High affinity antibodies that specifically bind to pepsinogen a

Abstract

The present disclosure provides antibodies that specifically bind to pepsinogen A, as well as compositions and kits comprising such antibodies. Furthermore, provided are polynucleotides encoding such antibodies, host cells expressing the antibodies, methods of producing such antibodies, and diagnostic methods using such antibodies.

Description

[0001] This invention provides novel antibodies against pepsinogen A, as well as compositions and kits comprising such antibodies (especially combinations thereof). Furthermore, it provides polynucleotides encoding such monoclonal antibodies, host cells expressing said antibodies, methods for producing such antibodies, and diagnostic methods using such antibodies. Background Technology

[0002] Alterations in gastric acid secretion levels are associated with the pathogenesis of various types of diseases originating in the upper gastrointestinal tract (GI). Classically, individuals with hyperacidity, particularly those infected with Helicobacter pylori, are more prone to duodenal ulcers, while those with hypoacidity face a higher risk of gastric cancer through the development of premalignant conditions (gastric atrophy) (El-Omar et al., 1995, 1997). Therefore, measuring gastric acid secretion levels has some clinical significance in estimating the risk of various upper GI diseases. However, given the significant discomfort and time-consuming nature of the procedure, direct measurement of gastric acid secretion levels has become less common today.

[0003] Pepsinogen (PG) I (A) is secreted solely by the fundic glands (Samloff 1971), while PG II (C) is secreted by the fundic glands, pyloric glands, and proximal duodenal mucosa (Samloff and Liebman 1973). Previous studies have shown that serum PG I (A) levels and / or I / II (A / C) ratios reflect the morphology (Samloff et al. 1982; Borch et al. 1989) and functional status of the gastric mucosa (Samloff et al. 1975; Nakanome et al. 1983; Miki et al. 1987; Feldman et al. 1988). Many studies have reported a significant correlation between serum PG levels and gastric acid secretion levels (Samloff et al. 1975; Nakanome et al. 1983; Miki et al. 1987; Haruma et al. 1993; Kinoshita et al. 1997; Feldman et al. 1988; Iijima et al. 2005; Derakhshan et al. 2006).

[0004] Although assays for detecting PGA and C are available on the market, there is still a need for improvement. There is a particular demand for higher precision, especially at low analyte concentrations, using smaller sample volumes, and providing faster results.

[0005] Therefore, there is a great need to identify high-affinity antibodies that specifically bind to pepsinogen A.

[0006] The invention described herein addresses the aforementioned needs. Attached Figure Description

[0007] The following figures are provided to aid in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the described procedures without departing from the spirit of the invention.

[0008] Figure 1 ELISA of the supernatant from mouse hybridoma culture. (e.g.) Figure 1 As shown, ELISA screening was performed using the supernatant from hybridoma culture.

[0009] (1) 96 / 384-well MTP coated with streptavidin (2) Recombinant PepA, (3) mAb sample (supernatant from hybridoma culture) (4) Anti-mouse-IgG POD-conjugate.

[0010] Figure 2 SPR (primary culture). Kinetic screening with exemplary kinetic characteristics of antibody / pepsinogen interactions. Binding to 150 nM PGA and PGC are shown. Superimposed normalized sensor plots of antibody interactions that are specific to PGA (top sensor plot) and do not cross-react with PGC (bottom sensor plot).

[0011] Figure 3SPR: Kinetic characteristics of mAbs: At 37°C, mAb2, mAb7, mAb8, and mAb6.7 bind to PGA5-V353L, PGA5-K222Q, PGA5-T265A, PGA5-E58K, PGA, and PGC. The concentration increase series is shown as c = 1.2 -450 nM, with a dilution factor of 3. A) mAb2 combined with PGA5-V353L, B) mAb2 combined with PGA5-K222Q, C) mAb2 combined with PGA5-T265A, D) mAb2 combined with PGA5-E58K, E) mAb2 combined with rec PGA, F) mAb2 combined with rec PGC, G) mAb8 combined with PGA5-V353L, H) mAb8 combined with PGA5-K222Q, I) mAb8 combined with PGA5-T265A, J) mAb8 combined with PGA5-E58K, K) mAb8 combined with rec PGA, L) mAb8 combined with rec PGC, M) mAb6.7 combined with PGA5-V353L, N) mAb6.7 combined with PGA5-K222Q, O) mAb6.7 combined with PGA5-T265A, P) mAb6.7 combined with PGA5-E58K, Q) mAb6.7 combined with rec PGA, R) mAb6.7 combined with rec PGC, S) mAb7 combined with PGA5-V353L, T) mAb7 combined with PGA5-K222Q, U) mAb7 combined with PGA5-T265A, V) mAb7 combined with PGA5-E58K, W) mAb7 combined with rec PGA, X) mAb7 combined with rec PGC.

[0012] The interactions are superimposed on the Langmuir 1:1 binding model (grey), with Rmax globally and RI=0. The interaction of antibody mAb6.7 corrects for mass transfer limitations.

[0013] Figure 4 Kinetic parameters of PGA and PGA with single-point mutations (PGA5-V353L, PGA5-K222Q, PGA5-T265A, PGA5-E58K) determined using mAb 2, mAb 8, mAb 6.7 and mAb 7, determined using Biacore.

[0014] Figure 5The comparison of VL and VR of mAB is shown: mAb 24, mAb 8, mAb6.7 and mAb7, and CDR are marked with boxes.

[0015] Figure 6 The developed Elecsys assay with mAb pairs was tested to determine whether it could also be used to determine the concentration of PGA subtypes (PGA5-V353L, PGA5-K222Q, PGA5-T265A, PGA5-E58K).

[0016] Figure 7 Determination of the limit of quantitation. Five runs were performed on one analyzer over two days, measuring serum samples from 10 individuals, with five replicates. This resulted in an intermediate precision CV ≤ 20%.

[0017] Figure 8 : Determination of the limit of quantitation. Five runs were performed on one analyzer over two days, measuring serum samples from 10 individuals, with five replicates. This resulted in an intermediate precision CV ≤ 20%. A graph of the coefficient of variation versus the mean concentration is presented. Detailed Implementation

[0018] Elements of the invention will now be described. These elements are set together with specific aspects and embodiments; however, it should be understood that they can be combined in any manner and in any number to create other aspects and embodiments.

[0019] The word “comprise” and its variants such as “comprises” and “comprising” should be understood to imply the inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0020] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a / an” and “the” include the plural referents.

[0021] Concentration, level, amount, and other numerical data may be expressed or presented in a “range” format herein. It should be understood that this range format is used solely for convenience and brevity, and therefore should be flexibly interpreted to include not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges covered by the range, as if each value and subrange were explicitly listed. For example, the numerical range “150 mg to 600 mg” should be interpreted to include not only the explicitly listed value of 150 mg to 600 mg, but also the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 150, 160, 170, 180, 190, 580, 590, 600 mg and subranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. The same principle applies to ranges that cite only one value. Furthermore, this interpretation applies regardless of the breadth of the range or characteristic.

[0022] When used in conjunction with numerical values, the term “about” means to cover a range of values ​​that has a lower limit of 5% less than the indicated value and an upper limit of 5% greater than the indicated value.

[0023] Generally, the described method is an in vitro method using a sample already obtained from the subject (i.e., providing a sample for the method, and the steps taken to obtain the sample from the subject are not included as part of the method). Therefore, the method may include the step of providing a biofluid sample from the subject.

[0024] As used herein, “providing,” “obtaining,” or “acquiring” can mean any means of acquiring a sample by “direct” or “indirect” means. Directly obtaining a sample means obtaining it through processing (e.g., performing physical methods such as extraction). Indirectly obtaining a sample means receiving it from another party or source (e.g., a third-party laboratory that directly obtained the sample).

[0025] As used herein, “affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is typically expressed by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

[0026] The terms "anti-pepsinogen A antibody" and "antibody specifically binding to pepsinogen A" refer to antibodies capable of specifically binding to pepsinogen A. In one aspect of the invention, the anti-pepsinogen A antibody binds to non-pepsinogen A proteins (e.g., pepsinogen C) to less than about 10% of its binding to pepsinogen A, as measured, for example, by surface plasmon resonance (SPR). In some aspects, antibodies binding to pepsinogen A have dissociation constants (KD) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, and ≤ 0.9 nM. When an antibody has a KD of 100 nM or less, it is said to "specifically bind" to the antigen (e.g., pepsinogen A).

[0027] As used herein, the terms “antibody,” “multiple antibodies,” and similar terms refer to whole immunoglobulin molecules and encompass naturally occurring antibody forms (including, but not limited to, IgG, IgA, IgM, and IgE) and recombinant antibody constructs, including, but not limited to, single-chain antibodies, chimeric antibodies, humanized antibodies, antibody fusion proteins, and multispecific antibodies; as well as antigen-binding fragments and derivatives of all of the foregoing. The terms “antibody,” “multiple antibodies,” and similar terms as used herein also refer to their antigen-binding fragments, which may be referred to herein as antibody-antigen-binding fragments and / or simply as antigen-binding fragments. These terms refer to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen (i.e., pepsinogen A), as known in the art, including, but not limited to, antigen-binding fragments containing an Fv domain, i.e., paired heavy and light chain variable domains, such as Fab, Fab', F(ab')2, and Fv fragments, and recombinant constructs, such as single-chain Fv domains referred to in the art as scFv. These terms also include antibody-antigen binding fragments containing a single unpaired heavy or light chain variable domain known in the art, which retains the ability to bind specifically and selectively to antigens as defined herein, including but not limited to single-domain antibodies (also referred to in the art as sdAb, dAb, and / or nanobodies) and VHH domains of camel-based heavy chains.

[0028] In some embodiments, the monoclonal antibody of the present invention may be a whole immunoglobulin, Fab, Fab', F(ab')2, Fv or scFv.

[0029] Antibodies can be polyclonal or monoclonal. In a preferred embodiment, the antibody of the present invention is monoclonal. As used herein, the term "monoclonal" refers to a population of antibody peptides or fragments thereof produced by a single B cell clone, containing only one antigen-binding site capable of responding to an immune response to a specific epitope of an antigen. This contrasts with "polyclonal" antibodies and compositions, which refer to a population of antibody peptides or antigen-binding fragments containing multiple antigen-binding sites. Modified forms of the monoclonal antibodies of the present invention are also included, such as their humanized or chimeric versions, and recombinant antibody constructs, such as antibody (or antigen-binding fragment) fusion proteins, wherein the antibody or antigen-binding fragment includes one or more additional domains, for example, for isolating and / or preparing recombinantly generated antibodies / fragments / constructs.

[0030] The term "epitope" refers to a site on a protein or non-protein antigen that binds to an anti-pepsinogen A antibody. Epitopes can be formed from continuous amino acid extensions (linear epitopes) or contain discontinuous amino acids (conformational epitopes), for example, due to folding of the antigen, i.e., spatial proximity via tertiary folding of protein antigens. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8 to 10 amino acids in a unique stereoconformation.

[0031] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using either the VH or VL domain from the antibody binding that antigen to screen for libraries with complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0032] As used in this article, the term "hypervariant region" or "HVR" refers to the regions in the antibody variable domain that are hypervariable in sequence and determine antigen-binding specificity, such as the "complementarity-determining region" ("CDR").

[0033] Typically, an antibody contains six CDRs; three in the VH region (CDR-H1, CDR-H2, CDR-H3) and three in the VL region (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs in this document include: (a) Hyperchromatic rings present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917(1987)); (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35 (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact sites present at amino acid residues 27-36 (L1), 46-55 (L2), 89-96 (L3), 30-35 (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

[0034] Unless otherwise stated, the CDR is determined according to the method described by Kabat et al. (ibid.). Those skilled in the art will understand that the CDR name can also be determined according to the methods described by Chothia (ibid.), McCallum (ibid.), or any other scientifically accepted naming system.

[0035] Unless otherwise specified, the amino acid residues of VH and VL in the antibodies of this invention are numbered according to Kabat. Those skilled in the art can convert the Kabat-based numbering to other numbering systems, such as Clothia, McCallum, etc.

[0036] "Frame" or "FR" refers to the variable domain residues other than the complementarity-determining region (CDR). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences usually appear in the VH (or VL) as follows: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0037] As used herein in the context of amino acids, the terms “substitution,” “exchange,” or “mutant” refer to the substitution of an amino acid at one position, and the introduction of one (or more) amino acids at different positions is explicitly not covered by the term “substitution.” As previously stated, this invention covers conserved or highly conserved amino acid substitutions as defined herein.

[0038] Amino acids in this article are spelled or abbreviated using one-letter or three-letter codes.

[0039] In the context of this invention, it refers to a variant of the sequence (e.g., a CDR modified by an amino acid substitution). These variants typically contain amino acid substitutions. Clearly, a variant CDR is a functional variant, i.e., having an amino acid sequence that may differ from the reference amino acid sequence, but which, in the case of the described heavy chain and / or light chain variable domains, exhibits or retains the same functional activity as the reference sequence. Specifically, as used herein, the term "same functional activity" means that the antibody or antibody-binding fragment of the invention comprising one or more variant CDRs will maintain a remarkably high association rate constant ka and / or affinity relative to binding to pepsinogen A.

[0040] As used in the context of this invention, "conservative amino acid substitution" means that an amino acid is substituted by another amino acid selected from the same physicochemical group, wherein the physicochemical group of the amino acid is: a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn, and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Acidic amino acids with a negative charge, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and if Pro is to be conservatively replaced, it is replaced by Ala.

[0041] As used in the context of this invention, "highly conserved amino acid substitution" refers to the following amino acid substitutions: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0042] "Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some respects, antibodies are purified to a purity greater than 95% or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0043] The terms “nucleic acid molecule,” “nucleic acid sequence,” or “polynucleotide” include any compound and / or substance comprising a nucleotide polymer. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate ester group. Typically, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically represented from 5' to 3'. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of antibodies used in this invention in vitro and / or in vivo (e.g., in a host). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. In embodiments, the nucleic acid / nucleic acid sequences of this invention can be isolated. Therefore, nucleic acid molecules can be, for example, nucleic acid molecules that have been synthesized according to conventional organic chemistry schemes, according to recombination methods, or semi-synthetically produced (e.g., by combining chemical synthesis and recombination methods). Those skilled in the art are familiar with the preparation and use of such nucleic acid molecules.

[0044] The "percentage of homology (%)" associated with a polypeptide / peptide and / or nucleic acid sequence or the amino acid sequence of a nucleic acid molecule describes the number of matches of the same amino acid or nucleic acid residues in two or more aligned sequences compared to the number of residues in the total length of the compared sequences (or the total comparison portion thereof). The percentage of identical residues can be determined when comparing and aligning two or more sequences or subsequences, either on a comparison window or in a specified region as measured using sequence comparison algorithms known in the art to obtain maximum correspondence, or when manually aligned and visually inspected. Non-limiting examples of algorithms used to determine sequence identity include, for example, those based on the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res. 25 (1997), 3389-3402), the CLUSTALW computer program (Thompson, Nucleic Acids Res. 2 (1994), 4673-4680), or FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., 85 (1988), 2444). Although the FASTA algorithm generally does not consider the deletion or addition of internal mismatches in the sequence, i.e., gaps, in its calculations, this can be manually corrected to avoid overestimating % sequence identity. However, CLUSTALW does consider sequence gaps in its identity calculations. The BLAST and BLAST 2.0 algorithms are also available (Altschul et al., Nucleic Acids Res., 25 (1977), 3389).

[0045] Preferably, the term "as shown" related to amino acid sequences and / or nucleic acid sequences or nucleic acid molecules may be replaced by "consisting of" or "consists of".

[0046] Unless otherwise specified, the term "pepsinogen A" as used herein refers to any naturally occurring pepsinogen A from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers "full-length" unprocessed pepsinogen A, as well as any form of pepsinogen A produced through cellular processing. The term also covers naturally occurring variants of pepsinogen A, such as splice variants or allelic variants. An exemplary amino acid sequence of pepsinogen A is shown in SEQ ID NO: 34. Furthermore, the term "pepsinogen A" also covers its isotypes, particularly variants containing amino acid substitutions of E58K, K222Q, T265A, or V353L (amino acid positions refer to SEQ ID NO: 34). Human pepsinogen A is shown in UniprotP0DJD9 (2012-02-22 v1).

[0047] Unless otherwise specified, the term "pepsinogen C" as used herein refers to any naturally occurring pepsinogen C from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers "full-length" unprocessed pepsinogen C, as well as any form of pepsinogen C produced through cellular processing. The term also covers naturally occurring variants of pepsinogen C, such as splice variants or allelic variants. An exemplary amino acid sequence of pepsinogen C is shown in SEQ ID NO: 69. Human pepsinogen C is shown in Uniprot P20142 (1991-02-01 v1).

[0048] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0049] As used herein, “immunoassay” is a recognized bioanalytical method in which the detection or quantification of an analyte depends on the reaction of the analyte with at least one analyte-specific binder to form an analyte:bindant complex. In the context of this invention, at least one of the at least one analyte-specific binders is an antibody of this invention. Specific embodiments of the “sandwich” immunoassay can be used with analytes having more than one recognition epitope. Therefore, sandwich assays require at least two binders linked to non-overlapping epitopes on the analyte. In a “heterogeneous sandwich immunoassay,” one binder functions to specifically capture the binder; this binder is immobilized on a solid phase (during the assay). A second analyte-specific binder is provided in a dissolved form in the liquid phase. Once the respective analyte binds to the first and second binders, a sandwich complex (bindant-1: analyte: binder-2) is formed. This sandwich complex is also referred to as a “detection complex.” In a detection complex, the analyte is sandwiched between binders; that is, in such a complex, the analyte represents a connecting element between the first and second binders.

[0050] The term "heterogeneous" (as opposed to "homogeneous") refers to two fundamental and independent steps in the assay process. In the first step, a label-containing detection complex is formed and immobilized, but unbound label remains surrounding the complex. The unbound label is removed from the immobilized detection complex before a label-dependent signal is determined, thus representing the second step. In contrast, a homogeneous assay generates an analyte-dependent detectable signal through a single incubation step and does not require a washing step.

[0051] In heterologous immunoassays, the solid phase is functionalized so that a functional capture binder (first binder) may be bound to its surface before contact with the analyte; or the surface of the solid phase is functionalized so that the first binder can be anchored after it reacts with the analyte. In the latter case, the anchoring process must not interfere with the binder's ability to specifically capture and bind the analyte. A second binder present in the liquid phase is used to detect the bound analyte. Thus, in heterologous immunoassays, the analyte is allowed to bind to both the first (capture) and second (detection) binders. This forms a "detection complex" in which the analyte is sandwiched between the capture binder and the detection binder. In a typical embodiment, the detection binder is labeled before contact with the analyte; alternatively, the label is specifically attached to the detection binder after the analyte has bound. In the case where the detection complex is immobilized on the solid phase, the amount of label detectable on the solid phase corresponds to the amount of analyte sandwiched in between. After removing the unbound label, the immobilized label indicating the presence and quantity of the analyte can be detected.

[0052] As used throughout this document, "detectable label" refers to a label that allows detection. According to embodiments of the invention, a detectable label is an enzyme or a light-emitting label, in which case it is fluorescent, luminescent, chemiluminescent, electrochemiluminescent, or radioactive. In a preferred embodiment, the label is an electrochemiluminescent label, in which case it is a tris(2,2'-bipyridine)ruthenium(II)-complex (Ru(bpy)). Since interference is caused by the three-dimensional structure of the label molecule and similar interfering molecules that attract autoantibodies, rather than by the signal emission mechanism of the label (such as, for example, light or radioactivity), all of the above-described labels can be used in this invention.

[0053] As used herein, "capture tag" refers to a tag that can immobilize a capture agent (e.g., an anti-pepsinogen A antibody having a capture tag attached thereto) onto a surface (e.g., magnetic particles such as microbeads). Non-limiting examples are members of binding pairs. Non-limiting examples of capture tags are biotin or derivatives thereof, which can interact with streptavidin or derivatives thereof. Various capture tags are well known in the art.

[0054] The methods described herein include providing a biological fluid sample (e.g., a blood sample) from a subject. The sample tested in the methods described herein is also referred to as a “test sample” or “sample”.

[0055] As used herein, the terms “biological (fluid) sample,” “test sample,” and “sample” are used interchangeably, and variations thereof refer to a sample obtained from or derived from a subject. For the purposes described herein, a sample is or includes a biological fluid (also referred to herein as a body fluid) sample.

[0056] As used in the context of this disclosure, "sample" can refer to a liquid sample that contains or is expected to contain pepsinogen A. Specifically, the sample can be a bodily fluid, such as, but not limited to, blood samples, cerebrospinal fluid, semen, saliva, or urine. In embodiments, the blood sample can be venous blood or capillary blood. In embodiments, the sample is a blood sample, such as whole blood, serum, or plasma. In embodiments, the sample is serum or plasma.

[0057] The sample is an in vitro sample and will be analyzed in vitro without being transferred back into the body.

[0058] Blood samples can be whole blood samples or processed blood samples, such as serum, plasma, etc. Methods for obtaining biological fluid samples (e.g., whole blood, serum, plasma, etc.) from subjects are well known in the art. For example, methods for obtaining blood samples from subjects are well known and include established techniques for venipuncture. The obtained blood samples can be further processed using standard techniques to obtain, for example, serum or plasma samples. Advantageously, methods for obtaining biological fluid samples from subjects are generally low-invasive or non-invasive.

[0059] A whole blood sample is defined as a blood sample drawn from the body without (substantially) removing any components (such as platelets or plasma). In other words, the relative proportions of the components in a whole blood sample are substantially the same as those in the blood within the body. In this context, "substantially the same" allows for very small variations in the relative proportions of whole blood components, such as at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, etc. Whole blood comprises both the cellular and fluid components of blood. Therefore, a whole blood sample can also be defined as a blood sample having (substantially) all of its cellular components in plasma, wherein the cellular components (i.e., the blood includes at least the essential white blood cells, red blood cells, and platelets) are intact.

[0060] Subjects may be referred to as patients in this document. The terms “subject,” “individual,” and “patient” are used interchangeably in this document and refer to animals, preferably mammals, and more generally humans.

[0061] In the following detailed description of the invention, numerous individual elements, characterizing features, techniques, and / or steps are disclosed. It will be readily appreciated that each of these is advantageous not only when considered or used individually, but also when considered and used in combination with each other. Therefore, to avoid excessive repetition and redundant paragraphs, this description avoids repeating every possible combination and arrangement. However, whether explicitly stated or not, it is understood that such combinations are entirely within the scope of the subject matter currently disclosed.

[0062] Unless otherwise specified, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations or equivalents of those techniques that would be obvious to one of ordinary skill in the art.

[0063] All amino acid sequences provided herein begin with the N-terminal residue and end with the C-terminal residue. As is customary in the art, the single-letter or three-letter code abbreviations used to identify amino acids in this invention correspond to those code abbreviations commonly used for amino acids.

[0064] Numerous documents, including patent applications and manufacturer's manuals, are referenced in this specification. The disclosures in these documents are considered irrelevant to the patentability of this invention and are incorporated herein by reference in their entirety. More specifically, all references are made as if each individual document were specifically and individually incorporated herein by reference.

[0065] In a first aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A and competes with an antibody consisting of: (i) the heavy chain variable domain (VH) shown in SEQ ID NO: 1; and (ii) the light chain variable domain (VL) shown in SEQ ID NO: 2.

[0066] Pepsinogen (PG)A (PG I) is secreted entirely by the fundic glands (Samloff 1971). Previous studies have shown that serum PG I levels and / or the PG I / II ratio (pepsinogen C (PG II)) reflect the morphology (Samloff et al. 1982; Borch et al. 1989) and functional status of the gastric mucosa (Samloff et al. 1975; Nakanome et al. 1983; Miki et al. 1987; Feldman et al. 1988). Numerous studies have reported significant correlations between serum PG values ​​and gastric acid secretion levels. Changes in gastric secretion levels are associated with the pathogenesis of various types of diseases originating in the upper gastrointestinal tract (GI). Classically, individuals with hyperacidity, especially those infected with Helicobacter pylori, are more prone to duodenal ulcers, while individuals with hypoacidity face a risk of developing gastric cancer through the development of pre-malignant conditions (gastric atrophy) (El-Omar et al. 1995, 1997). Therefore, measuring gastric acid secretion levels has clinical significance in estimating the risk of various upper GI tract diseases. However, given the significant discomfort and time-consuming nature of this procedure, direct measurement of gastric acid secretion levels has become less common. Therefore, antibodies that specifically bind to pepsinogen A are needed, as pepsinogen A is a useful indicator for measuring gastric acid secretion levels and estimating the risk of various upper GI tract diseases.

[0067] The inventors of this invention have identified high-affinity antibodies that specifically bind to pepsinogen A and compete with antibodies consisting of: (i) the heavy chain variable domain (VH) shown in SEQ ID NO: 1; and (ii) the light chain variable domain (VL) shown in SEQ ID NO: 2.

[0068] An antibody that competes with a reference anti-pepsinogen A antibody for binding to pepsinogen A is an antibody that blocks the binding of the reference anti-pepsinogen A antibody to its antigen by 50% or more in a competitive assay, and conversely, a reference antibody that blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. In examples, the antibody competes for binding to pepsinogen A when it binds to the same epitope as, for example, pepsinogen A. Competitive binding can be used to determine whether an antibody binds to the same epitope of pepsinogen A as the reference anti-pepsinogen A antibody or competes for binding with the reference anti-pepsinogen A antibody. For example, "antibody that binds to the same epitope as the reference anti-pepsinogen A antibody" means an antibody that blocks the binding of the reference anti-pepsinogen A antibody to its antigen by 50% or more in a competitive assay, and conversely, a reference antibody that blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Similarly, for example, to determine whether an antibody binds to the same epitope as a reference anti-pepsinogen A antibody, the reference antibody is saturated with pepsinogen A. After removing excess reference anti-pepsinogen A antibody, the binding ability of the anti-pepsinogen A antibody in question to pepsinogen A is evaluated. If the anti-pepsinogen A antibody binds to pepsinogen A after saturation binding with the reference anti-pepsinogen A antibody, it can be concluded that the anti-pepsinogen A antibody in question binds to a different epitope than the reference anti-pepsinogen A antibody. However, if the anti-pepsinogen A antibody in question does not bind to pepsinogen A after saturation binding with the reference anti-pepsinogen A antibody, then the anti-pepsinogen A antibody in question may bind to the same epitope as the reference anti-pepsinogen A antibody. To confirm whether the antibody in question binds to the same epitope or is simply hindered by steric hindrance, and thus competes with a reference anti-pepsinogen A antibody for binding without binding to the same epitope, standard experiments can be used (e.g., peptide mutation and binding assays using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). This assay should be performed in two settings: both antibodies are saturated antibodies. If, in both settings, only the first (saturated) antibody is able to bind to pepsinogen A, it can be concluded that the anti-pepsinogen A antibody in question and the reference anti-pepsinogen A antibody compete for binding to pepsinogen A.

[0069] In some respects, such as those measured in competitive binding assays, if one antibody inhibits the binding of another antibody by 1, 5, 10, 20, or 100 times at least 50%, at least 75%, at least 90%, or even 99% or higher, the two antibodies are considered to bind to the same or overlapping epitopes (see, for example, Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0070] To generate structural antibodies against pepsinogen A, mice can be immunized with recombinant pepsinogen A. Following immunization, the spleen of the immunized animal can be prepared and fused into myeloma cells.

[0071] These hybridoma cells can be cultured, and the responsiveness of their supernatant to recombinant pepsinogen A can be tested by ELISA.

[0072] Selected hybridoma clones can be cultured in a small-scale dialysis-fermentation apparatus and grown for several weeks with repeated changes of dialysis medium to produce IgG. The cell culture supernatant can be harvested, and the IgG can be purified using standard affinity chromatography.

[0073] These antibodies can be screened to determine whether they competitively bind to pepsinogen A. This screening can be performed as described above (e.g., using a competitive assay or competitive binding assay as described above) or using an automated Elecsys immunoassay (Elecsys® cobas® e411) via a sandwich immunoassay. For a sandwich immunoassay, two sets of antibody conjugates can be prepared. One set of antibodies can be labeled with one tag (e.g., ruthenium labeling), and the second set of antibodies can be labeled with a different tag (e.g., biotin labeling). Different combinations of antibodies labeled with different tags can be tested. Using this assay, antibodies that do not form a sandwich can be identified, and competitive binding can be identified based on sandwich compatibility (exemplary as described in Example 8).

[0074] In embodiments of the present invention, the antibody or antigen-binding fragment thereof according to the present invention comprises: (a) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4 or a variant thereof modified by one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5 or a variant thereof modified by one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6 or a variant thereof modified by one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7 or a variant thereof modified by one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8 or a variant thereof modified by one amino acid substitution.

[0075] In embodiments of the present invention, the antibody or antigen-binding fragment thereof according to the present invention comprises: b) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11 or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12 or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13 or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14 or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15 or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16 or a variant thereof modified with one amino acid substitution.

[0076] In embodiments of the present invention, the antibody or antigen-binding fragment thereof according to the present invention comprises: (c) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19 or a variant thereof modified by one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20 or a variant thereof modified by one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21 or a variant thereof modified by one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22 or a variant thereof modified by one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23 or a variant thereof modified by one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24 or a variant thereof modified by one amino acid substitution.

[0077] Surprisingly, the inventors have identified a family of antibodies that competitively and specifically bind to pepsinogen A. As shown in the accompanying examples (e.g., Example 8, Table 1), antibodies from this family (e.g., mAb 6.7, mAb 24, mAb 7) all competitively bind to pepsinogen A and exhibit excellent kinetic properties.

[0078] In embodiments of the invention, all amino acid substitutions are conserved amino acid substitutions. In a further embodiment, each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein these amino acid groups are... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0079] In a further specific embodiment, each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0080] In embodiments of the present invention, the antibody or antigen-binding fragment thereof according to the present invention comprises: (a) VH, which comprises: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 3; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 4; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 5; and VL, which comprises: CDR-L1 represented by the amino acid sequence of SEQ ID NO: 6; CDR-L2 represented by the amino acid sequence of SEQ ID NO: 7; and CDR-L3 represented by the amino acid sequence of SEQ ID NO: 8. In a further embodiment, the antibody or its antigen-binding fragment comprises: (b) VH, which comprises: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13; and VL, which comprises: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16. In a further embodiment, the antibody or its antigen-binding fragment comprises: (c) VH, which comprises: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 19; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 20; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 21; and VL, which comprises: CDR-L1 represented by the amino acid sequence of SEQ ID NO: 22; CDR-L2 represented by the amino acid sequence of SEQ ID NO: 23; and CDR-L3 represented by the amino acid sequence of SEQ ID NO: 24.

[0081] In embodiments of the present invention, the antibody or antigen-binding fragment binds to an epitope of pepsinogen A comprising the amino acid sequence shown in SEQ ID NO: 33. In a further embodiment, the antibody or antigen-binding fragment of the present invention binds to an epitope of pepsinogen A comprising the amino acid sequence shown in SEQ ID NO: 33.

[0082] The antibody of the present invention may have different frame regions with CDRs attached to the VH and VL domains.

[0083] The variable domain (VL) of a light chain can be composed of a framework region (FW) and a CDR, as shown in Equation I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) Furthermore, the heavy chain variable structural domain (VH) can be composed of FW and CDR, as shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II).

[0084] In embodiments of the present invention, VH comprises CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5; and VL comprises CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8; and FW comprises the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 39; The amino acid sequence of FW-L2, SEQ ID NO: 40; The amino acid sequence of FW-L3, SEQ ID NO: 41; The amino acid sequence of FW-L4, SEQ ID NO: 42; The amino acid sequence of FW-H1, SEQ ID NO: 35; The amino acid sequence of FW-H2, SEQ ID NO: 36; The amino acid sequence of FW-H3, SEQ ID NO: 37; The amino acid sequence of FW-H4, SEQ ID NO: 38.

[0085] In another preferred embodiment, VH comprises CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13; and VL comprises CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16; and FW comprises the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 47; The amino acid sequence of FW-L2, SEQ ID NO: 48; The amino acid sequence of FW-L3, SEQ ID NO: 49; The amino acid sequence of FW-L4, SEQ ID NO: 50; The amino acid sequence of FW-H1, SEQ ID NO: 43; The amino acid sequence of FW-H2, SEQ ID NO: 44; The amino acid sequence of FW-H3, SEQ ID NO: 45; The amino acid sequence of FW-H4, SEQ ID NO: 46.

[0086] In a preferred embodiment, VH comprises CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21; and VL comprises CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24; and FW comprises the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 55; The amino acid sequence of FW-L2, SEQ ID NO: 56; The amino acid sequence of FW-L3, SEQ ID NO: 57; The amino acid sequence of FW-L4, SEQ ID NO: 58; The amino acid sequence of FW-H1, SEQ ID NO: 51; The amino acid sequence of FW-H2, SEQ ID NO: 52; The amino acid sequence of FW-H3, SEQ ID NO: 53; The amino acid sequence of FW-H4, SEQ ID NO: 54.

[0087] In embodiments of the present invention, VH comprises CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5; and VL comprises CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8; and FW comprises the following amino acid sequences or variants thereof modified by substitution of up to three amino acids: The amino acid sequence of FW-L1, SEQ ID NO: 39; The amino acid sequence of FW-L2, SEQ ID NO: 40; The amino acid sequence of FW-L3, SEQ ID NO: 41; The amino acid sequence of FW-L4, SEQ ID NO: 42; The amino acid sequence of FW-H1, SEQ ID NO: 35; The amino acid sequence of FW-H2, SEQ ID NO: 36; The amino acid sequence of FW-H3, SEQ ID NO: 37; The amino acid sequence of FW-H4, SEQ ID NO: 38.

[0088] In another preferred embodiment, VH comprises CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13; and VL comprises CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16; and FW comprises the following amino acid sequences or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 47; The amino acid sequence of FW-L2, SEQ ID NO: 48; The amino acid sequence of FW-L3, SEQ ID NO: 49; The amino acid sequence of FW-L4, SEQ ID NO: 50; The amino acid sequence of FW-H1, SEQ ID NO: 43; The amino acid sequence of FW-H2, SEQ ID NO: 44; The amino acid sequence of FW-H3, SEQ ID NO: 45; The amino acid sequence of FW-H4, SEQ ID NO: 46.

[0089] In a preferred embodiment, VH comprises CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21; and VL comprises CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24; and FW comprises the following amino acid sequences or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 55; The amino acid sequence of FW-L2, SEQ ID NO: 56; The amino acid sequence of FW-L3, SEQ ID NO: 57; The amino acid sequence of FW-L4, SEQ ID NO: 58; The amino acid sequence of FW-H1, SEQ ID NO: 51; The amino acid sequence of FW-H2, SEQ ID NO: 52; The amino acid sequence of FW-H3, SEQ ID NO: 53; The amino acid sequence of FW-H4, SEQ ID NO: 54.

[0090] In embodiments of the invention, all amino acid substitutions are conserved amino acid substitutions. In a further embodiment, each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein these amino acid groups are... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0091] In a further specific embodiment, each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0092] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises: (i) VH, which comprises the sequence shown in SEQ ID NO: 1; and (ii) VL, which comprises the sequence shown in SEQ ID NO: 2.

[0093] In a further embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: (i) VH, which comprises the sequence shown in SEQ ID NO: 1; or (ii) VL, which comprises the sequence shown in SEQ ID NO: 2.

[0094] In another embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: (i) VH, which comprises the sequence shown in SEQ ID NO: 9; and (ii) VL, which comprises the sequence shown in SEQ ID NO: 10.

[0095] In a further embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: (i) VH, which comprises the sequence shown in SEQ ID NO: 9; or (ii) VL, which comprises the sequence shown in SEQ ID NO: 10.

[0096] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises: (i) VH, which comprises the sequence shown in SEQ ID NO: 17; and (ii) VL, which comprises the sequence shown in SEQ ID NO: 18.

[0097] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises: (i) VH, which comprises the sequence shown in SEQ ID NO: 17; or (ii) VL, which comprises the sequence shown in SEQ ID NO: 18.

[0098] In a second aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A and competes with an antibody composed of the following for binding to pepsinogen A. (i) The heavy chain variable structural domain (VH) shown in SEQ ID NO: 25; and (ii) The light chain variable domain (VL) shown in SEQ ID NO: 26.

[0099] In embodiments, the antibody or antigen-binding fragment of the present invention comprises: VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO:27 or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO:28 or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO:29 or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO:30 or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO:31 or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO:32 or a variant thereof modified with one amino acid substitution.

[0100] In embodiments of the invention, all amino acid substitutions are conserved amino acid substitutions. In a further embodiment, each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein these amino acid groups are... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0101] In a further specific embodiment, each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0102] In a further embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises: VH, which comprises: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 27; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 28; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 29; and VL, which comprises: CDR-L1 represented by the amino acid sequence of SEQ ID NO: 30; CDR-L2 represented by the amino acid sequence of SEQ ID NO: 31; and CDR-L3 represented by the amino acid sequence of SEQ ID NO: 32.

[0103] In a further embodiment, the antibody or antigen-binding fragment thereof according to the second aspect of the present invention comprises: a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), VH comprises CDR-H1 or a variant thereof with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 27; CDR-H2 or a variant thereof with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 28; and CDR-H3 or a variant thereof with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 29; and VL comprises CDR-L1 or a variant thereof with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 30; CDR-L2 or a variant thereof with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 31; and CDR-L3 or a variant thereof with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 32; and FW comprises the following amino acid sequences or variants thereof with at least 85% homology: The amino acid sequence of FW-L1, SEQ ID NO: 63; The amino acid sequence of FW-L2, SEQ ID NO: 64; The amino acid sequence of FW-L3, SEQ ID NO: 65; The amino acid sequence of FW-L4, SEQ ID NO: 66; The amino acid sequence of FW-H1, SEQ ID NO: 59; The amino acid sequence of FW-H2, SEQ ID NO: 60; The amino acid sequence of FW-H3, SEQ ID NO: 61; The amino acid sequence of FW-H4, SEQ ID NO: 62.

[0104] In a further embodiment, the antibody or antigen-binding fragment thereof according to the second aspect of the present invention comprises: a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), VH comprises CDR-H1 or a variant thereof modified with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 27; CDR-H2 or a variant thereof modified with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 28; and CDR-H3 or a variant thereof modified with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 29; and VL comprises CDR-L1 or a variant thereof modified with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 30; CDR-L2 or a variant thereof modified with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 31; and CDR-L3 or a variant thereof modified with one amino acid substitution as shown in the amino acid sequence of SEQ ID NO: 32; and FW comprises the following amino acid sequences or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 63; The amino acid sequence of FW-L2, SEQ ID NO: 64; The amino acid sequence of FW-L3, SEQ ID NO: 65; The amino acid sequence of FW-L4, SEQ ID NO: 66; The amino acid sequence of FW-H1, SEQ ID NO: 59; The amino acid sequence of FW-H2, SEQ ID NO: 60; The amino acid sequence of FW-H3, SEQ ID NO: 61; The amino acid sequence of FW-H4, SEQ ID NO: 62.

[0105] In embodiments of the invention, all amino acid substitutions are conserved amino acid substitutions. In a further embodiment, each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein these amino acid groups are... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0106] In a further specific embodiment, each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0107] In a further embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: (i) VH, which comprises the sequence shown in SEQ ID NO: 25; and (ii) VL, which comprises the sequence shown in SEQ ID NO: 26.

[0108] In a further embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises: (i) VH, which comprises the sequence shown in SEQ ID NO: 25; or (ii) VL, which comprises the sequence shown in SEQ ID NO: 26.

[0109] In embodiments of the second aspect and related embodiments, the antibody or its antigen-binding fragment is at a concentration of 0.6 nM or less. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0110] In embodiments of the second aspect and related embodiments, the antibody or its antigen-binding fragment is bound to pepsinogen A consisting of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A or V353L at 37°C for 21 minutes or longer t / 2-diss.

[0111] In embodiments of the second aspect and related embodiments, the association rate k of pepsinogen A composed of SEQ ID NO: 68 or its variants containing amino acid substitutions of E58K, K222Q, T265A or V353L is... a For at least 8.7x10 5 M -1 s -1 .

[0112] In a preferred embodiment, the antibody or its antigen-binding fragment according to the present invention is a monoclonal antibody.

[0113] In a further embodiment of the invention, the antibody or its antigen-binding fragment specifically binds to pepsinogen A, wherein pepsinogen A comprises SEQ ID NO: 34 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A, or V353L. In a further embodiment of the invention, the antibody or its antigen-binding fragment specifically binds to pepsinogen A, wherein pepsinogen A is composed of SEQ ID NO: 34 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A, or V353L.

[0114] As shown in the appended examples (e.g., Examples 5 and 9), the antibodies of the present invention are further characterized by high affinity for pepsinogen A comprising SEQ ID NO: 68 or variants thereof comprising amino acid substitutions of E58K, K222Q, T265A, or V353L. Therefore, the antibodies of the present invention are characterized by having an equilibrium dissociation constant K of 2 nM or less, or 1.5 nM or less, or 1 nM or less. D In a preferred embodiment, K is measured at 37°C. D The decrease in K D K is the preferred option. D The lower the value, the better.

[0115] In a preferred embodiment, the K-cell binding site of the antibody or its antigen-binding fragment according to the invention to pepsinogen A is determined by surface plasmon resonance spectroscopy. D The experimental setup is described in Example 9 of this paper.

[0116] Used to determine the equilibrium dissociation constant K D The method is known in the art.

[0117] In a preferred embodiment, K D It can be obtained through surface plasmon resonance spectroscopy (e.g., BIAcore) ® ) Measurement.

[0118] Used to determine K D The preferred temperature is 37°C.

[0119] In an embodiment, K can be determined using kinetic measurements that determine association and dissociation rates. D Therefore, in embodiments, these methods can be the same as those described below for determining k. a The same surface plasmon resonance method. This method may include corrections for mass transfer limitations.

[0120] In a preferred embodiment, K D Surface plasmon resonance spectroscopy (e.g., BIAcore) can be used. ® ) Measurement.

[0121] In a further embodiment, the antibody or antigen-binding fragment according to the invention has an antibody / antigen complex half-life of 20 minutes or longer at 37°C. / 2-diss .

[0122] Methods for determining the half-life of an antibody / antigen complex that binds to pepsinogen A are known in the art. Exemplary examples are described in the examples (particularly example 9).

[0123] The half-life of an antibody / antigen complex is determined by formula t. / 2 diss = ln(2) / (k d 60) Calculated in minutes.

[0124] As illustrated in the accompanying examples, the antibodies of the present invention are characterized by a particularly high association rate constant (kΩ) when binding to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L. a This rapid association rate makes the antibodies of this invention particularly suitable for high-throughput immunoassays and competitive assay formats.

[0125] Used to determine the association rate constant (k) for antibody binding to pepsinogen A. a The method is known in the art. Exemplary instances are described in the examples (especially example 9).

[0126] Therefore, in the embodiments, a further feature of the antibody or its antigen-binding fragment according to the invention is the association rate (kb) with pepsinogen A composed of SEQ ID NO: 68 or its variants containing amino acid substitutions of E58K, K222Q, T265A or V353L. a(At least 10) 5 M -1 s -1 Preferably at least 4.9x10 5 M -1 s -1 In a preferred embodiment, the association rate with pepsinogen A composed of SEQ ID NO: 68 or its variants containing amino acid substitutions of E58K, K222Q, T265A, or V353L is at least 4.9 x 10⁻⁶. 5 M -1 s - 1. In one embodiment, k is measured at 37°C. a The decrease in K D K is the preferred option. D The lower the value, the better.

[0127] Used to determine the association rate constant (k) for antibody binding to pepsinogen A. a The method is known in the art. Exemplary instances are described in the examples (especially example 9).

[0128] In a preferred embodiment, the association rate constant (k) between the antibody or its antigen-binding fragment according to the invention and pepsinogen A is determined by surface plasmon resonance spectroscopy. a ).

[0129] The surface plasmon resonance spectroscopy involves attaching or capturing an antibody or its antigen-binding fragment onto a CM5 sensor chip and injecting pepsinogen A, consisting of SEQ ID NO: 68, or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, as an analyte, wherein the determination is performed using HBS-ET buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (w / v) Tween 20®) at 37°C.

[0130] In a further embodiment of the invention, the antibody or its antigen-binding fragment does not specifically bind to pepsinogen C. This means that the antibody or its antigen-binding fragment binds to pepsinogen C with a KD greater than 100 nM. In a preferred embodiment, pepsinogen C consists of SEQ ID NO: 69.

[0131] In one embodiment, surface plasmon resonance spectroscopy is performed using a Biacore 8k or B4000 instrument.

[0132] Association constant ka [1 / Ms], dissociation rate constant k d [s -1 and dissociation equilibrium constant K D [M] can be calculated based on the Langmuir fitting model, for example, by using evaluation software corresponding to the instrument.

[0133] The antibodies and antigen-binding fragments of the present invention can be prepared using various techniques conventionally used in the art. For example, antibodies can be prepared by immunizing non-human animals (e.g., mice) with pepsinogen A, isolating and subsequently isolating antigen-reactive antibody-producing B cells. Preferred exemplary methods for producing antibodies according to the present invention using non-human animals are provided in the appended examples (see in particular Example 3). Selected clones for antibody production can be processed according to conventional methods for subsequent recombinant processing.

[0134] Another suitable method for generating or isolating the antibodies and antibody-antigen binding fragments of the present invention includes, but is not limited to, methods for selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, phage, ribosome, oligonucleotide, RNA, cDNA, or yeast display libraries) using targeted binding activity. For example, antibodies or antigen-binding fragments can be selected from such libraries by positive selection for specific binding to pepsinogen A. Display libraries are well known in the art and are available, for example, from a variety of commercial suppliers, including but not limited to Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinslied / Planeger, Germany), Biovation (Aberdeen, Scotland, UK), and Bioinvent (Lund, Sweden). Similarly, selected clones can be treated according to conventional methods for subsequent recombinant processing.

[0135] The antibodies of this invention are recombinantly expressible. Therefore, in some embodiments, the antibodies of this invention can be recombinant antibodies. Methods for generating recombinant antibodies are known in the art.

[0136] In a third aspect, the present invention relates to a polynucleotide or polynucleotide group encoding (i) the heavy chain variable domain of the antibody or its antigen-binding fragment according to the present invention, and / or (ii) The light chain variable domain of the antibody or its antigen-binding fragment according to the invention.

[0137] The present invention also provides a nucleic acid molecule encoding an antibody or any antigen-binding fragment thereof as defined above. In particular, a polynucleotide is provided encoding a heavy chain and / or light chain variable domain of an antibody that specifically binds to pepsinogen A as defined above. In some embodiments, the polynucleotide may include further sequences to ensure that not only the heavy chain and / or light chain variable domains are expressed, but also the remaining heavy chain and / or light chain constant regions are expressed, such that a full-length IgG antibody comprising the heavy chain and light chain variable domains of the present invention is expressed. Therefore, for each aspect and embodiment involving an antibody or antigen-binding fragment that specifically binds to pepsinogen A as described herein, a corresponding polynucleotide encoding the respective antibody or antigen-binding fragment is provided herein.

[0138] In a fourth aspect of the invention, a vector comprising the polynucleotides of the invention is provided herein. In particular, vectors comprising nucleic acid molecules encoding antibodies or antibody-antigen binding fragments of the invention are provided. As used herein, the term “vector” refers to a circular or linear nucleic acid molecule that can autonomously replicate in a host cell into which it has been introduced. Non-limiting examples of vectors suitable for use in the invention include viscera, plasmids (e.g., naked plasmids or those contained in liposomes), viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), and bacteriophages. However, many suitable vectors are provided in the art, the choice of which depends on the desired function. The development and use of suitable vectors are well documented in the art; see, for example, the techniques described in Sambrook and Russell, “Molecular Cloning, A Laboratory Manual”, Cold SpringHarbor Laboratory, NY (2001), and Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, NY (1989), (1994). The vectors used in connection with this invention comprise nucleic acid sequences that encode the full-length pepsinogen A antibody-antigen-binding fragment as disclosed herein. Therefore, for each aspect and embodiment involving an antibody or antigen-binding fragment that specifically binds to pepsinogen A as described herein, a vector comprising the corresponding polynucleotide encoding the respective antibody or antigen-binding fragment is provided herein.

[0139] Regarding the term "vector comprising..." as used herein, it will be understood in the art that the vector contains further nucleic acid sequences that are necessary and / or sufficient for the desired vector activity in the host cell, such as driving vector replication (and thereby encoding the nucleic acid sequence) and / or guiding the host cell to express the antibody or antigen-binding fragment of the present invention. Such further nucleic acid sequences include, but are not limited to, sequences that control vector replication and / or the expression of desired sequences in a particular cellular system. For example, the vector may contain a nucleic acid molecule encoding the antibody or antibody-antigen-binding fragment of the present invention, which is operatively linked to a regulatory sequence and / or under the control of a regulatory sequence. The term "regulatory sequence" refers to a DNA sequence that is necessary for the expression of the coding sequence operatively linked thereto. The term "control sequence" is intended to include at least all components that may be equally necessary for expression and may further include other advantageous components, for example, for allowing replication. As understood in the art, the nature of such regulatory and control sequences varies from host organism to host organism. For example, in prokaryotes, control sequences typically include a promoter, a ribosome binding site, and a terminator. In eukaryotes, control sequences typically include promoters, terminators, and in some cases, enhancers, transactivators, and / or transcription factors.

[0140] The vectors used in this invention are preferably expression vectors. Expression vectors are capable of directing the replication and expression of the nucleic acid molecules of this invention in host cells, and thus provide expression of heavy chain and / or light chain variable domains of antibodies that specifically bind to pepsinogen A, as disclosed herein. In some embodiments, the vector may contain further sequences to ensure expression not only of the heavy chain and light chain variable domains, but also of the remaining heavy chain and light chain constant regions, such that a full-length IgG antibody containing the heavy chain and light chain variable domains of this invention is expressed. Suitable expression vectors have been extensively described in the literature, and those skilled in the art can readily determine expression vectors suitable for a particular cell system using conventional methods. Preferably, the vectors disclosed herein comprise a recombinant polynucleotide (i.e., a nucleic acid sequence encoding a monoclonal antibody according to the invention) and an operatively linked expression control sequence. Vectors provided herein preferably further comprise a promoter. The vectors described herein may also comprise a selection marker gene and a replication source to ensure replication in the host. Furthermore, the vectors provided herein may also comprise a termination signal for transcription. Expression vectors known in the art can drive transient or constitutive expression in host cells.

[0141] The nucleic acid molecules and / or vectors of the present invention can be designed for transfection into prokaryotic or eukaryotic host cells by any means known in the art or described herein. Non-limiting examples of suitable methods include chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-glucan, nuclear transfection), non-chemical methods (electroporation, acoustic transfection, phototransfection, gene electrotransfer, hydrodynamic delivery, or transformation that occurs naturally after contacting cells with the nucleic acid molecules of the present invention), particle-based methods (gene gun, magnetic transfection, puncture infection), phage vector-based methods, and viral methods. For example, expression vectors derived from viruses (such as retroviruses, vaccinia virus, adeno-associated virus, herpesvirus, Semliki forest virus, or bovine papillomavirus) can be used to transfect nucleic acid molecules into target cell populations. Furthermore, baculovirus systems can also be used as vectors in eukaryotic expression systems targeting the nucleic acid molecules of the present invention.

[0142] The term "prokaryote" is intended to include all bacteria that can be transformed, transduced, or transfected with DNA or DNA or RNA molecules to express the proteins of the present invention. Prokaryotic hosts can include Gram-negative and Gram-positive bacteria, such as, for example, *Escherichia coli*, *Serratia typhimurium*, *Serratia marcescens*, *Corynebacterium* (glutamate-containing *Corynebacterium*), *Pseudomonas* (fluorescein-containing *Pseudomonas*), *Lactobacillus*, *Streptomyces*, *Salmonella*, and *Bacillus subtilis*. The term "eukaryote" refers to cells including yeast, higher plants, insects, and mammals. Non-limiting examples of mammalian host cells commonly used in this art include HeLa, HEK293, H9, Per.C6 and Jurkat cells, mouse NIH3T3, NS / 0, SP2 / 0 and C127 cells, COS cells (e.g. COS 1 or COS 7), CV1, quail QC1-3 cells, mouse L cells, mouse sarcoma cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells.

[0143] Therefore, in a fifth aspect, the present invention relates to a host cell comprising a polynucleotide according to the invention or a vector according to the invention. The host cell may be a prokaryotic cell or a eukaryotic cell. In a preferred embodiment, the host cell is a eukaryotic cell. In a particular embodiment, the cell is an HEK cell. In another particular embodiment, the host cell is a CHO cell.

[0144] When a recombinant expression vector encoding the heavy chain and / or light chain of an antibody according to the invention disclosed herein is introduced into a host cell, the antibody or antibody-antigen binding fragment is generated by culturing the host cell for a period of time sufficient to allow expression of the antibody or antigen binding fragment in the host cell, or preferably, to allow secretion of the antibody or antigen binding fragment into the culture medium in which the host cell is growing. The antibody and / or antigen binding fragment can be recovered from the culture medium using standard protein purification methods. Methods for purifying antibodies are well known in the art. Exemplary purification methods are described in the appended examples.

[0145] Therefore, in a seventh aspect, the present invention also provides a method for generating antibodies that specifically bind to pepsinogen A as disclosed herein. The method includes culturing the host cells of the present invention under suitable conditions and isolating the resulting antibodies. The isolated antibodies of the present invention can be obtained through purification steps, such as those described in the appended examples.

[0146] The invention further provides, in an eighth aspect, antibody or antigen-binding fragments that can be obtained by any of the methods disclosed herein.

[0147] The transformed host cells can be grown in a bioreactor and cultured according to techniques known in the art to achieve optimal cell growth. The antibodies and / or antibody-antigen binding fragments of the present invention can then be isolated from the cell fraction or growth medium by any conventional method, such as, but not limited to, affinity chromatography (e.g., using fusion tags, such as Strep-tag II or His6 tags), gel filtration (size exclusion chromatography), anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, high-performance liquid chromatography (HPLC), reversed-phase HPLC, or immunoprecipitation.

[0148] It is understood that variations of the above procedures are all within the scope of protection of this invention. For example, recombinant DNA technology can be used to remove or modify DNA sequences encoding antibodies and / or antibody-antigen binding fragments disclosed herein (e.g., encoding heavy chain and / or light chain variable domains as defined above). For example, recombinant DNA technology can be used to remove portions of the coding sequence that are not essential for maintaining specific and selective binding to the target antigen. Molecules expressed by such truncated DNA molecules are also covered by the antibodies of this invention.

[0149] Antibody derivatives can be produced, for example, by adding exogenous sequences to modify immunogenicity or reduce, enhance, or modify binding, affinity, association rate, dissociation rate, high affinity, specificity, half-life, or any other suitable property.

[0150] Humanized versions of the antibodies disclosed herein are also provided, comprising CDRs containing the heavy and / or light chains as described above. As is well known in the art, “humanization” (to produce a humanized version of the parent antibody) refers to the recombinant engineering of an antibody using a CDR derived from a non-human donor immunoglobulin, with both human frame domains and constant domains. During engineering, frame and / or CDR residues may be altered to maintain binding affinity and activity, such as specificity for pepsinogen A. Methods for humanizing antibodies are well known in the art, for example, as disclosed in: Queen et al., Proc. Natl. Acad Sci USA 86 (1989), 10029-10032; Hodgson et al., Bio / Technology 9 (1991) 421.

[0151] In a ninth aspect, the present invention provides a composition comprising the antibody of the present invention, the polynucleotide of the present invention, the vector of the present invention, or the host cell of the present invention. In a preferred embodiment, the composition is a diagnostic composition, i.e., a composition for use in diagnostic applications. In a preferred embodiment, the composition is used for an in vitro diagnostic assay for detecting pepsinogen A. In a preferred embodiment, the diagnostic composition may be a reagent for an immunoassay for detecting pepsinogen A. The diagnostic composition is preferably configured to allow detection of pepsinogen A in a sample obtained from a subject. The sample is preferably a blood sample (e.g., whole blood, serum, or plasma).

[0152] In a tenth aspect, the present invention provides a composition comprising a) An antibody or antigen-binding fragment thereof according to the first aspect of the invention or any embodiment related to the first aspect, and An antibody or an antigen-binding fragment thereof according to the second aspect of the invention or any embodiment related to the second aspect.

[0153] As shown in Example 6, all antibodies of the present invention are labeled with a single label. Two groups of antibodies were prepared. One group of antibodies was labeled with ruthenium, and the second group of antibodies was labeled with biotin. In Example 7, different combinations of antibodies were tested. A sample containing pepsinogen A was incubated with a ruthenium-labeled monoclonal pepsinogen A-specific IgG antibody and a biotinylated monoclonal pepsinogen A-specific IgG antibody to form a sandwich complex. As shown in Table 1, the inventors unexpectedly identified that the combined antibodies (e.g., the combination of mAb 2 with mAb 8, mAb 24, or mAb 6.7, i.e., antibodies binding to the epitopes of the exemplary antibodies) exhibited unexpectedly high signals compared to other combinations (e.g., mAb 6.7, mAb 24, or mAb 7 with mAb 8). Without being bound by theory, the epitopes bound by the antibodies appear to influence which antibody combinations are advantageous (see Table 2).

[0154] A composition is also provided comprising an antibody-associated polynucleotide, vector, or host cell related to the first aspect of the invention or an embodiment thereof, and an antibody-associated polynucleotide, vector, or host cell related to the second aspect of the invention or an embodiment thereof.

[0155] In one embodiment, the composition according to the invention is a diagnostic composition.

[0156] In particular, the present invention provides a composition comprising Antibodies or antigen-binding fragments thereof according to the first aspect or embodiments of the first aspect of the present invention; and An antibody or an antigen-binding fragment thereof according to a second aspect or an embodiment of the present invention; The composition is a diagnostic composition.

[0157] In an eleventh aspect, the present invention relates to the use of antibodies or antigen-binding fragments thereof according to the invention, and compositions according to the invention, for in vitro immunoassays, particularly for in vitro immunoassays to detect pepsinogen A in samples. Therefore, antibodies of the invention are also provided for use in immunoassays (e.g., heterologous immunoassays) to detect pepsinogen A.

[0158] The sample can be a bodily fluid, such as, but not limited to, blood, cerebrospinal fluid, semen, saliva, or urine. In this embodiment, the sample is a blood sample, such as whole blood, serum, or plasma. In this embodiment, the sample is serum or plasma.

[0159] In a twelfth aspect, the present invention provides an in vitro method for detecting pepsinogen A in a sample using the antibody of the present invention. The antibody may be part of a composition, particularly the diagnostic composition of the present invention.

[0160] The sample can be a bodily fluid, such as a blood sample, cerebrospinal fluid, semen, saliva, or urine. In this embodiment, the sample is a blood sample, such as whole blood, serum, or plasma. In this embodiment, the sample is serum or plasma.

[0161] The method for detection may include: (a) contacting a sample containing pepsinogen A with an antibody or an antigen-binding fragment thereof according to the invention to form a complex containing pepsinogen A and the antibody or an antigen-binding fragment thereof, and (b) detecting the complex formed in step (a) to detect pepsinogen A in the sample.

[0162] In one embodiment, the pepsinogen A in the sample contains a sequence that is at least 95% homologous to the sequence shown in SEQ ID NO: 34 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A or V353L.

[0163] In one specific embodiment, the method is a serum immunoassay, wherein the sample is a body fluid, preferably whole blood, serum or plasma.

[0164] In one specific embodiment, the antibody or its antigen-binding fragment has a detection marker attached thereto.

[0165] The detection marker linked to the antibody of the present invention can be any detection marker known in the art. For example, the detection marker can be selected from enzymes, luminescent markers (preferably fluorescent, luminescent, chemiluminescent, or electrochemiluminescent), or radioactive markers.

[0166] In a thirteenth aspect, the present invention provides an in vitro method for detecting pepsinogen A in a sample, the method comprising the following steps: 1) Contact the sample containing pepsinogen A with a first antibody or its antigen-binding fragment and a second antibody or its antigen-binding fragment, wherein... (a) The first antibody is (i) An antibody or an antigen-binding fragment thereof according to the first aspect or an embodiment of the first aspect of the invention; or (ii) An antibody or an antigen-binding fragment thereof according to the second aspect or an embodiment of the present invention; and (b) The second antibody specifically binds to pepsinogen A and does not compete with the first antibody for specific binding to pepsinogen A; 2) Incubate the mixture obtained in 1) to form a complex comprising pepsinogen A and a first antibody or an antigen-binding fragment thereof, and a second antibody or an antigen-binding fragment thereof, and 3) Detect pepsinogen A in the sample by detecting the formation of the complex defined in 2).

[0167] As discussed above, Example 6 demonstrates that the antibodies of the present invention are each labeled with a single label. Two groups of antibodies were prepared. One group of antibodies was labeled with ruthenium, and the second group of antibodies was labeled with biotin. In Example 7, different combinations of antibodies were tested. A sample containing pepsinogen A was incubated with a ruthenium-labeled monoclonal pepsinogen A-specific IgG antibody and a biotinylated monoclonal pepsinogen A-specific IgG antibody to form a sandwich complex. As shown in Table 1, the inventors unexpectedly identified that the combined antibodies (e.g., antibodies such as mAb 6.7 or antibodies binding to the same epitope (mAb 24 or 7) combined with mAb 2) exhibited a surprisingly high signal compared to other combinations (e.g., antibodies such as mAb 6.7 or antibodies binding to the same epitope (mAb 24 or 7) combined with mAb 8). It was quite unexpected which antibody combination provided a particularly good signal. Without being bound by theory, the epitopes that antibodies bind to appear to influence which antibody combinations are favorable (see Table 2).

[0168] In one embodiment of the thirteenth aspect of the invention (c), one of the first antibody and the second antibody includes a detection marker.

[0169] In a specific embodiment, a first antibody or its antigen-binding fragment that does not contain a detection marker, or a second antibody or its antigen-binding fragment that contains a capture marker, may be used.

[0170] As used herein, "capture tag" refers to a tag that can immobilize a capture agent (e.g., an antibody having a capture tag attached thereto according to the invention) onto a surface (e.g., magnetic particles such as microbeads). Non-limiting examples are members of binding pairs. A non-limiting example of a capture tag is biotin or a derivative thereof, which can interact with streptavidin or a derivative thereof. Various capture tags are well known in the art.

[0171] In one specific embodiment, the method further includes: adding particles capable of binding with the capture marker to the mixture of (1), and 2.1) separating the particles from the mixture before detecting the signal provided by the detection marker.

[0172] In one embodiment When the first antibody or its antigen-binding fragment is selected from aspect thirteen (i), then the second antibody is selected from aspect thirteen (ii); or When the first antibody or its antigen-binding fragment is selected from the thirteenth aspect (ii), the second antibody is selected from the thirteenth aspect (i).

[0173] In one embodiment, the pepsinogen A in the sample contains a sequence that is at least 95% homologous to the sequence shown in SEQ ID NO: 34 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A or V353L.

[0174] In a specific embodiment, the method is a serum immunoassay, wherein the sample is a body fluid, preferably whole blood, serum or plasma.

[0175] The detection marker linked to the antibody of the present invention can be any detection marker known in the art. For example, the detection marker can be selected from enzymes, luminescent markers (preferably fluorescent, luminescent, chemiluminescent, or electrochemiluminescent), or radioactive markers.

[0176] In a further embodiment, the limit of quantitation (LoQ) of the method of the present invention is 1.79 ng / mL at a 20% coefficient of variation (CV).

[0177] LoQ is defined as the minimum amount of analyte in a sample that can be accurately quantified with an intermediate precision CV of ≤ 20%.

[0178] In a further embodiment, the detection of pepsinogen A in the sample is achieved in 18 minutes or less.

[0179] In a fourteenth aspect, this document provides a kit comprising the antibody of the present invention or a composition comprising the antibody. In examples, the kit is a kit for the in vitro detection and / or quantification of pepsinogen A or its subtypes. In examples, the kit is an immunoassay kit. In examples, the kit is a kit for heterologous immunoassay.

[0180] In a fifteenth aspect, this document provides a kit comprising a composition according to a tenth aspect of the invention, or a first antibody or an antigen-binding fragment thereof, and a second antibody or an antigen-binding fragment thereof, wherein one of the first antibody or the second antibody or the antigen-binding fragment thereof is an antibody of the first aspect of the invention or an embodiment of the first aspect thereof, and the other of the first antibody or the second antibody or the antigen-binding fragment thereof is an antibody of the second aspect of the invention or an embodiment of the second aspect thereof.

[0181] As discussed above, Example 6 demonstrates that the antibodies of the present invention are each labeled with a single label. Two groups of antibodies were prepared. One group of antibodies was labeled with ruthenium, and the second group of antibodies was labeled with biotin. In Example 7, different combinations of antibodies were tested. A sample containing pepsinogen A was incubated with a ruthenium-labeled monoclonal pepsinogen A-specific IgG antibody and a biotinylated monoclonal pepsinogen A-specific IgG antibody to form a sandwich complex. As shown in Table 1, the inventors unexpectedly identified that the combined antibodies (e.g., antibodies such as mAb 6.7 or antibodies binding to the same epitope (mAb 24 or 7) combined with mAb 2) exhibited a surprisingly high signal compared to other combinations (e.g., antibodies such as mAb 6.7 or antibodies binding to the same epitope (mAb 24 or 7) combined with mAb 8). It was quite unexpected which antibody combination provided a particularly good signal. Without being bound by theory, the epitopes that antibodies bind to appear to influence which antibody combinations are advantageous (see Table 2). These advantageous antibody combinations can be included in the kit.

[0182] In one embodiment of the fifteenth aspect of the invention, one of the first antibody and the second antibody comprises a detection marker.

[0183] In a specific embodiment, a first antibody or its antigen-binding fragment that does not contain a detection marker, or a second antibody or its antigen-binding fragment that contains a capture marker, may be used.

[0184] As used herein, "capture tag" refers to a tag that can immobilize a capture agent (e.g., an antibody having a capture tag attached thereto according to the invention) onto a surface (e.g., magnetic particles such as microbeads). Non-limiting examples are members of binding pairs. A non-limiting example of a capture tag is biotin or a derivative thereof, which can interact with streptavidin or a derivative thereof. Various capture tags are well known in the art.

[0185] In one specific embodiment, the kit is an immunoassay kit. In another embodiment, the kit is a kit for heterologous immunoassay.

[0186] In a sixteenth aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment thereof comprising... (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 1; and / or (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 2.

[0187] As illustrated in the examples, the inventors unexpectedly identified a family of high-affinity antibodies that specifically bind to pepsinogen A. As shown in the accompanying examples (e.g., Example 8, Table 1), antibodies from this family (e.g., mAb 6.7, mAb 24, mAb 7) all specifically bind to pepsinogen A.

[0188] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 1; and (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 2.

[0189] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 1; or (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 2.

[0190] In a further embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 1; and (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 2.

[0191] In a further embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 1; or (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 2.

[0192] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 1; and (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 2.

[0193] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 1; or (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 2.

[0194] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 1; and (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 2.

[0195] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 1; or (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 2.

[0196] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, which contains the sequence shown in SEQ ID NO: 1; and (ii) VL, which contains the sequence shown in SEQ ID NO: 2.

[0197] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, which contains the sequence shown in SEQ ID NO: 1; or (ii) VL, which contains the sequence shown in SEQ ID NO: 2.

[0198] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH consisting of the sequence shown in SEQ ID NO: 1; and (ii) A VL consisting of the sequence shown in SEQ ID NO: 2.

[0199] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH consisting of the sequence shown in SEQ ID NO: 1; or (ii) A VL consisting of the sequence shown in SEQ ID NO: 2.

[0200] In a seventeenth aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment thereof comprising... (i) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified with one amino acid substitution; and (ii) VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified with one amino acid substitution.

[0201] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, comprising: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 3; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 4; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 5; and (ii) VL, comprising: CDR-L1 represented by the amino acid sequence of SEQ ID NO: 6; CDR-L2 represented by the amino acid sequence of SEQ ID NO: 7; and CDR-L3 represented by the amino acid sequence of SEQ ID NO: 8.

[0202] In embodiments of the invention, all amino acid substitutions are conserved amino acid substitutions. In a further embodiment, each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein these amino acid groups are... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0203] In a further specific embodiment, each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0204] In a further embodiment, the antibody or antigen-binding fragment thereof according to the seventeenth aspect of the present invention comprises The VL, consisting of the frame region (FW) and the CDR, is shown in Equation I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 39; The amino acid sequence of FW-L2, SEQ ID NO: 40; The amino acid sequence of FW-L3, SEQ ID NO: 41; The amino acid sequence of FW-L4, SEQ ID NO: 42; The amino acid sequence of FW-H1, SEQ ID NO: 35; The amino acid sequence of FW-H2, SEQ ID NO: 36; The amino acid sequence of FW-H3, SEQ ID NO: 37; The amino acid sequence of FW-H4, SEQ ID NO: 38.

[0205] In a further embodiment, the antibody or antigen-binding fragment thereof according to the seventeenth aspect of the present invention comprises: a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequence or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 39; The amino acid sequence of FW-L2, SEQ ID NO: 40; The amino acid sequence of FW-L3, SEQ ID NO: 41; The amino acid sequence of FW-L4, SEQ ID NO: 42; The amino acid sequence of FW-H1, SEQ ID NO: 35; The amino acid sequence of FW-H2, SEQ ID NO: 36; The amino acid sequence of FW-H3, SEQ ID NO: 37; The amino acid sequence of FW-H4, SEQ ID NO: 38.

[0206] In embodiments of the invention, all amino acid substitutions are conserved amino acid substitutions. In a further embodiment, each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein these amino acid groups are... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0207] In a further specific embodiment, each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0208] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, which contains the sequence shown in SEQ ID NO: 1; and (ii) VL, which contains the sequence shown in SEQ ID NO: 2.

[0209] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, which contains the sequence shown in SEQ ID NO: 1; or (ii) VL, which contains the sequence shown in SEQ ID NO: 2.

[0210] In further embodiments of the sixteenth or seventeenth aspect and related embodiments, the antibody or its antigen-binding fragment is expressed at 0.07 nM or less K. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0211] In further embodiments of the sixteenth or seventeenth aspect and related embodiments thereto, the antibody or its antigen-binding fragment is bound to pepsinogen A consisting of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L at 37°C for 313 minutes or longer t / 2-diss.

[0212] In further embodiments of the sixteenth or seventeenth aspect and related embodiments, the association rate k of the antibody binding or its antigen-binding fragment with pepsinogen A composed of SEQ ID NO: 68 or its variants containing amino acid substitutions of E58K, K222Q, T265A or V353L is... a For at least 4.9x10 5 M -1 s -1 .

[0213] In an eighteenth aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment thereof comprising... (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 9; and / or (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 10.

[0214] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 9; and (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 10.

[0215] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 9; and (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 10.

[0216] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 9; or (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 10.

[0217] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 9; and (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 10.

[0218] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 9; or (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 10.

[0219] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 9; and (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 10.

[0220] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 9; or (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 10.

[0221] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 9; and (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 10.

[0222] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 9; or (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 10.

[0223] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, which contains the sequence shown in SEQ ID NO: 9; and (ii) VL, which contains the sequence shown in SEQ ID NO: 10.

[0224] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, which contains the sequence shown in SEQ ID NO: 9; or (ii) VL, which contains the sequence shown in SEQ ID NO: 10.

[0225] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH consisting of the sequence shown in SEQ ID NO: 9; and (ii) A VL consisting of the sequence shown in SEQ ID NO: 10.

[0226] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH consisting of the sequence shown in SEQ ID NO: 9; or (ii) A VL consisting of the sequence shown in SEQ ID NO: 10.

[0227] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment thereof comprising... (i) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified with one amino acid substitution; and (ii) VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14 or a variant thereof modified by one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15 or a variant thereof modified by one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16 or a variant thereof modified by one amino acid substitution.

[0228] In embodiments of the invention, all amino acid substitutions are conserved amino acid substitutions. In a further embodiment, each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein these amino acid groups are... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0229] In a further specific embodiment, each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0230] In a further embodiment, the antibody or antigen-binding fragment of the present invention comprises: (i) VH, which comprises: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 11; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 12; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 13; and (ii) VL, which comprises: CDR-L1 represented by the amino acid sequence of SEQ ID NO: 14; CDR-L2 represented by the amino acid sequence of SEQ ID NO: 15; and CDR-L3 represented by the amino acid sequence of SEQ ID NO: 16.

[0231] The antibody of the present invention may have different frame regions with CDRs attached to the VH and VL domains.

[0232] The variable domain (VL) of a light chain can be composed of a framework region (FW) and a CDR, as shown in Equation I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) Furthermore, the heavy chain variable structural domain (VH) can be composed of FW and CDR, as shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II).

[0233] In a preferred embodiment, VH comprises CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13; and VL comprises CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16; and FW comprises the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 47; The amino acid sequence of FW-L2, SEQ ID NO: 48; The amino acid sequence of FW-L3, SEQ ID NO: 49; The amino acid sequence of FW-L4, SEQ ID NO: 50; The amino acid sequence of FW-H1, SEQ ID NO: 43; The amino acid sequence of FW-H2, SEQ ID NO: 44; The amino acid sequence of FW-H3, SEQ ID NO: 45; The amino acid sequence of FW-H4, SEQ ID NO: 46.

[0234] In a preferred embodiment, VH comprises CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13; and VL comprises CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16; and FW comprises the following amino acid sequences or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 47; The amino acid sequence of FW-L2, SEQ ID NO: 48; The amino acid sequence of FW-L3, SEQ ID NO: 49; The amino acid sequence of FW-L4, SEQ ID NO: 50; The amino acid sequence of FW-H1, SEQ ID NO: 43; The amino acid sequence of FW-H2, SEQ ID NO: 44; The amino acid sequence of FW-H3, SEQ ID NO: 45; The amino acid sequence of FW-H4, SEQ ID NO: 46.

[0235] In embodiments of the invention, all amino acid substitutions are conserved amino acid substitutions. In a further embodiment, each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein these amino acid groups are... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0236] In a further specific embodiment, each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0237] In further embodiments of the eighteenth aspect and related embodiments, the antibody or its antigen-binding fragment is at 0.9 nM or less K D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0238] In further embodiments of the eighteenth aspect and related embodiments, the association rate k of the antibody or its antigen-binding fragment with pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A or V353L is... a For at least 6x10 5 M -1 s -1 .

[0239] In a nineteenth aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment thereof comprising... (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 17; and / or (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 18.

[0240] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 17; or (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 18.

[0241] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 17; and (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 18.

[0242] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 17; or (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 18.

[0243] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 17; and (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 18.

[0244] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 17; or (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 18.

[0245] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 17; and (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 18.

[0246] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 17; or (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 18.

[0247] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 17; and (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 18.

[0248] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 17; or (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 18.

[0249] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, which contains the sequence shown in SEQ ID NO: 17; and (ii) VL, which contains the sequence shown in SEQ ID NO: 18.

[0250] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, which contains the sequence shown in SEQ ID NO: 17; or (ii) VL, which contains the sequence shown in SEQ ID NO: 18.

[0251] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH consisting of the sequence shown in SEQ ID NO: 17; and (ii) A VL consisting of the sequence shown in SEQ ID NO: 18.

[0252] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH consisting of the sequence shown in SEQ ID NO: 17; or (ii) A VL consisting of the sequence shown in SEQ ID NO: 18.

[0253] In a twentieth aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment thereof comprising... (i) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21, or a variant thereof modified with one amino acid substitution; and (ii) VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22 or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23 or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24 or a variant thereof modified with one amino acid substitution.

[0254] In the embodiments, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, comprising: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 19; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 20; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 21; and (ii) VL, comprising: CDR-L1 represented by the amino acid sequence of SEQ ID NO: 22; CDR-L2 represented by the amino acid sequence of SEQ ID NO: 23; and CDR-L3 represented by the amino acid sequence of SEQ ID NO: 24.

[0255] In a further embodiment, the antibody or antigen-binding fragment thereof according to the twentieth aspect of the present invention comprises: a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 55; The amino acid sequence of FW-L2, SEQ ID NO: 56; The amino acid sequence of FW-L3, SEQ ID NO: 57; The amino acid sequence of FW-L4, SEQ ID NO: 58; The amino acid sequence of FW-H1, SEQ ID NO: 51; The amino acid sequence of FW-H2, SEQ ID NO: 52; The amino acid sequence of FW-H3, SEQ ID NO: 53; The amino acid sequence of FW-H4, SEQ ID NO: 54.

[0256] In a further embodiment, the antibody or antigen-binding fragment thereof according to the twentieth aspect of the present invention comprises: a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequence or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 55; The amino acid sequence of FW-L2, SEQ ID NO: 56; The amino acid sequence of FW-L3, SEQ ID NO: 57; The amino acid sequence of FW-L4, SEQ ID NO: 58; The amino acid sequence of FW-H1, SEQ ID NO: 51; The amino acid sequence of FW-H2, SEQ ID NO: 52; The amino acid sequence of FW-H3, SEQ ID NO: 53; The amino acid sequence of FW-H4, SEQ ID NO: 54.

[0257] In embodiments of the invention, all amino acid substitutions are conserved amino acid substitutions. In a further embodiment, each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein these amino acid groups are... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0258] In a further specific embodiment, each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0259] In further embodiments of the nineteenth or twentieth aspect of the invention and related embodiments thereof, the antibody or its antigen-binding fragment is expressed at 0.9 nM or less K. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0260] In a further embodiment of the nineteenth or twentieth aspect of the invention and related embodiments thereof, the antibody or its antigen-binding fragment is bound at 37°C for 23 minutes or longer t / 2-diss to pepsinogen A consisting of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L.

[0261] In further embodiments of the nineteenth or twentieth aspect of the invention and related embodiments, the association rate k of the antibody or its antigen-binding fragment with pepsinogen A composed of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L is described. a For at least 5.2x10 5 M -1 s -1 .

[0262] In a twenty-first aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment thereof comprising... (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 25; and / or (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 26.

[0263] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 25; and / or (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 26.

[0264] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 25; and (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 26.

[0265] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 25; or (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 26.

[0266] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 25; and (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 26.

[0267] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 25; or (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 26.

[0268] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 25; and (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 26.

[0269] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 25; or (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 26.

[0270] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 25; and (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 26.

[0271] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 25; or (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 26.

[0272] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, which contains the sequence shown in SEQ ID NO: 25; and (ii) VL, which contains the sequence shown in SEQ ID NO: 26.

[0273] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, which contains the sequence shown in SEQ ID NO: 25; or (ii) VL, which contains the sequence shown in SEQ ID NO: 26.

[0274] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) VH consisting of the sequence shown in SEQ ID NO: 25; and (ii) A VL consisting of the sequence shown in SEQ ID NO: 26.

[0275] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises (i) A VH consisting of the sequence shown in SEQ ID NO: 25; or (ii) A VL consisting of the sequence shown in SEQ ID NO: 26.

[0276] In a twenty-second aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment thereof comprising... (i) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and (ii) VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30 or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31 or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32 or a variant thereof modified with one amino acid substitution.

[0277] In the embodiments, the antibody or its antigen-binding fragment according to the invention comprises (i) VH, comprising: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 27; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 28; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 29; and (ii) VL, comprising: CDR-L1 represented by the amino acid sequence of SEQ ID NO: 30; CDR-L2 represented by the amino acid sequence of SEQ ID NO: 31; and CDR-L3 represented by the amino acid sequence of SEQ ID NO: 32.

[0278] In a further embodiment, the antibody or antigen-binding fragment thereof according to the twenty-second aspect of the present invention comprises: a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 63; The amino acid sequence of FW-L2, SEQ ID NO: 64; The amino acid sequence of FW-L3, SEQ ID NO: 65; The amino acid sequence of FW-L4, SEQ ID NO: 66; The amino acid sequence of FW-H1, SEQ ID NO: 59; The amino acid sequence of FW-H2, SEQ ID NO: 60; The amino acid sequence of FW-H3, SEQ ID NO: 61; The amino acid sequence of FW-H4, SEQ ID NO: 62.

[0279] In a further embodiment, the antibody or antigen-binding fragment thereof according to the twenty-second aspect of the present invention comprises: a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequence or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 63; The amino acid sequence of FW-L2, SEQ ID NO: 64; The amino acid sequence of FW-L3, SEQ ID NO: 65; The amino acid sequence of FW-L4, SEQ ID NO: 66; The amino acid sequence of FW-H1, SEQ ID NO: 59; The amino acid sequence of FW-H2, SEQ ID NO: 60; The amino acid sequence of FW-H3, SEQ ID NO: 61; The amino acid sequence of FW-H4, SEQ ID NO: 62.

[0280] In embodiments of the invention, all amino acid substitutions are conserved amino acid substitutions. In a further embodiment, each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein these amino acid groups are... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0281] In a further specific embodiment, each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0282] In further embodiments of the twenty-first or twenty-second aspect of the present invention and related embodiments thereof, the antibody or its antigen-binding fragment is expressed at a concentration of 0.6 nM or less. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0283] In a further embodiment of the twenty-first or twenty-second aspect of the invention and related embodiments thereof, the antibody or its antigen-binding fragment is bound at 37°C for 21 minutes or longer t / 2-diss to pepsinogen A consisting of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L.

[0284] In further embodiments of the twenty-first or twenty-second aspect of the invention and related embodiments, the association rate k of the antibody or its antigen-binding fragment with pepsinogen A composed of SEQ ID NO: 68 or its variants comprising amino acid substitutions of E58K, K222Q, T265A or V353L is described. a For at least 8.7x10 5 M -1 s -1 .

[0285] In a preferred embodiment, the antibody or its antigen-binding fragment according to the present invention is a monoclonal antibody.

[0286] In a further embodiment of the invention, the antibody or its antigen-binding fragment specifically binds to pepsinogen A, wherein pepsinogen A comprises SEQ ID NO: 34 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A, or V353L. In a further embodiment of the invention, the antibody or its antigen-binding fragment specifically binds to pepsinogen A, wherein pepsinogen A is composed of SEQ ID NO: 34 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A, or V353L.

[0287] As shown in the appended examples (e.g., Examples 5 and 9), the antibodies of the present invention are further characterized by high affinity for pepsinogen A composed of SEQ ID NO: 68 or variants thereof comprising amino acid substitutions of E58K, K222Q, T265A, or V353L. Therefore, the antibodies of the present invention may be characterized by having an equilibrium dissociation constant K of 2 nM or less, 1.5 nM or less, or 1 nM or less. D In a preferred embodiment, K is measured at 37°C. D The decrease in K D K is the preferred option. D The lower the value, the better.

[0288] In a preferred embodiment, surface plasmon resonance spectroscopy is used to determine the binding of the antibody or its antigen-binding fragment according to the invention to pepsinogen A and its isoforms containing amino acid substitutions of E58K, K222Q, T265A, or V353L. D The experimental setup is described in Example 9 of this paper.

[0289] Used to determine the equilibrium dissociation constant K D The method is known in the art.

[0290] In a preferred embodiment, K D It can be obtained through surface plasmon resonance spectroscopy (e.g., BIAcore) ® ) Measurement.

[0291] Used to determine K D The preferred temperature is 37°C.

[0292] In an embodiment, K can be determined using kinetic measurements that determine association and dissociation rates. D Therefore, in embodiments, these methods can be the same as those described below for determining k. a The same surface plasmon resonance method. This method may include corrections for mass transfer limitations.

[0293] In a preferred embodiment, K D Surface plasmon resonance spectroscopy (e.g., BIAcore) can be used. ® ) Measurement.

[0294] In a further embodiment, the antibody or antigen-binding fragment according to the invention has an antibody / antigen complex half-life of 20 minutes or longer at 37°C. / 2-diss .

[0295] Methods for determining the half-life of an antibody / antigen complex that binds to pepsinogen A are known in the art. Exemplary examples are described in the examples (particularly example 9).

[0296] The half-life of an antibody / antigen complex is determined by formula t. / 2 diss = ln(2) / (k d 60) Calculated in minutes.

[0297] As illustrated in the accompanying examples, the antibodies of the present invention are characterized by a particularly high association rate constant (kΩ) when binding to pepsinogen A comprising SEQ ID NO: 34 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A, or V353L. a This rapid association rate makes the antibodies of this invention particularly suitable for high-throughput immunoassays and competitive assay formats.

[0298] Used to determine the association rate constant (k) for antibody binding to pepsinogen A. a The method is known in the art. Exemplary instances are described in the examples (especially example 9).

[0299] Therefore, in the embodiments, a further feature of the antibody or its antigen-binding fragment according to the invention is the association rate (kb) with pepsinogen A composed of SEQ ID NO: 68 or its variants containing amino acid substitutions of E58K, K222Q, T265A or V353L. a (For at least 10) 5 M -1 s -1 Preferably at least 4.9x10 5 M -1 s -1 In a preferred embodiment, the association rate with pepsinogen A composed of SEQ ID NO: 68 or its variants containing amino acid substitutions of E58K, K222Q, T265A, or V353L is at least 4.9 x 10⁻⁶. 5 M -1 s - 1. In one embodiment, k is measured at 37°C. a .

[0300] The association rate constant (k) used to determine the binding of an antibody to pepsinogen A (or its variants) is used to determine the binding rate constant. a The method is known in the art. Exemplary instances are described in the examples (especially example 9).

[0301] In a preferred embodiment, the association rate constant (k) between the antibody or its antigen-binding fragment according to the invention and pepsinogen A or its variant is determined by surface plasmon resonance spectroscopy. a ).

[0302] The surface plasmon resonance spectroscopy involves attaching or capturing an antibody or its antigen-binding fragment onto a CM5 sensor chip and injecting pepsinogen A, consisting of SEQ ID NO: 68, or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, as an analyte, wherein the determination is performed using HBS-ET buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (w / v) Tween 20®) at 37°C.

[0303] In a further embodiment of the invention, the antibody or its antigen-binding fragment does not specifically bind to pepsinogen C. This means that the antibody or its antigen-binding fragment binds to pepsinogen C with a KD greater than 100 nM. In a preferred embodiment, pepsinogen C consists of SEQ ID NO: 69.

[0304] In one embodiment, surface plasmon resonance spectroscopy is performed using a Biacore 8k or B4000 instrument.

[0305] Association constant k a [1 / Ms], dissociation rate constant k d [s -1 and dissociation equilibrium constant K D [M] can be calculated based on the Langmuir fitting model, for example, by using evaluation software corresponding to the instrument.

[0306] The antibodies and antigen-binding fragments of the present invention can be prepared using various techniques conventionally used in the art. For example, antibodies can be prepared by immunizing non-human animals (e.g., mice) with pepsinogen A, isolating and subsequently isolating antigen-reactive antibody-producing B cells. Preferred exemplary methods for producing antibodies according to the present invention using non-human animals are provided in the appended examples (see in particular Example 3). Selected clones for antibody production can be processed according to conventional methods for subsequent recombinant processing.

[0307] Another suitable method for generating or isolating the antibodies and antibody-antigen binding fragments of the present invention includes, but is not limited to, methods for selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, phage, ribosome, oligonucleotide, RNA, cDNA, or yeast display libraries) using targeted binding activity. For example, antibodies or antigen-binding fragments can be selected from such libraries by positive selection for specific binding to pepsinogen A. Display libraries are well known in the art and are available, for example, from a variety of commercial suppliers, including but not limited to Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinslied / Planeger, Germany), Biovation (Aberdeen, Scotland, UK), and Bioinvent (Lund, Sweden). Similarly, selected clones can be treated according to conventional methods for subsequent recombinant processing.

[0308] The antibodies of this invention are recombinantly expressible. Therefore, in some embodiments, the antibodies of this invention can be recombinant antibodies. Methods for generating recombinant antibodies are known in the art.

[0309] In a twenty-third aspect, the present invention relates to a polynucleotide or polynucleotide group encoding (i) The heavy chain variable domain of the antibody or its antigen-binding fragment according to the present invention, and (ii) The light chain variable domain of the antibody or antigen-binding fragment according to the invention.

[0310] The present invention also provides a nucleic acid molecule encoding an antibody or any antigen-binding fragment thereof as defined above. In particular, a polynucleotide is provided encoding a heavy chain and / or light chain variable domain of an antibody that specifically binds to pepsinogen A as defined above. In some embodiments, the polynucleotide may include further sequences to ensure that not only the heavy chain and / or light chain variable domains are expressed, but also the remaining heavy chain and / or light chain constant regions are expressed, such that a full-length IgG antibody comprising the heavy chain and light chain variable domains of the present invention is expressed. Therefore, for each aspect and embodiment involving an antibody or antigen-binding fragment that specifically binds to pepsinogen A as described herein, a corresponding polynucleotide encoding the respective antibody or antigen-binding fragment is provided herein.

[0311] In a twenty-fourth aspect, this document provides vectors comprising the polynucleotides of the present invention. In particular, vectors comprising nucleic acid molecules encoding antibodies or antibody-antigen binding fragments of the present invention are provided. As used herein, the term “vector” refers to a circular or linear nucleic acid molecule that can autonomously replicate in a host cell into which it has been introduced. Non-limiting examples of vectors suitable for use in the present invention include viscera, plasmids (e.g., naked plasmids or those contained in liposomes), viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), and bacteriophages. However, many suitable vectors are provided in the art, the choice of which depends on the desired function. The development and use of suitable vectors are well documented in the art; see, for example, the techniques described in Sambrook and Russell, “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, NY (2001), and Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, NY (1989), (1994). The vectors used in connection with this invention comprise nucleic acid sequences that encode the full-length pepsinogen A antibody-antigen-binding fragment as disclosed herein. Therefore, for each aspect and embodiment involving an antibody or antigen-binding fragment that specifically binds to pepsinogen A as described herein, a vector comprising the corresponding polynucleotide encoding the respective antibody or antigen-binding fragment is provided herein.

[0312] Regarding the term "vector comprising..." as used herein, it will be understood in the art that the vector contains further nucleic acid sequences that are necessary and / or sufficient for the desired vector activity in the host cell, such as driving vector replication (and thereby encoding the nucleic acid sequence) and / or guiding the host cell to express the antibody or antigen-binding fragment of the present invention. Such further nucleic acid sequences include, but are not limited to, sequences that control vector replication and / or the expression of desired sequences in a particular cellular system. For example, the vector may contain a nucleic acid molecule encoding the antibody or antibody-antigen-binding fragment of the present invention, which is operatively linked to a regulatory sequence and / or under the control of a regulatory sequence. The term "regulatory sequence" refers to a DNA sequence that is necessary for the expression of the coding sequence operatively linked thereto. The term "control sequence" is intended to include at least all components that may be equally necessary for expression and may further include other advantageous components, for example, for allowing replication. As understood in the art, the nature of such regulatory and control sequences varies from host organism to host organism. For example, in prokaryotes, control sequences typically include a promoter, a ribosome binding site, and a terminator. In eukaryotes, control sequences typically include promoters, terminators, and in some cases, enhancers, transactivators, and / or transcription factors.

[0313] The vectors used in this invention are preferably expression vectors. Expression vectors are capable of directing the replication and expression of the nucleic acid molecules of this invention in host cells, and thus provide expression of heavy chain and / or light chain variable domains of antibodies that specifically bind to pepsinogen A, as disclosed herein. In some embodiments, the vector may contain further sequences to ensure expression not only of the heavy chain and light chain variable domains, but also of the remaining heavy chain and light chain constant regions, such that a full-length IgG antibody containing the heavy chain and light chain variable domains of this invention is expressed. Suitable expression vectors have been extensively described in the literature, and those skilled in the art can readily determine expression vectors suitable for a particular cell system using conventional methods. Preferably, the vectors disclosed herein comprise a recombinant polynucleotide (i.e., a nucleic acid sequence encoding a monoclonal antibody according to the invention) and an operatively linked expression control sequence. Vectors provided herein preferably further comprise a promoter. The vectors described herein may also comprise a selection marker gene and a replication source to ensure replication in the host. Furthermore, the vectors provided herein may also comprise a termination signal for transcription. Expression vectors known in the art can drive transient or constitutive expression in host cells.

[0314] The nucleic acid molecules and / or vectors of the present invention can be designed for transfection into prokaryotic or eukaryotic host cells by any means known in the art or described herein. Non-limiting examples of suitable methods include chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-glucan, nuclear transfection), non-chemical methods (electroporation, acoustic transfection, phototransfection, gene electrotransfer, hydrodynamic delivery, or transformation that occurs naturally after contacting cells with the nucleic acid molecules of the present invention), particle-based methods (gene gun, magnetic transfection, puncture infection), phage vector-based methods, and viral methods. For example, expression vectors derived from viruses (such as retroviruses, vaccinia virus, adeno-associated virus, herpesvirus, Semliki forest virus, or bovine papillomavirus) can be used to transfect nucleic acid molecules into target cell populations. Furthermore, baculovirus systems can also be used as vectors in eukaryotic expression systems targeting the nucleic acid molecules of the present invention.

[0315] The term "prokaryote" is intended to include all bacteria that can be transformed, transduced, or transfected with DNA or DNA or RNA molecules to express the proteins of the present invention. Prokaryotic hosts can include Gram-negative and Gram-positive bacteria, such as, for example, *Escherichia coli*, *Serratia typhimurium*, *Serratia marcescens*, *Corynebacterium* (glutamate-containing *Corynebacterium*), *Pseudomonas* (fluorescein-containing *Pseudomonas*), *Lactobacillus*, *Streptomyces*, *Salmonella*, and *Bacillus subtilis*. The term "eukaryote" refers to cells including yeast, higher plants, insects, and mammals. Non-limiting examples of mammalian host cells commonly used in this art include HeLa, HEK293, H9, Per.C6 and Jurkat cells, mouse NIH3T3, NS / 0, SP2 / 0 and C127 cells, COS cells (e.g. COS 1 or COS 7), CV1, quail QC1-3 cells, mouse L cells, mouse sarcoma cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells.

[0316] Therefore, in a twenty-fifth aspect, the present invention relates to a host cell comprising a polynucleotide according to the invention or a vector according to the invention. The host cell may be a prokaryotic cell or a eukaryotic cell. In a preferred embodiment, the host cell is a eukaryotic cell. In a particular embodiment, the cell is an HEK cell. In another particular embodiment, the host cell is a CHO cell.

[0317] When a recombinant expression vector encoding the heavy chain and / or light chain of an antibody according to the invention disclosed herein is introduced into a host cell, the antibody or antibody-antigen binding fragment is generated by culturing the host cell for a period of time sufficient to allow expression of the antibody or antigen binding fragment in the host cell, or preferably, to allow secretion of the antibody or antigen binding fragment into the culture medium in which the host cell is growing. The antibody and / or antigen binding fragment can be recovered from the culture medium using standard protein purification methods. Methods for purifying antibodies are well known in the art. Exemplary purification methods are described in the appended examples.

[0318] Therefore, in its twenty-sixth aspect, the present invention also provides a method for generating antibodies that specifically bind to pepsinogen A as disclosed herein. The method includes culturing the host cells of the present invention under suitable conditions and isolating the resulting antibodies. The isolated antibodies of the present invention can be obtained through purification steps, for example as described in the appended examples.

[0319] The invention further provides, in its twenty-seventh aspect, antibody or antigen-binding fragments that can be obtained by any of the methods disclosed herein.

[0320] The transformed host cells can be grown in a bioreactor and cultured according to techniques known in the art to achieve optimal cell growth. The antibodies and / or antibody-antigen binding fragments of the present invention can then be isolated from the cell fraction or growth medium by any conventional method, such as, but not limited to, affinity chromatography (e.g., using fusion tags, such as Strep-tag II or His6 tags), gel filtration (size exclusion chromatography), anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, high-performance liquid chromatography (HPLC), reversed-phase HPLC, or immunoprecipitation.

[0321] It is understood that variations of the above procedures are all within the scope of protection of this invention. For example, recombinant DNA technology can be used to remove or modify DNA sequences encoding antibodies and / or antibody-antigen binding fragments disclosed herein (e.g., encoding heavy chain and / or light chain variable domains as defined above). For example, recombinant DNA technology can be used to remove portions of the coding sequence that are not essential for maintaining specific and selective binding to the target antigen. Molecules expressed by such truncated DNA molecules are also covered by the antibodies of this invention.

[0322] Antibody derivatives can be produced, for example, by adding exogenous sequences to modify immunogenicity or reduce, enhance, or modify binding, affinity, association rate, dissociation rate, high affinity, specificity, half-life, or any other suitable property.

[0323] Humanized versions of the antibodies disclosed herein are also provided, comprising CDRs containing the heavy and / or light chains as described above. As is well known in the art, “humanization” (to produce a humanized version of the parent antibody) refers to the recombinant engineering of an antibody using a CDR derived from a non-human donor immunoglobulin, with both human frame domains and constant domains. During engineering, frame and / or CDR residues may be altered to maintain binding affinity and activity, such as specificity for pepsinogen A. Methods for humanizing antibodies are well known in the art, for example, as disclosed in: Queen et al., Proc. Natl. Acad Sci USA 86 (1989), 10029-10032; Hodgson et al., Bio / Technology 9 (1991) 421.

[0324] In a twenty-eighth aspect, the present invention provides a composition comprising the antibody of the present invention, the polynucleotide of the present invention, the vector of the present invention, or the host cell of the present invention. In a preferred embodiment, the composition is a diagnostic composition, i.e., a composition for use in diagnostic applications. In a preferred embodiment, the composition is used for an in vitro diagnostic assay for detecting pepsinogen A. In a preferred embodiment, the diagnostic composition may be a reagent for an immunoassay for detecting pepsinogen A. The diagnostic composition is preferably configured to allow detection of pepsinogen A in a sample obtained from a subject. The sample is preferably a blood sample (e.g., whole blood, serum, or plasma).

[0325] In a twenty-ninth aspect, the present invention provides a composition comprising a) An antibody or antigen-binding fragment thereof according to the sixteenth, seventeenth, eighteenth, nineteenth, or twentieth aspect of the invention or according to any embodiment relating to any of these methods, and An antibody or antigen-binding fragment thereof according to the twenty-first or twenty-second aspect of the present invention or any embodiment related to the twenty-first or twenty-second aspect.

[0326] A composition is also provided comprising a polynucleotide, vector, or host cell associated with an antibody of the sixteenth, seventeenth, eighteenth, nineteenth, or twentieth aspect of the invention or any embodiment associated with any of these aspects, and a polynucleotide, vector, or host cell associated with an antibody of the twenty-first or twenty-second aspect of the invention or any embodiment associated with the twenty-first or twenty-second aspect.

[0327] In one embodiment, the composition according to the invention is a diagnostic composition.

[0328] In particular, the present invention provides a composition comprising Antibodies or antigen-binding fragments thereof according to the sixteenth, seventeenth, eighteenth, nineteenth, or twentieth aspects of the invention or according to any embodiment relating to any of these methods; and An antibody or an antigen-binding fragment thereof according to the twenty-first or twenty-second aspect of the present invention or according to any embodiment related to the twenty-first or twenty-second aspect; The composition is a diagnostic composition.

[0329] As shown in Example 6, all antibodies of the present invention are labeled with a single label. Two groups of antibodies were prepared. One group of antibodies was labeled with ruthenium, and the second group of antibodies was labeled with biotin. In Example 7, different combinations of antibodies were tested. A sample containing pepsinogen A was incubated with a ruthenium-labeled monoclonal pepsinogen A-specific IgG antibody and a biotinylated monoclonal pepsinogen A-specific IgG antibody to form a sandwich complex. As shown in Table 1, the inventors unexpectedly identified that the combined antibodies (e.g., the combination of mAb 2 with mAb 8, mAb 24, or mAb 6.7, i.e., antibodies binding to the epitopes bound by the exemplary antibodies) exhibited unexpectedly high signals compared to other combinations (e.g., mAb 6.7, mAb 24, or mAb 7 with mAb 8). It was quite unexpected which antibody combination provided a particularly good signal. Without being bound by theory, it appears that the epitopes bound by the antibodies influence which antibody combinations are advantageous (see Table 2).

[0330] In a thirtieth aspect, the present invention relates to the use of antibodies or antigen-binding fragments thereof according to the invention, and compositions according to the invention, for in vitro immunoassays, particularly for in vitro immunoassays to detect pepsinogen A in samples. Therefore, antibodies of the invention are also provided for use in immunoassays (e.g., heterologous immunoassays) to detect pepsinogen A.

[0331] The sample can be a bodily fluid, such as, but not limited to, blood, cerebrospinal fluid, semen, saliva, or urine. In this embodiment, the sample is a blood sample, such as whole blood, serum, or plasma. In this embodiment, the sample is serum or plasma.

[0332] The method for detection may include: (a) contacting a sample containing pepsinogen A with an antibody or an antigen-binding fragment thereof according to the invention to form a complex containing pepsinogen A and the antibody or an antigen-binding fragment thereof, and (b) detecting the complex formed in step (a) to detect pepsinogen A in the sample.

[0333] In one embodiment, the pepsinogen A in the sample contains a sequence that is at least 95% homologous to the sequence shown in SEQ ID NO: 34 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A or V353L.

[0334] In one specific embodiment, the method is a serum immunoassay, wherein the sample is a body fluid, preferably whole blood, serum or plasma.

[0335] In one specific embodiment, the antibody or its antigen-binding fragment has a detection marker attached thereto.

[0336] The detection marker linked to the antibody of the present invention can be any detection marker known in the art. For example, the detection marker can be selected from enzymes, luminescent markers (preferably fluorescent, luminescent, chemiluminescent, or electrochemiluminescent), or radioactive markers.

[0337] In its thirty-first aspect, the present invention provides an in vitro method for detecting pepsinogen A in a sample, the method comprising the following steps: 1) Contact the sample containing pepsinogen A with a first antibody or its antigen-binding fragment and a second antibody or its antigen-binding fragment, wherein... (a) The first antibody is (i) an antibody or antigen-binding fragment thereof according to the sixteenth, seventeenth, eighteenth, nineteenth, or twentieth aspect of the invention or according to any embodiment relating to any of these methods; or (ii) An antibody or antigen-binding fragment thereof according to the twenty-first or twenty-second aspect of the invention or any embodiment related to the twenty-first or twenty-second aspect; and (b) The second antibody specifically binds to pepsinogen A and does not compete with the first antibody for specific binding to pepsinogen A; and 2) Incubate the mixture obtained in 1) to form a complex comprising pepsinogen A and the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment; and 3) Detect pepsinogen A in the sample by detecting the formation of the complex as defined in 2).

[0338] As shown in Example 6, all antibodies of the present invention are labeled with a single label. Two groups of antibodies were prepared. One group of antibodies was labeled with ruthenium, and the second group of antibodies was labeled with biotin. In Example 7, different combinations of antibodies were tested. A sample containing pepsinogen A was incubated with ruthenium-labeled monoclonal pepsinogen A-specific IgG antibody and biotinylated monoclonal pepsinogen A-specific IgG antibody to form a sandwich complex. As shown in Table 1, the inventors unexpectedly identified that, compared to other combinations (e.g., combinations of mAb 2 with mAb 8, mAb 24, or mAb 6.7), the combined antibodies (e.g., antibodies binding to the epitopes of the exemplary antibodies) exhibited unexpectedly high signals. Which antibody combinations provided particularly good signals was quite surprising. Without being bound by theory, the epitopes bound by the antibodies appear to influence which antibody combinations are advantageous (see Table 2). These advantageous antibody combinations can be used in in vitro methods for detecting pepsinogen A in samples.

[0339] In one embodiment of the thirty-first aspect of the present invention, (c) one of the first antibody and the second antibody comprises a detection marker.

[0340] In a specific embodiment, a first antibody or its antigen-binding fragment that does not contain a detection marker, or a second antibody or its antigen-binding fragment that contains a capture marker, may be used.

[0341] As used herein, "capture tag" refers to a tag that can immobilize a capture agent (e.g., an antibody having a capture tag attached thereto according to the invention) onto a surface (e.g., magnetic particles such as microbeads). Non-limiting examples are members of binding pairs. A non-limiting example of a capture tag is biotin or a derivative thereof, which can interact with streptavidin or a derivative thereof. Various capture tags are well known in the art.

[0342] In one specific embodiment, the method further includes: adding particles capable of binding with the capture marker to the mixture of (1), and 2.1) separating the particles from the mixture before detecting the signal provided by the detection marker.

[0343] In one embodiment of the thirty-first aspect, the present invention When the first antibody or its antigen-binding fragment is selected from (i), then the second antibody is selected from (ii); or When the first antibody or its antigen-binding fragment is selected from (ii), the second antibody is selected from (i).

[0344] In one embodiment, the pepsinogen A in the sample contains a sequence that is at least 95% homologous to the sequence shown in SEQ ID NO: 34 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A or V353L.

[0345] In a specific embodiment, the method is a serum immunoassay, wherein the sample is a body fluid, preferably whole blood, serum or plasma.

[0346] The detection marker linked to the antibody of the present invention can be any detection marker known in the art. For example, the detection marker can be selected from enzymes, luminescent markers (preferably fluorescent, luminescent, chemiluminescent, or electrochemiluminescent), or radioactive markers.

[0347] In a further embodiment, the limit of quantitation (LoQ) of the method of the present invention is 1.79 ng / mL at 20% CV.

[0348] LoQ is defined as the minimum amount of analyte in a sample that can be accurately quantified with an intermediate precision CV of ≤ 20%.

[0349] In a further embodiment, the detection of pepsinogen A in the sample is achieved in 18 minutes or less.

[0350] In a thirty-second aspect, this document provides a kit comprising the antibody of the present invention or a composition comprising the antibody. In examples, the kit is a kit for the in vitro detection and / or quantification of pepsinogen A. In examples, the kit is an immunoassay kit. In examples, the kit is a kit for heterologous immunoassay.

[0351] In a thirty-third aspect, this document provides a kit comprising a composition according to the twenty-ninth aspect of the invention, or a first antibody or an antigen-binding fragment thereof, and a second antibody or an antigen-binding fragment thereof, wherein one of the first antibody or the second antibody or the antigen-binding fragment thereof is an antibody or an antigen-binding fragment thereof according to the sixteenth, seventeenth, eighteenth, nineteenth, or twentieth aspect of the invention or any embodiment related to any of these methods, and the other of the first antibody or the second antibody or the antigen-binding fragment thereof is an antibody or an antigen-binding fragment thereof according to the twenty-first or twenty-second aspect of the invention or any embodiment related to the twenty-first or twenty-second aspect.

[0352] As shown in Example 6, all antibodies of the present invention are labeled with a single label. Two groups of antibodies were prepared. One group of antibodies was labeled with ruthenium, and the second group of antibodies was labeled with biotin. In Example 7, different combinations of antibodies were tested. A sample containing pepsinogen A was incubated with a ruthenium-labeled monoclonal pepsinogen A-specific IgG antibody and a biotinylated monoclonal pepsinogen A-specific IgG antibody to form a sandwich complex. As shown in Table 1, the inventors unexpectedly identified that the combined antibodies (e.g., the combination of mAb 2 with mAb 8, mAb 24, or mAb 6.7, i.e., antibodies binding to the epitopes bound by the exemplary antibodies) exhibited unexpectedly high signals compared to other combinations (e.g., mAb 6.7, mAb 24, or mAb 7 with mAb 8). It was quite unexpected which antibody combination provided a particularly good signal. Without being bound by theory, it appears that the epitopes bound by the antibodies influence which antibody combinations are advantageous (see Table 2).

[0353] In one embodiment of the thirty-third aspect of the present invention, one of the first antibody and the second antibody comprises a detection marker.

[0354] In a specific embodiment, a first antibody or its antigen-binding fragment that does not contain a detection marker, or a second antibody or its antigen-binding fragment that contains a capture marker, may be used.

[0355] As used herein, "capture tag" refers to a tag that can immobilize a capture agent (e.g., an antibody having a capture tag attached thereto according to the invention) onto a surface (e.g., magnetic particles such as microbeads). Non-limiting examples are members of binding pairs. A non-limiting example of a capture tag is biotin or a derivative thereof, which can interact with streptavidin or a derivative thereof. Various capture tags are well known in the art.

[0356] In one specific embodiment, the kit is an immunoassay kit. In another embodiment, the kit is a kit for heterologous immunoassay.

[0357] In one embodiment, the kit is used to detect and / or quantify pepsinogen A.

[0358] In a thirty-fourth aspect, the present invention provides a kit comprising a first antibody or an antigen-binding fragment thereof, and a second antibody or an antigen-binding fragment thereof. a) Wherein the first antibody or its antigen-binding fragment is It specifically binds to pepsinogen A and competes with antibodies or their antigen-binding fragments that bind to pepsinogen A. (i) The heavy chain variable structural domain (VH) shown in SEQ ID NO: 1; and (ii) The light chain variable domain (VL) shown in SEQ ID NO: 2; and (b) wherein the second antibody or its antigen-binding fragment is An antibody or its antigen-binding fragment that specifically binds to pepsinogen A and competes with pepsinogen A for binding, wherein the antibody or its antigen-binding fragment consists of the following components: (iii) The heavy chain variable structural domain (VH) shown in SEQ ID NO: 25; and (iv) The light chain variable domain (VL) shown in SEQ ID NO: 26.

[0359] In one embodiment, the second antibody or its antigen-binding fragment does not compete with the first antibody or its antigen-binding fragment for binding to pepsinogen A.

[0360] As shown in Example 6, all antibodies of the present invention are labeled with a single label. Two groups of antibodies were prepared. One group of antibodies was labeled with ruthenium, and the second group of antibodies was labeled with biotin. In Example 7, different combinations of antibodies were tested. A sample containing pepsinogen A was incubated with ruthenium-labeled monoclonal pepsinogen A-specific IgG antibody and biotinylated monoclonal pepsinogen A-specific IgG antibody to form a sandwich complex. As shown in Table 1, the inventors unexpectedly identified that, compared to other combinations (e.g., combinations of mAb 2 with mAb 8, mAb 24, or mAb 6.7), the combined antibodies (e.g., antibodies binding to the epitopes of the exemplary antibodies) exhibited unexpectedly high signals. Which antibody combinations provided particularly good signals was quite surprising. Without being bound by theory, the epitopes bound by the antibodies appear to influence which antibody combinations are advantageous (see Table 2). These advantageous antibody combinations can be used in in vitro methods for detecting pepsinogen A in samples.

[0361] In one embodiment, the kit according to the invention comprises a first antibody or an antigen-binding fragment thereof and a second antibody or an antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment thereof comprises... (a) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified with one amino acid substitution, and The second antibody or its antigen-binding fragment contains (d) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

[0362] In another embodiment, the kit according to the invention comprises a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment thereof, the first antibody or antigen-binding fragment thereof comprising... (b) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16, or a variant thereof modified with one amino acid substitution; and The second antibody or its antigen-binding fragment contains (d) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

[0363] In another embodiment, the kit according to the invention comprises a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment thereof, the first antibody or antigen-binding fragment thereof comprising... (c) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24, or a variant thereof modified with one amino acid substitution; and The second antibody or its antigen-binding fragment contains (d) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

[0364] In one embodiment, the kit according to the invention comprises a first antibody or an antigen-binding fragment thereof and a second antibody or an antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment thereof comprises... (a) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified with one amino acid substitution, or The second antibody or its antigen-binding fragment contains (d) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

[0365] In another embodiment, the kit according to the invention comprises a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment thereof, the first antibody or antigen-binding fragment thereof comprising... (b) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16, or a variant thereof modified with one amino acid substitution; or The second antibody or its antigen-binding fragment contains (d) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

[0366] In another embodiment, the kit according to the invention comprises a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment thereof, the first antibody or antigen-binding fragment thereof comprising... (c) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24, or a variant thereof modified with one amino acid substitution; or The second antibody or its antigen-binding fragment contains (d) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

[0367] In a thirty-fifth aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A and competes with an antibody consisting of: (i) the heavy chain variable domain (VH) shown in SEQ ID NO: 1; and (ii) the light chain variable domain (VL) shown in SEQ ID NO: 2.

[0368] Pepsinogen (PG)A (PG I) is secreted entirely by the fundic glands (Samloff 1971). Previous studies have shown that serum PGI levels and / or the PGI / II ratio reflect the morphology (Samloff et al. 1982; Borch et al. 1989) and functional status of the gastric mucosa (Samloff et al. 1975; Nakanome et al. 1983; Miki et al. 1987; Feldman et al. 1988). Numerous studies have reported significant correlations between serum PG values ​​and gastric acid secretion levels. Changes in gastric secretion levels are associated with the pathogenesis of various types of diseases originating in the upper gastrointestinal tract (GI). Classically, individuals with hyperacidity, particularly those infected with Helicobacter pylori, are more prone to duodenal ulcers, while individuals with hypoacidity face a risk of developing gastric cancer through the development of pre-malignant conditions (gastric atrophy) (El-Omar et al. 1995, 1997). Therefore, measuring gastric acid secretion levels has certain clinical significance in estimating the risk of various diseases of the upper GI. However, given the significant discomfort and time-consuming nature of this procedure, direct measurement of gastric acid secretion levels has become less common. Therefore, antibodies that specifically bind to pepsinogen A are needed, as pepsinogen A is a useful indicator for measuring gastric acid secretion levels and estimating the risk of various upper GI tract diseases.

[0369] The inventors of this invention have identified high-affinity antibodies that specifically bind to pepsinogen A and compete with antibodies consisting of: (i) the heavy chain variable domain (VH) shown in SEQ ID NO: 1; and (ii) the light chain variable domain (VL) shown in SEQ ID NO: 2.

[0370] In embodiments of the present invention, the antibody or antigen-binding fragment thereof according to the present invention comprises: (a) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4 or a variant thereof modified by one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5 or a variant thereof modified by one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6 or a variant thereof modified by one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7 or a variant thereof modified by one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8 or a variant thereof modified by one amino acid substitution.

[0371] In embodiments of the present invention, the antibody or antigen-binding fragment thereof according to the present invention comprises: b) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11 or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12 or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13 or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14 or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15 or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16 or a variant thereof modified with one amino acid substitution.

[0372] In embodiments of the present invention, the antibody or antigen-binding fragment thereof according to the present invention comprises: (c) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19 or a variant thereof modified by one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20 or a variant thereof modified by one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21 or a variant thereof modified by one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22 or a variant thereof modified by one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23 or a variant thereof modified by one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24 or a variant thereof modified by one amino acid substitution.

[0373] The inventors were surprised to identify a family of antibodies that competitively and specifically bind to pepsinogen A. As shown in the accompanying examples (e.g., Example 8, Table 1), antibodies from this family (e.g., mAb 6.7, mAb 24, mAb 7) all competitively bind to pepsinogen A.

[0374] In a thirty-sixth aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A and competes with an antibody composed of the following for binding to pepsinogen A. (i) The heavy chain variable structural domain (VH) shown in SEQ ID NO: 25; and (ii) The light chain variable domain (VL) shown in SEQ ID NO: 26.

[0375] In one embodiment, the antibody or antigen-binding fragment according to the invention comprises: VH, which comprises: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27 or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28 or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29 or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

[0376] In a thirty-eighth aspect, the present invention relates to a polynucleotide or polynucleotide group encoding a polynucleotide sequence. (i) The heavy chain variable domain of the antibody or its antigen-binding fragment according to the present invention, and (ii) The light chain variable domain of the antibody or its antigen-binding fragment according to the invention.

[0377] This article further provides a vector comprising the polynucleotide of the present invention.

[0378] In a further aspect, the present invention relates to a host cell comprising a polynucleotide according to the invention or a vector according to the invention, and expressing an antibody or an antigen-binding fragment according to the invention.

[0379] The host cell can be a prokaryotic cell or a eukaryotic cell. In a preferred embodiment, the host cell is a eukaryotic cell. In a particular embodiment, the cell is an HEK cell. In another particular embodiment, the host cell is a CHO cell.

[0380] When a recombinant expression vector encoding the heavy chain and / or light chain of an antibody according to the invention disclosed herein is introduced into a host cell, the antibody or antibody-antigen binding fragment is generated by culturing the host cell for a period of time sufficient to allow expression of the antibody or antigen binding fragment in the host cell, or preferably, to allow secretion of the antibody or antigen binding fragment into the culture medium in which the host cell is growing. The antibody and / or antigen binding fragment can be recovered from the culture medium using standard protein purification methods. Methods for purifying antibodies are well known in the art. Exemplary purification methods are described in the appended examples.

[0381] Therefore, in a further aspect, the present invention provides a method for generating antibodies that specifically bind to pepsinogen A as disclosed herein. The method includes culturing the host cells of the present invention under suitable conditions and isolating the resulting antibodies. The isolated antibodies of the present invention can be obtained through purification steps, such as those described in the appended examples.

[0382] In one aspect, the present invention further provides antibody or antigen-binding fragments that can be obtained by any of the methods disclosed herein.

[0383] In a further aspect, the present invention provides a composition comprising the antibody of the present invention, the polynucleotide of the present invention, the vector of the present invention, or the host cell of the present invention. In a preferred embodiment, the composition is a diagnostic composition, i.e., a composition for use in diagnostic applications. In a preferred embodiment, the composition is used for an in vitro diagnostic assay for detecting pepsinogen A. In a preferred embodiment, the diagnostic composition may be a reagent for an immunoassay for detecting pepsinogen A. The diagnostic composition is preferably configured to allow detection of pepsinogen A in a sample obtained from a subject. The sample is preferably a blood sample (e.g., whole blood, serum, or plasma).

[0384] In a further aspect, the present invention provides a composition comprising a) An antibody or antigen-binding fragment thereof according to the thirty-fifth aspect of the invention or any embodiment related to the thirty-fifth aspect of the invention, and An antibody or an antigen-binding fragment thereof according to the thirty-sixth aspect of the invention or any embodiment related to the thirty-sixth aspect of the invention.

[0385] A composition is also provided comprising a polynucleotide, a vector, or a host cell associated with an antibody of the thirty-fifth aspect of the present invention or an embodiment thereof, and a polynucleotide, a vector, or a host cell associated with an antibody of the thirty-sixth aspect of the present invention or an embodiment thereof.

[0386] In a further aspect, the present invention relates to the use of antibodies or antigen-binding fragments thereof according to the invention, and compositions according to the invention, for in vitro immunoassays, particularly for in vitro immunoassays to detect pepsinogen A in samples. Therefore, antibodies of the invention are also provided for use in immunoassays (e.g., heterologous immunoassays) to detect pepsinogen A.

[0387] In a further aspect, the present invention provides an in vitro method for detecting pepsinogen A in a sample using the antibody of the present invention. The antibody may be part of a composition, particularly the diagnostic composition of the present invention.

[0388] The sample can be a bodily fluid, such as a blood sample, cerebrospinal fluid, semen, saliva, or urine. In this embodiment, the sample is a blood sample, such as whole blood, serum, or plasma. In this embodiment, the sample is serum or plasma.

[0389] The method for detection may include: (a) contacting a sample containing pepsinogen A with an antibody or an antigen-binding fragment thereof according to the invention to form a complex containing pepsinogen A and the antibody or an antigen-binding fragment thereof, and (b) detecting the complex formed in step (a) to detect pepsinogen A in the sample.

[0390] In a thirty-ninth aspect, the present invention provides an in vitro method for detecting pepsinogen A in a sample, the method comprising the following steps: 1) Contact the sample containing pepsinogen A with a first antibody or its antigen-binding fragment and a second antibody or its antigen-binding fragment, wherein... (a) The first antibody is (i) An antibody or an antigen-binding fragment thereof according to the thirty-fifth aspect or an embodiment of the present invention; or (ii) An antibody or an antigen-binding fragment thereof according to the thirty-sixth aspect or an embodiment of the present invention; and (b) The second antibody specifically binds to pepsinogen A and does not compete with the first antibody for specific binding to pepsinogen A; 2) Incubate the mixture obtained in 1) to form a complex comprising pepsinogen A and a first antibody or an antigen-binding fragment thereof, and a second antibody or an antigen-binding fragment thereof, and 3) Detect pepsinogen A in the sample by detecting the formation of the complex defined in 2).

[0391] In one embodiment, the present invention relates to a kit according to the thirty-fourth aspect, wherein a first antibody or antigen-binding fragment comprises... (a) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified with one amino acid substitution; or (b) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16, or a variant thereof modified with one amino acid substitution; or (c) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22 or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23 or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24 or a variant thereof modified with one amino acid substitution.

[0392] In one embodiment, the present invention provides a kit according to a thirty-fourth aspect of the invention, wherein the second antibody or its antigen-binding fragment comprises... VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

[0393] In one embodiment, the present invention provides a kit according to a thirty-fourth aspect of the invention, wherein the first antibody or antigen-binding fragment comprises (a) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified with one amino acid substitution; or (b) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16, or a variant thereof modified with one amino acid substitution; or (c) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24, or a variant thereof modified with one amino acid substitution; and / or The second antibody or its antigen-binding fragment contains (d) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

[0394] In a further aspect, the present invention provides an in vitro method for detecting pepsinogen A in a sample, the method comprising the following steps: 1) Contact the sample containing pepsinogen A with a first antibody or its antigen-binding fragment and a second antibody or its antigen-binding fragment, wherein... (a) The first antibody is (i) An antibody or an antigen-binding fragment thereof according to the first aspect or an embodiment of the first aspect of the invention; or (ii) An antibody or an antigen-binding fragment thereof according to the second aspect or an embodiment of the present invention; and (b) The second antibody specifically binds to pepsinogen A and does not compete with the first antibody for specific binding to pepsinogen A; 2) Incubate the mixture obtained in 1) to form a complex comprising pepsinogen A and a first antibody or an antigen-binding fragment thereof, and a second antibody or an antigen-binding fragment thereof, and 3) Detect pepsinogen A in the sample by detecting the formation of the complex defined in 2). (c) Optionally quantify the amount of pepsinogen A in the sample.

[0395] In a further aspect, the present invention provides an in vitro method for detecting pepsinogen A in a sample, the method comprising the following steps: 1) Contact the sample containing pepsinogen A with a first antibody or its antigen-binding fragment and a second antibody or its antigen-binding fragment, wherein... (a) The first antibody is (i) an antibody or antigen-binding fragment thereof according to the sixteenth, seventeenth, eighteenth, nineteenth, or twentieth aspect of the invention or according to any embodiment relating to any of these methods; or (ii) An antibody or an antigen-binding fragment thereof according to the nineteenth aspect or an embodiment of the present invention; and (b) The second antibody specifically binds to pepsinogen A and does not compete with the first antibody for specific binding to pepsinogen A; and 2) Incubate the mixture obtained in 1) to form a complex comprising pepsinogen A and the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment; and 3) Detect pepsinogen A in the sample by detecting the formation of the complex defined in 2). (c) Optionally quantify the amount of pepsinogen A in the sample.

[0396] In a further aspect, the present invention provides an in vitro method for detecting and / or quantifying pepsinogen A in a sample, the method comprising: (a) Contacting a sample containing pepsinogen A with an antibody or antigen-binding fragment thereof according to the invention to form a complex comprising pepsinogen A and said antibody or antigen-binding fragment thereof, and (b) Detect the complex formed in step (a) to detect pepsinogen A in the sample, and (c) Optionally quantify the amount of pepsinogen A in the sample.

[0397] This invention also relates to the following items: 1. An antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof specifically binding to pepsinogen A and competing with an antibody composed of the following for binding to pepsinogen A. (i) The heavy chain variable structural domain (VH) shown in SEQ ID NO: 1; and (ii) The light chain variable domain (VL) shown in SEQ ID NO: 2, preferably wherein the antibody or its antigen-binding fragment binds to the same epitope as the antibody composed of the heavy chain variable domain (VH) shown in SEQ ID NO: 1 and the light chain variable domain (VL) shown in SEQ ID NO: 2.

[0398] 2. The antibody or its antigen-binding fragment as described in Project 1, comprising... (a) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified with one amino acid substitution; or (b) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16, or a variant thereof modified with one amino acid substitution; or (c) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22 or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23 or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24 or a variant thereof modified with one amino acid substitution.

[0399] 3. The antibody or its antigen-binding fragment as described in Project 2, wherein all amino acid substitutions are conserved amino acid substitutions.

[0400] 4. The antibody or its antigen-binding fragment according to Project 3, wherein one of the conserved amino acid substitutions is an amino acid replaced by another amino acid selected from the same group, wherein the amino acid group is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0401] 5. The antibody or antigen-binding fragment thereof according to item 3 or 4, wherein one of the conserved amino acid substitutions is selected from the following highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0402] 6. Based on any one of items 1 to 4, the antibody or its antigen-binding fragment contains... (a) VH, comprising: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 3; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 4; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 5; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8; or (b) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16; or (c) VH, comprising: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 19; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 20; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 21; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24.

[0403] 7. An antibody or antigen-binding fragment thereof according to any one of items 1 to 6, wherein the antibody or antigen-binding fragment thereof binds to an epitope of pepsinogen A containing the amino acid sequence shown in SEQ ID NO: 33.

[0404] 8. An antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the antibody comprises a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), The VH and VL contain the sequence according to (a), and the FW contains the following amino acid sequence or a variant thereof that is at least 85% homologous to it: The amino acid sequence of FW-L1, SEQ ID NO: 39; The amino acid sequence of FW-L2, SEQ ID NO: 40; The amino acid sequence of FW-L3, SEQ ID NO: 41; The amino acid sequence of FW-L4, SEQ ID NO: 42; The amino acid sequence of FW-H1, SEQ ID NO: 35; The amino acid sequence of FW-H2, SEQ ID NO: 36; The amino acid sequence of FW-H3, SEQ ID NO: 37; The amino acid sequence of FW-H4, SEQ ID NO: 38; or The VH and VL contain the sequence according to (b), and the FW contains the following amino acid sequence or a variant thereof that is at least 85% homologous to it: The amino acid sequence of FW-L1, SEQ ID NO: 47; The amino acid sequence of FW-L2, SEQ ID NO: 48; The amino acid sequence of FW-L3, SEQ ID NO: 49; The amino acid sequence of FW-L4, SEQ ID NO: 50; The amino acid sequence of FW-H1, SEQ ID NO: 43; The amino acid sequence of FW-H2, SEQ ID NO: 44; The amino acid sequence of FW-H3, SEQ ID NO: 45; The amino acid sequence of FW-H4, SEQ ID NO: 46; or The VH and VL contain the sequence as described in (c), and the FW contains the following amino acid sequence or a variant thereof that is at least 85% homologous to it: The amino acid sequence of FW-L1, SEQ ID NO: 55; The amino acid sequence of FW-L2, SEQ ID NO: 56; The amino acid sequence of FW-L3, SEQ ID NO: 57; The amino acid sequence of FW-L4, SEQ ID NO: 58; The amino acid sequence of FW-H1, SEQ ID NO: 51; The amino acid sequence of FW-H2, SEQ ID NO: 52; The amino acid sequence of FW-H3, SEQ ID NO: 53; The amino acid sequence of FW-H4, SEQ ID NO: 54.

[0405] 9. An antibody or antigen-binding fragment thereof according to any one of items 1 to 8, wherein the antibody comprises a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), The VH and VL contain the sequence according to (a), and the FW contains the following amino acid sequence or a variant thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 39; The amino acid sequence of FW-L2, SEQ ID NO: 40; The amino acid sequence of FW-L3, SEQ ID NO: 41; The amino acid sequence of FW-L4, SEQ ID NO: 42; The amino acid sequence of FW-H1, SEQ ID NO: 35; The amino acid sequence of FW-H2, SEQ ID NO: 36; The amino acid sequence of FW-H3, SEQ ID NO: 37; The amino acid sequence of FW-H4, SEQ ID NO: 38; or The VH and VL contain the sequence according to (b), and the FW contains the following amino acid sequence or a variant thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 47; The amino acid sequence of FW-L2, SEQ ID NO: 48; The amino acid sequence of FW-L3, SEQ ID NO: 49; The amino acid sequence of FW-L4, SEQ ID NO: 50; The amino acid sequence of FW-H1, SEQ ID NO: 43; The amino acid sequence of FW-H2, SEQ ID NO: 44; The amino acid sequence of FW-H3, SEQ ID NO: 45; The amino acid sequence of FW-H4, SEQ ID NO: 46; or The VH and VL contain the sequence according to (c), and the FW contains the following amino acid sequence or a variant thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 55; The amino acid sequence of FW-L2, SEQ ID NO: 56; The amino acid sequence of FW-L3, SEQ ID NO: 57; The amino acid sequence of FW-L4, SEQ ID NO: 58; The amino acid sequence of FW-H1, SEQ ID NO: 51; The amino acid sequence of FW-H2, SEQ ID NO: 52; The amino acid sequence of FW-H3, SEQ ID NO: 53; The amino acid sequence of FW-H4, SEQ ID NO: 54.

[0406] 10. The antibody or its antigen-binding fragment as described in Item 9, wherein all amino acid substitutions are conserved amino acid substitutions.

[0407] 11. The antibody or antigen-binding fragment thereof according to Item 10, wherein one of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein the amino acid group is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0408] 12. The antibody or antigen-binding fragment thereof according to item 10 or 11, wherein one of the conserved amino acid substitutions is selected from highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0409] 13. Based on any one of items 1 to 12, the antibody or its antigen-binding fragment contains... (i) VH, which contains the sequence shown in SEQ ID NO: 1; and / or (ii) VL, which contains the sequence shown in SEQ ID NO: 2.

[0410] 14. Based on any of the antibodies or antigen-binding fragments from items 1 to 12, It contains (i) VH, which contains the sequence shown in SEQ ID NO: 9; and / or (ii) VL, which contains the sequence shown in SEQ ID NO: 10.

[0411] 15. Based on any of the antibodies or antigen-binding fragments from items 1 to 12, It contains (i) VH, which contains the sequence shown in SEQ ID NO: 17; and / or (ii) VL, which contains the sequence shown in SEQ ID NO: 18.

[0412] 16. An antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof specifically binding to pepsinogen A and competing with an antibody composed of the following for binding to pepsinogen A. (i) The heavy chain variable structural domain (VH) shown in SEQ ID NO: 25; and (ii) The light chain variable domain (VL) shown in SEQ ID NO: 26, preferably wherein the antibody or its antigen-binding fragment binds to the same epitope as the antibody composed of the heavy chain variable domain (VH) shown in SEQ ID NO: 25 and the light chain variable domain (VL) shown in SEQ ID NO: 26.

[0413] 17. The antibody or antigen-binding fragment thereof as described in Item 16, comprising... VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

[0414] 18. The antibody or its antigen-binding fragment as described in Item 17, wherein all amino acid substitutions are conserved amino acid substitutions.

[0415] 19. The antibody or antigen-binding fragment thereof according to item 17 or 18, wherein one of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein the amino acid group is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0416] 20. An antibody or antigen-binding fragment thereof according to any one of items 18 or 19, wherein each of the conserved amino acid substitutions is a highly conserved amino acid substitution selected from the following: a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0417] 21. Based on any one of items 16 to 20, the antibody or its antigen-binding fragment contains... VH, comprising: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 27; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 28; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 29; and VL, comprising: CDR-L1 with the amino acid sequence of SEQ ID NO: 30; CDR-L2 with the amino acid sequence of SEQ ID NO: 31; and CDR-L3 with the amino acid sequence of SEQ ID NO: 32.

[0418] 22. An antibody or antigen-binding fragment thereof according to any one of items 16 to 21, wherein the antibody comprises a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 63; The amino acid sequence of FW-L2, SEQ ID NO: 64; The amino acid sequence of FW-L3, SEQ ID NO: 65; The amino acid sequence of FW-L4, SEQ ID NO: 66; The amino acid sequence of FW-H1, SEQ ID NO: 59; The amino acid sequence of FW-H2, SEQ ID NO: 60; The amino acid sequence of FW-H3, SEQ ID NO: 61; The amino acid sequence of FW-H4, SEQ ID NO: 62.

[0419] 23. An antibody or antigen-binding fragment thereof according to any one of items 16 to 22, wherein the antibody comprises a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequence or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 63; The amino acid sequence of FW-L2, SEQ ID NO: 64; The amino acid sequence of FW-L3, SEQ ID NO: 65; The amino acid sequence of FW-L4, SEQ ID NO: 66; The amino acid sequence of FW-H1, SEQ ID NO: 59; The amino acid sequence of FW-H2, SEQ ID NO: 60; The amino acid sequence of FW-H3, SEQ ID NO: 61; The amino acid sequence of FW-H4, SEQ ID NO: 62.

[0420] 24. The antibody or its antigen-binding fragment as described in Item 23, wherein all amino acid substitutions are conserved amino acid substitutions.

[0421] 25. The antibody or antigen-binding fragment thereof according to item 24, wherein one of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein the amino acid group is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0422] 26. The antibody or antigen-binding fragment thereof according to item 24 or 25, wherein one of the conserved amino acid substitutions is selected from highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0423] 27. Based on any one of items 16 to 26, the antibody or its antigen-binding fragment contains... (i) VH, which contains the sequence shown in SEQ ID NO: 25; and / or (ii) VL, which contains the sequence shown in SEQ ID NO: 26.

[0424] 28. An antibody or antigen-binding fragment thereof according to any one of items 17 to 27, wherein the antibody or antigen-binding fragment thereof is expressed at a Kc of 0.6 nM or less. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0425] 29. An antibody or antigen-binding fragment thereof according to any one of items 17 to 28, wherein the antibody or antigen-binding fragment thereof is bound to pepsinogen A consisting of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L at t / 2-diss for 21 minutes or longer at 37°C.

[0426] 30. The antibody or antigen-binding fragment thereof according to any one of items 17 to 29, wherein the association rate k of pepsinogen A composed of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L is... a For at least 8.7x10 5 M -1 s -1 .

[0427] 31. An antibody or an antigen-binding fragment thereof according to any one of items 1 to 30, wherein the antibody is a monoclonal antibody.

[0428] 32. The antibody or antigen-binding fragment thereof according to any one of items 1 to 31, wherein the pepsinogen A comprises SEQ ID NO: 34 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L, preferably wherein the pepsinogen A is composed of SEQ ID NO: 34 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L.

[0429] 33. An antibody or antigen-binding fragment thereof according to any one of items 1 to 27 and 31 to 32, wherein the antibody or antigen-binding fragment thereof is expressed in K+ of 2 nM or less, or 1.5 nM or less, or 1 nM or less. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0430] 34. An antibody or antigen-binding fragment thereof according to any one of items 1 to 27 and 31 to 33, wherein the antibody or antigen-binding fragment thereof binds to the pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A or V353L at 37°C for 20 minutes or longer t / 2-diss.

[0431] 35. An antibody or antigen-binding fragment thereof according to any one of items 1 to 27 and 31 to 34, wherein the association rate k of pepsinogen A composed of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L is specified.a For at least 10 5 M -1 s -1 Preferably at least 4.9x10 5 M -1 s -1 .

[0432] 36. The antibody or antigen-binding fragment thereof according to any one of items 28, 30, and 33 to 35, wherein the K D and / or the k a It was determined by surface plasmon resonance spectroscopy.

[0433] 37. The antibody or antigen-binding fragment thereof as described in Item 36, wherein the surface plasmon resonance spectroscopy comprises attaching or capturing the antibody or antigen-binding fragment thereof onto a CM5 sensor chip and injecting pepsinogen A consisting of SEQ ID NO:68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A or V353L as an analyte, wherein the determination is performed using HBS-ET buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (w / v) Tween 20®) at a temperature of 37°C.

[0434] 38. An antibody or an antigen-binding fragment thereof according to any one of items 1 to 37, wherein the antibody does not specifically bind to pepsinogen C.

[0435] 39. An antibody or antigen-binding fragment thereof according to any one of items 36 to 38, wherein the surface plasmon resonance spectrum is performed using a Biacore 8k or B4000 instrument.

[0436] 40. An antibody or antigen-binding fragment thereof according to any one of items 36 to 39, wherein the K D and / or the k a This determination involves fitting surface plasmon resonance data using the Langmuir fitting model.

[0437] 41. A polynucleotide or polynucleotide sequence encoding (i) the heavy chain variable domain of the antibody or its antigen-binding fragment according to any one of items 1 to 40, and / or (ii) The light chain variable domain of any antibody or antigen-binding fragment thereof according to any one of items 1 to 40.

[0438] 42. A vector comprising a polynucleotide or polynucleotide group as described in item 41.

[0439] 43. A host cell comprising a polynucleotide or polynucleotide group according to item 41 or a vector according to item 42, and expressing an antibody or an antigen-binding fragment thereof according to any one of items 1 to 40.

[0440] 44. The host cell as described in item 43, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

[0441] 45. The host cell according to item 44, wherein the host cell is a eukaryotic cell, wherein the eukaryotic cell is a HEK293 cell or a CHO cell.

[0442] 46. ​​A method for producing an antibody or an antigen-binding fragment thereof according to any one of items 1 to 40, comprising culturing a host cell according to any one of items 43 to 45 and isolating the antibody or the antigen-binding fragment thereof.

[0443] 47. An antibody or antigen-binding fragment thereof according to any one of items 1 to 40, which may be obtained by the method according to item 46.

[0444] 48. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of items 1 to 40, a polynucleotide or polynucleotide group according to item 41, a vector according to item 42, or a host cell according to any one of items 43 to 45.

[0445] 49. A composition comprising a) An antibody or antigen-binding fragment thereof according to any one of items 1 to 16 or according to any one of items 31 to 40 or 47 belonging to any one of items 1 to 16; and The antibody or antigen-binding fragment thereof is based on any one of items 17 to 30 or any one of items 31 to 40 or 47 which are subordinate to any one of items 17 to 30; b) A polynucleotide or polynucleotide group according to any one of items 41 belonging to any one of items 1 to 16 or according to any one of items 31 to 40 or 47 belonging to any one of items 1 to 16; and According to item 38 belonging to any one of items 17 to 30, or according to any one of items 31 to 40 or 47 belonging to any one of items 17 to 30; c) A carrier according to item 42 belonging to any one of items 1 to 16, or according to any one of items 31 to 40 or 47 belonging to any one of items 1 to 16; and The carrier according to item 42, which is subordinate to any one of items 17 to 30, or according to any one of items 31 to 40 or 47, which are subordinate to any one of items 17 to 30; or d) The host cell according to any one of items 43 to 45 belonging to any one of items 1 to 16, or according to any one of items 31 to 40 or 47 belonging to any one of items 1 to 16; and The host cell is any one of items 43 to 45 belonging to any one of items 17 to 30 or any one of items 31 to 40 or 47 belonging to any one of items 17 to 30.

[0446] 50. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of items 1 to 40 or 47, wherein the composition is a diagnostic composition.

[0447] 51. A composition comprising An antibody or antigen-binding fragment thereof according to any one of items 1 to 16 or according to any one of items 31 to 40 or 47 belonging to any one of items 1 to 16; and The antibody or antigen-binding fragment thereof is based on any one of items 17 to 30 or any one of items 31 to 40 or 47 which are subordinate to any one of items 17 to 30; The composition is a diagnostic composition.

[0448] 52. Use of the antibody or antigen-binding fragment thereof according to any one of items 1 to 40 or 47, or the composition according to item 50 or 51, for use in an in vitro immunoassay, particularly for an in vitro immunoassay to detect pepsinogen A in a sample.

[0449] 53. The use of the antibody or its antigen-binding fragment as described in item 52, wherein the immunoassay is a heterologous immunoassay.

[0450] 54. The antibody or antigen-binding fragment thereof as described in item 52 or 53, wherein the sample used for the immunoassay is a sample prepared from blood, plasma or serum.

[0451] 55. An in vitro method for detecting pepsinogen A in a sample, the method comprising: (a) Contacting a sample containing pepsinogen A with an antibody or an antigen-binding fragment thereof according to any one of items 1 to 40 or 47 to form a complex comprising pepsinogen A and said antibody or an antigen-binding fragment thereof, and (b) Detect the complex formed in step (a) to detect pepsinogen A in the sample.

[0452] 56. The method according to item 55, wherein the method is a serum immunoassay, wherein the sample is a body fluid, preferably whole blood, serum or plasma.

[0453] 57. The method according to item 55 or 56, wherein the antibody or its antigen-binding fragment has a detection marker attached thereto.

[0454] 58. The method according to item 57, wherein the detection label is selected from enzymes, luminescent labels (preferably fluorescent, luminescent, chemiluminescent, electrochemiluminescent) or radioactive.

[0455] 59. An in vitro method for detecting pepsinogen A in a sample, the method comprising the following steps: 1) Contact the sample containing pepsinogen A with a first antibody or its antigen-binding fragment and a second antibody or its antigen-binding fragment, wherein... (a) The first antibody is (i) an antibody or antigen-binding fragment thereof according to any one of items 1 to 16 or according to any one of items 31 to 40 or 47 belonging to any one of items 1 to 16; or (ii) An antibody or an antigen-binding fragment thereof according to any one of items 17 to 30 or according to any one of items 31 to 40 or 47 which are subordinate to any one of items 17 to 30; (b) The second antibody specifically binds to pepsinogen A and does not compete with the first antibody for specific binding to pepsinogen A; 2) Incubate the mixture obtained in 1) to form a complex comprising pepsinogen A and a first antibody or an antigen-binding fragment thereof, and a second antibody or an antigen-binding fragment thereof, and 3) Detect pepsinogen A in the sample by detecting the formation of the complex defined in 2).

[0456] 60. The method according to item 59, wherein (c) one of the first antibody and the second antibody contains a detection marker.

[0457] 61. The method according to item 60, wherein the first antibody or its antigen-binding fragment that does not contain the detection marker, or the second antibody or its antigen-binding fragment that contains the capture marker.

[0458] 62. The method according to item 60, wherein the method further comprises: adding particles capable of binding with the capture marker to the mixture of (1), and 2.1) separating the particles from the mixture before detecting the signal provided by the detection marker.

[0459] 63. The method according to any one of items 59 to 62, wherein When the first antibody or its antigen-binding fragment is selected from (i), then the second antibody is selected from (ii); or When the first antibody or its antigen-binding fragment is selected from (ii), the second antibody is selected from (i).

[0460] 64. The method according to any one of items 59 to 63, wherein the method is a serum immunoassay, wherein the sample is a body fluid, preferably whole blood, serum or plasma.

[0461] 65. The method according to any one of items 59 to 64, wherein the detection label is selected from enzymes, luminescent labels (preferably fluorescent, luminescent, chemiluminescent, electrochemiluminescent) or radioactive.

[0462] 66. The method according to any one of items 59 to 65, wherein the limit of quantification (LoQ) is 1.79 ng / mL at 20% CV.

[0463] 67. The method according to any one of items 59 to 66, wherein the detection of pepsinogen A in the sample is achieved in 18 minutes or less.

[0464] 68. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of items 1 to 40 or 47.

[0465] 69. A reagent kit comprising 1) The composition according to item 51, or 2) A first antibody or its antigen-binding fragment thereof, and a second antibody or its antigen-binding fragment thereof, wherein one of the first antibody or the second antibody or its antigen-binding fragment is a) An antibody or antigen-binding fragment thereof according to any one of items 1 to 16 or according to any one of items 31 to 40 or 47 belonging to any one of items 1 to 16; And the first antibody or the second antibody or the other of its antigen-binding fragments is b) An antibody or antigen-binding fragment thereof according to any one of items 17 to 30 or according to any one of items 31 to 40 or 47 which are subordinate to any one of items 17 to 30.

[0466] 70. The kit according to item 69, wherein the first antibody or the second antibody or the antigen-binding fragment thereof contains a detection marker.

[0467] 71. The kit according to item 70, wherein the first antibody or its antigen-binding fragment that does not contain the detection marker, or the second antibody or its antigen-binding fragment that contains the capture marker.

[0468] 72. The kit according to any one of items 68 to 71, wherein the kit is used to detect pepsinogen A.

[0469] 73. The kit according to any one of items 68 to 72 is an immunoassay kit.

[0470] 74. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 1; and / or (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 2.

[0471] 75. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 1; and / or (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 2.

[0472] 76. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 1; and / or (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 2.

[0473] 77. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 1; and / or (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 2.

[0474] 78. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 1; and / or (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 2.

[0475] 79. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) VH, which contains the sequence shown in SEQ ID NO: 1; and / or (ii) VL, which contains the sequence shown in SEQ ID NO: 2.

[0476] 80. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH consisting of the sequence shown in SEQ ID NO: 1; and / or (ii) A VL consisting of the sequence shown in SEQ ID NO: 2.

[0477] 81. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified with one amino acid substitution; and (ii) VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified with one amino acid substitution.

[0478] 82. The antibody or antigen-binding fragment thereof as described in item 81, comprising... (i) VH, comprising: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 3; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 4; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 5; and (ii) VL, comprising: CDR-L1 represented by the amino acid sequence of SEQ ID NO: 6; CDR-L2 represented by the amino acid sequence of SEQ ID NO: 7; and CDR-L3 represented by the amino acid sequence of SEQ ID NO: 8.

[0479] 83. The antibody or its antigen-binding fragment as described in Item 81, wherein all amino acid substitutions are conserved amino acid substitutions.

[0480] 84. The antibody or antigen-binding fragment thereof according to item 83, wherein one of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein the amino acid group is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0481] 85. The antibody or antigen-binding fragment thereof according to item 84, wherein one of the conserved amino acid substitutions is selected from the following highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0482] 86. The antibody or antigen-binding fragment thereof according to any one of items 74 to 85, wherein the antibody or antigen-binding fragment thereof binds to an epitope of pepsinogen A comprising the amino acid sequence shown in SEQ ID NO: 33.

[0483] 87. Based on any of items 81 to 86, the antibody or its antigen-binding fragment, The antibody contains a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 39; The amino acid sequence of FW-L2, SEQ ID NO: 40; The amino acid sequence of FW-L3, SEQ ID NO: 41; The amino acid sequence of FW-L4, SEQ ID NO: 42; The amino acid sequence of FW-H1, SEQ ID NO: 35; The amino acid sequence of FW-H2, SEQ ID NO: 36; The amino acid sequence of FW-H3, SEQ ID NO: 37; The amino acid sequence of FW-H4, SEQ ID NO: 38.

[0484] 88. The antibody or antigen-binding fragment thereof according to any one of items 81 to 87, The antibody contains a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequence or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 39; The amino acid sequence of FW-L2, SEQ ID NO: 40; The amino acid sequence of FW-L3, SEQ ID NO: 41; The amino acid sequence of FW-L4, SEQ ID NO: 42; The amino acid sequence of FW-H1, SEQ ID NO: 35; The amino acid sequence of FW-H2, SEQ ID NO: 36; The amino acid sequence of FW-H3, SEQ ID NO: 37; The amino acid sequence of FW-H4, SEQ ID NO: 38.

[0485] 89. The antibody or its antigen-binding fragment as described in Item 88, wherein all amino acid substitutions are conserved amino acid substitutions.

[0486] 90. The antibody or antigen-binding fragment thereof according to item 89, wherein one of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from the same group, wherein the amino acid group is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0487] 91. The antibody or antigen-binding fragment thereof according to item 90, wherein one of the conserved amino acid substitutions is selected from the following highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0488] 92. According to any one of items 74 to 91, the antibody or its antigen-binding fragment contains... (i) VH, which contains the sequence shown in SEQ ID NO: 1; and / or (ii) VL, which contains the sequence shown in SEQ ID NO: 2.

[0489] 93. An antibody or antigen-binding fragment thereof according to any one of items 74 to 92, wherein the antibody or antigen-binding fragment thereof is expressed in K+ of 0.07 nM or less. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0490] 94. An antibody or antigen-binding fragment thereof according to any one of items 74 to 93, wherein the antibody or antigen-binding fragment thereof is bound to pepsinogen A consisting of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L at 37°C for 313 minutes or longer t / 2-diss.

[0491] 95. The antibody or antigen-binding fragment thereof according to any one of items 74 to 94, wherein the association rate k of pepsinogen A composed of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L is... a For at least 4.9x10 5 M -1 s -1 .

[0492] 96. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 9; and / or (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 10.

[0493] 97. The antibody or antigen-binding fragment thereof as described in item 96, comprising... (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 9; and / or (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 10.

[0494] 98. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 9; and / or (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 10.

[0495] 99. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 9; and / or (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 10.

[0496] 100. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 9; and / or (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 10.

[0497] 101. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) VH, which contains the sequence shown in SEQ ID NO: 9; and / or (ii) VL, which contains the sequence shown in SEQ ID NO: 10.

[0498] 102. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH consisting of the sequence shown in SEQ ID NO: 9; and / or (ii) A VL consisting of the sequence shown in SEQ ID NO: 10.

[0499] 103. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified with one amino acid substitution; and (ii) VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14 or a variant thereof modified by one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15 or a variant thereof modified by one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16 or a variant thereof modified by one amino acid substitution.

[0500] 104. The antibody or antigen-binding fragment thereof as described in item 103, comprising... (i) VH, comprising: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 11; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 12; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 13; and (ii) VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16.

[0501] 105. The antibody or its antigen-binding fragment as described in Item 103, wherein all amino acid substitutions are conserved amino acid substitutions.

[0502] 106. The antibody or antigen-binding fragment thereof according to item 105, wherein one of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein the amino acid group is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0503] 107. The antibody or antigen-binding fragment thereof according to item 106, wherein one of the conserved amino acid substitutions is selected from the following highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0504] 108. Based on any of items 103 to 107, the antibody or its antigen-binding fragment, The antibody contains a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 47; The amino acid sequence of FW-L2, SEQ ID NO: 48; The amino acid sequence of FW-L3, SEQ ID NO: 49; The amino acid sequence of FW-L4, SEQ ID NO: 50; The amino acid sequence of FW-H1, SEQ ID NO: 43; The amino acid sequence of FW-H2, SEQ ID NO: 44; The amino acid sequence of FW-H3, SEQ ID NO: 45; The amino acid sequence of FW-H4, SEQ ID NO: 46.

[0505] 109. The antibody or antigen-binding fragment thereof according to any one of items 103 to 108, The antibody contains a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequence or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 47; The amino acid sequence of FW-L2, SEQ ID NO: 48; The amino acid sequence of FW-L3, SEQ ID NO: 49; The amino acid sequence of FW-L4, SEQ ID NO: 50; The amino acid sequence of FW-H1, SEQ ID NO: 43; The amino acid sequence of FW-H2, SEQ ID NO: 44; The amino acid sequence of FW-H3, SEQ ID NO: 45; The amino acid sequence of FW-H4, SEQ ID NO: 46.

[0506] 110. The antibody or its antigen-binding fragment as described in Item 109, wherein all amino acid substitutions are conserved amino acid substitutions.

[0507] 111. The antibody or antigen-binding fragment thereof according to item 110, wherein each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein the group of amino acids is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0508] 112. The antibody or antigen-binding fragment thereof according to item 111, wherein one of the conserved amino acid substitutions is selected from the following highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0509] 113. An antibody or antigen-binding fragment thereof according to any one of items 96 to 112, wherein the antibody or antigen-binding fragment thereof is expressed at a Kc of 0.9 nM or less. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0510] 114. The antibody or antigen-binding fragment thereof according to any one of items 96 to 113, wherein the association rate k of binding to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L is specified. a For at least 6x10 5 M -1 s -1 .

[0511] 115. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising... (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 17; and (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 18.

[0512] 116. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 17; and / or (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 18.

[0513] 117. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising... (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 17; and / or (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 18.

[0514] 118. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising... (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 17; and / or (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 18.

[0515] 119. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 17; and / or (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 18.

[0516] 120. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) VH, which contains the sequence shown in SEQ ID NO: 17; and / or (ii) VL, which contains the sequence shown in SEQ ID NO: 18.

[0517] 121. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH consisting of the sequence shown in SEQ ID NO: 17; and / or (ii) A VL consisting of the sequence shown in SEQ ID NO: 18.

[0518] 122. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21, or a variant thereof modified with one amino acid substitution; and (ii) VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22 or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23 or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24 or a variant thereof modified with one amino acid substitution.

[0519] 123. The antibody or antigen-binding fragment thereof as described in item 122, comprising... (i) VH, comprising: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 19; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 20; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 21; and (ii) VL, comprising: CDR-L1 represented by the amino acid sequence of SEQ ID NO: 22; CDR-L2 represented by the amino acid sequence of SEQ ID NO: 23; and CDR-L3 represented by the amino acid sequence of SEQ ID NO: 24.

[0520] 124. The antibody or its antigen-binding fragment as described in Item 122, wherein all amino acid substitutions are conserved amino acid substitutions.

[0521] 125. The antibody or antigen-binding fragment thereof according to item 124, wherein one of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein the amino acid group is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0522] 126. The antibody or antigen-binding fragment thereof according to item 125, wherein one of the conserved amino acid substitutions is selected from the following highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0523] 127. Based on any of the antibodies or antigen-binding fragments from items 122 to 126, The antibody contains a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 55; The amino acid sequence of FW-L2, SEQ ID NO: 56; The amino acid sequence of FW-L3, SEQ ID NO: 57; The amino acid sequence of FW-L4, SEQ ID NO: 58; The amino acid sequence of FW-H1, SEQ ID NO: 51; The amino acid sequence of FW-H2, SEQ ID NO: 52; The amino acid sequence of FW-H3, SEQ ID NO: 53; The amino acid sequence of FW-H4, SEQ ID NO: 54.

[0524] 128. Based on any of items 122 to 127, the antibody or its antigen-binding fragment, The antibody contains a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequence or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 55; The amino acid sequence of FW-L2, SEQ ID NO: 56; The amino acid sequence of FW-L3, SEQ ID NO: 57; The amino acid sequence of FW-L4, SEQ ID NO: 58; The amino acid sequence of FW-H1, SEQ ID NO: 51; The amino acid sequence of FW-H2, SEQ ID NO: 52; The amino acid sequence of FW-H3, SEQ ID NO: 53; The amino acid sequence of FW-H4, SEQ ID NO: 54.

[0525] 129. The antibody or its antigen-binding fragment as described in Item 128, wherein all amino acid substitutions are conserved amino acid substitutions.

[0526] 130. The antibody or antigen-binding fragment thereof according to item 129, wherein one of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from the same group, wherein the amino acid group is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0527] 131. The antibody or antigen-binding fragment thereof according to item 130, wherein one of the conserved amino acid substitutions is selected from the following highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0528] 132. An antibody or antigen-binding fragment thereof according to any one of items 115 to 131, wherein the antibody or antigen-binding fragment thereof is expressed at a Kc of 0.9 nM or less. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0529] 133. An antibody or antigen-binding fragment thereof according to any one of items 115 to 132, wherein the antibody or antigen-binding fragment thereof is bound to pepsinogen A consisting of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L at 37°C for 23 minutes or longer t / 2-diss.

[0530] 134. The antibody or antigen-binding fragment thereof according to any one of items 115 to 133, wherein the association rate k of binding to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L is specified. a For at least 5.2x10 5 M -1 s -1 .

[0531] 135. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) a heavy-chain variable domain (VH) that is at least 80% homologous to the VH shown in SEQ ID NO: 25; and / or (ii) Light chain variable domains (VL) that are at least 80% homologous to the VL shown in SEQ ID NO: 26.

[0532] 136. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH that is at least 85% homologous to the VH shown in SEQ ID NO: 25; and / or (ii) A VL that is at least 85% homologous to the VL shown in SEQ ID NO: 26.

[0533] 137. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising... (i) A VH that is at least 90% homologous to the VH shown in SEQ ID NO: 25; and / or (ii) A VL that is at least 90% homologous to the VL shown in SEQ ID NO: 26.

[0534] 138. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising... (i) A VH that is at least 95% homologous to the VH shown in SEQ ID NO: 25; and / or (ii) A VL that is at least 95% homologous to the VL shown in SEQ ID NO: 26.

[0535] 139. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising... (i) A VH that is at least 99% homologous to the VH shown in SEQ ID NO: 25; and / or (ii) A VL that is at least 99% homologous to the VL shown in SEQ ID NO: 26.

[0536] 140. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising... (i) VH, which contains the sequence shown in SEQ ID NO: 25; and / or (ii) VL, which contains the sequence shown in SEQ ID NO: 26.

[0537] 141. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising: (i) A VH consisting of the sequence shown in SEQ ID NO: 25; and / or (ii) A VL consisting of the sequence shown in SEQ ID NO: 26.

[0538] 142. An antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen A, the antibody or antigen-binding fragment comprising... (i) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and (ii) VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30 or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31 or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32 or a variant thereof modified with one amino acid substitution.

[0539] 143. The antibody or antigen-binding fragment thereof as described in item 142, wherein the antibody or antigen-binding fragment thereof comprises (i) VH, comprising: CDR-H1 represented by the amino acid sequence of SEQ ID NO: 27; CDR-H2 represented by the amino acid sequence of SEQ ID NO: 28; and CDR-H3 represented by the amino acid sequence of SEQ ID NO: 29; and (ii) VL, comprising: CDR-L1 represented by the amino acid sequence of SEQ ID NO: 30; CDR-L2 represented by the amino acid sequence of SEQ ID NO: 31; and CDR-L3 represented by the amino acid sequence of SEQ ID NO: 32.

[0540] 144. The antibody or its antigen-binding fragment as described in Item 142, wherein all amino acid substitutions are conserved amino acid substitutions.

[0541] 145. The antibody or antigen-binding fragment thereof according to item 144, wherein one of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein the amino acid group is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0542] 146. The antibody or antigen-binding fragment thereof according to item 144 or 145, wherein one of the conserved amino acid substitutions is selected from highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0543] 147. Based on any of items 142 to 146, the antibody or its antigen-binding fragment, The antibody contains a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequences or variants thereof that are at least 85% homologous to them: The amino acid sequence of FW-L1, SEQ ID NO: 63; The amino acid sequence of FW-L2, SEQ ID NO: 64; The amino acid sequence of FW-L3, SEQ ID NO: 65; The amino acid sequence of FW-L4, SEQ ID NO: 66; The amino acid sequence of FW-H1, SEQ ID NO: 59; The amino acid sequence of FW-H2, SEQ ID NO: 60; The amino acid sequence of FW-H3, SEQ ID NO: 61; The amino acid sequence of FW-H4, SEQ ID NO: 62.

[0544] 148. Based on any of items 142 to 147, the antibody or its antigen-binding fragment, The antibody contains a VL consisting of a frame region (FW) and a CDR, as shown in Formula I: (FW-L1) - (CDR-L1) - (FW-L2) - (CDR-L2) - (FW-L3) - (CDR-L3) - (FW-L4) (Formula I) And VH, composed of FW and CDR, is shown in Equation II: (FW-H1) - (CDR-H1) - (FW-H2) - (CDR-H2) - (FW-H3) - (CDR-H3) - (FW-H4) (Formula II), FW contains the following amino acid sequence or variants thereof modified with up to three amino acid substitutions: The amino acid sequence of FW-L1, SEQ ID NO: 63; The amino acid sequence of FW-L2, SEQ ID NO: 64; The amino acid sequence of FW-L3, SEQ ID NO: 65; The amino acid sequence of FW-L4, SEQ ID NO: 66; The amino acid sequence of FW-H1, SEQ ID NO: 59; The amino acid sequence of FW-H2, SEQ ID NO: 60; The amino acid sequence of FW-H3, SEQ ID NO: 61; The amino acid sequence of FW-H4, SEQ ID NO: 62.

[0545] 149. The antibody or antigen-binding fragment thereof as described in item 148, wherein all amino acid substitutions are conserved amino acid substitutions.

[0546] 150. The antibody or antigen-binding fragment thereof according to item 149, wherein each of the conserved amino acid substitutions is an amino acid substituted with another amino acid selected from its group, wherein the group of amino acids is... a) Nonpolar hydrophobic amino acids, composed of Gly, Ala, Val, Leu, Ile, Phe, Try, Trp and Met; b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln; c) Positively charged basic amino acids, composed of Arg, Lys, and His; and d) Negatively charged amino acids, composed of Asp and Glu. If Cys is to be conservatively replaced, it is replaced by Ser or Ala, and If Pro is to be conservatively replaced, it is replaced by Ala.

[0547] 151. The antibody or antigen-binding fragment thereof according to item 149 or 150, wherein one of the conserved amino acid substitutions is selected from highly conserved amino acid substitutions. a) Replace Ala with Val, Leu, Ile, or Gly; b) Replace Arg with Lys; c) Replace Asn with Gln; d) Replace Asp with Glu; e) Replace Cys with Ser; f) Replace Gln with Asn; g) Replace Glu with Asp; h) Replace Gly with Ala; i) Replace His with Arg; j) Replace Ile with Leu, Val, or Ala; k) Replace Leu with Ile, Val, or Ala; l) Replace Lys with Arg; m) Replace Met with Leu, Ile, or Val; n) Replace Phe with Tyr or Trp; o) Replace Pro with Ala; p) Replace Ser with Thr; q) Replace Thr with Ser; r) Replace Trp with Phe or Tyr; s) Replace Tyr with Phe or Trp; and t) Replace Val with Leu, Ile, or Ala.

[0548] 152. An antibody or antigen-binding fragment thereof according to any one of items 135 to 151, wherein the antibody or antigen-binding fragment thereof is expressed at a Kc of 0.6 nM or less. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0549] 153. An antibody or antigen-binding fragment thereof according to any one of items 135 to 152, wherein the antibody or antigen-binding fragment thereof is bound to pepsinogen A consisting of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L at 37°C for 21 minutes or longer t / 2-diss.

[0550] 154. The antibody or antigen-binding fragment thereof according to any one of items 135 to 153, wherein the association rate k of binding to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L is specified. a For at least 8.7x10 5 M -1 s -1 .

[0551] 155. An antibody or an antigen-binding fragment thereof according to any one of items 74 to 154, wherein the antibody is a monoclonal antibody.

[0552] 156. The antibody or antigen-binding fragment thereof according to any one of items 74 to 155, wherein the pepsinogen A comprises SEQ ID NO: 34 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L, preferably wherein the pepsinogen A consists of SEQ ID NO: 34 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L.

[0553] 157. An antibody or antigen-binding fragment thereof according to any one of items 74 to 92, 96 to 112, 115 to 131 and 135 to 156, wherein the antibody or antigen-binding fragment thereof is in the form of a K+ of 2 nM or less, or 1.5 nM or less, or 1 nM or less. D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein the K D It was measured at 37℃.

[0554] 158. An antibody or antigen-binding fragment thereof according to any one of items 74 to 92, 96 to 112, 115 to 131 and 135 to 157, wherein the antibody or antigen-binding fragment thereof binds to the pepsinogen A composed of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L at 37°C for 20 minutes or longer t / 2-diss.

[0555] 159. An antibody or antigen-binding fragment thereof according to any one of items 74 to 92, 96 to 112, 115 to 131 and 135 to 158, wherein the association rate k of pepsinogen A composed of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L is... a For at least 10 5 M -1 s -1 Preferably at least 4.9x10 5 M -1 s -1 .

[0556] 160. The antibody or antigen-binding fragment thereof according to any one of items 157 to 159, wherein the K D and / or the k a Determined by surface plasmon resonance spectroscopy.

[0557] 161. The antibody or antigen-binding fragment thereof as described in Item 160, wherein the surface plasmon resonance spectroscopy comprises attaching or capturing the antibody or antigen-binding fragment thereof onto a CM5 sensor chip and injecting pepsinogen A consisting of SEQ ID NO: 68 or a variant thereof comprising amino acid substitutions of E58K, K222Q, T265A or V353L as an analyte, wherein the determination is performed using HBS-ET buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (w / v) Tween 20®) at 37°C.

[0558] 162. An antibody or an antigen-binding fragment thereof according to any one of items 74 to 161, wherein the antibody does not specifically bind to pepsinogen C.

[0559] 163. An antibody or antigen-binding fragment thereof according to any one of items 160 to 162, wherein the surface plasmon resonance spectrum is performed using a Biacore 8k or B4000 instrument.

[0560] 164. An antibody or antigen-binding fragment thereof according to any one of items 160 to 163, wherein the K D And / or the determination of ka includes fitting surface plasmon resonance data using a Langmuir fitting model.

[0561] 165. A polynucleotide or polynucleotide sequence encoding... (i) the heavy chain variable domain of any antibody or antigen-binding fragment thereof according to any one of items 74 to 164, and / or (ii) The light chain variable domain of an antibody or antigen-binding fragment according to any one of items 74 to 164.

[0562] 166. A vector comprising a polynucleotide or polynucleotide group as described in item 165.

[0563] 167. A host cell comprising a polynucleotide or polynucleotide group according to item 165 or a vector according to item 166, and expressing an antibody or an antigen-binding fragment thereof according to any one of items 74 to 164.

[0564] 168. The host cell as described in item 167, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

[0565] 169. The host cell according to item 168, wherein the host cell is a eukaryotic cell, wherein the eukaryotic cell is a HEK293 cell or a CHO cell.

[0566] 170. A method for producing an antibody or an antigen-binding fragment thereof according to any one of items 74 to 164, comprising culturing a host cell according to any one of items 167 to 169 and isolating the antibody or the antigen-binding fragment thereof.

[0567] 171. The antibody or antigen-binding fragment thereof according to any one of items 74 to 164, which may be obtained by the method according to item 170.

[0568] 172. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of items 74 to 164 or 171, a polynucleotide or polynucleotide group according to item 165, a vector according to item 166, or a host cell according to any one of items 167 to 169.

[0569] 173. A composition comprising a) An antibody or antigen-binding fragment thereof according to any one of items 74 to 134 or according to any one of items 155 to 164 or 171 which are subordinate to any one of items 74 to 134; And an antibody or antigen-binding fragment thereof according to any one of items 135 to 154 or according to any one of items 155 to 164 or 171 which are subordinate to any one of items 135 to 154; b) A polynucleotide or polynucleotide group according to any one of items 165 belonging to any one of items 74 to 134 or according to any one of items 155 to 164 or 171 belonging to any one of items 74 to 134; And a polynucleotide or polynucleotide group according to any one of items 165 belonging to any one of items 135 to 154 or according to any one of items 155 to 164 or 171 belonging to any one of items 135 to 154; c) A carrier according to item 166, which is subordinate to any one of items 74 to 134, or according to any one of items 155 to 164 or 171, which are subordinate to any one of items 74 to 134; and a carrier according to item 166, which is subordinate to any one of items 135 to 154, or according to any one of items 155 to 164 or 171, which is subordinate to any one of items 135 to 154; or d) Host cells according to any one of items 167 to 169 belonging to any one of items 74 to 134 or according to any one of items 155 to 164 or 171 belonging to any one of items 74 to 134; And the host cell according to any one of items 167 to 169 of any item belonging to any one of items 135 to 154 or according to any one of items 155 to 164 or 171 of any item belonging to any one of items 135 to 154.

[0570] 174. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of items 74 to 164 or 171, wherein the composition is a diagnostic composition.

[0571] 175. A composition comprising An antibody or antigen-binding fragment thereof according to any one of items 74 to 134 or according to any one of items 155 to 164 or 171 belonging to any one of items 74 to 134; and The antibody or antigen-binding fragment thereof is based on any one of items 135 to 154 or any one of items 155 to 164 or 171 which are subordinate to any one of items 135 to 154; The composition is a diagnostic composition.

[0572] 176. Use of the antibody or antigen-binding fragment thereof according to any one of items 74 to 164 or 171, or the composition according to item 174 or 175, for use in an in vitro immunoassay, particularly for an in vitro immunoassay to detect pepsinogen A in a sample.

[0573] 177. The use of the antibody or its antigen-binding fragment as described in item 176, wherein the immunoassay is a heterologous immunoassay.

[0574] 178. The antibody or antigen-binding fragment thereof as described in item 176 or 177, wherein the sample used for the immunoassay is a sample prepared from blood, plasma or serum.

[0575] 179. An in vitro method for detecting pepsinogen A in a sample, the method comprising: (a) Contacting a sample containing pepsinogen A with an antibody or an antigen-binding fragment thereof according to any one of items 74 to 164 or 171 to form a complex comprising pepsinogen A and said antibody or an antigen-binding fragment thereof, and (b) Detect the complex formed in step (a) to detect pepsinogen A in the sample.

[0576] 180. The method according to item 179, wherein the method is a serum immunoassay, wherein the sample is a body fluid, preferably whole blood, serum or plasma.

[0577] 181. The method according to item 179 or 180, wherein the antibody or its antigen-binding fragment has a detection marker attached thereto.

[0578] 182. The method according to item 181, wherein the detection label is selected from enzymes, luminescent labels (preferably fluorescent, luminescent, chemiluminescent, electrochemiluminescent) or radioactive.

[0579] 183. An in vitro method for detecting pepsinogen A in a sample, the method comprising the following steps: 1) Contact the sample containing pepsinogen A with a first antibody or its antigen-binding fragment and a second antibody or its antigen-binding fragment, wherein... (a) The first antibody is (i) an antibody or antigen-binding fragment thereof according to any one of items 74 to 134 or according to any one of items 155 to 164 or 171 belonging to any one of items 74 to 134; or (ii) An antibody or antigen-binding fragment thereof according to any one of items 135 to 154 or according to any one of items 155 to 164 or 171 which are subordinate to any one of items 135 to 154; (b) The second antibody specifically binds to pepsinogen A and does not compete with the first antibody for specific binding to pepsinogen A; and 2) Incubate the mixture obtained in 1) to form a complex comprising pepsinogen A and the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment; and 3) Detect pepsinogen A in the sample by detecting the formation ...

Claims

1. A kit comprising a first antibody or an antigen-binding fragment thereof and a second antibody or an antigen-binding fragment thereof. a) Wherein the first antibody or its antigen-binding fragment is It specifically binds to pepsinogen A and competes with antibodies or their antigen-binding fragments that bind to pepsinogen A. (i) The heavy chain variable structural domain (VH) shown in SEQ ID NO: 1; and (ii) The light chain variable domain (VL) shown in SEQ ID NO: 2; and (b) wherein the second antibody or its antigen-binding fragment is An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen A and competes with pepsinogen A for binding, said antibody or antigen-binding fragment thereof comprising the following components: (iii) The heavy chain variable structural domain (VH) shown in SEQ ID NO: 25; and (iv) The light chain variable domain (VL) shown in SEQ ID NO: 26; and The second antibody or its antigen-binding fragment does not compete with the first antibody or its antigen-binding fragment for binding to pepsinogen A.

2. The kit according to claim 1, wherein the first antibody or antigen-binding fragment comprises... (a) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified with one amino acid substitution; or (b) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16, or a variant thereof modified with one amino acid substitution; or (c) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24, or a variant thereof modified with one amino acid substitution; and / or The second antibody or its antigen-binding fragment contains (d) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

3. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment specifically binding to pepsinogen A and competing with an antibody composed of the following for binding to pepsinogen A. (i) The heavy chain variable structural domain (VH) shown in SEQ ID NO: 1; and (ii) The light chain variable domain (VL) shown in SEQ ID NO:

2.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the antibody or antigen-binding fragment thereof comprises... (a) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified with one amino acid substitution; or (b) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 14, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 15, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 16, or a variant thereof modified with one amino acid substitution; or (c) VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 19, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 20, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 21, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 22 or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 23 or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 24 or a variant thereof modified with one amino acid substitution.

5. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment specifically binding to pepsinogen A and competing with an antibody composed of the following for binding to pepsinogen A. (i) The heavy chain variable structural domain (VH) shown in SEQ ID NO: 25; and (ii) The light chain variable domain (VL) shown in SEQ ID NO:

26.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof comprises... VH, comprising: CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 27, or a variant thereof modified with one amino acid substitution; CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 28, or a variant thereof modified with one amino acid substitution; and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 29, or a variant thereof modified with one amino acid substitution; and VL, comprising: CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 30, or a variant thereof modified with one amino acid substitution; CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 31, or a variant thereof modified with one amino acid substitution; and CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 32, or a variant thereof modified with one amino acid substitution.

7. The antibody or antigen-binding fragment thereof according to any one of claims 3 to 6, wherein the antibody or antigen-binding fragment thereof is in the form of 2 nM or less, or 1.5 nM or less, or 1 nM or less K D It binds to pepsinogen A composed of SEQ ID NO: 68 or a variant thereof containing amino acid substitutions of E58K, K222Q, T265A, or V353L, preferably wherein said K D It was measured at 37℃.

8. A polynucleotide or polynucleotide sequence encoding... (i) the heavy chain variable domain of the antibody or its antigen-binding fragment according to any one of claims 3 to 7, and / or (ii) The light chain variable domain of the antibody or antigen-binding fragment according to any one of claims 3 to 7.

9. A vector comprising the polynucleotide or polynucleotide group according to claim 8.

10. A host cell comprising the polynucleotide or polynucleotide group according to claim 8 or the vector according to claim 9, and expressing an antibody or an antigen-binding fragment thereof according to any one of claims 3 to 7.

11. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 3 to 7, the method comprising culturing a host cell according to claim 10 and isolating the antibody or the antigen-binding fragment thereof.

12. The antibody or antigen-binding fragment thereof according to any one of claims 3 to 7, which can be obtained by the method according to claim 11.

13. A composition comprising a) The antibody or its antigen-binding fragment according to any one of claims 3 to 7 or 12, the polynucleotide or polynucleotide sequence according to claim 8, the vector according to claim 9, or the host cell according to claim 10, or b) An antibody or antigen-binding fragment thereof according to claim 3 or 4, or according to claim 7 or 12 which is dependent on claim 3 or 4; and The antibody or its antigen-binding fragment according to claim 5 or 6, or according to claim 7 or 12 which are dependent on claim 5 or 6.

14. The antibody or antigen-binding fragment thereof according to any one of claims 3 to 7 or 12, the composition according to claim 13, or the kit according to claim 1 or 2 is used for... in vitro Immunoassays, especially those used to detect pepsinogen A in samples in vitro Applications of immunoassay.

15. A method for detecting pepsinogen A in a sample. in vitro The method includes (a) Contacting a sample containing pepsinogen A with an antibody or an antigen-binding fragment thereof according to any one of claims 3 to 7 or 12 to form a complex comprising pepsinogen A and said antibody or an antigen-binding fragment thereof, and (b) Detect the complex formed in step (a) to detect pepsinogen A in the sample.

16. A method for detecting pepsinogen A in a sample. in vitro The method includes the following steps: 1) Contact the sample containing pepsinogen A with a first antibody or its antigen-binding fragment and a second antibody or its antigen-binding fragment, wherein... (a) The first antibody is (i) The antibody or antigen-binding fragment thereof according to claim 3 or 4, or according to claim 7 or 12 which are dependent on claim 3 or 4, or (ii) An antibody or antigen-binding fragment thereof according to claim 5 or 6, or according to claim 7 or 12 which is dependent on claim 5 or 6; and (b) The second antibody specifically binds to pepsinogen A and does not compete with the first antibody for specific binding to pepsinogen A; 2) Incubate the mixture obtained in 1) to form a complex comprising pepsinogen A and the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment; and 3) Detect pepsinogen A in the sample by detecting the formation of the complex as defined in 2).

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