds to an epitope according to SEQ ID NO: 9 or 10. In one embodiment, the antibody "Ab4" binds to an epitope according to SEQ ID NO: 11 or 12. In one embodiment, the antibody "Ab5" binds to an epitope according to SEQ ID NO: 13 or 14. In one embodiment, the antibody "Ab6" binds to an epitope according to SEQ ID NO: 15 or 16. In one embodiment, the antibody "Ab7" binds to an epitope according to SEQ ID NO: 17 or 18.

[0059] In some embodiments, preferred combinations of antibodies used in the method of the present invention are as follows: - Ab1 and Ab2 or Ab3, preferably Ab1 and Ab2, - Ab2 and Ab1, Ab4, Ab3, Ab5 or Ab6, preferably Ab2 and Ab3, - Ab4 and Ab2, Ab3 and Ab1, Ab2, or Ab7, - Ab5 and Ab2, - Ab6 and Ab2, and - Ab7 and Ab3.

[0060] Embodiments having a specific epitope and / or antibody sequence: In one embodiment, the first and / or second antibody includes VH and VL domains containing sequences according to SEQ ID NOs: 136 and 137 (Ab1), 151 and 152 (Ab2), 166 and 167 (Ab3), 181 and 182 (Ab4), 196 and 197 (Ab5), 211 and 212 (Ab6), or 226 and 227 (Ab7), respectively.

[0061] In embodiments, the first and second antibodies may be any combination of the antibodies described above (or elsewhere in this specification).

[0062] While the following specific combinations of embodiments are preferred, the invention is not limited to any one or more of these specific combinations.

[0063] Ab2 and Ab3: In one embodiment, the first and second epitopes are different and selected from SEQ ID NO: 7 or 8 (epitope of Ab2) and SEQ ID NO: 9 or 10 (epitope of Ab3). The donor label contains a terbium cryptotate or chelate, and the acceptor label contains a fluorescent dye having excitation and emission spectra that match the excitation and fluorescence spectra of terbium.

[0064] Spectrum (in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplified cryptote emission (TRACE) reaction, preferably by the method of claim 7).

[0065] In one embodiment, the first and second antibodies are different, and each antibody contains six CDRs. According to the following sequence, H-CDR1 corresponds to sequence number 153 or 154. H-CDR2 follows sequence number 155 or 156, H-CDR3 follows sequence number 157 or 158, L-CDR1 follows sequence number 159 or 160, L-CDR2 follows sequence number 161 or 162, L-CDR3 follows sequence number 163(Ab2), and H-CDR1 follows sequence number 168 or 169, H-CDR2 follows sequence number 170 or 171, H-CDR3 follows sequence number 172 or 173, L-CDR1 follows sequence number 174 or 175, L-CDR2 follows sequence number 176 or 177, and L-CDR3 follows sequence number 178 (Ab3).

[0066] Here, the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye having an excitation and emission spectrum that matches the excitation and emission spectrum of terbium (in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplified cryptotate emission (TRACE) reaction, preferably by the reaction described in claim 7).

[0067] In one embodiment, the first antibody and the second antibody are different and contain VH and VL. Domains conforming to sequence numbers 151 and 152 (Ab2), and sequence numbers 166 and 167 (Ab3).

[0068] Ab2 and Ab4: In one embodiment, the first and second epitopes are different and selected from SEQ ID NO: 7 or 8 (epitope of Ab2) and SEQ ID NO: 11 or 12 (epitope of Ab4). The donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye.

[0069] It has excitation and emission spectra that are compatible with those of terbium. Spectrum (in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplified cryptote emission (TRACE) reaction, preferably by the method of claim 7).

[0070] In one embodiment, the first and second antibodies are different, and each antibody contains six CDRs. The 30 sequences follow this pattern: H-CDR1 corresponds to sequence number 153 or 154. H-CDR2 follows sequence number 155 or 156, H-CDR3 follows sequence number 157 or 158, L-CDR1 follows sequence number 159 or 160, L-CDR2 follows sequence number 161 or 162, L-CDR3 follows sequence number 163(Ab2), and H-CDR1 is according to SEQ ID NO: 183 or 184, H-CDR2 is according to SEQ ID NO: 185 or 186, H-CDR3 is according to SEQ ID NO: 187 or 188, L-CDR1 is according to SEQ ID NO: 189 or 190, L-CDR2 is according to SEQ ID NO: 191 or 192, and L-CDR3 is according to SEQ ID NO: 193 (Ab4), where the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye having an excitation and emission spectrum compatible with the excitation and emission spectrum of terbium (in time-resolved fluorescence resonance energy transfer (TR-FRET) reactions and / or time-resolved amplified cryptotate emission (TRACE) reactions, preferably according to claim 7).

[0071] In one embodiment, the first and second antibodies differ and contain VH and VL domains according to SEQ ID NOs: 151 and 152 (Ab2), and SEQ ID NOs: 181 and 182 (Ab4), respectively.

[0072] Ab1 and Ab2: In one embodiment, the first and second epitopes are different, such as sequence number 5 or 6 (epitope of Ab1), and Sequence ID: 7 or 8 (epitope of Ab2), the donor label contains terbium cryptotate or chelate, and the acceptor label is a fluorescent dye having excitation and emission spectra compatible with the excitation and emission spectra of terbium in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction. and / or comprising a time-resolved amplification cryptotate luminescence (TRACE) reaction, preferably according to claim 7.

[0073] In one embodiment, the first antibody and the second antibody are different, each having six CDR sequences according to the following: H-CDR1 corresponds to sequence number 138 or 139, H-CDR2 corresponds to sequence number 140 or 141, H-CDR3 corresponds to sequence number 142 or 143, and L-CDR1 corresponds to sequence number 144 or 145. L-CDR2 follows sequence number 146 or 147, and L-CDR3 follows sequence number 148. According to (Ab1), and H-CDR1 corresponds to sequence number 153 or 154, H-CDR2 corresponds to sequence number 155 or 156, H-CDR3 corresponds to sequence number 157 or 158, L-CDR1 corresponds to sequence number 159 or 160, L-CDR2 corresponds to sequence number 161 or 162, and L-CDR3 corresponds to sequence number 163. (Ab2), Here, the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye having an excitation and emission spectrum compatible with the excitation and emission spectrum of terbium (preferably according to claim 7 in time-resolved fluorescence resonance energy transfer (TR-FRET) reactions and / or time-resolved amplified cryptotate emission (TRACE) reactions).

[0074] In one embodiment, the first and second antibodies are different and contain VH and VL domains according to SEQ ID NOs. 136 and 137 (Ab1), 151 and 152 (Ab2).

[0075] Ab1 and Ab3: In one embodiment, the first and second epitopes are different, such as sequence number 5 or 6 (epitope of Ab1), and selected from Sequence ID No. 9 or 10 (epitope of Ab3), and the donor label contains a terbium cryptotate or chelate, and the acceptor label contains a fluorescent dye having an excitation-emission spectrum that matches the excitation and emission spectrum of terbium (time-resolved fluorescence resonance energy transfer (TR-FRET) reaction, and / or in a time-resolved amplification cryptote emission (TRACE) reaction, preferably according to claim 7).

[0076] In one embodiment, the first antibody and the second antibody are different, each having six CDR sequences according to the following:

[0077] H-CDR1 follows SEQ ID NO: 138 or 139, H-CDR2 follows SEQ ID NO: 140 or 141, H-CDR3 follows SEQ ID NO: 142 or 143, L-CDR1 follows SEQ ID NO: 144 or 145, L-CDR2 follows SEQ ID NO: 146 or 147, L-CDR3 follows SEQ ID NO: 148 (Ab1), and

[0078] H-CDR1 follows sequence number 168 or 169, H-CDR2 follows sequence number 170 or 171,

[0079] H-CDR3 follows sequence number 172 or 173, L-CDR1 follows sequence number 174 or 175, L-CDR2 follows sequence number 176 or 177, L-CDR3 follows sequence number 178 (Ab3),

[0080] Here, the donor label contains terbium cryptotate or chelate, and the acceptor label contains a fluorescent dye having excitation and emission spectra compatible with terbium.

[0081] Excitation and emission spectra (in time-resolved fluorescence resonance energy transfer (TR-FRET) reactions and / or time-resolved amplified cryptotate emission (TRACE) reactions, preferably according to claim 7).

[0082] In one embodiment, the first and second antibodies are different, comprising VH and VL domains (Ab1) according to SEQ ID NOs: 136 and 137, and VH and VL domains (Ab3) according to SEQ ID NOs: 166 and 167.

[0083] In this embodiment, the antibodies used in the embodiments described above (or elsewhere in this specification) may be defined by the epitopes to which the antibodies bind. For example, the epitopes to which Ab1-4 bind are unique and novel epitopes not found in the prior art, enabling unexpectedly effective sensitivity in MR-proADM detection in TRACE-based detection systems. In the art, there is no indication of using antigens and epitopes as disclosed with respect to the antibodies of the present invention, and the enhancement of the TRACE signal obtained using the labeling and detection systems described herein was a surprise.

[0084] In embodiments, the antibodies used in the embodiments described above (or elsewhere in this specification) may be defined by the CDR sequence of each antibody disclosed herein, with or without complete specific VH and VL sequences, or in combination thereof.

[0085] The sequences, for example, the CDR sequences of the antibodies Ab1, Ab2, Ab3, and Ab4, are disclosed in the sequence listing of this application.

[0086] In embodiments, antibodies may be defined by a combination of CDR sequences of each antibody, and sequences having at least 70%, 80%, 90%, or 95% sequence identity with respect to specific VH and VL sequences of each antibody.

[0087] Embodiments using surrogate antibody sequences: In this embodiment, the first and / or second antibody comprises VH and VL domains, which include sequences corresponding to SEQ ID NOs: 21 and 22 (Ab1), 39 and 40 (Ab2), 54 and 55 (Ab3), 68 and 69 (Ab4), 84 and 85 (Ab5), 102 and 103 (Ab6), or 119 and 120 (Ab7), respectively.

[0088] In one embodiment, the first and second antibodies are different and comprise VH and VL domains (Ab2) according to SEQ ID NOs: 39 and 40 and VH and VL domains (Ab3) according to SEQ ID NOs: 54 and 55, and the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye having excitation and emission spectra compatible with terbium.

[0089] Excitation and emission spectra (time-resolved fluorescence resonance energy transfer) (In TR-FRET reactions and / or time-resolved amplification cryptotate luminescence (TRACE) reactions).

[0090] In one embodiment, the first and second antibodies differ, each containing VH and VL domains (Ab2) according to SEQ ID NOs: 39 and 40, and VH and VL domains (Ab4) according to SEQ ID NOs: 68 and 69, respectively, and the donor label contains a terbium cryptotate or chelate, while the acceptor label contains a fluorescent dye with excitation and emission spectra compatible with terbium.

[0091] Excitation and emission spectra (in time-resolved fluorescence resonance energy transfer (TR-FRET) reactions and / or time-resolved amplified cryptotate emission (TRACE) reactions).

[0092] In one embodiment, the first and second antibodies are different, each having VH and VL domains, and conform to SEQ ID NOs: 21 and 22 (Ab1), and SEQ ID NOs: 54 and 55 (Ab3).

[0093] Here, the donor label contains terbium cryptotate or chelate, and the acceptor label contains This includes fluorescent dyes with excitation and fluorescence spectra compatible with terbium (in time-resolved fluorescence resonance energy transfer (TR-FRET) reactions and / or time-resolved amplification cryptotate fluorescence (TRACE) reactions).

[0094] In one embodiment, the first antibody and the second antibody are different and contain VH and VL.

[0095] Domains according to Sequence IDs 21 and 22 (Ab1), and domains according to Sequence IDs 39 and 40 (Ab2), wherein the donor label contains a terbium cryptotate or chelate, and the acceptor label contains a fluorescent dye with excitation and emission spectra compatible with terbium (in time-resolved fluorescence resonance energy transfer (TR-FRET) and / or time-resolved amplified cryptotate emission (TRACE) reactions).

[0096] In some embodiments, the antibodies used in the embodiments described above (or elsewhere in this specification) may be defined by the CDR sequence of each antibody disclosed herein, either in combination with or independently of the complete specific VH and VL sequences.

[0097] In an embodiment, the antibody may be defined by the following combination of CDR sequences. A sequence having at least 70%, 80%, 90%, or 95% sequence identity with respect to each antibody and the specific VH and VL sequences of each antibody.

[0098] Yet another embodiment: The present invention also includes embodiments corresponding to any combination of any one or more of Ab1 to Ab7, preferably the combinations disclosed herein.

[0099] As shown in the following examples, different antibody and fluorescent dye molecule combinations, particularly the preferred combinations disclosed herein, have been shown to produce relatively larger signals measured in relative fluorescence units (RFU) compared to the conventional B·R·A·H·M·S MR-proADM assay. These characteristics mean that larger signal emission may allow for lower detection limits, intra-assay precision, and / or improved functional assay sensitivity compared to conventional assays.

[0100] Furthermore, there is no decrease in accuracy, Since absolute MR-proADM concentration measurement shows high agreement with existing assays, the present invention can achieve similarly reliable accuracy in the measurement of MR-proADM and may offer advantages through more sensitive detection.

[0101] In the embodiment, the antibodies and fluorescent dyes used enable an improvement in the detection limit compared to conventional assay methods based on lower conventional alternative antibodies (e.g., polyvalent antibodies) and / or alternative fluorescent dyes.

[0102] For example, in an embodiment, the lower detection limit of the measurement method of the present invention is measured using the method described in reference Morgenthaler (Clinical Chemistry 51:10; 1823-1829, 2005), for example, using horse serum (mean value plus 2 standard deviations of measurements with mean relative light units of 20), which is less than 0.08 nmol / L, preferably less than 0.05 nmol / L, more preferably less than 0.01 nmol / L, or 0.005 nmol / L.

[0103] In this embodiment, the antibody and fluorescent dye molecules used enable improved intra-assay accuracy compared to conventional assays based on conventional alternative antibodies (e.g., polyclonal antibodies) and / or alternative fluorescent dye molecules.

[0104] For example, the intra-assay precision of the assay of the present invention is less than 5%, preferably less than 4%, 3%, 2%, or 1%, when multiple (e.g., 10 to 30) human EDTA plasma samples covering a concentration range of 0.01 to 20.00 nmol / L are measured in multiple parallel measurements (e.g., 5 to 15 times) using the method described in reference Morgenthaler (Clinical Chemistry 51:10; 1823-1829, 2005).

[0105] In some embodiments, the antibodies and fluorescent dye molecules used enable improved sensitivity of functional assays compared to assays based on conventional antibodies (e.g., polyclonal antibodies) and / or conventional fluorescent dye molecules.

[0106] For example, in embodiments of the present invention, the functional assay sensitivity of this measurement method is improved compared to the prior art when using the method described in reference Morgenthaler (Clinical Chemistry 51:10; 1823-1829, 2005). For example, the functional assay sensitivity can be evaluated as an MR-proADM concentration with an inter-assay precision of 20%.

[0107] In one aspect, the present invention further relates to analytical, detection, and / or diagnostic kits for the following purposes:

[0108] Uses in the methods of the present invention, including the following: - A first monoclonal antibody and a second monoclonal antibody, or fragments thereof, which bind to the first and second epitopes of MR-proADM, respectively, and - One or more donor labels containing rare earth cryptotes or chelates, preferably containing lanthanide ions, more preferably containing terbium chelates or cryptotes, and This method uses one or more acceptor labels containing a fluorescent dye or a chemiluminescent dye, wherein, The excitation and emission spectra of the acceptor label match those of the donor label (preferably according to claim 7 in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplification cryptotate emission (TRACE) reaction). Here, the label is either physically adjacent to the antibody in the kit or conjugated to the antibody.

[0109] In one embodiment, the kit includes the following: a. Detection reagents for measuring the level of precursor ADM (proADM) or its fragments. In patient samples, for example, using antibodies described herein, and b. Reference data, particularly reference values, thresholds, or cutoff values, for any disease associated with MR-proADM, or when MR-proADM may be used as a diagnostic, prognostic, or treatment guideline marker, wherein such reference data is preferably stored on a computer-readable medium and / or made available in a computer-executable format, configured to compare the determined level of proADM or its fragments with the following: Cutoff value, d. Furthermore, a detection reagent (preferably PCT, D-dimer, troponin, IP10, IL6, body weight and / or age) for measuring the level of at least one additional parameter or biomarker, or fragment thereof, in the patient sample, and reference data (e.g., a reference level for the risk threshold or cutoff value of the at least one additional biomarker, preferably PCT) The reference data is preferably stored on a computer-readable medium and / or used in the form of computer-executable code, such as an algorithm configured to compare the measurement level of at least one additional biomarker or fragment thereof with a threshold or cutoff value.

[0110] The present invention has a wide range of clinical applications, including, but not limited to, the determination of infections, adverse events, death, organ dysfunction or injury, and the diagnosis of the severity and prognosis of the risk of developing serious health conditions in patients. This includes both rule-in and rule-out of medical conditions, or risk assessment and determination of treatment effectiveness (responders / non-responders) in patients.

[0111] By using the kit and method of the present invention, it becomes possible to make decisions regarding patient diagnosis, prognosis, or treatment guidance based on objective criteria such as reference data from the kit.

[0112] The kit's reference data can be provided on computer executable code configured to compare measurements of ProADM or its fragments with the aforementioned reference range.

[0113] In some embodiments, the features of the kit can be considered to include features used to characterize the method of the present invention, and vice versa.

[0114] Further measurable markers in the context of the present invention include lactate and CRP. Clinical scores that can be evaluated in the context of the present invention include the SI score (systemic inflammation score), SOFA, qSOFA, SIRS, SAPS II, APACHE II, CRB-65, and PSI.

[0115] In the context of the method of the present invention, it has been shown that measuring proADM or a portion of its fragments, as well as at least one clinical score such as SOFA, PSI, or CRB-65, can provide even greater accuracy regarding the prognosis of subsequent conditions requiring hospitalization. In yet another embodiment, at least one additional biomarker is PCT, lactate, at least one marker of rhabdomyolysis, such as creatine kinase (CK), lactate dehydrogenase (LDH), creatinine, myoglobin, aldolase, troponin, carbonic anhydrase type 3, fatty acid-binding protein (FABP), transaminase, or potassium.

[0116] A detection reagent (antibody) for measuring the level of proADM or its fragments, and optionally a detection reagent for measuring the levels of PCT, lactate, and / or C-reactive protein or its fragments, is preferably selected from those necessary to carry out the method. For example, an antibody against MR-proADM, appropriate labeling (such as fluorescent labeling), preferably two different fluorescent labels suitable for application in the KRYPTOR assay.

[0117] Sample collection tube The embodiments and features disclosed with respect to the methods of the present invention also apply to the kits of the present invention, and vice versa.

[0118] In one embodiment of the method described herein, the method further includes the step of comparing the measured level of proADM or its fragments with a reference level, a threshold in the patient, and / or a population mean corresponding to proADM or its fragments.

[0119] The aforementioned comparison is performed on a computer processor using computer-executable code.

[0120] The method of the present invention may be partially performed by computer. For example, the step of comparing the level of a detected marker (e.g., proADM or its fragments) with a reference level can be performed on a computer system. The measured marker levels can be combined with the levels of other markers and / or subject parameters to calculate a score that indicates diagnosis, prognosis, prediction, risk assessment, and / or risk stratification. For example, the measured values ​​can be entered either manually by a healthcare professional or automatically from below. The device on which each marker level was measured may be recorded in a computer system.

[0121] Computer systems may be installed directly in clinical settings (e.g., primary care, ICU, emergency department) or remotely via computer networks (e.g., the internet or specialized medical cloud systems), and can be combined with other IT systems and platforms, such as hospital information systems (HIS), as needed. Typically, the computer system stores values ​​(e.g., marker levels, age, blood pressure, weight, sex, or parameters such as clinical assessment score systems like SOFA, qSOFA, and BMI) on a computer-readable storage medium and calculates scores based on predefined and / or pre-stored reference levels and values. The resulting scores are displayed and / or printed to the user (usually a medical professional such as a physician). Alternatively, or in addition, relevant prognosis, diagnosis, evaluation, treatment guidance, patient management guidance, or stratification may be displayed and / or printed to the user (usually a medical professional such as a physician).

[0122] In one embodiment of the present invention, a software system equipped with a machine learning algorithm can be used, preferably using data from an electronic health record (EHR), to identify hospitalized patients at risk of sepsis, severe sepsis, and septic shock. The machine learning method can be trained using a random forest classifier with EHR data from patients (e.g., biomarker expression, vital signs, and demographic information).

[0123] Machine learning is a type of artificial intelligence that gives computers the ability to learn complex patterns in data without being explicitly programmed, unlike simpler rule-based systems.

[0124] Previous research has focused on using electronic medical record data to issue alerts for general deterioration of clinical conditions. In one embodiment of the present invention, proADM level processing may be incorporated into appropriate software for comparison with existing datasets, and for example, proADM levels may be processed in machine learning software to support the diagnosis of adverse event occurrence and prognosis prediction.

[0125] The use of MR-proADM antibodies in combination with other biomarker assays (e.g., for PCT or CRP) can be performed in a single multiplex assay or in two separate assays for patient-derived samples. Samples may be related to the same substance, different samples, or different samples.

[0126] Assays used for detection and measurement MR-proADM and, for example, PCT, may be the same or different, and immunoassays may be used to measure any of the above markers. A more detailed description of appropriate assays is provided below.

[0127] Furthermore, functional assay sensitivity can be measured to indicate a statistically significant value that can be used as a reference value or cutoff value according to established techniques.

[0128] Each laboratory can independently establish the functional sensitivity of an assay according to clinically relevant protocols. "Functional sensitivity" can be defined as the concentration at which the coefficient of variation (CV) is 20% (or another predetermined %CV, or the accuracy of the cross-assay), and therefore serves as an indicator of the assay's accuracy at low concentrations of the analyte. The coefficient of variation (or functional assay sensitivity) is a standardized standard deviation (SD) that allows for comparison of variability assessments regardless of the magnitude of the analyte concentration and is applicable at least across the majority of the assay's working range.

[0129] Furthermore, methods based on ROC analysis can be used to determine statistically significant differences between two clinical patient groups. Receiver operational characteristic (ROC) curves measure the efficiency of classification by the model's fit probability and sort response levels. ROC curves are also useful in setting judgment criteria in diagnostic tests. A higher degree of curvature from the diagonal indicates a better fit. If a logistic fit has more than one response level, a generalized ROC curve is generated. In such a plot, there is a curve corresponding to each response level, and that curve is the ROC curve for that level versus all other levels.

[0130] Software capable of enabling this type of analysis to set appropriate reference levels and cutoff values ​​is available, such as SAS's JMP 12, JMP 13, and Statistical Discovery.

[0131] Cutoff values ​​can be similarly determined for other biomarkers used in combination with MR-proADM. Literature for determining appropriate cutoff values ​​is available to those skilled in the art; for example, Philipp Schuetz et al. (BMC Medicine. 2011; 9:107) found that PCT was highly sensitive in ruling out infection at a cutoff value of 0.1 ng / mL. Terence Chan et al. (Expert Rev. Mol. Diagn. 2011; 11(5), 487-496) describe that indicators such as positive likelihood ratios and negative likelihood ratios calculated based on sensitivity and specificity are also useful in evaluating the effectiveness of diagnostic tests. The values ​​are generally graphed as receiver-operated characteristic curves (ROC curves) for multiple cutoff values ​​(CV). The area under the curve values ​​is used to determine the CV that is most diagnostically relevant. This literature describes the variability of CV (cutoff value) (which depends on the measurement method and study design) and appropriate methods for determining cutoff values.

[0132] The population mean of MR-proADM or its fragments can also be used as a reference value. For example, by using the population mean of MR-proADM, a patient sample can be compared to a control group. The control group preferably includes 10, 20, 30, 40, or 50 or more subjects.

[0133] In one embodiment of the present invention, the cutoff value for MR-proADM can be a value in the range of 0.005 to 50.00 nmol / L in a plasma sample for any diagnostic, prognostic, or therapeutic guideline statement. In some embodiments, the cutoff level is in the range of 0.01 to 30, 0.05 to 20, 0.1 to 15, or 0.1 to 10 ng / mL. Any value within these ranges may be considered an appropriate cutoff value.

[0134] In one embodiment of the present invention, coupled PCT measurement may be used in parallel with or in combination with the measurement method of the present invention.

[0135] In embodiments, the PCT cutoff value may be in the range of 0.01 to 100.00 ng / mL in a serum sample, for example, when using the Luminex MAC Pix E-Bioscience assay or the B·R·A·H·M·S PCT-Kryptor assay. In preferred embodiments, the PCT cutoff level is in the range of 0.01 to 100, 0.05 to 50, 0.1 to 20, or 0.1 to 2 ng / mL, most preferably >0.05 It may be up to 0.5 ng / mL. Any value within these ranges may be considered an appropriate cutoff value. For example, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL may be used. In some embodiments, the PCT level in healthy subjects is approximately 0.05 ng / mL.

[0136] Embodiments related to the antibody of the present invention and its applications: In view of the prior art, another technical problem of the present invention was to provide alternative or improved binders suitable as diagnostic, therapeutic and / or prophylactic reagents for identifying, quantifying and / or measuring MR-proADM and / or medical conditions associated with MR-proADM.

[0137] Another object of the present invention is to provide an antibody or antibody fragment with improved properties.

[0138] Another object of the present invention is to provide an antibody or antibody fragment with improved affinity for the MR-proADM epitope.

[0139] Another object of the present invention is to provide an antibody or antibody fragment with improved detection sensitivity for MR-proADM.

[0140] Another object of the present invention is to provide an antibody or antibody fragment that reduces unwanted and nonspecific cross-reactivity with non-target proteins.

[0141] Therefore, the present invention also relates to monoclonal antibodies or antibody fragments having the characteristic of binding to the intermediate region pro-adrenomedullin (MR-proADM).

[0142] The antibodies described herein may be used in embodiments of the methods, kits, and other aspects of the invention.

[0143] In some embodiments, the antibody or fragment thereof is a mouse-derived antibody, preferably a mouse antibody. For example, the antibody or fragment thereof may include, or be composed of, an amino acid sequence isolated after immunization of a mouse subject.

[0144] In some embodiments, the antibody or its fragment binds to the MR-proADM epitope in the sequence according to SEQ ID NO: 3.

[0145] In embodiments of the present invention, an antibody or its fragment binds to an MR-proADM epitope according to one or more of SEQ ID NOs: 5 to 18.

[0146] In one embodiment, antibody "Ab1" binds to an epitope according to SEQ ID NO: 5 or 6. In one embodiment, antibody "Ab2" binds to an epitope according to SEQ ID NO: 7 or 8.

[0147] In one embodiment, the antibody "Ab3" binds to an epitope according to SEQ ID NO: 9 or 10.

[0148] In one embodiment, antibody "Ab4" binds to an epitope according to SEQ ID NO: 11 or 12. In one embodiment, antibody "Ab5" binds to an epitope according to SEQ ID NO: 13 or 14. In one embodiment, antibody "Ab6" binds to an epitope according to SEQ ID NO: 15 or 16. In one embodiment, antibody "Ab7" binds to an epitope according to SEQ ID NO: 17 or 18.

[0149] In one embodiment, an antibody or antibody fragment is defined by one or more complementarity-determining regions (CDRs) as described herein, preferably six CDR regions, i.e., three from heavy chain variable regions and three from light chain variable regions. The CDRs described herein may be used in a specific combination found, or in any combination that results in binding to the target protein while maintaining the target protein.

[0150] In some embodiments, the antibody comprises the following: A single heavy chain (variable VH) domain, wherein the VH domain contains the following complementarity-determining region (CDR) sequence:

[0151] H-CDR1 is selected from sequence numbers: 138, 139, 153, 154, 168, 169, 183, 184, 198, 199, 213, 214, 228, or 229, or a CDR sequence having at least 80%, preferably 90%, identity with any of the above CDR sequences.

[0152] H-CDR2 is selected from sequence numbers: 140, 141, 155, 156, 170, 171, 185, 186, 200, 201, 215, 216, 230, or 231, or a CDR sequence having at least 80%, preferably 90%, identity with any of the above CDR sequences.

[0153] H-CDR3 is a CDR sequence and a light-chain variable (VL) domain selected from sequence numbers: 142, 143, 157, 158, 172, 173, 187, 188, 202, 203, 217, 218, 232, or 233, or having at least 80%, preferably 90%, identity to any of the aforementioned CDR sequences, wherein the VL domain includes the following complementarity-determining region (CDR) sequences:

[0154] - L-CDR1 is a CDR sequence selected from sequence numbers 144, 145, 159, 160, 174, 175, 189, 190, 204, 205, 219, 220, 234, or 235, or a CDR sequence having at least 80%, preferably 90%, identity with any of the above CDR sequences. - L-CDR2 is selected from sequence numbers: 146, 147, 161, 162, 176, 177, 191, 192, 206, 207, 221, 222, 236 or 237, or a CDR sequence having at least 80%, preferably 90%, identity with any of the aforementioned CDR sequences, and - L-CDR3 is selected from sequence numbers: 148, 163, 178, 193, 208, 223, or 238, or A CDR sequence having at least 80%, preferably 90%, identity with any of the aforementioned CDR sequences.

[0155] In this embodiment, the antibody comprises the following: A single heavy chain (variable VH) domain, wherein the VH domain contains the following complementarity-determining region (CDR) sequence: - H-CDR1 is selected from sequence numbers: 138, 153, 168, 183, 198, 213, or 228. - H-CDR2 is selected from sequence numbers: 140, 155, 170, 185, 200, 215, or 230. - H-CDR3 is selected from sequence numbers: 142, 157, 172, 187, 202, 217, or 232. and a light chain variable (VL) domain, the VL domain comprising the following complementarity-determining region (CDR) sequence: - L-CDR1 is selected from sequence numbers: 144, 159, 174, 189, 204, 219, or 234. - L-CDR2 is selected from sequence numbers: 146, 161, 176, 191, 206, 221, or 236. - L-CDR3 is selected from sequence numbers: 148, 163, 178, 193, 208, 223, or 238.

[0156] In some embodiments, the antibody comprises the following: A single heavy chain (variable VH) domain, wherein the VH domain contains the following complementarity-determining region (CDR) sequence: - H-CDR1 is selected from sequence numbers: 139, 154, 169, 184, 199, 214, or 229. - H-CDR2 is selected from sequence numbers: 141, 156, 171, 186, 201, 216, or 231. - H-CDR3 is selected from sequence numbers: 143, 158, 173, 188, 203, 218, or 233. and a light chain variable (VL) domain, the VL domain comprising the following complementarity-determining region (CDR) sequence: - L-CDR1 is selected from sequence numbers: 145, 160, 175, 190, 205, 220, or 235. - L-CDR2 is selected from sequence numbers: 147, 162, 177, 192, 207, 222, or 237. - L-CDR3 is selected from sequence numbers: 148, 163, 178, 193, 208, 223, or 238.

[0157] In embodiments of the present invention, the antibody or antibody fragment comprises a heavy chain variable (VH) domain, the VH domain comprising a sequence having at least 70%, preferably 80%, and more preferably 90% identity with any of sequence numbers: 136, 151, 166, 181, 196, 211, or 226. It also comprises a light chain variable (VL) domain, the VL domain comprising a sequence having at least 70%, preferably 80%, and more preferably 90% identity with any of sequence numbers: 137, 152, 167, 182, 197, 212, or 227.

[0158] In some embodiments, the antibody or antibody fragment of the present invention has a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH and VL comprise a CDR sequence according to the following:

[0159] [Table 1-1]

[0160] [Table 1-2]

[0161] In embodiments of the present invention, in any given CDR, the first CDR sequence listed above can be combined with any other first CDR sequence. In some embodiments, in any given CDR, the second CDR sequence listed above can be combined with any other second CDR sequence.

[0162] In some embodiments, the antibody or antibody fragment of the present invention has VH and VL domains containing sequences according to SEQ ID NOs: 136 and 137 (Ab1), 151 and 152 (Ab2), 166 and 167 (Ab3), 181 and 182 (Ab4), 196 and 197 (Ab5), 211 and 212 (Ab6), or 226 and 227 (Ab7), respectively.

[0163] In some embodiments, the CDR sequences shown above are presented with alternative sequences, and each CDR may be selected from one, two, or three similar sequences. In some embodiments of the present invention, these alternative examples originate from alternative annotations of variable sequences, and the definition of the CDR sequences is found using, for example, imgt or Kabat CDR prediction models.

[0164] In some embodiments, any combination of six CDRs, each containing at least one of H-CDR1-3 and L-CDR1-3, obtained from any given selection or combination of CDR annotation models, is considered to be included in the present invention and disclosed herein.

[0165] Embodiments for surrogate antibody sequences: In some embodiments, the antibody comprises the following: A single heavy chain (variable VH) domain, wherein the VH domain contains the following complementarity-determining region (CDR) sequence: - H-CDR1 is selected from sequence numbers: 23, 24, 25, 41, 42, 56, 57, 58, 70, 71, 86, 87, 88, 104, 105, 121, 122, or 123, or a CDR sequence having at least 80%, preferably 90%, identity with any one of the aforementioned CDR sequences. - H-CDR2 is selected from sequence numbers: 26, 27, 28, 43, 44, 59, 72, 73, 74, or 89, 90, 91, 106, 107, 124, 125, or 126, or a CDR sequence having at least 80%, preferably 90%, identity with any of the above CDR sequences. - H-CDR3 is selected from sequence numbers: 29, 30, 31, 45, 60, 75, 92, 93, 108, 109, 110, 127, or 128, or any CDR sequence having at least 80%, preferably 90%, identity with the aforementioned CDR sequences. and a light chain variable (VL) domain, the VL domain comprising the following complementarity-determining region (CDR) sequence: aL-CDR1 is selected from sequence numbers: 32, 33, 46, 47, 61, 62, 76, 77, 94, 95, or 111, 112, 129, or 130, or a CDR sequence having at least 80%, preferably 90%, identity with any of the above CDR sequences. bL-CDR2 is selected from SEQ ID NOs: 34, 35, 48, 49, 63, 64, 78, 79, 80, 96, 97, 113, 114, 131, 132, or at least 80%, preferably 90% Any CDR sequence that is identical to any of the following, cL-CDR3 is selected from sequence numbers: 36, 50, 51, 65, 81, 98, 99, 115, 116, or 133. Alternatively, a CDR sequence having at least 80%, preferably 90%, identity with any of the above-mentioned CDR sequences.

[0166] In some embodiments, the antibody comprises the following: A single heavy chain (variable VH) region, the VH region includes the following complementarity-determining region (CDR) sequence: - H-CDR1 is selected from sequence numbers: 23, 24, 25, 41, 42, 56, 57, 58, 70, 71, 86, 87, 88, 104, 105, 121, 122, or 123. - H-CDR2 is selected from sequence numbers: 26, 27, 28, 43, 44, 59, 60, 72, 73, 74, 89, 90, 91, 106, 107, 124, 125 or 126. - H-CDR3 is selected from sequence numbers: 29, 30, 31, 45, 60, 75, 92, 93, 108, 109, 110, 127 or 128. and a light chain variable (VL) domain, the VL domain comprising the following complementarity-determining region (CDR) sequence: dL-CDR1 is selected from sequence numbers: 32, 33, 46, 47, 61, 62, 76, 77, 78, 94, 95, 111, 112, 129, or 130. eL-CDR2 is selected from sequence numbers: 34, 35, 48, 49, 63, 64, 78, 79, 80, 96, 97, 113, 114, 131, 132. fL-CDR3 is selected from sequence numbers: 36, 50, 51, 65, 81, 98, 99, 115, 116, or 133. In some embodiments, the antibody or antibody fragment of the present invention comprises a heavy chain variable (VH) domain, the VH domain comprising a sequence having at least 70% identity with SEQ ID NOs: 21, 39, 54, 68, 84, 102, or 119. and a light chain variable (VL) domain, the VL domain containing a sequence having at least 70% identity to any of sequence numbers: 22, 40, 55, 69, 85, 103, or 120.

[0167] In some embodiments, the antibody or antibody fragment of the present invention has a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH and VL domains conform to the following CDR sequence: - H-CDR1 conforms to Sequence ID: 23, 24, or 25, - H-CDR2 conforms to Sequence ID: 26 or 28, - H-CDR3 conforms to Sequence ID: 29 or 31, - L-CDR1 conforms to sequence number: 32 or 33, - L-CDR2 conforms to Sequence ID: 34 or 35, and - L-CDR3 conforms to Sequence ID: 36, (Alternative Ab1)

[0168] or - H-CDR1 conforms to Sequence ID: 41 or 42, - H-CDR2 conforms to Sequence ID: 43 or 44, - H-CDR3 conforms to sequence number 45, - L-CDR1 conforms to Sequence ID: 46 or 47, - L-CDR2 conforms to Sequence ID: 48 or 49, and - L-CDR3 conforms to Sequence ID: 50 or 51 (Alternative Ab2)

[0169] or - H-CDR1 conforms to Sequence ID: 56, 57, or 58, - H-CDR2 conforms to Sequence ID: 59 or 59, - H-CDR3 conforms to Sequence ID: 60, - L-CDR1 conforms to sequence number: 61 or 62, - L-CDR2 conforms to Sequence ID: 63 or 64, and - L-CDR3 conforms to Sequence ID: 65, (Alternative Ab3)

[0170] or - H-CDR1 conforms to Sequence ID: 70 or 71, - H-CDR2 conforms to Sequence ID: 72, 73, or 74, - H-CDR3 conforms to Sequence ID: 75, - L-CDR1 conforms to sequence number: 76 or 77, - L-CDR2 conforms to Sequence ID: 78, 79, or 80, and - L-CDR3 conforms to Sequence ID: 81, (alternative Ab4)

[0171] or - H-CDR1 is the one that conforms to Sequence ID: 86, 87, or 88. - H-CDR2 conforms to sequence number: 89, 90, or 91, - H-CDR3 conforms to sequence number 92 or 93, - L-CDR1 conforms to sequence number: 94 or 95, - L-CDR2 conforms to sequence number 96 or 97, and - L-CDR3 conforms to sequence number 98 or 99 (alternative Ab5)

[0172] or - H-CDR1 conforms to Sequence ID: 104 or 105, - H-CDR2 conforms to sequence number 106 or 107, - H-CDR3 conforms to sequence number: 108, 109, or 110, - L-CDR1 conforms to sequence number: 111 or 112, - L-CDR2 conforms to sequence number: 113 or 114, and - L-CDR3 conforms to sequence number 115 or 116 (alternative Ab6)

[0173] or - H-CDR1 conforms to sequence number: 121, 122, or 123, - H-CDR2 conforms to sequence number: 124, 125, or 126, - H-CDR3 conforms to sequence number 127 or 128, - L-CDR1 conforms to sequence number: 129 or 130, - L-CDR2 conforms to sequence number: 131 or 132, and - L-CDR3 conforms to Sequence ID: 133 (alternative Ab7).

[0174] In some embodiments of the present invention, the antibody or antibody fragment of the invention comprises VH and VL domains containing sequences according to SEQ ID NOs: 21 and 22, 39 and 40, 54 and 55, 68 and 69, 84 and 85, 102 and 103, or 119 and 120, respectively.

[0175] Further embodiments: Those skilled in the art can determine antibody binding affinity to CDR sequences and sequence mutations with any given sequence identity to a specific sequence, and can obtain a series of CDR sequences with a given sequence identity that maintain the antibody binding properties described herein without requiring excessive effort.

[0176] If the sequences are of a length that does not reasonably produce sequence mutations that would result in, for example, 90% sequence identity, a person skilled in the art can determine and appropriately adjust or exclude these sequences. Considering the detailed information presented in the examples regarding the desired binding properties of the antibody, any given antibody with sufficient sequence identity can be determined without excessive effort.

[0177] Regarding L-CDR2 sequences, in some embodiments, they contain three amino acids or consist of only these. In some embodiments, mutations in the amino acid sequence are possible, for example, one of the three amino acids is substituted from the original sequence while the linkage is maintained. Such mutants have 2 / 3 amino acid identity with respect to the original sequence, in particular the following:

[0178] Conservative substitutions are also included in the scope of this invention. Specific L-CDR2 sequences are shown below.

[0179] In one embodiment, the combination of VH and VL is the same as that described in the original isolated antibody.

[0180] In one embodiment, the antibody or antibody fragment comprises VH and VL domains having a specific CDR sequence described in the isolated antibody, and is further characterized by a framework sequence having identity with the specific isolated VH and VL sequences described herein.

[0181] For example, the CDR sequence is a sequence defined by an isolated antibody, and outside the identified CDR sequence, the antibody contains adjacent framework sequences and has the following characteristics: - Having at least 70%, preferably at least 80% or at least 90%, more preferably at least 95% or complete sequence identity with respect to the corresponding non-CDR portions of the variable domain described herein, e.g., SEQ ID NOs: 136 and 137 (Ab1), 151 and 152 (Ab2), 166 and 167 (Ab3), 181 and 182 (Ab4), 196 and 197 (Ab5), 211 and 212 (Ab6), or 226 and 227 (Ab7), respectively, for the VH and VL sequences.

[0182] For example, when using an alternative sequence, the CDR sequence is identical to that identified in the isolated antibody, and in the portion of the antibody other than the specified CDR sequence, it includes adjacent framework sequences. - Having at least 70%, preferably at least 80% or at least 90%, more preferably at least 95% or complete sequence identity with respect to the non-CDR portion of the variable domain described herein. For example, corresponding to the VH and VL sequences of SEQ ID NOs: 21 and 22, 39 and 40, 54 and 55, 68 and 69, 84 and 85, 102 and 103, or 119 and 120, respectively.

[0183] In embodiments of the present invention, the amino acid sequence mutation used to obtain the antibody may occur in either the CDR region or the framework region of the original antibody, where the framework region refers to a region located within the variable domain of a protein belonging to the immunoglobulin superfamily, which is not as "variable" as the CDR region. CDR sequence. In preferred embodiments, any sequence variant that maintains one or more of the binding properties disclosed herein is included in the present invention.

[0184] In one embodiment, Ab1 may be defined by a combination of one or more, preferably all six, CDR sequences according to the sequence, sequence alignment, or sequence formula shown below.

[0185] As follows:

[0186] [ka]

[0187] In the embodiments and sequence alignments described above, each X (considered to be a variable position in the sequence) may be any amino acid, or preferably the amino acid shown at each variable position (when the aligned sequence is read "longitudinal" at each position), and more preferably a conservative amino acid substitution at each variable position (as disclosed in the following examples). In embodiments, any CDR sequence may be defined solely by amino acids. No mutations are found throughout the entire alignment. In embodiments, this means that the CDR can be defined using only amino acids that do not contain X residues from the general sequence at the bottom of each alignment, i.e., amino acids that do not change within the alignment. In one embodiment, Ab2 may be defined by one or more, preferably all six, combinations of CDR sequences conforming to the following sequences, sequence alignments, or sequence formulas:

[0188] [ka]

[0189] In the embodiments and sequence alignments described above, each X (considered a variable position in the sequence) may be any amino acid, preferably an amino acid listed at each variable position (when the sequence alignment at each position is read "longitudinally"), or preferably a conservative amino acid substitution at any variable position (as illustrated below). In embodiments, any CDR sequence may be defined only by amino acids that do not exhibit mutations throughout the entire alignment. In embodiments, this may relate to a general sequence at the bottom of the alignment that does not contain an X residue. That is, only amino acids that do not change throughout the entire sequence in the alignment may be used to define a CDR. In one embodiment of the present invention, Ab3 may be defined by a combination of one or more, preferably all six, CDR sequences according to the following sequences, sequence alignments, or sequence formulas.

[0190] [ka]

[0191] In the embodiments and sequence alignments described above, each X (considered a variable position in the sequence) can be any amino acid, but is preferably set to the amino acid indicated at each variable position (when the alignment is read longitudinally), or preferably to the position of a conservative amino acid substitution at any variable site (as illustrated below). In embodiments, any CDR sequence may be defined only by amino acids in which no mutations are observed throughout the alignment. In embodiments, this may relate to a general sequence shown at the bottom of each alignment and may be defined using only amino acids that do not contain X residues, i.e., amino acids that do not change between sequences within the alignment. CDR.

[0192] In one embodiment of the present invention, Ab4 may be defined by a combination of one or more, preferably all six, CDR sequences according to the following sequences, sequence alignments, or sequence formulas.

[0193] [ka]

[0194] In the embodiments and sequence alignments described above, each X (considered a variable position in the sequence) can be any amino acid, but is preferably set to the amino acid indicated at each variable position (when the alignment is read longitudinally), or preferably to the position of a conservative amino acid substitution in any variable region (as illustrated below). In embodiments, any CDR sequence may be defined only by amino acids in which no mutations are observed throughout the alignment. In embodiments, this may relate to a general sequence shown at the bottom of each alignment and may be defined using only amino acids that do not contain X residues, i.e., amino acids that do not change between sequences within the alignment. CDR.

[0195] In this embodiment, Ab5 may be defined by a combination of one or more, preferably all six, CDR sequences, according to the following sequences, sequence alignments, or sequence formulas.

[0196] [ka]

[0197] In the embodiments and alignments described above, each X (considered a variable position in the sequence) may be any amino acid. Alternatively, preferably, each variable position may be one of the amino acids listed (reading each position "vertically"), or preferably, any conserved amino acid substitution (as illustrated below) for each variable position. In embodiments, any CDR sequence may be defined only by amino acids that do not undergo mutation throughout the entire alignment. In embodiments, this may relate to a general sequence at the bottom of the alignment that does not contain an X residue. That is, only amino acids that do not change throughout the entire sequence in the alignment may be used to define a CDR.

[0198] In one embodiment, Ab6 may be defined by a combination of one or more, preferably all six, CDR sequences, according to a sequence, sequence alignment, or the following sequence formula. The following formula:

[0199] [ka]

[0200] In the embodiments and sequence arrangements described above, each X (considered to be a variable position in the sequence) can be any amino acid. Preferably, it is the amino acid at each variable position as shown when the sequence arrangement at each variable position is read longitudinally, or any conservative amino acid substitution at each variable position (as illustrated below). In embodiments, any CDR sequence may be defined solely by amino acids. A sequence alignment in which no mutations are observed throughout the entire sequence. In embodiments, this may relate to a common sequence at the bottom of the alignment that does not contain an X residue. That is, only amino acids that do not change throughout the entire sequence within the alignment may be used to define a CDR. In one embodiment of the present invention, Ab7 may be defined by a combination of one or more, preferably all six, CDR sequences, according to a sequence, sequence alignment, or the following sequence formula. The following formula:

[0201] [ka]

[0202] In the embodiments and sequence alignments described above, each X (considered a variable position in the sequence) can be any amino acid, but preferably the amino acid indicated at each variable position (when the alignment is read longitudinally), or preferably a conservative amino acid substitution at each arbitrary variable position (as illustrated below). In embodiments, any CDR sequence may be defined only by amino acids that do not undergo mutation throughout the entire alignment. In embodiments, this may relate to a general sequence at the bottom of the alignment that does not contain an X residue. That is, only amino acids that do not change throughout the entire sequence in the alignment may be used to define a CDR.

[0203] In some embodiments, the antibody or antibody fragment of the present invention has a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH and VL comprise a CDR sequence according to the following:

[0204] [Table 2-1]

[0205] [Table 2-2]

[0206] In some embodiments, preferred CDR combinations are listed in the table below using Kabat CDR notes.

[0207] [Table 3]

[0208] In some embodiments, preferred CDR combinations are shown in the table below using IMGT CDR annotations.

[0209] [Table 4]

[0210] It was quite surprising that the specific antibodies described herein, preferably defined by the CDR regions of the VL and VH regions involved in binding, or by the VH and VL sequences disclosed herein, exhibited the properties shown in the examples and possessed binding properties that enabled the desired therapeutic and / or diagnostic effects.

[0211] In general, any changes made to the CDR area can be considered a characteristic of the CDR.

[0212] A modified CDR sequence, considered independently of the entire framework sequence, may be considered a defining feature of the present invention and may apply within or independently of the entire framework region described herein. For example, the CDR sequences identified above may be considered to define the features of the present invention regardless of the surrounding framework sequence. In some embodiments, sequence mutations based on the degree of sequence identity of VH and VL antibody sequences can be used in combination with specific CDR sequences.

[0213] All possible modification combinations for any potential mutant residue proposed herein are included in the present invention. By combining one or more of these substitutions, variants exhibiting the desired binding properties can be generated.

[0214] The original isolated antibody. The antibodies or parts thereof described herein contain sequences having at least 70%, 75%, 80%, 85%, preferably 90% or 95%, sequence identity with the further disclosed sequences.

[0215] As is evident from the overall disclosure of the present invention, in embodiments, several changes in the variable region sequence, including mutations in the CDR sequence, are described below.

[0216] It is possible based on various factors, such as the method used to measure the antibody sequence.

[0217] Sequences, the CDR annotation methods detailed below, or other factors. Those skilled in the art can combine the various CDR sequences of the present invention without particular difficulty and without loss of utility or significant loss.

[0218] The characteristics shown herein. Any CDR sequence shown in the sequence listing can be combined to form any combination of six CDRs including H-CDR1-3 and L-CDR1-3.

[0219] Yet another aspect of the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antibody fragment described herein.

[0220] Yet another aspect of the present invention relates to a host cell (e.g., a bacterial cell or a mammalian cell) that can produce an antibody or antibody fragment or that contains a nucleic acid molecule described herein.

[0221] Yet another aspect of the present invention relates to an analytical, detection, and / or diagnostic kit or composition suitable for use in an analytical, detection, and / or diagnostic assay, said kit or composition comprising an antibody or antibody fragment described herein.

[0222] Yet another aspect of the present invention relates to a pharmaceutical composition comprising an antibody or antibody fragment described herein and a pharmaceutically acceptable carrier.

[0223] In some embodiments, the antibody or antibody fragment of the present invention exhibits strong affinity for the MR-proADM target.

[0224] In some embodiments, the affinity of an antibody or fragment for a target can be quantitatively determined by surface plasmon resonance (SPR) measurements, for example, by using the KD value determined using SPR. In some embodiments, the antibody or antibody fragment preferably has affinity, which is measured using the KD value. The KD value is determined preferably using SPR and is <500 nM, preferably <100 nM, <50 nM, <10 nM, more preferably <5 nM, <2 nM, <1 nM, more preferably <0.1 nM, or <0.01 nM.

[0225] In alternative nomenclature, in some embodiments, the antibody or antibody fragment has affinity, which is preferably measured using the KD value. The KD value is preferably measured using an SPR of 5.0E-07, preferably 1.0E-07, 5.0E-08, 1.0E-08, more preferably 7.0E-09, 5.0E-09, 3.0E-09, 1.0E-09 or lower.

[0226] The KD value indicates the equilibrium dissociation constant between an antibody and its antigen, which is expressed as the koff / kon ratio. KD and affinity are inversely related. The KD value is related to the antibody concentration (the amount of antibody required for a particular experiment); therefore, the lower the KD value (lower concentration), the higher the affinity. Examples of KD values ​​are as follows: KD value: Molar concentration (sensitivity) E-04~E-06, micromoles (μM) E-07~E-09 Nanomoles (nM)

[0227] Antibodies whose affinity is indicated by KD. While nanomolar values ​​may be beneficial, they are not always predictable, especially in the production of monoclonal antibodies.

[0228] A further aspect of the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antibody fragment described herein.

[0229] In one embodiment, the present invention relates to a preferably isolated nucleic acid molecule selected from the group consisting of the following. a) Nucleic acid molecules containing nucleotide sequences - Encoding an isolated antibody or antibody fragment as described herein, - Encodes an amino acid sequence according to a heavy chain variable (VH) domain, wherein the VH domain contains a sequence having at least 70%, preferably 80%, and more preferably 90% identity with any of sequence numbers: 136, 151, 166, 181, 196, 211, or 226, or encodes an amino acid sequence according to a light chain variable (VL) domain. The VL domain contains a sequence having at least 70% identity, preferably 80% identity, and more preferably 90% identity, with any of sequence numbers: 137, 152, 167, 182, 197, 212, or 227. or - When alternative sequences are used, they encode an amino acid sequence corresponding to the heavy chain variable (VH) domain, and the VH domain contains a sequence that has at least 70%, preferably 80%, and more preferably 90% identity with any of sequence numbers: 21, 39, 54, 68, 84, 102, or 119. Alternatively, it encodes an amino acid sequence corresponding to the light chain (VL) domain. The VL domain is sequence numbers 22, 40, 55, 69, 85, 103, or 120, or b) A sequence having at least 70% identity, preferably 80% identity, and more preferably 90% identity with respect to any one nucleic acid molecule complementary to the nucleotide sequence according to a), or c) A nucleic acid molecule containing a nucleotide sequence having sufficient sequence identity to be functionally similar / equivalent to a nucleotide sequence conforming to a) or b), d) A nucleic acid molecule that, as a result of the genetic code, has degenerated into a nucleotide sequence following a) to c). e) A nucleic acid molecule that follows the nucleotide sequence of a) to d), which is modified by deletion, addition, substitution, translocation, inversion and / or insertion, and is functionally similar to or equivalent to the nucleotide sequence of a) to d).

[0230] A further aspect of the present invention relates to a host cell, such as a bacterial cell or a mammalian cell, which contains the nucleic acid molecules described herein and is capable of producing an antibody or antibody fragment.

[0231] Another aspect of the present invention relates to a pharmaceutical composition containing the isolated antibody or antibody fragment, or nucleic acid molecule, or host cell described herein, together with a pharmaceutically acceptable carrier.

[0232] Accordingly, the present invention as described herein includes monoclonal production and functional characterization of monoclonal antibodies that bind to MR-proADM. The properties of the antibodies and their corresponding sequences are described in detail herein. Surprisingly, the monoclonal antibodies described herein enable improved detection sensitivity of MR-proADM compared to established immunoassays.

[0233] As illustrated in the following examples, when evaluating plasma samples with a constant concentration of the target antigen, the antibodies described herein enable the acquisition of a larger signal compared to established conventional methods. Furthermore, these antibodies exhibit high specificity and do not cross-reactivity with untargeted proteins. These antibodies are used in SPR Because it exhibits outstanding and surprisingly high affinity when evaluated using [this method], it provides further unexpected and beneficial properties of the antibody of the present invention.

[0234] [Detailed description of the invention] The present invention relates to a method for measuring intermediate region proadrenomedullin. The present invention comprises two labeled monoclonal antibodies that bind to (preferably different) epitopes of MR-proADM in a sample, and detects a signal by the binding of both antibodies to MR-proADM. The present invention further relates to monoclonal antibodies or antibody fragments that bind to the intermediate region pro-adrenomedullin (MR-proADM). The present invention further relates to diagnostic applications and methods, for example, in vitro methods for measuring the following:

[0235] Measuring MR-proADM in a sample using the antibody or antibody fragment of the present invention. Various related aspects and embodiments are encompassed by the present invention.

[0236] Assays and time-resolved energy transfer: In some embodiments, the antibody of the present invention can be conjugated to one or more labels, such as a fluorescent label, and preferably can be conjugated to two distinct fluorescent labels suitable for application in the KRYPTOR (trademark) assay.

[0237] In some embodiments, the KRYPTOR assay is based on the TRACE technology, which is non-radiative energy transfer from a donor, which is a cage structure (cryptate) having a europium ion at the center, to an acceptor. The proximity of the donor (cryptate) and acceptor in the formed immune complex, and the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor enhance the fluorescence signal on the one hand and extend the lifetime of the acceptor signal on the other hand.

[0238] This enables the measurement of time-delayed fluorescence.

[0239] When the sample to be measured is excited with a nitrogen laser of an appropriate wavelength, the donor (cryptate) emits a long-lived fluorescence signal on the order of milliseconds, while the acceptor generates a short-lived signal on the order of nanoseconds. When both components bind in the immune complex, both signal amplification and an extension of the lifetime of the acceptor signal occur, and its lifetime is in the microsecond range. This delayed acceptor signal is proportional to the concentration of the analyte being measured. Specific fluorescence proportional to the antigen concentration is obtained by dual selection. That is, spectroscopic (separation depending on wavelength) and temporal (time-resolved measurement) options. This enables exclusive measurement of only the signal emitted from the immune complex, and fluctuations in the light transmittance of the medium can be corrected in real time by the ratio between two wavelengths.

[0240] In this embodiment, the measurement method is homogeneous and does not require separation or washing steps. Therefore, it is possible to acquire data without interrupting the immunoassay.

[0241] In other embodiments, the antibody may be used by a method selected from luminescence immunoassay (LIA), radioimmunoassay (RIA), chemiluminescence immunoassay and fluorescence immunoassay, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), luminescence-based bead array, magnetic bead array, protein microarray assay, and rapid test formats.

[0242] Rapid testing methods include, for example, immunochromatography test strips, rarecryptate assays, and automated systems / analytes.

[0243] Antibodies can be used in a homogeneous system method, in which an antibody or a sandwich complex formed by multiple antibodies and a marker (e.g., proADM or a fragment thereof, which is the target of detection) is suspended in a liquid phase. In this case, if two antibodies are used, it is preferable that both antibodies be labeled with a component of the detection system.

[0244] When both antibodies are integrated into a single sandwich, it generates or triggers a signal.

[0245] These techniques are embodied particularly in fluorescence enhancement methods or fluorescence quenching detection methods. A particularly preferred aspect relates to the use of paired detection reagents, such as those described below.

[0246] U.S. Patent No. 4,882,733A, European Patent No. 0,180,492,B1, or European Patent No. 0,539,477,B1, and the prior art cited herein.

[0247] In this way, it becomes possible to directly detect only the reaction product containing both labeled components within a single immunocomplex in the reaction mixture.

[0248] For example, such technology is offered under the brand names "TRACE" (Trademark) (Time Resolved Amplified Cryptate Emission) or "KRYPTOR" (Trademark) in the form of implementing the above teachings. Application: Therefore, in a particularly preferred aspect, the method described herein is carried out using a diagnostic device.

[0249] For example, the levels of the proADM protein or its fragments, and / or other markers of the method provided herein, are measured. In a particularly preferred aspect, the diagnostic device is the KRYPTOR.

[0250] In one embodiment of the method described herein, the method is an immunoassay, Here, the measurement is performed in a homogeneous or heterogeneous phase. In one embodiment of the method described herein, a first antibody and a second antibody (preferably at least one, more preferably both, are antibodies described herein) are dispersed in a liquid reaction mixture. A first labeling component, which is part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, is bound to the first antibody. Furthermore, the second labeling component of the labeling system is bound to the second antibody. This generates a measurable signal that allows detection of the resulting sandwich complex in the measurement solution after both antibodies have bound to the target MR-proADM or its fragment.

[0251] In one embodiment of the method described herein, the labeling system comprises a combination of a rare earth cryptotate or chelate and a fluorescent or chemiluminescent dye, particularly a cyanine-type dye.

[0252] For example, two types of cages have been developed to chelate lanthanides and enable labeling of target receptors. (i) The chelate shows high affinity for europium and terbium ions, but complex formation is reversible, and Mn 2+ Mg 2+ Ca 2+ (ii) Cryptates, on the other hand, offer greater stability for the following reasons:

[0253] Terbium and europium cannot be released after complex formation.

[0254] As an example of cryptotate structure, terbium cryptotate is shown below.

[0255] [ka]

[0256] When excited, the half-life of lanthanum-based fluorescence is in the range of approximately 1 millisecond, compared to several nanoseconds for conventional fluorescent dyes. TR-FRET and TRACE utilize this strategy.

[0257] Introducing a time delay (typically around 50 μs) between excitation and detection of the fluorescence signal allows for the distinction between short-lived and long-lived fluorescence.

[0258] Furthermore, by detecting the fluorescence signal, it is possible to distinguish between short-lived fluorescence and long-lasting fluorescence. Therefore, all short-lived fluorescence generated by the medium or biological sample, or fluorescence caused by direct excitation of acceptors, is removed by time delay. Only long-lived fluorescence, which is generated from the following involving donors or acceptors, is included.

[0259] The FRET process is measured after a time delay.

[0260] Both europium and terbium cryptotes are excited at 300–350 nm. Both exhibit significant stoke shifts and possess complex emission spectra with multiple fluorescence peaks. For example, europium cryptotate trisbipyridine [TBP(Eu)] shows four major fluorescence peaks at 585, 605, 620, and 700 nm, while europium pyridine bisbipyridine (Eu-PBP) has two major peaks.

[0261] Europium and terbium cryptotes exhibit peaks at 595 nm and 615 nm, with two smaller peaks at 680 nm and 705 nm. Terbium cryptotes also show four fluorescence peaks at wavelengths of approximately 490, 550, 585, and 620 nm (Figure 2C). This makes europium and terbium cryptotes compatible with deep red Cy5 and DY647-like fluorescent dyes. Furthermore, due to the emission peak around 490 nm, terbium cryptotes are also compatible with fluorescein-like fluorescent dyes as acceptors.

[0262] Non-limiting examples of acceptor fluorescent dyes are as follows (excerpted from www.abcam.com / technical):

[0263] [Table 5]

[0264] antibody: As used herein, “antibody” generally refers to a protein consisting of one or more polypeptides substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene.

[0265] When the term "antibody" is used, it is assumed that the term also refers to "antibody fragments." Recognized immunoglobulin genes include numerous immunoglobulin variable region genes, in addition to the constant region genes of kappa, lambda, alpha, gamma, delta, epsilon, and mu. The light chain is classified as either a kappa or lambda chain. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which defines the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0266] The basic structural units of immunoglobulins (antibodies) are known to consist of tetramers or dimers. Each tetramer is composed of two sets of identical polypeptide chains, each set having one "light chain" (L) (approximately 25 kD) and one "heavy chain" (H) (approximately 50-70 kD). The N-terminus of each chain defines a variable region consisting of approximately 100-110 or more amino acids, which is mainly involved in antigen recognition.

[0267] The terms "variable light chain" and "variable heavy chain" refer to these variable regions of the light and heavy chains, respectively. Optionally, an antibody or its immunological portion can be chemically conjugated to another protein or expressed as a fusion protein.

[0268] In embodiments, the antibody is specific to MR-proADM or binds specifically to MR-proADM. “Specific binding” is understood by those skilled in the art, who are well aware of the various experimental techniques that can be used to verify binding and binding specificity. While some degree of cross-reactivity or background binding may be unavoidable in many protein-protein interactions, this does not impair the “specificity” of the binding between the antibody and the epitope. The term “against” is similarly applied when considering the term “specifically” in understanding the interaction between the antibody and the epitope.

[0269] In some embodiments, an antibody is considered specific if it has affinity for the target molecule, e.g., MR-proADM. The reactivity to MR-proADM or its fragment is at least 50 times higher, preferably 100 times higher, and most preferably at least 1000 times higher, than the reactivity to other molecules in the sample containing the target molecule. Those skilled in the art are well aware of methods for developing and selecting antibodies having a given specificity.

[0270] In the context of the present invention, monoclonal antibodies are preferred.

[0271] The antibodies or antibody-conjugated fragments described herein bind to a specifically defined marker or the fragment.

[0272] In particular, the antibody or antibody-binding fragment binds to the peptide of MR-proADM as defined herein. Therefore, the peptide as defined herein may also be an epitope to which the antibody specifically binds. Furthermore, the antibody or antibody-binding fragment is used in the methods and kits of the present invention that specifically bind to MR-proADM.

[0273] The antibodies of the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies or chimeric antibodies, single variable region fragments (ssFv), single domain antibodies (such as nanobody-derived VHH fragments), single-chain fragments (scFv), Fab fragments, F(ab')2 fragments, fragments produced by Fab expression libraries, anti-idiotype antibodies, and any of the above epitope-binding fragments, or combinations thereof, provided that they retain their original binding properties.

[0274] Furthermore, the method of the present invention may also utilize mini-antibodies and polyvalent antibodies such as diabodies, triabodies, tetravalent antibodies, and peptabodies. The immunoglobulin molecules of the present invention may be any class (i.e., IgG, IgE, IgM, IgD, IgA) or a subclass of immunoglobulin molecules. Accordingly, the term “antibody” as used herein also includes whole antibodies, or antibodies and antibody fragments produced by modification of de novo synthesized antibodies using recombinant DNA technology.

[0275] The present invention further relates to the use of the antibody or its fragment, such as a variable region, described herein as a recognition molecule or affinity reagent suitable for selective binding to a target. The affinity reagent, antibody or its fragment according to the present invention may be PEGylated, where PEGylation refers to covalently bonding a polyethylene glycol (PEG) polymer chain to the antibody of the present invention. PEGylation can be achieved by conventional methods by incubating a reactive PEG derivative with the target molecule. PEGylation of an antibody potentially masks its drug from the host immune system, reducing its immunogenicity and antigenicity. Alternatively, increasing the hydrodynamic size of the drug may reduce renal clearance, potentially prolonging circulating time.

[0276] The variable region of an antibody refers to the variable region of the light chain or the variable region of the heavy chain, either individually or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions.

[0277] The CDRs of each chain are held in close proximity to each other by the FR, and together with the CDR of the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining the CDR: (1) an approach based on interspecific sequence variability (i.e., Kabat et al. Sequences of Immunologically Important Proteins, 5th edition (published 1991, National Institutes of Health, Bethesda, Maryland)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Allazikani et al. (1997) J. Molec. Biol. 273:927-948). As used herein, CDR may refer to a CDR defined by either one of these methods, a combination of both methods, or other appropriate methods.

[0278] In some embodiments, the present invention provides an antibody comprising at least one CDR.

[0279] At least two, at least three, or more CDRs are substantially identical to at least one, at least two, at least three, or more CDRs of the antibody of the present invention. Other embodiments include antibodies having at least two, three, four, five, or six CDRs which are substantially identical to at least two, three, four, five, or six CDRs of the antibody of the present invention, or antibodies derived from the antibody of the present invention. In some embodiments, at least one, two, three, four, five, or six CDRs are at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one, two, or three CDRs of the antibody of the present invention.

[0280] For the purposes of this invention, binding specificity and / or overall activity are generally retained, but the degree may vary (may be higher or lower) compared to the aforementioned antibody.

[0281] The half-life and potential cytotoxicity of an antibody depend primarily on the interaction between the Fc domain and various Fc gamma receptors. In the case of antibody half-life, the neonatal Fc receptor (FcRn) plays a major role. This receptor is expressed on cell types and tissues capable of taking up serum proteins into recycling endosomes, such as certain cell monocytes and vascular endothelial cells. Within endosomes, the pH decreases to approximately 6, and under these conditions, antibodies can bind to FcRn. This interaction protects the antibody from degradation until it is released into the bloodstream, when its binding to the receptor is again inhibited by physiological pH (Roopenian and Akilesh (2007) Nat Rev Immunol 7:715-725).

[0282] The higher an antibody's affinity for FcRn at pH 6, the longer its half-life becomes. Mutations in the Fc fragment known to stabilize this interaction are summarized in Presta (2008, Curr Opin Immunol 20:460-470).

[0283] The present invention may further include "MR-proADM binders" based on the structure and / or sequence described herein. Or relating to "affinity reagents" or other "binding agents".

[0284] Sequence mutation: Sequence variants of nucleic acids, proteins, and antibodies claimed in the claims (e.g., defined by the claimed % sequence identity) that maintain the characteristics of the invention are also included in the scope of the invention. Such variants exhibit alternative sequences but maintain essentially the same binding properties as the specific sequence provided, and are known as functional analogs or functionally similar. Sequence identity refers to the percentage of identical nucleotides or amino acids when sequence alignment is performed.

[0285] Those with ordinary skill in the art will understand that, as a result of the degeneracy of the genetic code, there are numerous nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides have minimal identity or sequence identity with any nucleotide sequence of a native gene. Nevertheless, polynucleotides that differ due to differences in codon usage are also given particular consideration in this invention. Deletions, substitutions, and other modifications of sequences that correspond to described sequence identity are also included in this invention.

[0286] Modifications of protein sequences that may result from substitutions are also included in the scope of the present invention. Substitutions as defined herein are modifications made to the amino acid sequence of a protein, in which one or more amino acids are replaced by the same number of (different) amino acids to produce a protein, the protein containing an amino acid sequence different from that of the primary protein, and preferably without significantly altering the function of the protein.

[0287] Like additions, substitutions can be natural or artificial. As is well known to those skilled in the art, amino acid substitutions may not have a significant impact on the function of a protein. This is especially true when the modification concerns “conservative” amino acid substitutions, i.e., substitutions for other amino acids with similar properties. Such “conservative” amino acids may be natural or synthetic amino acids for size reasons, and their charge, polarity, and stereochemistry may not significantly affect the structure and function of the protein of the present invention.

[0288] In many cases, many amino acids can be substituted with conserved amino acids without having a detrimental effect on protein function.

[0289] Generally, nonpolar amino acids include Gly, Ala, Val, Ile, Leu; nonpolar aromatic amino acids include Phe, Trp, Tyr; and neutral polar amino acids include Ser, Thr, Cys, Gln, Asn, Met, and Positively charged amino acids include lysine (Lys), arginine (Arg), and histidine (His); negatively charged amino acids include aspartic acid (Asp) and Glu represents a group of conserved amino acids. This list is not exhaustive. For example, alanine (Ala), glycine (Gly), serine (Ser), and sometimes cysteine ​​(Cys) are well known to be interchangeable even though they belong to different taxa.

[0290] A substituted variant is one in which at least one amino acid residue is substituted within the antibody molecule.

[0291] Then, a different residue is inserted at that position. While highly variable regions are of most interest in introducing substitutional mutations, modifications to the framework region (FR) are also considered. If such substitutions result in changes in biological activity, they are shown as "exemplary substitutions" in the table below, or explained as described later, and more substantial changes may be made. The following amino acid classes may be introduced and the products screened.

[0292] [Table 6]

[0293] Substantial modification of the biological properties of an antibody is achieved by selecting substitutions that have a significantly different effect on (a) the structure of the polypeptide backbone in the substituted region, e.g., sheet or helix structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chains. Conserved amino acid substitutions are not limited to naturally occurring amino acids, but also include synthetic amino acids. Commonly used synthetic amino acids include omega amino acids of various chain lengths, and their neutral, nonpolar analogs: cyclohexylalanine; citrulline and methionine sulfoxides (both neutral, nonpolar analogs), phenylglycine (aromatic neutral analog), cysteic acid (negatively charged analog), and ornithine (positively charged amino acid analog). As with naturally occurring amino acids, this list is not exhaustive and represents only a few examples of substitutions well known in this technique.

[0294] Antibody production: The antibodies of the present invention can be produced by any suitable method, for example, by introducing an expression vector containing the coding sequence of the antibody of the present invention into a host cell. The expression vector or recombinant plasmid is generated by operably conjugating these coding sequences of the antibody to a conventional regulatory sequence that can control replication and expression within the host cell and / or secretion from the host cell.

[0295] The control sequence may include a promoter sequence (e.g., CMV promoter) or a signal sequence that can be derived from other known antibodies. Similarly, a second expression vector may be generated that has DNA sequences encoding the light or heavy chain of a complementary antibody. In certain embodiments, this second expression vector is identical to the first expression vector, except with respect to the coding sequence and selection marker. This is to ensure that each polypeptide chain is expressed as functionally as possible. Alternatively, the coding sequences for both the heavy and light chains of the antibody may be present on a single vector.

[0296] Selected host cells are co-transduced with both the first and second vectors (or simply transduced with a single vector) using conventional methods to produce transduced host cells containing both recombinant or artificial light and heavy chains according to the present invention. The transduced cells are then cultured using conventional techniques to produce the modified antibodies of the present invention. Antibodies containing both recombinant heavy and / or light chains are screened from the cultures using appropriate assays such as ELISA or RIA. Other antibodies can be produced using similar conventional techniques.

[0297] The cloning vectors used in the method for preparing the compositions of the present invention and in the subcloning process can be selected by those skilled in the art. For example, conventional pUC series cloning vectors can be used. One vector, pUC19, is commercially available. The components of such vectors, such as replicons, selection genes, enhancers, promoters, signal sequences, etc., can be obtained from commercial sources, naturally occurring sources, or synthesized by known methods used for inducing the expression and / or secretion of recombinant DNA products in selected hosts. Many other suitable expression vectors of the prior art known for the expression of mammals, bacteria, insects, yeasts, and fungi can also be selected for this purpose.

[0298] The present invention also encompasses cell lines transduced with recombinant plasmids containing the coding sequence of the antibody of the present invention. The host cells useful for cloning and other operations of these cloning vectors are also conventional.

[0299] Host cells or cell lines suitable for expressing the antibodies of the present invention include the following: Mammalian cells (NS0, Sp2 / 0, CHO (e.g., DG44), COS, HEK, fibroblasts (e.g., 3T3)), and myeloma cells, which can express this molecule in, for example, CHO cells or myeloma cells. Using human cells, it is possible to modify this molecule with a human-derived glycosylation pattern. Alternatively, other prokaryotic or eukaryotic cell lines can be used. Methods for selecting and transforming, culturing, amplifying, and screening appropriate mammalian host cells, as well as for producing and purifying the product, are well known to those skilled in the art.

[0300] The present invention provides a method for producing an antibody of the present invention that binds to MR-proADM, the method comprising the following steps: providing a first vector encoding the heavy chain of the antibody; providing a second vector encoding the light chain of the antibody; transforming mammalian host cells (e.g., CHO) with the first and second vectors; culturing the host cells from step (c) under conditions that promote the secretion of the antibody from the host cells into the culture medium; and recovering the secreted antibody from step (d). Once expressed, the antibody can be evaluated for desired binding properties using the method described herein.

[0301] Antibodies and their use in immunoassays In some embodiments, the level of MR-proADM or its fragments can be measured by any assay that measures the concentration of the marker using the antibody of the present invention.

[0302] In particular, the antibody-based immunoassay method of the present invention can be used as illustrated in the attached examples. In this specification, "immunoassay method" means a biochemical test that measures the presence or concentration of a macromolecule / polypeptide in a solution by the following method. Use of antibodies, antibody-conjugated fragments, or immunoglobulins. Furthermore, antibodies or antibody-conjugated fragments may, in some embodiments, be used in the methods and kits of the present invention to specifically bind to proADM or its fragments, and optionally to other markers of the present invention, such as PCT. Exemplary immunoassays include luminescence immunoassay (LIA), radioimmunoassay (RIA), chemiluminescence immunoassay, fluorescence immunoassay, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), fluorescence-emitting bead arrays, magnetic bead arrays, protein microarray assays, rapid test formats, and rare cryptotate assays. Additionally, assays suitable for point-of-care testing or rapid test formats, such as immunochromatography strip tests, can be used. Automated immunoassays, such as the KRYPTOR® assay, are also intended.

[0303] In one aspect of the present invention, the method is an immunoassay and comprises the following steps: a) The sample, i. A primary antibody or antigen-binding fragment or derivative thereof that is specific to the primary epitope of the proADM, ii. The step of contacting the proADM with a second antibody or antigen-binding fragment or derivative thereof that is specific to the second epitope; and b) A step of detecting the binding of two antibodies or their antigen-binding fragments or derivatives thereof to the proADM.

[0304] In some embodiments, the first and second antibodies may be dispersed in a liquid reaction mixture. Here, the first labeling component, which is part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, is bound to the first antibody, and the second labeling component of the labeling system is bound to the second antibody, so that both antibodies are bound to the proADM or its fragments and then detected, generating a measurable signal in the measurement solution that enables detection of the resulting sandwich complex. The labeling system may include a rare earth cryptotate or chelate in combination with a fluorescent or chemiluminescent dye, particularly a cyanine-type dye.

[0305] The terms "detection reagent" and "binding agent" used herein include any reagent suitable for measuring the markers described herein.

[0306] Such exemplary detection reagents include, for example, ligands, such as antibodies or fragments thereof that specifically bind to the peptide or epitope of the marker described herein. Such ligands may be used in the immunoassays described herein. Other reagents used in immunoassays for measuring the level of the marker may also be included in the kit and are considered herein as detection reagents. Detection reagents may also relate to reagents used to detect the marker or fragment by mass spectrometry (MS) based methods. Thus, such detection reagents may also be reagents used when preparing a sample for MS analysis, such as enzymes, chemicals, and buffers. A mass spectrometer can also be considered a detection reagent. Detection reagents according to the present invention can also be used in calibration solutions, for example, to measure and compare the levels of the marker.

[0307] In some embodiments, the antibody may be immobilized on a solid support. After the addition of the sample solution, MR-proADM in the patient's sample binds to the solid-phase antibody or a fragment thereof of the present invention. For example, MR-proADM obtained from the patient's serum and bound to the solid phase may then be detected using labeling or labeling reagents and quantified as necessary, preferably using antibodies against MR-proADM further described herein. Thus, according to the present invention, the detection of MR-proADM in this method is achieved using labeling reagents by the widely known ELISA (enzyme-linked immunosorbent assay) technique. Accordingly, the labeling according to the present invention includes an enzyme that catalyzes a chemical reaction measured by optical means, particularly a chromogenic substrate, chemiluminescence, or a fluorescent dye. In another preferred embodiment, the autoantibody is labeled with a weak radioactive substance in radioimmunoassay (RIA) and detected by measuring the resulting radioactivity.

[0308] The term "immunoassay" encompasses techniques that include, but are not limited to, the following methods: Immunoassays (EIA) such as enzyme amplification immunoassay (EMIT), enzyme-linked immunosorbent assay (ELISA), antigen capture ELISA, sandwich ELISA, IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunoradioassay (IRMA), fluorescence-polarized immunoassay (FPIA), lateral flow assay (LFA), and chemiluminescence assay (CL).

[0309] In another preferred embodiment of the present invention, a soluble or solid-bound antibody, or a fragment thereof, is used to bind to MR-proADM. In a second reaction step, yet another antibody, such as the antibody of the present invention against MR-proADM, is used, and these yet another antibody is labeled for detection. The advantage of this embodiment is that it uses ELISA technology, which is commonly available in laboratory facilities, and allows for the cost-effective establishment of the detection according to the present invention. Furthermore, the antibody may be bound to fluorescein isothiocyanate (FITC) in a detectable form. Very similar to the ELISA described above, FITC technology is a system available in many locations and therefore allows for the smooth and low-cost establishment of the detection method of the present invention as a laboratory routine.

[0310] Examples of enzymes known in the art for indirect labeling include horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, and urease. A horseradish peroxidase detection system can, for example, use the chromogenic substrate tetramethylbenzidine (TMB) to produce a soluble product detectable at 450 nm in the presence of hydrogen peroxide. An alkaline phosphatase detection system can, for example, use the chromogenic substrate p-nitrophenyl phosphate to produce a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can use the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) to produce a soluble product detectable at 410 nm.

[0311] In another embodiment of the present invention, an antibody or fragment of the present invention conforming to one or more sequences disclosed herein is bound to a solid phase. The binding of the antibody or fragment of the present invention conforming to one or more sequences disclosed herein to the solid phase may be performed via a spacer. Any compound having suitable structural and functional prerequisites for spacer function can be used as a spacer, as long as it does not alter the binding behavior of the antibody or fragment with MR-proADM, which would adversely affect the present invention.

[0312] In another preferred embodiment of the present invention, the molecule is α-aminocarboxylic acid and its homooligomers and heterooligomers, α,ω-aminocarboxylic acid and its branched homo or heterooligomers, other amino acids, and linear and branched homo or heterooligomers; amino-oligoalkoxyalkylamines; maleimidocarboxylic acid derivatives; alkylamine oligomers; 4-alkylphenyl derivatives; 4-oligoalkoxyphenyl or 4-oligoalkoxyphenyloxy derivatives; 4-oligoalkyl-mercaptophenyl or 4-oligoalkylmercaptophenyloxy derivatives; 4-oligoalkylaminophenyl or 4-oligoalkylaminophenyloxy derivatives; (oligoalkylbenzyl)phenyl or 4-(oligoalkylbenzyl)phenyloxy derivatives, and 4-(oligoalkoxybenzyl)phenyl or 4-(oligoalkoxybenzyl)phenyloxy derivatives; trityl derivatives; benzyloxyaryl or benzylalkyl derivatives; xanthene-3-yloxyalkyl derivatives; (4-alkylphenyl) or {-(4-alkylphenoxy)alkanoic acid derivatives; containing linkers or spacers selected from the group consisting of oligoalkylphenoxyalkyl or oligoalkoxyphenoxyalkyl derivatives, carbamate derivatives, amines, trialkylsilyl or dialkylalkoxysilyl derivatives, alkyl or aryl derivatives, or combinations thereof.

[0313] In another embodiment of the present invention, the antibody or fragment thereof is immobilized. More specifically, the antibody or fragment thereof, bound to a solid phase of the present invention, is bound to organic, inorganic, synthetic and / or polymer mixtures, preferably agarose, cellulose, silica gel, polyamide and / or polyvinyl alcohol. In the sense of the present invention, immobilization is understood to include various methods and techniques for immobilizing the antibody or fragment thereof on a specific carrier, for example, according to WO 99 / 56126 or WO 02 / 26292. For example, immobilization helps to stabilize the peptide, thereby preventing its activity from being reduced or adversely affected by biological, chemical, or physical exposure, particularly during storage or single-batch use. Immobilization of the antibody or fragment thereof enables repeated use under routine technical or clinical conditions. Furthermore, a sample—preferably a blood component—can be reacted with at least one of the antibodies or fragments thereof in a continuous manner. In the sense of the present invention, three basic methods for immobilization can be used. (i) Crosslinking: In crosslinking, antibodies are immobilized on each other, but this does not adversely affect their activity. Advantageously, as a result of such crosslinking, they are no longer soluble. (ii) Binding to carriers: Binding to carriers occurs, for example, via adsorption, ionic bonding, or covalent bonding. Such binding can occur within microbial cells, liposomes, and other membrane-bound or open structures. Advantageously, these antibodies are not adversely affected by such fixation processes. For example, the use of carrier-bound antibodies multiple times or consecutively is advantageous in clinical diagnosis and treatment. (iii) Inclusion: Inclusion in the sense of the present invention proceeds within a semipermeable membrane, particularly in the form of a gel, fibril, or fiber. Preferably, the encapsulated antibody is separated from the surrounding sample solution by the semipermeable membrane, but interaction with the virus is still possible. Various methods are available for immobilization, such as adsorption to an inert or charged inorganic or organic carrier.

[0314] Another method involves covalent bonding to a support material. Furthermore, the support may have reactive groups that form ispolar bonds with the amino acid side chains. Suitable groups in antibodies are carboxyl groups, hydroxyl groups, and sulfide groups, particularly the terminal amino group of lysine. Aromatic groups offer the possibility of diazo bonding. Advantageously, Many antibodies can be directly covalently bonded to polyacrylamide resin.

[0315] The present invention also relates to a diagnostic kit for determining MR-proADM, comprising antibodies conforming to one or more sequences disclosed herein. The diagnostic kit optionally includes instructions relating to how to combine the contents of the kit and / or providing a preparation for detecting the viral infection. For example, the instructions may be provided in the form of a manual or other media that provides information to the user regarding the type of method by which the substances are used. Obviously, the information does not necessarily have to be provided in the form of a package insert, and may be provided, for example, via the internet. One example of the advantageous effect of such a kit for a patient is that they can understand the actual state of the disease without having to consult directly with a doctor.

[0316] Monitoring of diagnosis, prognosis, and treatment: In embodiments, antibodies or fragments thereof may be used in diagnostic, prognostic, treatment guidance, risk stratification, or other diagnostic applications to predict the presence or risk of developing medical conditions associated with MR-proADM.

[0317] In this specification, “diagnosis” in the context of the present invention relates to recognition and (early) detection of a clinical condition. The assessment of severity may also be included in the term “diagnosis.”

[0318] "Prognosis" refers to predictions of the subject's outcome or specific risks. This may include estimates of the subject's likelihood of recovery or the likelihood of adverse consequences.

[0319] The reagents and methods of the present invention can also be used for monitoring, treatment monitoring, treatment guidance, and / or treatment management. "Monitoring" refers to tracking the progress of a patient and potential complications, for example, analyzing the progression of the healing process or the impact of specific treatments or therapies on the patient's health.

[0320] In the context of this invention, the terms “treatment monitoring” or “treatment control” refer to monitoring and / or adjusting the therapeutic treatment of the patient, for example, by obtaining feedback on the effectiveness of the treatment. In this specification, the term “treatment guidance” refers to applying a specific treatment, therapeutic action, or medical intervention based on the value / level of one or more biomarkers and / or clinical parameters and / or clinical scores. This includes adjusting or discontinuing treatment.

[0321] In this invention, the terms “risk assessment” and “risk stratification” relate to classifying subjects into different risk groups according to their future prognosis. Risk assessment also relates to stratification for the application of preventive and / or therapeutic measures. In particular, the term “treatment stratification” relates to classifying or dividing patients into different groups, such as risk groups or treatment groups that receive therapeutic measures according to specific different classifications.

[0322] The term "treatment stratification" also refers to classifying or separating patients who have an infection or symptoms of an infection into groups that do not require specific treatment measures.

[0323] The sensitivity and specificity of diagnostic and / or prognostic tests are not determined solely by the analytical "quality" of the test, but also depend on the definition of what constitutes an abnormal outcome. In practice, the receiver operational characteristic curve (ROC curve) is typically calculated by plotting the values ​​of a variable and their relative frequencies in the "normal" group (i.e., clearly healthy individuals without infection) and the "disease" group (e.g., subjects with infection). For a particular marker (e.g., ADM), the concentration distributions of marker levels in subjects with and without the disease or condition will likely overlap. In such situations, the test may not perfectly distinguish between normal and disease with 100% accuracy, and the overlapping region may indicate where the test fails to distinguish between normal and disease. A threshold is selected, below which the test is considered abnormal, above which the test is considered normal, or below or above the threshold the test may indicate a specific condition (e.g., infection). The region below the ROC curve is a measure of the probability of correctly identifying a condition and the perceived measurement. The ROC curve can be used even when test results do not necessarily provide precise numerical values. As long as the results can be ranked, an ROC curve can be constructed. For example, test results for a “disease” sample may be ranked according to their degree (e.g., 1) (1=low, 2=normal, and 3=high). This ranking can be correlated with the results of the “normal” population, and an ROC curve can be constructed. These methods are well known in the art; see, for example, Hanley et al., 1982, Radiology 143:29-36. Preferably, the threshold may be selected such that the area under the ROC curve is greater than about 0.5, more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. In this context, the term “about” refers to ±5% of a given measurement.

[0324] The horizontal axis of the ROC curve represents (1-specificity), which increases as the false positive rate increases. The vertical axis of the curve represents sensitivity, which increases with increasing true positive rates. Therefore, for a specific selected cutoff, the value of (1-specificity) can be determined, and the corresponding sensitivity can be obtained. The area under the ROC curve is an indicator of the probability that the measured marker level will allow for correct identification of a disease or condition. Therefore, the area under the ROC curve can be used to determine the effectiveness of the test.

[0325] Sample type: In this specification, the term “sample” refers to a biological sample obtained or isolated from a patient or subject. In this specification, “sample” may also refer to a sample of bodily fluids or tissues obtained for the purpose of analysis, diagnosis, prognosis, or evaluation of a subject of interest, such as a patient.

[0326] Preferably, in this specification, the sample is a bodily fluid sample such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural fluid, cells, cell extracts, tissue samples, any tissue samples from the upper or lower respiratory tract, tissue biopsies, or fecal samples. In particular, the sample is blood, plasma, or serum.

[0327] In this invention, "plasma" refers to the supernatant obtained after centrifuging blood containing an anticoagulant, which is almost entirely free of cells. Typical anticoagulants include calcium ion-binding compounds such as EDTA and citrate, and thrombin inhibitors such as heparins and hirudin. Cell-free plasma can be obtained by centrifuging anticoagulated blood (e.g., citrate-treated blood, EDTA-treated blood, or heparin-treated blood) for at least 15 minutes at 2000-3000 g for several minutes.

[0328] In the context of this invention, "serum" refers to the liquid component of whole blood collected after blood coagulation. Serum can be obtained as the supernatant when coagulated blood (blood clot) is centrifuged.

[0329] In this specification, "urine" refers to the bodily fluid secreted by the kidneys through the process of urine production (urination) and expelled through the urethra.

[0330] Adrenomedullin: In the context of the present invention, "measuring the level of proADM or its fragments," etc., is understood to preferably refer to any means of measuring MR-proADM or its fragments. The fragment can have any length, for example, at least about 5, 10, 20, 30, 40, 50, or 100 amino acids, as long as the fragment allows for a clear measurement of the level of MR-proADM. In a particularly preferred embodiment of the present invention, "measuring the level of proADM" refers to measuring the level of intermediate region pro-adrenomedullin (MR-proADM). MR-proADM is a fragment and / or region of proADM.

[0331] Adrenomedullin (ADM), a peptide, was discovered as an antihypertensive peptide consisting of amino acids isolated from 52 human pheochromocytoma cells (Kitamura et al., 1993). Adrenomedullin (ADM) is encoded as a precursor peptide consisting of 185 amino acids ("preproadrenomedullin" or "pre proADM"). An example amino acid sequence of ADM is shown in Sequence ID No. 1.

[0332] ADM contains positions 95-146 of the pre-proADM amino acid sequence and is its splice product. "Pro-adrenomedullin" ("proADM" SEQ ID NO: 2) refers to the precursor proADM with the signal sequence (amino acid residues 1-21) removed, i.e., amino acid residues 22-185 of the precursor proADM. "Intermediate region pro-adrenomedullin (MR-proADM)" refers to amino acids 42-95 of pre-proADM. An example amino acid sequence of MR-proADM is shown in SEQ ID NO: 3.

[0333] In this specification, peptides and fragments of pre-proADM or MR-proADM can be used in the methods described herein. For example, the peptide or fragment may contain amino acids 22-41 of pre-proADM (PAMP peptide) or amino acids 95-146 of pre-proADM (mature adrenomedjuline, including a biologically active form, also known as bio-ADM, SEQ ID NO: 4).

[0334] The C-terminal fragment of proADM (amino acids 153-185 of pre-proADM) is called adrenotensin. A fragment of the proADM peptide or a fragment of MR-proADM can contain, for example, at least about 5, 10, 20, or 30 amino acids. Therefore, a fragment of proADM may be selected from the group consisting of, for example, MR-proADM, PAMP, adrenotensin, and mature adrenomedullin, and preferably, in this specification, the fragment is MR-proADM.

[0335] The measurement of these various MR-proADMs or their fragments also includes measuring and / or detecting specific subregions of these molecules using antibodies or other affinity reagents against specific parts of these molecules.

[0336] Other biomarkers or parameters: Therefore, the methods and kits of the present invention may include measuring at least one additional biomarker, marker, clinical score and / or parameter in addition to ADM. In this specification, “parameter” means a characteristic, feature, or measurable factor that helps define a particular system. Parameters are important elements in health and physiological assessments, such as the risk of disease, disability, or clinical condition, and are preferably related to organ dysfunction. Furthermore, parameters are defined as characteristics that are measured and evaluated as indicators of objectively normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. Exemplary parameters include the Pneumonia Severity Index (PSI), Acute Physiological and Chronic Health Assessment II (APACHE II), Simplified Acute Physiological Score (SAPS II score), Sequential Organ Failure Assessment Score (SOFA score), Quick Sequential Organ Failure Assessment Score (qSOFA), Body Mass Index (BMI), Weight, Age, Sex, IGS II, Fluid Intake, White Blood Cell Count, Sodium, Potassium, Body Temperature, Blood Pressure, Dopamine, Bilirubin, Respiratory Rate, Oxygen Partial Pressure, World Federation of Neurosurgical Societies (WFNS) Grading, Glasgow Coma Scale (GCS), and CURB-65. The group may be selected from those including pneumonia severity score, pneumonia severity index (PSI), age, sex, family history, ethnicity, weight, body mass index (BMI), cystoscopy report, white blood cell count, lymphocyte count, imaging studies such as CT scan, PET scan, and X-ray, blood pressure, heart rate, antihypertensive treatment, fluid intake, wheezing, body temperature, presence or absence of diabetes, blood glucose levels, and (current) smoking habits.

[0337] These parameters may also be evaluated in combination with the methods described herein, contributing to improvements in assay execution and diagnostic outcomes.

[0338] The present invention offers the following advantages over conventional methods: the methods and kits of the present invention are rapid, objective, easy to use, and highly accurate. The methods and kits of the present invention relate to markers and clinical scores that can be measured in a way that is readily measurable on a daily basis, because levels of proADM, PCT, D-dimer, troponin, copeptin, lactate, and C-reactive protein can be measured in blood samples that are normally collected, or in other biological fluids or samples obtained from subjects.

[0339] As used herein, the terms “marker,” “surrogate,” “prognostic marker,” “factor,” “biomarker,” or “biological marker” are used interchangeably and relate to quantifiable biological markers (e.g., the concentration or fragment of a particular protein or enzyme, the concentration or fragment of a particular hormone, or the presence of a biological substance or fragment thereof, which are indicators of health and physiological assessments, e.g., disease / disability / clinical condition risk, preferably adverse events). A marker or biomarker is an objectively measurable feature that is evaluated as an indicator of a normal biological process, a pathological process, or a ~.

[0340] Pharmacological response biomarkers to therapeutic interventions may be measured in samples (as blood, plasma, urine, or tissue specimens).

[0341] At least one additional marker and / or parameter of the subject may be selected from the group including lactate levels in the sample, procalcitonin (PCT) levels in the sample, and the subject's sequential organ failure assessment score (SOFA score).

[0342] Optionally, the following tests may be performed on the subject: quick SOFA score, Simplified Acute Physiology Score (SAPSII), Acute Physiology and Chronic Disease Assessment II (APACHE II) score, soluble Fms-like tyrosine kinase-1 (sFlt-1), histone H2A, histone H2B, histone H3, histone H4, calcitonin, endothelin-1 (ET-1), arginine vasopressin (AVP), atrial natriuretic peptide (ANP), neutrophil gelatinase-related lipocalin (NGAL), troponin, brain natriuretic peptide (BNP), C-reactive protein (CRP), pancreatic stone protein (PSP), trigger receptor 1 (TREM1) expressed on myeloid cells, interleukin-6 (IL-6), interleukin-1, interleukin-24 (IL-24), interleukin-22 (IL-22), interleukin-20 (IL-20), and others. IL, presepsin (sCD14-ST), lipopolysaccharide-binding protein (LBP), alpha-1-antitrypsin, matrix metalloproteinase 2 (MMP2), metalloproteinase 8 (MMP8), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 7 (MMP7), placental growth factor (PlGF), chromogranin A, S100A protein, S100B protein, and tumor necrosis factor α (TNFα), neopterin, α-1-antitrypsin, pro-arginine vasopressin (AVP, proAVP) Or Copeptin), procalcitonin, atrial natriuretic peptide (ANP, pro-ANP), endothelin-1, CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13,CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, IL8 / CXCL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM1 9A4, FAM19A5, CLCF1, CNTF, IL11, IL31, IL6, leptin, LIF, OSM, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, IFNA8, IFNA5 / I FNaG, IFNω / IFNW1, BAFF, 4-1BBL, TNFSF8, CD40LG, CD70, CD95L / CD178, EDA-A1, TNFSF14, LTA / TNFB, LTB, TNFα, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TNFSF4, TRAIL, IP-10, IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33, or a group consisting of fragments thereof. Furthermore, markers include membrane microparticles, platelet count, mean platelet volume (MPV), sCD14-ST, prothrombinase, antithrombin and / or antithrombin activity, positive protein 18 (CAP18), von Willebrand factor (vWF) cleavage protease, lipoprotein CRP, fibrinogen, fibrin, B2GP1, GPIIb-IIIa, undenatured fibrin D-dimer, platelet factor 4, histones, and combinations with PT assays.

[0343] In some embodiments, the biomarkers analyzed in combination with MR-proADM using the antibodies described herein are PCT, TRAIL, and / or IP-10, which are preferably used in combination with MR-proADM, more preferably by measurement in the sample or on the same analytical system.

[0344] In this specification, "procalcitonin" or "PCT" refers to a peptide or fragment thereof containing amino acid residues 1-116, 2-116, and 3-116 of the procalcitonin peptide. PCT is a peptide precursor of the hormone calcitonin. Therefore, the length of the procalcitonin fragment is at least 12 amino acids, preferably 50 or more amino acids, and more preferably 110 or more amino acids. PCT may include post-translational modifications such as glycosylation, lipidization, or derivatization. Procalcitonin is a precursor of calcitonin and katacalcin. Therefore, under normal conditions, circulating PCT levels are very low (< approximately 0.05 ng / ml).

[0345] The PCT level in the subject's sample can be measured by the immunoassay described herein. As used herein, the level of "ribonucleic acid or deoxyribonucleic acid" encoding "procalcitonin" or "PCT" can also be measured. Methods for measuring PCT are well known to those skilled in the art. For example, this can be done using products available from Thermo Fisher Scientific / B·R·A·H·M·S GmbH.

[0346] Other markers can also be used in combination with the measurement of MR-proADM using the antibody of the present invention.

[0347] Pharmaceutical composition: In some embodiments, the present invention relates to a pharmaceutical composition comprising the antibody or a fragment thereof and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier in the sense of the present invention may be a non-toxic material that does not significantly interfere with the action of the present invention in such a way that it adversely affects the efficacy of the biological activity of the antibody thereof, without causing any harmful effects.

[0348] Clearly, the properties of the carrier depend on the route of administration. Such compositions may include, in addition to the active ingredient and carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other substances well known in the art. Formulations of pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as are the development of appropriate dosages and treatment plans for use in various treatment regimens, including oral, parenteral, intravenous, subcutaneous, nasal, inhalation, and intramuscular administration, for the use of the specific compositions described herein.

[0349] A pharmaceutical product or pharmaceutical composition containing an active ingredient (antibody or antibody fragment) may exist in a form suitable for oral administration, such as tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, or elixirs. Compositions intended for oral administration may be prepared according to any method known in the prior art with respect to the manufacture of pharmaceutical products.

[0350] Such a composition may contain one or more chemical substances belonging to the following group: This invention provides a formulation that is excellent as a medicine and easy to take, containing sweeteners, flavorings, colorings, and preservatives. The tablets contain the active ingredient mixed with non-toxic and pharmaceutically acceptable excipients and are suitable for tablet manufacturing.

[0351] These excipients may include, for example, calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulators and disintegrants, such as corn starch or alginate; binders, such as starch, gelatin, or acacia; and lubricants, such as inert diluents like magnesium stearate, stearic acid, or talc. The tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, providing a longer-lasting effect. For example, time-delaying materials such as glyceryl monostearate or glyceryl distearate may be used. These may also be coated. The present invention also relates to pharmaceutical compositions for topical administration, oral ingestion, inhalation, or injection into the skin, subcutaneously, or intravenously. Those skilled in the art are familiar with the carriers and additives required for specific use forms.

[0352] Pharmaceuticals (also called pharmaceutical compositions) containing an active ingredient (antibody or antibody fragment) may be provided in a form suitable for injection. Dosage forms including injection include, but are not limited to, subcutaneous (under the skin), intramuscular (intramuscular), intravenous (intravenous), or subarachnoid (perimal spinal) administration. Antibody therapy is typically administered intravenously, which is a preferred method of administration in this invention.

[0353] Excipients, as examples of pharmaceutically acceptable carriers for liquid formulations for injection, are known in the art and can be appropriately selected by those skilled in the art. Excipients are used to improve the broad stability of protein and peptide-based formulations, reducing protein dynamics and motility, enhancing the conformational stability of monoclonal antibodies (mAbs) in particular at high concentrations, and suppressing interface-dependent aggregation. Excipients typically protect proteins by inhibiting aggregation and adsorbing to the gas-liquid interface.

[0354] For example, the use of surfactants (e.g., polysorbate 20 and 80), carbohydrates (e.g., cyclodextrin derivatives), and amino acids (e.g., arginine and histidine) helps prevent aggregation through this mechanism. Cyclodextrins have been reported to stabilize commercially available antibody-based drugs in hydrogel formulations. Some generally recognized safe (GRAS) excipients include Pluronic F68, trehalose, glycine, and amino acids such as arginine, glycine, glutamate, and histidine, which are found in many commercially available protein therapy products. For example, bevacizumab, 25 mg / mL contains trehalose dihydrate, sodium phosphate, and polysorbate 20. The excipients of trastuzumab for subcutaneous administration, 600 mg, are rHuPH20, histidine hydrochloride, histidine, anhydrous trehalose, polysorbate 20, methionine, and water for injection.

[0355] When a therapeutically effective amount of the active ingredient (antibody or antibody fragment) of the present invention is administered intravenously, intradermally, or subcutaneously, the active ingredient may exist in the form of a solution, preferably an aqueous solution that does not contain pyrogens and is acceptable for parenteral administration.

[0356] The preparation of such parenterally acceptable solutions, with due consideration to pH, isotonicity, stability, etc., is within the scope of the skills of the art. Preferred pharmaceutical compositions for intravenous, intradermal, or subcutaneous injection should contain, in addition to the active ingredient, an isotonic vehicle such as physiological saline, Ringer's solution, glucose injection, glucose and physiological saline, lactate Ringer's solution, or other vehicles known in the art.

[0357] The pharmaceutical composition of the present invention may contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art.

[0358] The present invention also relates to administering a therapeutically effective dose of the antibodies described herein in the treatment of a subject having a medical disorder described herein. As used herein, the term “therapeutably effective dose” means the total amount of each active ingredient in the pharmaceutical composition or method that is sufficient to demonstrate a meaningful patient benefit. The amount of active ingredients in the pharmaceutical composition of the present invention is determined according to the nature and severity of the disease being treated, as well as the content of the patient’s previous treatments. More doses may be administered until the patient achieves the optimal therapeutic effect, at which point the dose is not increased further.

[0359] The dose of antibody administered clearly depends on numerous technically well-known factors, including the chemical properties of the antibody, the pharmaceutical formulation, the patient's weight, body surface area, age, and sex, as well as the timing and route of administration. For adults, the dose may be exemplary in the range of 0.001 μg to 1 g per day, preferably 0.1 μg to 100 mg per day, more preferably 1 μg to 100 mg per day, and even more preferably 5 μg to 10 mg per day. For continuous infusion, the dose is, for example, in the range of 0.01 μg to 100 mg, preferably in the range of 1 μg to 10 mg / min per kg of body weight.

[0360] Medical indications: In one aspect of the present invention, the antibodies or antibody fragments of the present invention described herein are used to treat medical conditions associated with MR-proADM. For example, they are used in medical indications where MR-proADM levels are overexpressed or present in blood samples at levels higher than, for example, the mean value in a healthy human population, or at other appropriate cutoff values ​​compared to healthy subjects.

[0361] The terms "patient" or "subject" as used herein may be vertebrates. In the context of this invention, the term "subject" includes both humans and animals, particularly mammals, and other microorganisms.

[0362] In the present invention, "treatment" or "therapy" generally means obtaining a desired pharmacological and / or physiological effect. This effect may be preventive in the sense of completely or partially preventing the disease and / or symptoms, or it may be achieved in the sense of partially or completely curing the disease and / or the adverse effects of the disease by, for example, reducing the risk that the subject will have the disease or symptoms.

[0363] In the present invention, “treatment” includes any treatment for a disease or condition in a mammal, particularly a human, and includes, for example, the following treatments (a) to (c): (a) prevention of the onset of a disease, condition or symptom in a patient; (b) suppression of symptoms, i.e., prevention of the progression of symptoms; (c) improvement of symptoms of a condition, i.e., induction of regression of the disease or symptom.

[0364] Diagnosis, sepsis, and infections: In embodiments of the present invention, this method may be used for the diagnosis, prognosis, treatment management, risk stratification, and / or treatment of sepsis, severe sepsis, and / or septic shock.

[0365] In some embodiments, this method can be used for diagnosis, prognosis, treatment management, and / or risk stratification for assessing patient severity in clinical settings (general practitioners, emergency departments (EDs), intensive care units (ICUs), and wards). This includes, but is not limited to, rule-in / out survival rates (mortality), complications, companion diagnostics for monitoring treatment (e.g., responders / non-responders), and / or monitoring or prediction of disease progression.

[0366] In the context of this invention, “sepsis” refers to a systemic response to infection. Alternatively, sepsis may be considered as SIRS and a confirmed infection or combination of infections. Sepsis may be characterized as a clinical syndrome defined by both the presence of infection and a systemic inflammatory response (Levy MM et al., 2001 SCCM / ESICM / ACCP / ATS / SIS International Conference on the Definition of Sepsis). Critical Care Medicine April 2003; vol. 31(4): 1250-6. As used herein, the term “sepsis” includes, but is not limited to, sepsis, severe sepsis, and septic shock.

[0367] As used herein, the term “sepsis” includes, but is not limited to, sepsis, severe sepsis, and septic shock. Severe sepsis refers to sepsis accompanied by organ dysfunction, hypoperfusion abnormalities, or septic hypotension. Hypoperfusion abnormalities include lactic acidosis, oliguria, and acute altered consciousness. Septic hypotension is defined as a systolic blood pressure of less than approximately 90 mmHg or more than approximately 40 mmHg below normal, in the absence of other causes of hypotension (e.g., cardiogenic shock). Septic shock is defined as severe sepsis accompanied by septic hypotension that persists despite adequate fluid resuscitation, and further characterized by the presence of hypoperfusion abnormalities or organ dysfunction (Bone et al., CHEST 101(6): 1644-55, 1992).

[0368] The term "sepsis" may be defined as life-threatening organ dysfunction caused by a poorly controlled host response to infection. In clinical practice, organ dysfunction is preferably represented by a Sequential Organ Failure Assessment (SOFA) score of 2 or more, which is associated with an in-hospital failure rate of less than 10%, and is associated with an in-hospital failure rate of less than 10%. Septic shock, particularly severe septic shock, can be defined as a subset of sepsis in which particularly severe circulatory, cellular, and metabolic abnormalities are associated with a higher mortality risk than sepsis alone. Patients with septic shock are clinically identified by the use of vasopressors to maintain mean arterial pressure above 65 mmHg and by serum lactate levels greater than 2 mmol / L (or >18 mg / dl in the absence of hypovolemia).

[0369] As used herein, the term “sepsis” refers to all possible stages in the development of sepsis. The term “sepsis” also includes severe sepsis or septic shock according to the SEPSIS-2 definition (Bone et al., 2009). The term “sepsis” also includes subjects who meet the SEPSIS-3 definition (Singer et al., 2016). As used herein, the term “sepsis” refers to all possible stages in the development of sepsis.

[0370] In embodiments of the present invention, this method may be used for the diagnosis, prognosis assessment, treatment management, or risk stratification of infectious or contagious diseases.

[0371] As used herein, “infection” means, within the scope of the present invention, a pathological process caused by the invasion of pathogenic or potentially pathogenic factors / pathogens, microorganisms and / or microbial communities into tissues or bodily fluids that are normally sterile, and preferably relates to infections by bacteria, viruses, fungi and / or parasites.

[0372] Furthermore, infection-related complications can be “hospital-acquired” infections. Hospital-acquired infections (HAIs) are infections transmitted within hospitals and other healthcare facilities. They are sometimes called healthcare-associated infections (HAIs or HCAIs) to emphasize both hospital-based and non-hospital settings. Such infections can be transmitted in hospitals, nursing homes, rehabilitation facilities, outpatient clinics, or other clinical settings. Hospital-acquired infections can spread to susceptible patients in a variety of ways within the clinical setting. Healthcare workers can spread infections through contaminated equipment, bedding, or droplets. Infections may originate from the external environment, other infected patients, potentially infected staff, or in some cases, the source of infection may be unidentifiable. In some cases, the microorganisms originate from the patient’s own skin microbiota and develop opportunistically after surgery or other procedures that compromise the skin’s protective barrier. Even if a patient develops an infection from their own skin, it is still considered a hospital-acquired infection because it occurs within a healthcare setting.

[0373] Furthermore, subjects with infectious diseases may be infected from multiple sources simultaneously. For example, a subject with an infectious disease may have a mixed infection including bacterial and viral infections, viral and fungal infections, bacterial and fungal infections, bacterial, fungal and viral infections, or one or more infections described herein, for example, a secondary infection (super infection) in which one or more bacterial infections combine with other infections. Multiple viral infections and / or one or more fungal infections.

[0374] Medical treatment: In the context of the present invention, the terms “medical treatment” or “treatment” may mean treatment with the antibodies of the present invention, or subsequent treatment following the use of the antibodies of the present invention in a diagnostic or other analytical assay.

[0375] The term “treatment” encompasses a variety of treatments and therapeutic strategies, including, but is not limited to, anti-inflammatory strategies, administration of therapeutic antibodies, ADM antagonists such as siRNA or DNA, extracellular blood purification, or prevention of cytokines by apheresis, dialysis, or removal of harmful substances via adsorbents.

[0376] This includes adequate oxygen supply to the body through cytokine storms, removal of inflammatory mediators, plasma apheresis, administration of vitamins such as vitamin C, surgical procedures, emergency surgery, mechanical and non-mechanical ventilation, such as focus defocal treatment, transfusion of blood products, colloidal fluid administration, organ replacement such as kidney or liver replacement, antibiotic treatment, invasive mechanical ventilation, non-invasive mechanical ventilation, use of vasopressors, fluid therapy, apheresis, and organ replacement.

[0377] Protection. Those skilled in the art will be able to determine which of the treatments described herein require administration in a hospital setting.

[0378] Those skilled in the art can also determine what diseases and their severity require treatments that are only (or primarily) available in a hospital setting, such as an emergency department (ED) or intensive care unit (ICU).

[0379] Further therapeutic applications of the present invention include the administration of cells or cell products such as stem cells, blood, or plasma, stabilization of the patient's circulation, and protection of the endothelial glycocalyx. For example, this includes achieving normal blood volume and preventing or treating excessive or insufficient blood volume through optimal fluid management strategies. Furthermore, vasopressors and catecholamines, as well as albumin or heparanase inhibitors, are useful for treatments supporting the circulatory system and endothelium, carried out via unfractionated heparin or heparin reacetylated after desulfation.

[0380] Furthermore, the medical procedures of the present invention include, but are not limited to, blood coagulation stabilization, antifibrinolytic therapy, iNOS inhibitors, anti-inflammatory agents such as hydrocortisone, sedatives and analgesics, and insulin.

[0381] Mechanical ventilation is an effective means of promoting proper gas exchange and ventilation, and is intended to save lives in cases of severe hypoxemia. Mechanical ventilation refers to assisting or stimulating a subject's breathing. Mechanical ventilation can be selected from a group that includes mechanical ventilation, manual ventilation, extracorporeal membrane oxygenation (ECMO), and non-invasive ventilation (NIV). Mechanical ventilation relates to a method of mechanically assisting or replacing spontaneous breathing. This may involve a device called a ventilator. Mechanical ventilation may be high-frequency oscillatory ventilation or partial fluid ventilation.

[0382] In a preferred embodiment, the term “medical treatment” or “treatment” includes antibiotic treatment such as intravenous antibiotics, oral antibiotics, or topical antibiotics. In a more preferred embodiment,

[0383] The terms "medical procedure" or "procedure" include intravenous administration. The antibiotic treatment and medical procedures administered also include methods to prevent hypothermia, such as the use of warmed intravenous fluids and heated blankets.

[0384] Medical procedures include wound cleaning, application of local anesthetics, and wound closure techniques, whether or not tourniquets are used for bleeding control and hemostasis, as well as wound management, including skin adhesive tapes, tissue adhesives, sutures, staples, and wound dressings.

[0385] The medical procedure of the present invention is antibiotic treatment, and if an infection is detected and diagnosed or prognosed by the method of the present invention, one or more "antibiotics" or "antibiotic agents" may be administered.

[0386] Antibiotics or antimicrobial agents The present invention may also include antifungal or antiviral compounds used to treat diagnosed infections or sepsis.

[0387] General terms: In this specification, the terms “comprising” and “including” or their grammatical variations are understood to identify a described feature, integer, procedure, or component, but not to impose the possibility of adding one or more additional features, integers, procedures, components, or groups thereof. This term includes the terms “consisting of” and “consisting essentially of.”

[0388] Therefore, in this specification, the terms "consisting essentially of," "including," and "having" mean that any additional components (such as features, elements, steps, etc.) may be present. The term "consisting of" means that no other components (such as features, elements, steps, etc.) are present.

[0389] The phrase "essentially consisting of" or its grammatical variations thereof, as used herein, shall be interpreted as identifying the described feature, number, step, or component, but shall not preclude the addition of one or more additional features, numbers, steps, components, or groups thereof, provided that the additional features, numbers, steps, components, or groups thereof do not substantially alter the basic and novel characteristics of the claimed composition, apparatus, or method.

[0390] Therefore, the term “substantially consisting of” means that certain additional components (or similar features, elements, steps, etc.) may be present that do not substantially affect the essential properties of the composition, apparatus, or method. In other words, the term “consisting essentially of” (and herein, “comprising substantially” which may be used interchangeably with the term) allows for the presence of other components in the composition, apparatus, or method in addition to essential components (or similarly essential features, elements, steps, etc.), and applies when the presence of the other components does not substantially affect the essential properties of the apparatus or method.

[0391] The term “method” refers to methods, means, techniques and procedures for achieving a particular task, including, but not limited to, methods, means, techniques and procedures known or readily developable from methods, means, techniques and procedures known by chemical, biological and biophysical engineers.

[0392] [Table 7-1]

[0393] [Table 7-2]

[0394] [Table 7-3]

[0395] [Table 7-4]

[0396] [Table 7-5]

[0397] Table 7-6

[0398] Table 7-7

[0399] Table 7-8

[0400] Table 7-9

[0401] Table 7-10

[0402] Table 7-11

[0403] Table 7-12

[0404] Table 7-13

[0405] Table 7-14

[0406] Table 7-15

[0407] [Table 7-16]

[0408] [Table 7-17]

[0409] [drawing] The present invention is demonstrated through the illustrated embodiments disclosed herein. The presented figures illustrate certain non-limiting embodiments and are not intended to limit the scope of the invention. [Brief explanation of the drawing]

[0410] [Figure 1] Figure 1: SPR measurement for MR-proADM antigen 1. [Figure 2] Figure 2: SPR measurement for MR-proADM antigen 2. [Figure 3] Figure 3: Overview of MR-proADM measurement [Figure 4] Figure 4: Data obtained from MR-proADM measurements [Figure 5] Figure 5: Data obtained from MR-proADM measurements. [Figure 6] Figure 6: Comparative quantitative measurement of MR-proADM in two assays [Figure 7] Figure 7: Comparative quantitative measurement of MR-proADM in two assays

[0411] [Detailed description of the drawing] [Figure 1] SPR measurement for MR-proADM antigen 1. Surface plasmon resonance (SPR) was used to measure the affinity of antibodies Ab2, Ab3, and Ab5 for the MR-proADM antigen. [Figure 2] SPR measurement for MR-proADM antigen 2. The affinity of antibodies Ab2, Ab3, and Ab5 for the MR-proADM antigen was measured using surface plasmon resonance (SPR). [Figure 3] Overview of MR-proADM measurement. The antibody of the present invention was used in immunoluminometric assays to measure function and compare it with established methods, including the B·R·A·H·M·S MR-proADM KRYPTOR assay, which is based on a polyclonal antibody against MR-proADM. [Figure 4] Data obtained from MR-proADM measurement. Overview of MR-proADM measurement. The antibodies of the present invention were used in immunoluminometric assays to evaluate their function and compare with established methods, including the B·R·A·H·M·S MR-proADM KRYPTOR assay, which is an existing method based on polyclonal antibodies against MR-proADM. Samples were provided from healthy patients (low MR-proADM), and quality control samples were performed in parallel using samples containing 4 nmol / L MR-proADM in EDTA-treated or heparinized plasma and analyzed using various combinations of the antibodies of the present invention. The antibodies were appropriately labeled (Tb = terbium donor, AF = Alexa fluorescent dye acceptor) and analyzed using an immunoluminometric assay system. [Figure 5] This shows an excerpt from Figure 11 at the 30-minute mark, highlighting its useful points. The combination of Ab2 and Ab3 compared to an existing assay based on labeled polyclonal antibodies (B·R·A·H·M·S MR-proADM KRYPTOR assay). [Figure 6] Two individual comparative studies were conducted using the Ac2-Tb / Ab3-AF assay format with 57 EDTA plasma samples (KG0007) to compare the established B·R·A·H·M·S MR-proADM assay with the novel (inventive invention) assay "MR-proADM 2.0 mAb". Quantitative MR-proADM measurements were performed on each sample to compare the performance of the two assays. [Figure 7] A comparative study of the established B·R·A·H·M·S MR-proADM assay with the novel (inventive invention) assay "MR-proADM 2.0 mAb" was conducted using the Ac2-Tb / Ab3-AF assay format, with 29 EDTA plasma samples (KG0166), in two individual comparisons. Quantitative MR-proADM measurements were performed for each sample to compare the performance of the two assays. [Examples]

[0412] The present invention is demonstrated by the examples disclosed herein. The provided examples represent specific embodiments and are not intended to limit the scope of the invention.

[0413] These embodiments are non-limiting examples and are intended to provide illustrations and technical support for carrying out the present invention.

[0414] The following examples describe the characterization of various monoclonal antibodies obtained by immunizing mice with MR-proADM fragments. These examples further show antibody sequencing and the associated variable domain sequences of the antibodies.

[0415] The examples further illustrate the use of antibodies when measuring MR-proADM in plasma samples using an immunoluminometric assay.

[0416] Immunization in mice: Mice were immunized according to established protocols. For example, mice were immunized by administering purified antigens (e.g., SEQ ID NOs: 5-18, preferably SEQ ID NOs: 6, 8, 10, 12, 14, 16, or 18) every other week.

[0417] In mice, soluble protein antigens are used, typically in doses of 50–100 μg per immunization. Adjuvants (e.g., Freund, Ribi, Hunter's TiterMax, ImmunEasy, or aluminum) are mixed with the immunogen during the first two immunizations. A complete Freund's adjuvant is used for the first immunization. Subsequent immunizations are performed in phosphate-buffered saline (PBS) or saline, with or without an incomplete Freund's adjuvant mixture. Each animal can receive a total of six injections over the course of a six-week immunization schedule. Once adequate antibody titers have formed against the target antigen, boost immunizations and blood collections are performed periodically to obtain serum.

[0418] Production of mouse monoclonal antibodies: Standard methods were used to generate monoclonal antibodies using established hybridoma technology. This technology typically combines two steps: the first step involves appropriate immunization (see above), and the second step involves the fusion of B lymphocytes with immortalized Mayleuma cells to create a hybrid capable of producing immortal antibody molecules in cell culture.

[0419] The generated hybridoma cells are recloned and diluted to obtain a stable monoclonal cell line that secretes the desired monoclonal antibody into the culture supernatant. The supernatant is tested for antigen specificity to MR-proADM, for example, by enzyme immunosorbent assay (ELISA). After selecting the appropriate cell clone, the cells are transferred to large-scale culture.

[0420] Culture is used to produce larger quantities of the desired antibody molecule. Antibody purification is routinely performed by affinity chromatography.

[0421] Determination of antibody sequences: Antibody sequences were determined using next-generation sequencing (ngs). Hybridomas were provided to sequencing providers, total RNA was extracted, and DNA libraries were created using cRT-PCR.

[0422] Subsequently, the cDNA library was sequenced using next-generation sequencing (Illumina HiSeq sequencer). Contigs were then ligated, and antibody sequences were identified. Sequences of known abnormal (non-functional) antibody genes frequently found in hybridoma cell lines were not included in the analysis.

[0423] Alternatively, the antibody sequence was determined by peptide mapping using liquid chromatography. De novo protein sequencing using mass spectrometry-coupled chromatography (LC / MS) is an established method for determining protein sequences using mass spectrometry. The advantage of this method is that it does not require prior information on the peptide's amino acid sequence and avoids the limitations of methods that rely on protein sequence databases. De novo protein sequencing analysis yielded results that allowed for the direct identification of CDR sequences from the acquired mass spectrometry data. LC-MS analysis was performed according to an established protocol. Using the identified sequences, a conventional bottom-up search of the DDA data was performed using FragPipe software.

[0424] Based on next-generation (nucleic acid) and de novo (protein) sequencing, antibody sequences were evaluated according to standard methods using three methods: CDR and variable region annotation, for example, with Protein Metrics software or Geneious antibody annotation software. Antibodies have predictable secondary structures. Standard schemes exist for annotating the CDR region of linear protein sequences, the most common of which is called the Chothia method (Al-Lazikani, B. et al., 1997; Journal of Molecular Biology, 273(4), 927-948) or Kabat (Kabat EA, et al., 1991, U.S. Patent) (Department of Health and Human Services, Public Health Service, National Institutes of Health). These have been widely adopted in sequence-based schemes for antibody residue numbering. In preferred embodiments, Kabat CDR annotation is used, for example, in sequence numbers 134-203.

[0425] In some embodiments, any CDR sequence shown herein can be combined with any other CDR sequence. In some embodiments, CDR sequences obtained from the same annotation method can be combined with each other to form, for example, a combination of three L-CDRs or three H-CDRs. In some embodiments, CDR sequences obtained from different annotation methods can be combined with each other if they are obtained from the same VH or VL sequence to form, for example, a combination of three L-CDRs or three H-CDRs. In some embodiments, CDR annotations can be combined with each other using methods 1, 2, or 3 from any one or more of the same VH or VL sequences to form, for example, a combination of three L-CDRs or three H-CDRs. The obtained antibody sequences are shown in the sequence listing above.

[0426] Antibody stability: The stability and aggregation of seven antibodies were evaluated using nanoDSF measurement. No aggregation was observed under the following conditions:

[0427] [Table 8]

[0428] Furthermore, the thermal stability of the protein was measured using a Prometheus NT.48 instrument by monitoring changes in autofluorescence (derived from tyrosine or phenylalanine residues) under a temperature gradient. To measure the melting point (Tm) when half of the protein is spread out, the ratio of fluorescence intensity (F350 / F330) can be plotted against temperature.

[0429] Protein samples were subjected to a linear temperature increase (2°C / min, from 20°C to 90°C), and fluorescence at 350 nm and 330 nm was collected at a rate of 10 data points per minute. The midpoint of the transition was automatically determined from the first derivative of the fluorescence ratio (F350 / F330).

[0430] Antibody concentration: 200 μg / mL, buffer: PBS, excitation light intensity: 90%, temperature rise rate: 2.0°C / min. Agglutination analysis was performed simultaneously by light scattering under the same temperature change. The scattering onset temperature corresponds to the onset of agglutination.

[0431] [Table 9]

[0432] Ab7 appears relatively unstable, but within acceptable limits (Tm1 = 58.8 °C compared to 63-69 °C for other Abs). Agglutination by scattering (light scattering) shows an aggregation tendency according to Ac4 > Ac1 > Ac5 > Ac7 > Ac6. Surprisingly, no aggregation was detected for Ac2 and Ac3. For Ac1, Ac4, and Ac5, the aggregation temperature starts before the first Tm temperature. All antibodies showed sufficient stability and aggregation characteristics for the assay of interest.

[0433] Aggregation and Tm analysis were repeated under different conditions. Antibody spread and aggregation were evaluated using NanoDSF based on antibody concentration.

[0434] 1 mg / mL, buffer: PBS, excitation intensity: 40%, temperature gradient: 2.0°C / min, capillary high sensitivity. Protein thermal stability was measured using a Prometheus NT.48 instrument by monitoring changes in autofluorescence (derived from tyrosine or phenylalanine residues) under a temperature gradient. To measure the melting point (Tm) when half of the protein is spread out, the ratio of fluorescence intensities (F350 / F330) can be plotted against temperature. Protein samples were subjected to a linear temperature increase (2°C / min, from 20°C to 90°C), and fluorescence at 350 nm and 330 nm was collected at a rate of 10 data points per minute. The midpoint of the transition was automatically determined from the first derivative of the fluorescence ratio (F350 / F330). Aggregation analysis was simultaneously measured by light diffusion at the same temperature change. The scattering onset temperature corresponds to the onset of aggregation.

[0435] [Table 10]

[0436] The melting point (fluorescence) and stability assessments were consistent with previous experiments. Positively, each batch of Ab2 and Ab3 showed the same profile, confirming consistent characterization. Under these conditions, Ab5 appears stable. All antibodies exhibited sufficient stability and aggregation characteristics, making them suitable for the target assay.

[0437] Affinity measurement of identified antibodies: Using surface plasmon resonance (SPR), the antibody affinity of Ab2, Ab3, and Ab5 to MR-proADM targets was measured, for example. Two individually prepared MR-proADM solutions were used.

[0438] The target was used to test affinity. The test antigen consisted of a synthetic peptide containing the following:

[0439] The complete sequence of MR-proADM, as described in Sequence ID No. 3. The peptide was retained at 1 mg / mL (195.5 μM) in phosphate buffer with 0.1% BSA-free protease, 6 mmol / L EDTA, and pH 7.0, with a mass of 5113.8 g / mol.

[0440] Ab2, Ab3, and Ab5 interact with antigen 1 and antigen 2, respectively. Their binding affinities are as follows:

[0441] [Table 11]

[0442] The antibody showed reproducible results across multiple tests and preparations of multiple antigens. Additional experiments using the remaining antibodies are currently underway.

[0443] Additional experiments were repeated to evaluate the affinity of the invented antibodies. As an example, surface plasmon resonance was again used to measure the antibody affinity of Ab2, Ab3, and Ab5 against the MR-proADM target. The test antigen was a synthetic peptide containing MR-proADM (45-92 amino acids of pre-proADM), as shown in SEQ ID NO: 3.

[0444] SPR analysis was performed using a T200 instrument in PBS-pH 7.4 containing 0.05% P20 surfactant (Cytiva) at 25°C. For binding measurements, Ac was captured (1000-2000 RU) on mouse anti-Fc antibody immobilized on a CM5S dextran sensor chip. Anti-Fc antibody diluted in acetate buffer (pH 5) was covalently immobilized (10000-13000 RU) on the CM5S sensor chip using the EDC / NHS activation method according to the manufacturer's instructions (Kit mouse anti-Fc Cytiva). 100 nM antigen peptide was injected at 50 μL / min (injection time = 120 seconds). After dissociation in running buffer for 400 seconds, the sensor surface was regenerated using 30 μL of Gly-HCl pH 1.7. KD values ​​were calculated using the Langmuir 1:1 fitting model (BiaEvaluation3.2, Cytiva). All sensorgrams were corrected before fitting evaluation by subtracting low signals from the control reference surface (without immobilized protein) and blank buffer injection.

[0445] [Table 12]

[0446] Testing of the functional properties of identified antibodies: The invented antibody was used in immunoluminescence assays to evaluate its function and compare it with existing methods such as B·R·A·H·M·S MR-.

[0447] "proADM KRYPTOR" assay using polyclonal antibodies against MR-proADM As seen in Figure 3-5, the invented antibody shows improved signaling compared to the "B·R·A·H·M·S MR-proADM KRYPTOR" (trademark) assay when analyzing plasma samples containing a certain amount of MR-proADM target. Samples were provided from healthy patients (low MR-proADM), and quality control samples were run in parallel. Samples containing 4 nmol / L MR-proADM in EDTA or heparinized plasma samples were also analyzed using different combinations of the antibody of the present invention. The antibody was appropriately labeled (Tb = terbium donor, AF = lexafluorophore receptor) and analyzed using an immunoluminometric assay system.

[0448] The experimental conditions are as follows: Twelve types of monoclonal antibodies (mAbs) have been tested, with approximately 140 pairs tested on Pherastar. - Conjugated concentration: L4: 0.3 μg / mL - AF: 3 μg / mL - Buffer conditions: TRIS 0.1M, pH 7, mouse IgG 0.1 mg / mL, BSA 0.1%, bovine IgG 0.5 mg / mL. - 7 samples per condition (EDTA, HP, QC) - T0, 15 min, 30 min, 60 min. Sample volume: 26 μL for B·R·A·H·M·S MR-proADM KRYPTOR, and 50 μL for the new (improved) MR-proADM 2.0 mAb couple.

[0449] The measurement was performed by determining the "unitless" relative fluorescence units (RFU). This is essentially a measurement of the fluorescence signal emitted from the fluorescent dye used in the assay.

[0450] As can be deduced from Figure 3-5, detection was achieved with the following antibody combinations. signal: - Ab1 together with Ab2 or Ab3 - Ab2 using Ab1, Ab4, Ab3, Ab5, or Ab6 - Ab4 and Ab2, - Ab3 is used with Ab1, Ab2, or Ab7. - Ab5 and Ab2, - Ab6 and Ab2, and - Ab7 and Ab3.

[0451] In particular, beneficial combinations were identified when using Ab1, Ab2, Ab3, Ab4, and Ab5 in various combinations. Improvements in RFU measurements were confirmed compared to the "B·R·A·H·M·S MR-proADM KRYPTOR" (trademark) control.

[0452] Figure 5 shows an excerpt of the complete data, highlighting the beneficial results obtained by using the combination of Ab2 and Ab3.

[0453] Optimizing antibody pairing: The experiments described above were repeated using the most promising antibody pairs, with different combinations of donor and acceptor fluorophores, and with EDTA and heparinized plasma samples at various proADM concentrations.

[0454] The conditions under which it was used are as follows: - KRYPTOR Gold KG0198 + Comparison Method B·R·A·H·M·S MR-proADM KRYPTOR - Conjugated concentrations: Terbium (Tb) 0.3 μg / mL - AF 3 μg / mL - Buffer conditions: TRIS 0.1M, pH 7, mouse IgG 0.1 mg / mL, BSA 0.1%, bovine IgG 0.5 mg / mL - Sample volume: 26 μL for B·R·A·H·M·S MR-proADM KRYPTOR, and 50 μL for the novel (inventive) assay "MR-proADM 2.0 mAb" antibody pair. - 59 minutes of analysis time

[0455] [Table 13]

[0456] As the results clearly show, the combination of monoclonal antibodies Ab2 and Ab3 demonstrated superior results in both donor and acceptor fluorophore configurations, confirming superior detection performance compared to conventional polyclonal antibody-based MR-proADM assays. Signal intensity was remarkably improved by up to approximately 10 times compared to conventional methods.

[0457] Buffer fluctuation: The experiments described above were repeated using the most promising antibody pairs, with different combinations of donor and acceptor fluorophores, and with samples of EDTA plasma and heparin plasma at different proADM concentrations and different buffer components.

[0458] In particular, both TRIS buffer and phosphate buffer were evaluated. The conditions used were as follows: - 2 Systems + Comparison Method B·R·A·H·M·S MR-proADM KRYPTOR - Terbium (Tb): 0.3 μg / mL - AF: 3 μg / mL - TRIS 0.1M, pH 7, mouse IgG 0.1 mg / mL, BSA 0.1%, bovine IgG 0.5 mg / mL (freshly prepared) - Phosphate buffer (0.1 M, pH 7), mouse IgG 0.1 mg / mL, BSA 0.1%, bovine IgG 0.5 mg / mL (freshly prepared) - Sample volume: 26 μL for B·R·A·H·M·S MR-proADM KRYPTOR, 50 μL for new measurements. Antibody pair for assay "MR-proADM 2.0 mAb" (related to the invention) - Analysis time 29 minutes.

[0459] [Table 14]

[0460] As the results clearly show, the combination of monoclonal antibodies Ab2 and Ab3 demonstrated excellent results in both Tris buffer and phosphate buffer, regardless of the configuration using donor and acceptor fluorophores. Mutations in fluorescent dyes: Further analyses were conducted to evaluate the performance differences when various fluorescent dyes were used. The above experiments were repeated using donor fluorophores of europium cryptotate ("-K") or terbium cryptotate ("-Tb") bound to Ab2, and comparisons were made with existing products in both EDTA and heparinized plasma samples. The conditions used were as follows: Europium cryptotate / AF647 (PO4 + KF600mM buffer) vs. Terbium / AF (PO4 buffer) vs. B·R·A·H·M·S MR-proADM KRYPTOR - MR-proADM reference material, EDTA, and heparinized plasma pool - Sample volume: 26 μL for B·R·A·H·M·S MR-proADM KRYPTOR, 50 μL for new measurements. (Inventive) assay "MR-proADM 2.0 mAb" antibody pair - Analysis time 29 minutes.

[0461] [Table 15]

[0462] As the results show, europium-labeled Ab2 demonstrated superior performance, showing improvement compared to QC samples measured in both novel and conventional assays. Furthermore, terbium-labeled Ab2 resulted in further signal enhancement in both assays.

[0463] Europium assays using established assays and novel monoclonal antibodies. As can be observed from the results, while europium-labeled Ab2 shows a similar signal level in plasma samples compared to existing assays using polyclonal antibodies, terbium-labeled Ab2 shows further signal enhancement in plasma patient samples compared to europium-labeled Ab2.

[0464] Additional comparison with existing assays: The established B·R·A·H·M·S MR-proADM assay and a new (proprietary) assay, "MR-proADM 2.0 mAb," were compared using the Ac2-Tb / Ab3-AF assay format. The comparison was conducted in two phases, using 57 EDTA plasma samples (KG0007; Figure 6) and 29 EDTA plasma samples (KG0166; Figure 7). Quantitative MR-proADM measurements were performed for each sample, and the performance of the two assay methods was compared. The results showed very good agreement between the two assays when calculating MR-proADM levels in EDTA plasma samples. In each of the two comparisons, Spearman correlation coefficients (rs) were achieved at 0.993 and 0.987, and Fisher's exact probability test values ​​were 0.989–0.996 and 0.972–0.994, respectively.

Claims

1. A method for measuring intermediate region pro-adrenomedullin (MR-proADM) in a sample in vitro, comprising the following: - The sample, i. A first monoclonal antibody or fragment thereof that is specific to the first epitope of MR-proADM, and ii. Contacting with a second monoclonal antibody or fragment specific to the second epitope of MR-proADM; and - To detect the binding of the first and second monoclonal antibodies or their fragments to the aforementioned MR-proADM, - The method, characterized in that the first and second antibodies are labeled with a detectable label, and the signal generated by the label is modulated when the first and second antibodies bind to the antibody-MR-proADM complex.

2. The method according to claim 1, wherein a first antibody and a second antibody are dispersed in a liquid reaction mixture (homogeneous immunoassay).

3. The method according to any one of the preceding claims, wherein the first antibody is labeled with a donor label and the second antibody is labeled with an acceptor label.

4. The method according to any one of the preceding claims, wherein the proximity of the donor and acceptor labels in the antibody-MR-proADM complex and the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor enhance the signal and / or extend the signal lifetime, enabling the measurement of time-delayed fluorescence.

5. The method according to any one of the preceding claims, wherein the donor label comprises a rare earth cryptotate or chelate, preferably a lanthanide ion, and the acceptor label comprises a fluorescent or chemiluminescent dye.

6. The method according to any one of the preceding claims, wherein the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye having an excitation and emission spectrum that matches the excitation and emission spectrum of the terbium, wherein the excitation and fluorescence spectrum of the terbium is in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplification cryptotate emission (TRACE) reaction.

7. The method according to any one of the preceding claims, wherein the donor label comprises a terbium cryptotate or chelate having an emission spectrum at 485-495, 540-550, 585-595 and / or 615-625 nm, and the acceptor label comprises a fluorescent dye having an excitation spectrum corresponding to the emission spectrum of the donor label and having an emission spectrum different from the emission spectrum of the donor label, wherein the wavelength range of the emission spectrum is preferably 500-570 nm, more preferably 510-560 nm, or 515-555 nm (green), or 580-770 nm, preferably 600-780, or 670-690 nm (red).

8. A method according to any one of the preceding claims, wherein the first and / or second antibody or fragment thereof is a murine antibody, preferably a mouse antibody, wherein the antibody or fragment thereof comprises, for example, an amino acid sequence isolated after immunization of a murine subject.

9. The method according to any one of the preceding claims, wherein the first and second epitopes are different from each other and are selected from MR-proADM epitopes according to Sequence IDs 5 to 18.

10. The method according to any one of the preceding claims, wherein the first and / or second antibody comprises VH and VL domains containing sequences according to SEQ ID NOs. 136 and 137 (Ab1), 151 and 152 (Ab2), 166 and 167 (Ab3), 181 and 182 (Ab4), 196 and 197 (Ab5), 211 and 212 (Ab6), or 226 and 227 (Ab7), respectively.

11. A method according to any one of the preceding claims, wherein the first and second epitopes are different and selected from SEQ ID NO: 7 or 8 (epitope of Ab2) and SEQ ID NO: 9 or 10 (epitope of Ab3), and the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye that matches the excitation and emission spectra of terbium, wherein the excitation and emission spectra are in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplification of cryptotate emission (TRACE) reaction, preferably according to claim 7.

12. The method according to any one of the preceding claims, wherein the first and second antibodies are different and each comprises the following six CDR sequences: H-CDR1 corresponds to sequence number 153 or 154. H-CDR2 follows sequence number 155 or 156, H-CDR3 follows sequence number 157 or 158, L-CDR1 follows sequence number 159 or 160, L-CDR2 follows sequence number 161 or 162, L-CDR3 follows sequence number 163 (Ab2), and H-CDR1 follows sequence number 168 or 169, H-CDR2 follows sequence number 170 or 171, H-CDR3 follows sequence number 172 or 173, L-CDR1 follows sequence number 174 or 175, L-CDR2 follows sequence number 176 or 177, and L-CDR3 follows sequence number 178 (Ab3). The method according to any one of claims 1 to 10, wherein the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye having an excitation and emission spectrum that matches the excitation and emission spectrum of terbium, wherein the excitation and emission spectrum is in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplification of cryptotate fluorescence (TRACE) reaction, and preferably according to claim 7.

13. The method according to any one of the preceding claims, wherein the first and second antibodies are different and include VH and VL domains corresponding to the VH and VL domains according to SEQ ID NOs. 151 and 152 (Ab2) and SEQ ID NOs. 166 and 167 (Ab3).

14. The first and second epitopes are different and selected from sequence numbers 7 or 8 (epitope of Ab2) and sequence number 11 or 12 (epitope of Ab4), A method according to any one of claims 1 to 10, wherein the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye that conforms to the excitation and emission spectra of terbium, wherein the excitation and emission spectra of terbium are those in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplification cryptotate emission (TRACE) reaction, preferably according to claim 7.

15. The method according to any one of claims 1 to 10, wherein the first and second antibodies are different and each has the following six CDR sequences: H-CDR1 corresponds to sequence number 153 or 154. H-CDR2 follows sequence number 155 or 156, H-CDR3 follows sequence number 157 or 158, L-CDR1 follows sequence number 159 or 160, L-CDR2 follows sequence number 161 or 162, L-CDR3 follows sequence number 163 (Ab2), and H-CDR1 follows sequence number 183 or 184, H-CDR2 follows sequence number 185 or 186, H-CDR3 follows sequence number 187 or 188, L-CDR1 follows sequence number 189 or 190, L-CDR2 follows sequence number 191 or 192, and L-CDR3 follows sequence number 193 (Ab4). The method according to any one of claims 1 to 10, wherein the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye having an excitation and emission spectrum that matches the excitation and emission spectrum of terbium, wherein the excitation and emission spectrum is in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplification of cryptotate fluorescence (TRACE) reaction, and preferably according to claim 7.

16. The method according to any one of the preceding claims, wherein the first and second antibodies are different and include VH and VL domains according to SEQ ID NOs. 151 and 152 (Ab2), and SEQ ID NOs. 181 and 182 (Ab4).

17. The method according to any one of claims 1 to 10, wherein the first and second epitopes are different and selected from SEQ ID NO: 5 or 6 (epitope of Ab1) and SEQ ID NO: 7 or 8 (epitope of Ab2), the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye having an excitation and emission spectrum that matches the excitation and emission spectrum of terbium, wherein the excitation and emission spectrum is that of a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplified cryptotate emission (TRACE) reaction, preferably according to claim 7.

18. The method according to any one of claims 1 to 10, wherein the first and second antibodies are different and each has the following six CDR sequences: H-CDR1 follows sequence number 138 or 139, H-CDR2 follows sequence number 140 or 141, H-CDR3 follows sequence number 142 or 143, L-CDR1 follows sequence number 144 or 145, L-CDR2 follows sequence number 146 or 147, L-CDR3 follows sequence number 148 (Ab1), and H-CDR1 corresponds to sequence number 153 or 154. H-CDR2 is according to sequence number 155 or 156. H-CDR3 is according to sequence number 157 or 158. L-CDR1 corresponds to sequence number 159 or 160. L-CDR2 follows sequence number 161 or 162, and L-CDR3 follows sequence number 163 (Ab2). The method according to any one of claims 1 to 10, wherein the donor label comprises a terbium cryptotate or chelate, and the acceptor label comprises a fluorescent dye having an excitation and emission spectrum that matches the excitation and emission spectrum of terbium, wherein the excitation and emission spectrum is in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplification of cryptotate fluorescence (TRACE) reaction, and preferably according to claim 7.

19. The method according to any one of the preceding claims, wherein the first antibody and the second antibody are different and include VH and VL domains according to SEQ ID NOs. 136 and 137 (Ab1) and 151 and 152 (Ab2).

20. An analytical, detection, and / or diagnostic kit used in a method described in any one of the preceding claims, comprising: - A first monoclonal antibody and a second monoclonal antibody, or fragments thereof, which bind to the first and second epitopes of MR-proADM, respectively, and - One or more donor labels comprise a rare earth cryptotate or chelate, preferably a lanthanide ion, more preferably a terbium chelate or cryptotate, and the acceptor label comprises a fluorescent or chemiluminescent dye, the excitation and emission spectra of the acceptor label match those of the donor label, and the excitation and emission spectra of the acceptor label are those of a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplified cryptotate emission (TRACE) reaction, preferably according to claim 7, - The label is positioned in physical proximity to the antibody in the kit and / or is bound to the antibody.

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