Rabbit antibodies against human immunoglobulin G

Abstract

This invention provides a monoclonal antibody that is universally specific to human IgG but does not bind to monkey IgG. [Solution] An anti-human IgG antibody obtained from rabbits, its antigen-binding portion, and a method for using the antibody and portion are provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Patent Application No. 63 / 033073, filed on June 1, 2020, and this disclosure is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on 24 May 2021, is named 022548_WO063_SL.txt and has a size of 84,819 bytes. [Background technology]

[0003] Therapeutic monoclonal antibodies (mAbs) have become one of the fastest-growing drug classes in recent years, approved for the treatment of a wide range of conditions, from cancer to autoimmune diseases. Preclinical pharmacokinetic characterization of these therapeutic monoclonal antibodies is often performed in non-human primates to demonstrate efficacy and safety before initiating clinical trials. Cynomolgus monkeys are a preferred non-human primate for such preclinical studies because they often exhibit sufficient levels of cross-reactivity with therapeutic antibody targets (Non-Patent Literature 1). However, cynomolgus immunoglobulins also exhibit high sequence homology with human immunoglobulins. The high level of IgG protein sequence homology means that the lack of high-quality reagent antibodies capable of distinguishing human therapeutic molecules in serum from cynomolgus immunoglobulins poses a significant challenge for the bioanalytical measurement of human therapeutic antibodies in non-human primate serum samples (Non-Patent Literature 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Iwasaki et al., Drug Metab Pharmacokinet. (2019) 34:55-63. [Non-Patent Document 2] Stubenrauch et al., J Pharm Biomed Anal. (2009) 49:1003-8 [Overview of the project] [Problems that the invention aims to solve]

[0005] Currently, the evaluation of the pharmacokinetics (PK) and pharmacodynamics (PD) of therapeutic antibodies in preclinical trials relies on drug-specific anti-idiotype antibodies, which require significant effort and time to develop. Each drug candidate requires its own anti-idiotype antibody. There are few general-purpose reagents available that can detect all human IgG-based therapeutic antibodies in preclinical trials. Therefore, to accurately measure the levels of human IgG-derived therapeutic mAbs in non-human primates during preclinical trials, it is necessary to develop monoclonal antibodies that are universally specific to human IgG but do not bind to monkey IgG.

[0006] This disclosure provides an isolated monoclonal antibody or its antigen-binding moiety that specifically binds to human IgG, wherein the antibody or moiety includes heavy chain complementarity-determining regions (CDRs) 1-3 and light chain CDRs 1-3, respectively, containing SEQ ID NOs. 27-32, SEQ ID NOs. 33-38, SEQ ID NOs. 39-44, SEQ ID NOs. 45-50, SEQ ID NOs. 51-56, or SEQ ID NOs. 57-62. The antibody is a rabbit antibody or can be modified from such a molecule (e.g., mouse, rat, or human). (Includes chimeric antibodies with non-rabbit Fc domains). [Means for solving the problem]

[0007] In some embodiments, the antibody or portion comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) containing SEQ ID NOs: 13 and 19, SEQ ID NOs: 14 and 20, SEQ ID NOs: 15 and 21, SEQ ID NOs: 16 and 22, SEQ ID NOs: 17 and 23, or SEQ ID NOs: 18 and 24, respectively. The antibody may be a rabbit antibody or modified from such a molecule.

[0008] In some embodiments, the antibody comprises the heavy chain constant region amino acid sequence of SEQ ID NO: 25 and / or the light chain constant region amino sequence of SEQ ID NO: 26. In further embodiments, the antibody comprises a heavy chain and a light chain having the amino acid sequences of SEQ ID NOs: 63 and 69, SEQ ID NOs: 64 and 70, SEQ ID NOs: 65 and 71, SEQ ID NOs: 66 and 72, SEQ ID NOs: 67 and 73, or SEQ ID NOs: 68 and 74, respectively, with or without the leader sequence.

[0009] In certain embodiments, the antibody or antigen-binding moiety includes a detectable label.

[0010] This disclosure also provides compositions or kits comprising the monoclonal antibody or antigen-binding moiety in an aqueous buffer solution.

[0011] In other embodiments, the Disclosure provides isolated nucleic acid molecules encoding the heavy chain, light chain, or both of the monoclonal antibody or antigen-binding moiety. In some embodiments, the nucleic acid molecules include SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and / or 12. In further embodiments, the nucleic acid molecules include SEQ ID NOs: 1 and 7, SEQ ID NOs: 2 and 8, SEQ ID NOs: 3 and 9, SEQ ID NOs: 4 and 10, SEQ ID NOs: 5 and 11, or SEQ ID NOs: 6 and 12. Expression constructs comprising nucleic acid molecules and host cells (e.g., mammalian cells) comprising nucleotide sequences encoding the heavy and light chains of the monoclonal antibody or antigen-binding moiety are also provided herein. The Disclosure also provides a method for producing an antibody or its antigen-binding moiety, comprising the steps of culturing host cells under conditions that enable expression of the heavy and light chains of the antibody or moiety, and isolating the antibody or moiety from the cultured cells or the supernatant of a cell culture.

[0012] In another embodiment, the Disclosure provides a method for detecting human IgG or a fragment thereof in a sample, comprising the step of contacting the sample with one or more monoclonal antibodies or antigen-binding moieties described herein. The sample (e.g., a tissue sample or biopsy sample such as a blood, serum, or plasma sample) may be obtained from an animal administered, for example, an antibody or fragment thereof (e.g., a Fab or F(ab')2 fragment) containing a human IgG constant region (e.g., a human IgG1, IgG2, IgG3, or IgG4 constant region). The animal may be a non-human primate such as a cynomolgus monkey or a rhesus monkey.

[0013] Other features, purposes, and advantages of the present invention will become apparent in the following detailed description. However, it should be understood that the detailed description illustrates, but is for illustrative purposes only, and not limiting, embodiments and aspects of the present invention. Various modifications and changes within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawing]

[0014] [Figure 1] This is a panel of Biacore sensogram graphs showing the binding of six rabbit recombinant antibody clones to whole human IgG1 (hIgG1), whole human IgG4 (hIgG4), human Fab (hFab), and cynomolgus monkey IgG (cynoIgG). [Figure 2A] These figures show the alignment of the heavy chain (Figure 2A) and light chain (Figure 2B) amino acid sequences of six rabbit recombinant antibody clones using the Clustal Omega and Kabat numbering schemes, respectively. The CDR (see also Zhang and Ho, MABS (2017) 9(3): pp. 419-429) is underlined and in bold. The leader sequence, the beginning of the variable domain, and the beginning of the constant region are marked as shown. [Figure 2B] These figures show the alignment of the heavy chain (Figure 2A) and light chain (Figure 2B) amino acid sequences of six rabbit recombinant antibody clones using the Clustal Omega and Kabat numbering schemes, respectively. The CDR (see also Zhang and Ho, MABS (2017) 9(3): pp. 419-429) is underlined and in bold. The leader sequence, the beginning of the variable domain, and the beginning of the constant region are marked as shown. [Figure 2C] This figure shows a phylogenetic tree of the VH and VL sequences of six rabbit antibody clones. [Figure 3A] This figure shows the epitope determination of six rabbit anti-hIgG clones using the Biacore competitive assay (Figure 3A) and Biacore kinetic assay sensogram (Figure 3B). Antibodies MCA5748G (Bio-Rad), 19B1, 11F9, and mouse anti-hIgG mAb (Southern Biotech catalog number 9042-01) were first biotinylated and captured on the Biacore SA chip at 500-600 RU. Eight 1:2 serial dilutions of (80-0 nM) hIgG1 antibody (isatuximab; "isa") were mixed with each of the competitive antibodies (240 nM) before use. If the pre-mixed competitive antibody binds to hIgG1 at the same site as the captured antibody on the chip surface, hIgG1 cannot bind to the captured antibody. [Figure 3B]Figure showing epitope determination of six rabbit anti-hIgG clones by Biacore competition assay (Figure 3A) and Biacore kinetic assay sensorgram (Figure 3B). Antibodies MCA5748G (Bio-Rad), 19B1, 11F9 and mouse anti-hIgG mAb (Southern Biotech catalog number 9042-01) were first biotinylated and captured at 500 - 600 RU on a Biacore SA chip. hIgG1 antibody (isatuximab; "isa") serially diluted 1:2 eight times (80 - 0 nM) was mixed with each of the competing antibodies (240 nM) prior to use. If the pre-mixed competing antibody binds to hIgG1 at the same site as the antibody captured on the chip surface, hIgG1 cannot bind to the captured antibody. [Figure 4A] Figure showing a overlay graph of hIgG4 in a Gyrolab (trademark) immunoassay using clone 16F5 as the capture reagent, and in an assay matrix containing 0%, 4%, 10% and 25% cynomolgus monkey serum (Figure 4A); Figure showing a standard curve graph of hIgG4 in an assay matrix containing 4% cynomolgus monkey serum (Figure 4B); Figure showing quality control in the same assay matrix containing 4% cynomolgus monkey serum (Figure 4C). Clone 16F5 was biotinylated and used as the capture reagent. Human IgG4 antibody (hIgG4) serving as an assay standard was diluted 1:4 in the range of 1200 - 0.30 ng / mL. Alexa Fluor647 conjugated goat anti-human IgG was used for detection. [Figure 4B]A figure showing a overlay graph of hIgG4 in a Gyrolab™ immunoassay using clone 16F5 as the capture reagent, and in an assay matrix containing 0%, 4%, 10% and 25% cynomolgus monkey serum (Figure 4A); a figure showing a standard curve graph of hIgG4 in an assay matrix containing 4% cynomolgus monkey serum (Figure 4B); a figure showing quality control in the same assay matrix containing 4% cynomolgus monkey serum (Figure 4C). Clone 16F5 was biotinylated and used as the capture reagent. Human IgG4 antibody (hIgG4) that functions as an assay standard was diluted 1:4 in the range of 1200 - 0.30 ng / mL. Alexa Fluor 647 conjugated goat anti-human IgG was used for detection. [Figure 4C] A figure showing a overlay graph of hIgG4 in a Gyrolab™ immunoassay using clone 16F5 as the capture reagent, and in an assay matrix containing 0%, 4%, 10% and 25% cynomolgus monkey serum (Figure 4A); a figure showing a standard curve graph of hIgG4 in an assay matrix containing 4% cynomolgus monkey serum (Figure 4B); a figure showing quality control in the same assay matrix containing 4% cynomolgus monkey serum (Figure 4C). Clone 16F5 was biotinylated and used as the capture reagent. Human IgG4 antibody (hIgG4) that functions as an assay standard was diluted 1:4 in the range of 1200 - 0.30 ng / mL. Alexa Fluor 647 conjugated goat anti-human IgG was used for detection. [Figure 5A] A figure showing a comparison of different clones used as capture reagents for the detection of hIgG4 and hFab. Figure 5A is a figure showing standard curves of all six rabbit anti-hIgG mAb clones, as well as Gyrolab™ and MCA5748G reagents, for the detection of hIgG4. Figure 5B is a figure showing the same set of capture reagents used for the detection of hIgG Fab. It should be noted that hFab cannot be detected by the Gyrolab™ reagent or by the MCA5748G reagent. [Figure 5B]This figure shows a comparison of different clones used as capture reagents for the detection of hIgG4 and hFab. Figure 5A shows standard curves for the detection of hIgG4 for all six rabbit anti-hIgG mAb clones, as well as for the Gyrolab® and MCA5748G capture reagents. Figure 5B shows the same set of capture reagents used for the detection of hIgG Fab. It should be noted that hFab is not detectable by either the Gyrolab® or MCA5748G capture reagent. [Figure 6A] This figure shows a comparison of four antibodies used in precipitation and acid dissociation (PandA) assays. The dynamic range and sensitivity of two rabbit anti-human IgG clones, 2C5 and 11G5, were compared with a commercially available mouse anti-Fc mAb, clone JDC-10 (Southern Biotech, catalog no. 9040-01). pAb: Rabbit polyclonal anti-drug antibody (drug: monoclonal antibody with human IgG4 constant region). ECL: Electrochemiluminescence. [Figure 6B] This figure shows the results of the PandA assay for clones 2C5 and 11G5 in human, rat, monkey, and mouse serum matrices. Pos ctrl: Positive control. [Figure 6C] This figure shows the results of the PandA assay for clones 2C5 and 11G5 in human, rat, monkey, and mouse serum matrices. Pos ctrl: Positive control. [Modes for carrying out the invention]

[0015] This disclosure provides rabbit monoclonal antibodies that bind to human immunoglobulin G and its Fab fragments with high affinity but do not bind to IgG in non-human primates such as monkeys (e.g., cynomolgus monkeys or rhesus monkeys) at detectable levels. These rabbit antibodies are particularly useful as reagents for detecting human IgG or its fragments in preclinical pharmaceutical studies of therapeutic antibodies based on human IgG in cynomolgus monkeys or other non-human primates. For example, these rabbit antibodies can be used to study therapeutic antibodies that are full human IgG antibodies, humanized IgG antibodies, chimeric antibodies having a human IgG constant region, and their Fab fragments. These rabbit antibodies can also be used in preclinical immunohistochemical studies and in the development of manufacturing processes for therapeutic antibodies based on human IgG (e.g., all antibodies, including monospecific, bispecific, and trispecific antibodies, as well as their Fab fragments).

[0016] This rabbit monoclonal antibody binds to three unique epitopes on human IgG, and these epitopes are further distinct from those bound by the commercially available mouse anti-hIgG antibody MCA5748G. The rabbit monoclonal antibody exhibits several advantages over traditional mouse monoclonal antibodies. These advantages include higher binding affinity and specificity, as well as more diverse epitope recognition. The rabbit immune system is evolutionarily different from that of rodents, employing different mechanisms to generate, diversify, and optimize the affinity of the antibodies it produces. In addition, the rabbit immune system retains the ability to recognize smaller, non-immunogenic epitopes in mice and produce a strong immune response. Therefore, the rabbit antibodies described herein are advantageous over mouse antibodies.

[0017] Rabbit anti-hIgG antibodies This disclosure provides antibodies that specifically (i.e., with high affinity) bind to human IgG and its antigen-binding moieties (e.g., Fab and F(ab')2). These antibodies do not bind to immunoglobulins (such as IgG) of other species commonly used in preclinical trials (e.g., mouse, rat, rabbit, non-human primate, or dog) at detectable levels.

[0018] In this specification, the term "affinity" refers to a measure of the attractiveness between an antigen and an antibody. The intrinsic attractive effect of an antibody to an antigen is expressed by the binding affinity equilibrium constant (K) of a particular antibody-antigen interaction. D It is generally expressed as ). High binding affinity, i.e., K D An antibody is said to bind specifically to an antigen if its strength is ≤100 nM (for example, ≤10 nM or ≤1 nM). For example, K D The binding affinity constant can be measured by surface plasmon resonance (Biacore®) using, for example, Biacore® T200 from Biacore. The binding affinity of a specific antibody-antigen interaction can also be shown by a standard concentration-response curve using, for example, Gyrolab® xPlore from Gyros Protein Technologies. In some embodiments, in the Biacore assay, this antibody is K D It binds to human IgG and Fab fragments derived therefrom at a M content of 2 nM or less, but does not show detectable binding to cynomolgus monkey IgG.

[0019] The antibodies exemplified herein bind to three different epitopes on hIgG and its Fab fragment. Herein, the term “epitope” refers to a portion of an antigen (determinant) that specifically binds to an antibody or a related molecule, such as a bispecific binding molecule. Epitope determinants generally consist of a group of chemically active surfaces of molecules, such as amino acids or carbohydrate or sugar side chains, and generally possess specific three-dimensional structural properties and specific charge properties. Epitopes can be “linear” or “conformal.” In linear epitopes, all interaction sites between a protein (e.g., antigen) and an interacting molecule (such as an antibody) are linearly located along the primary amino acid sequence of the protein. In conformal epitopes, interaction sites are located between amino acid residues on the protein that are far apart from each other in the primary amino acid sequence. Once a desired epitope on an antigen is determined, it is possible to generate an antibody against that epitope using techniques well known to those skilled in the art. For example, antibodies against linear epitopes are produced by immunizing animals with a peptide containing the amino acid residues of the linear epitope. Antibodies against conformational epitopes are produced by immunizing animals with a minidomain containing the relevant amino acid residues of the conformational epitope. Antibodies against specific epitopes are also produced by immunizing animals with a target molecule (e.g., IgG or Fab) or a related portion and screening for binding to the epitope.

[0020] By using methods known to those skilled in the art, including but not limited to competitive assays, epitope binning, and alanine scanning, it is possible to determine whether an antibody binds to the same epitope or competes for binding with the anti-hIgG antibody of this disclosure. In some embodiments, the anti-hIgG antibody of this disclosure is bound to hIgG under saturated conditions, and then the ability of the test antibody to bind to hIgG is measured. If the test antibody can bind to hIgG simultaneously with the reference anti-IgG antibody, the test antibody is bound to a different epitope than the reference anti-IgG antibody. However, if the test antibody cannot bind to hIgG simultaneously, the test antibody binds to the same epitope as the anti-IgG antibody of this disclosure, an overlapping epitope, or an adjacent epitope. This experiment can be performed, for example, by ELISA, RIA, BIACORE®, SPR, Biolayer Interference, or Fluoroscopy. This can be performed using itchometry. The above competition method can be used in two directions to test whether an anti-hIgG antibody cross-competes with another anti-IgG antibody, namely, whether a known antibody blocks the test antibody and vice versa. Competition experiments can be performed, for example, using a Biacore® T200 instrument.

[0021] The antigen-binding moieties of anti-hIgG antibodies disclosed herein can be used in place of complete antibodies. The term “antigen-binding moiety” refers to one or more parts or fragments of an antibody that have the ability to specifically bind to an antigen (e.g., human IgG or a fragment thereof). Examples of antigen-binding moieties include, but are not limited to, (i) Fab fragments: monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments: bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of one arm of the antibody; (v) dAb fragments consisting of a VH domain; and (vi) isolated complementarity-determining regions (CDRs) that have the ability to specifically bind to an antigen. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these domains can be linked by a synthetic linker that allows the VL and VH domains to pair up to form a single protein chain (known as single-stranded Fv(scFv)) that forms a monovalent molecule using recombination methods. Other forms of single-stranded antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to pair the two domains on the same chain is used, so the domains are forced to pair with complementary domains on another chain, resulting in two antigen-binding sites. Antibody moieties such as Fab and F(ab')2 fragments can be prepared from whole antibodies using conventional techniques such as papain or pepsin digestion of the whole antibody or recombinant DNA technology.

[0022] This antibody binds to one, more than, or all of the human IgG subtypes hIgG1, hIgG2, hIgG3, and hIgG4. In certain embodiments, this antibody binds to all of the aforementioned subtypes. The antibodies described herein are understood to be capable of binding to humanized and / or chimeric antibodies containing human IgG-derived sequences.

[0023] In some embodiments, the Disclosure provides an anti-hIgG monoclonal antibody or its antigen-binding moiety, wherein the heavy chain CDR1-3 and light chain CDR1-3 comprise SEQ ID NOs. 27-32, 33-38, 39-44, 45-50, 51-56, or 57-62, respectively. This antibody framework can be obtained from antibodies of rabbits or another species (e.g., mouse, human, or rat).

[0024] In some embodiments, the disclosure provides an anti-hIgG monoclonal antibody or its antigen-binding moiety, wherein the heavy chain variable domain (VH) and light chain variable domain (VL) comprise SEQ ID NOs. 13 and 19, 14 and 20, 15 and 21, 16 and 22, 17 and 23, or 18 and 24, respectively. The constant region of this antibody can be obtained from an antibody of a rabbit or another species (e.g., mouse, human, or rat).

[0025] In some embodiments, this disclosure is: a) Heavy chain (HC) containing the amino acid sequences of SEQ ID NOs. 13 and 25, and light chain (LC) containing the amino acid sequences of SEQ ID NOs. 19 and 26; b) HC containing the amino acid sequences of SEQ ID NOs. 14 and 25, and LC containing the amino acid sequences of SEQ ID NOs. 20 and 26; c) HC containing the amino acid sequences of SEQ ID NOs. 15 and 25, and LC containing the amino acid sequences of SEQ ID NOs. 21 and 26; d) HC containing the amino acid sequences of SEQ ID NOs. 16 and 25, and SEQ ID NOs. 22 and 2 LC containing 6 amino acid sequences; e) HC containing the amino acid sequences of SEQ ID NOs. 17 and 25, and LC containing the amino acid sequences of SEQ ID NOs. 23 and 26; or f) HC containing the amino acid sequences of SEQ ID NOs. 18 and 25, and LC containing the amino acid sequences of SEQ ID NOs. 24 and 26. This provides an anti-hIgG monoclonal antibody containing the above.

[0026] In some embodiments, the anti-hIgG antibody or antigen-binding moiety has a VH amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs. 13, 14, 15, 16, 17, or 18.

[0027] In some embodiments, the anti-hIgG antibody or antigen-binding moiety has a VL amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs. 19, 20, 21, 22, 23, or 24.

[0028] In some embodiments, the anti-hIgG antibody or antigen-binding moiety has VH and VL amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs. 13 and 19, 14 and 20, 15 and 21, 16 and 22, 17 and 23, or 18 and 24, respectively.

[0029] In some embodiments, the anti-hIgG antibody has HC and LC containing SEQ ID NOs. 63 and 69, 64 and 70, 65 and 71, 66 and 72, 67 and 73, or 68 and 74, respectively, with or without the leader sequence.

[0030] In some embodiments, the anti-hIgG antibody or antigen-binding moiety of the Disclosure comprises the HCDR1-3 and LCDR1-3, VH and VL or HC and LC amino acid sequences of antibody 2C5, 9E6, 11F9, 11G5, 16F5 or 19B1.

[0031] The assignment of amino acid numbers and CDRs may follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)). See also Zhang above.

[0032] The anti-hIgG antibodies or antigen-binding portions of the present disclosure can be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibody or portion thereof is derivatized such that IgG binding is not adversely affected by the derivatization or labeling. For example, the antibodies or antibody portions of the present disclosure can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or another method) to one or more other molecular entities such as another antibody or a detectable label or tag. Examples include radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophore, phycoerythrin, or Alexa Fluor® dyes), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal-binding domains, epitope tags), and magnetic agents such as gadolinium chelates, but are not limited thereto. In some embodiments, the label is added by spacer arms of various lengths to reduce potential steric hindrance.

[0033] The anti-hIgG antibody and antigen-binding moiety of this disclosure are useful for detecting and / or measuring levels of human IgG or Fab in animal-derived samples (e.g., non-human primates such as cynomolgus monkeys or rhesus monkeys). In some embodiments, the antibody and antigen-binding moiety can be used to detect and / or measure levels of human IgG or Fab in human-derived samples. Suitable detection and measurement methods include immunological methods such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, and immunohistochemistry. In some embodiments, the antibody and antigen-binding moiety can be used to detect and / or measure levels of human IgG or Fab in human-derived samples in preclinical or clinical immunohistochemistry (IHC) studies.

[0034] The rabbit antibodies described herein can bind to different epitopes and can therefore be used alone or in pairs to detect human IgG in any host animal to meet the needs of therapeutic monoclonal antibody development in preclinical trials. For example, antibodies 16F5, 11F9, and 11G5 / 19B1 / 2C5 / 9E6 bind to three different epitopes, which are also different from the epitope of MCA5748G. Therefore, a pair of antibodies selected from those that bind to two different epitopes can be used together to increase assay sensitivity and specificity, for example. For example, 16F5 can be used with 11F9; 16F5 or 11F9 can be used with 11G5, 19B1, 2C5, or 9E6; and MCA5748G can be used with any one of 16F5, 11F9, 11G5, 19B1, 2C5, and 9E6. The paired antibodies are labeled differently.

[0035] Production of anti-hIgG antibodies This anti-hIgG antibody can be produced using a well-known hybridoma technique, which involves fusing rabbit B cells that produce the target antibody with immortalized cells to form an antibody-producing hybridoma cell line.

[0036] Alternatively, the hIgG antibody or its antigen-binding moiety may be prepared by recombinant technology using host cells containing nucleotide sequences encoding the heavy and light chains of the antibody or moiety. Therefore, this disclosure also provides nucleic acid molecules and sequences encoding the anti-IgG antibody or its antigen-binding moiety described herein. The nucleotide sequences encoding the heavy and light chain amino acid sequences are introduced into host cells using two different vectors or the same vector. They are expressed under transcriptional regulation of one promoter or two separate promoters.

[0037] In some embodiments, the nucleic acid molecule includes a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to (i) the nucleotide sequence encoding SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; or (ii) the nucleotide sequence encoding SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

[0038] In the context of amino acid and nucleic acid sequences, the term “percent sequence identity” refers to the residues that are identical in two sequences when aligned to the maximum extent possible. For example, the length of a sequence identity comparison spans at least about 9 nucleotides, typically at least about 18 nucleotides, more commonly at least about 24 nucleotides, generally at least about 28 nucleotides, more commonly at least about 32 nucleotides, preferably at least about 36, 48, or more nucleotides. There are many different algorithms known to those skilled in the art that can be used to measure the identity of amino acid and nucleotide sequences. For example, Polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, programs from Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, and Wisconsin. For example, FASTA, including programs FASTA2 and FASTA3, provides best overlap region alignment and percent sequence identity between the query and search sequences (see, e.g., Pearson, Methods Enzymol. (1990) 183:63-98; Pearson, Methods Mol. Biol. (2000) 132:185-219; Pearson, Methods Enzymol. (1996) 266:227-58; and Pearson, J. Mol. Biol. (1998) 276:71-84; incorporated herein by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with default parameters (word size 6 and NOPAM factor in the score matrix), or using Gap with default parameters provided in GCG Version 6.1, which are incorporated herein by reference.

[0039] In certain embodiments, the disclosure provides a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequences of SEQ ID NOs: 1 and 7, 2 and 8, 3 and 9, 4 and 10, 5 and 11, or 6 and 12.

[0040] In any of the embodiments described above, the nucleic acid molecule is isolated. A nucleic acid molecule referred herein as “isolated” or “purified” is (1) isolated from the nucleic acid of genomic DNA or cellular RNA of its source of origin; and / or (2) is a nucleic acid that does not exist in nature.

[0041] In further embodiments, the disclosure provides vectors suitable for expressing one or both of the chains of the antibodies or their antigen-binding portions described herein. In this specification, the term “vector” means a nucleic acid molecule capable of transporting another ligated nucleic acid. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA to which an additional DNA fragment is ligated. Furthermore, certain vectors are capable of directing the expression of a gene that is operationally ligated. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).

[0042] This disclosure provides a vector comprising a nucleic acid molecule encoding the heavy chain, light chain, or both the heavy chain and light chain of an anti-hIgG antibody or its antigen-binding moiety as described herein. The vector may further comprise an expression regulatory sequence.

[0043] In this specification, the term “regulatory sequence” means a polynucleotide sequence necessary to influence the expression and processing of a ligated coding sequence. Regulatory sequences include appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak common sequences); sequences that enhance protein stability; and, if necessary, sequences that enhance protein secretion. The nature of such regulatory sequences varies depending on the host organism; in prokaryotes, such regulatory sequences generally include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such regulatory sequences generally include promoters and transcription termination sequences. The term “regulatory sequence” is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0044] In some embodiments, the nucleic acid molecules described herein include a nucleotide sequence encoding the VH domain of the anti-IgG antibody or antigen-binding moiety described herein, in-frame connected to a nucleotide sequence encoding a heavy chain constant region derived from any source. Similarly, the nucleic acid molecules described herein may include a nucleotide sequence encoding the VL domain of the anti-IgG antibody or antigen-binding moiety described herein, in-frame connected to a nucleotide sequence encoding a light chain constant region derived from any source.

[0045] In further embodiments of this disclosure, nucleic acid molecules encoding VH and / or VL are “converted” into full-length antibody genes. In some embodiments, nucleic acid molecules encoding VH or VL domains are converted into full-length antibody genes by insertion into an expression vector already encoding a heavy chain constant (CH) or light chain constant (CL) region, respectively, such that the VH portion is operatively ligated to the CH portion in the vector and / or the VL portion is operatively ligated to the CL portion in the vector. In another embodiment, nucleic acid molecules encoding VH and / or VL domains are converted into full-length antibody genes by ligating, for example, ligating the nucleic acid molecule encoding the VH and / or VL domains to a nucleic acid molecule encoding the CH and / or CL region using standard molecular biological techniques. The full-length heavy chain and / or light chain encoding nucleic acid molecules are then expressed from introduced cells to isolate anti-IgG antibodies.

[0046] In some embodiments, the framework region is mutated such that the resulting framework region has the amino acid sequence of the corresponding germ cell gene. Mutations can be made within the framework region or constant region, for example, to increase the half-life of an anti-IgG antibody. See, for example, PCT Publication WO00 / 09560. Mutations in the framework region or constant region can also be made to alter the immunogenicity of the antibody and / or to provide a covalent or non-covalent site to another molecule. According to this disclosure, an antibody may have mutations in any one or more of the CDR of the variable domain or the framework region, or in the constant region.

[0047] This disclosure also provides antibody compositions and antibodies and methods for producing the antigen-binding moieties described herein. In some embodiments, this disclosure relates to a method for producing an anti-IgG antibody or antigen-binding moiety described herein, comprising the steps of: preparing recombinant host cells containing a heavy chain or a nucleotide sequence encoding the antigen-binding moiety and a light chain or a nucleotide sequence encoding the antigen-binding moiety of an anti-IgG antibody or antigen-binding moiety described herein; culturing the host cells under conditions suitable for the expression of the antibody or antigen-binding moiety; and isolating the obtained antibody or antigen-binding moiety. The antibody or antigen-binding moiety produced by such expression in such recombinant host cells is referred to herein as a “recombinant” antibody or antigen-binding moiety. This disclosure also provides progeny of such host cells and the antibody or antigen-binding moiety produced therefrom.

[0048] In this specification, the term “recombinant host cell” (or simply “host cell”) means a cell into which a recombinant expression vector has been introduced. By definition, recombinant host cells do not exist in nature. This disclosure provides, for example, host cells that may contain the vectors described herein. This disclosure also provides, for example, host cells containing the anti-IgG antibody described herein or its heavy chain or the nucleotide sequence encoding its antigen-binding portion, its light chain or the nucleotide sequence encoding its antigen-binding portion, or both. It should be understood that “recombinant host cell” and “host cell” mean not only a specific target cell but also its progeny. Because some modifications may exist in later generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included in the scope of the term “host cell” as described herein. do.

[0049] Suitable mammalian, plant, bacterial, or yeast host cells can be transfected using anti-IgG antibodies and nucleic acid molecules encoding their antigen-binding moieties, as well as vectors containing these nucleic acid molecules. Transformation can be carried out by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known to those skilled in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors.

[0050] Antibodies expressed by different cell lines or in transgenic animals are likely to have different glycosylation patterns. However, all antibodies encoded by nucleic acid molecules provided herein, or containing amino acid sequences provided herein, are part of this disclosure regardless of their glycosylation state, and more generally, regardless of whether or not they have undergone post-translational modifications.

[0051] Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Typical methods and materials are described below, but similar or equivalent methods and materials may also be used in the implementation or testing of this disclosure. In case of any inconsistency, this specification shall prevail, including definitions. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, bacteriology, genetics, analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used by those skilled in the art. Enzyme reactions and purification techniques shall be performed according to the manufacturer's specifications, as generally achieved by those skilled in the art or as described herein. Furthermore, unless contextually required, singular terms shall include plural forms and plural terms shall include singular forms. Throughout this specification and its embodiments, variations of the words “have” and “comprise,” or “has,” “having,” “comprises,” or “comprising,” will be understood to mean that they encompass the integer or group of integers described, but do not exclude any other integer or group of integers. All publications and other references referenced herein are incorporated by reference in their entirety. Many documents are cited herein, but this citation does not authorize any of these documents to form part of the general knowledge common to those skilled in the art.

[0052] Examples are provided below to better illustrate this disclosure. These examples are for illustrative purposes only and should not be construed as limiting the scope of this disclosure in any way. [Examples]

[0053] The following example describes an experiment in which rabbits were immunized with human IgG Fab, splenocytes were isolated, and these were used to sort human IgG-specific B cells. Six human IgG and Fab-specific mAb clones were obtained using cynomolgus monkey IgG as a counter-screening agent. These clones demonstrated superior binding affinity and targeted different epitopes than the only commercially available mouse-derived anti-hIgG mAb clone, MCA5748G. These rabbit anti-hIgG mAb clones were evaluated by the Gyrolab® assay and found to be suitable for use as capture reagents in comprehensive pharmacokinetic assays in the presence of cynomolgus monkey serum. The materials and methods are as follows:

[0054] Chemicals and reagents The therapeutic antibodies used in these experiments were the humanized therapeutic monoclonal antibody IgG1 (hIgG1), the humanized development candidate monoclonal antibody IgG4 (hIgG4), and the internal research reagent human Fab (hFab). Cynomolgus monkey IgG (cynoIgG) was purified from cynomolgus monkey serum purchased from Innovative Research (Novi, MI 48377) by protein A affinity purification. The mouse anti-human IgG monoclonal antibody MCA5748G (Stubenrauch et al., J Pharm Biomed Anal. (2009) 49:1003-1008) was purchased from BioRad Laboratories (Hercules, CA). Cell culture medium and phosphate-buffered saline (PBS) were purchased from ThermoFisher Scientific (Waltham, MA). All other chemicals were of analytical quality.

[0055] ELISA for determining binding specificity The specificity of antibody clones for binding to human immunoglobulin (IgG) was evaluated using enzyme-linked immunosorbent assay (ELISA) with cynoIgG as a control. ELISA was performed at room temperature on ThermoFisher Scientific (Waltham, MA) microtiter plates pre-coated with hIgG1, hIgG4, or cynoIgG in PBS for 1 hour. After washing three times with phosphate-buffered saline-polysorbate 20 (Tween 20), the plates were blocked with PBS / 3% bovine serum albumin for 1 hour. The plates were then washed again and incubated with anti-human IgG antibody clones for 1 hour. After one more washing step, the bound antibodies were detected with Southern Biotech (Birmingham, AL) horseradish peroxidase (HRP) conjugated anti-rabbit IgG antibody according to the manufacturer's instructions.

[0056] Biacore assay for determining binding specificity and kinetics The specificity of rabbit anti-human IgG monoclonal antibodies was evaluated using a second assay system described elsewhere (Chu et al., Sci Rep. (2015) 4:7360). These experiments were performed on a Biacore® T200 instrument (Biacore, Uppsala, Sweden) using Biacore streptavidin or CM5 sensor chips. Antibody coating of streptavidin chips was achieved by injecting biotinylated target antibodies amine-coupled using ThermoFisher Scientific (Waltham, MA) EZ-Link® Amine-PEG11-Biotin reagent according to the manufacturer's instructions. For CM5 chips, the target antigen or antibody was coupled to the chip surface by standard amine coupling using a Biacore amine coupling kit. Unless otherwise specified, all binding and kinetic assays were performed at 25°C in HBS-EP+ buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% volume / volume Surfactant P20). Dissociation determinants (KD) were calculated using a 1:1 Langmuir fitting model with Biacore BIAevaluation software V4.1.

[0057] Rabbit immunization and B cell cloning Wu et al., Nat Cancer (2020) 1: pp. 86-98, hIgG Two rabbits were immunized using Fab with a total of five antigen injections. Complete Freund's adjuvant (CFA) was used for the primary injection, and incomplete Freund's adjuvant (IFA) was used for four booster immunizations. Both CFA and IFA were manufactured by ThermoFisher Scientific (Waltham, MA). Serum titers were measured using antigen swabs. Rabbits were monitored using ELISA with a protein-based approach. Rabbits with higher ELISA titers were selected for splenectomy.

[0058] New splenocytes were isolated from the spleen for the isolation of B cells. Approximately 1.2 × 10⁻⁶ cells were isolated. 8 Individual splenocytes were cultured overnight in a special B-cell medium customized by Yurogen (Worcester, MA) prior to sorting. Splenocytes were enriched with antigen-recognizing B cells by processing them using Yurogen's SMab® platform. Antigen-sorted B cells were seeded at 1 cell / well in 96-well plates and cultured for 10–14 days.

[0059] Antigen-recognizing B cell clones were identified and confirmed using direct ELISA coated with hIgG4, and purified cynoIgG was used in ELISA for counter-screening. Antigen-specific B cell clones were ranked and selected according to the positive / negative ELISA signal ratio, and the heavy and light chains of the IgG coding sequence were amplified by RT-PCR. The heavy and light chain PCR products were combined and used to directly transfect HEK293F cells. Subsequently, transiently expressed recombinant rabbit IgG clones were further confirmed for specific binding to hIgG1, hIgG4, and hFab by ELISA and Biacore binding assays. After confirming specific binding to hIgG1, hIgG4, and hFab, PCR products from selected positive B cells were cloned into mammalian expression vectors to expand antibody production in HEK293F cells. Recombinant rabbit mAb clones produced by HEK293F transfection were purified using protein A chromatography for further evaluation.

[0060] Gyrolab® assay Gyrolab® xPlore from Gyros Protein Technologies (Uppsala Sweden), Bioaffy 1000nL CD, Rexxip A, and Rexxip F buffers were used in all experiments (Fraley et al., Bioanalysis (2013) 5:1765-74). Biotinylated capture antibodies were diluted to 0.1-0.2 μg / μL with Rexxip A buffer, and Bioaffy The streptavidin bead column was run through the CD microstructure. Standard curve and quality control (QC) samples were prepared by spiking hIgG4 or hFab within the indicated range in Rexxip A buffer containing varying amounts of cynomolgus monkey serum. Standard curve samples, QC samples, dummy samples, and assay reagents were added to PCR plates and loaded into a Gyrolab® instrument. One replica of the standard curve, QC sample, or dummy sample was placed into two CD microstructures using a Gyros instrument and then run through the bead column. Alexa fluor 647-labeled goat anti-human IgG(Fc) antibody, purchased from Southern Biotech (Birmingham, AL), was used as the detection reagent at 2 μg / mL in Rexxip F buffer. The streptavidin beads were pre-wetted by running a PBS washing solution containing 0.01% volume / volume Tween-20 through the column before each step, and any unbound reagents were washed off after each step of the assay. Sample concentrations were determined by data acquisition at 1% photomultiplication levels. Results were analyzed using the Gyrolab® Evaluator Program with manufacturer-specified 5-parameter fitting and 1 / Y2 weighting. [Examples]

[0061] Isolation of human IgG-specific rabbit antibody clones This example describes an experiment in which six rabbit antibody clones that recognize both human IgG and Fab but not cynomolgus monkey IgG were developed using rabbit B cell cloning technology.

[0062] Hybridoma screening and presentation methodology were used in the development of rabbit monoclonal antibodies. However, both methods have several drawbacks: hybridoma technology has low cell fusion efficiency, and the presentation method loses the natural congeneral pairs of heavy and light chains (Zhang et al., Front Immunol. (2017) 8:494). To overcome these problems, antibody gene cloning technology (or single B cell cloning) based on single B cells has recently been developed (Seeber et al., PLoS ONE (2014) 9:e86184; Zielonka and Krah (eds.), Genotype Phenotype Coupling. Methods in Molecular Biology, Vol. 2070, Humana, New York, NY, Rashidian et al., "Single B Cell Cloning and Production of Rabbit Monoclonal Antibodies").

[0063] In short, single B cell cloning consists of (i) isolating a specific single B cell from peripheral blood or lymphoid tissue by antigen-based FACS sorting, (ii) growing and expanding the single B cell for two weeks, (iii) amplifying and sequencing the antibody gene by performing RT-PCR using antibody-specific primers, (iv) cloning the antibody gene into an expression vector and producing recombinant monoclonal antibody in a mammalian cell line (e.g., HEK293, CHO cells), and (v) purifying the recombinant monoclonal antibody and evaluating it by ELISA and other in vitro assays.

[0064] Rabbits were immunized using human IgG1 Fab, and the resulting splenocytes were sorted with biotinylated hIgG1, a humanized total IgG1 molecule. A total of 530 primary B cells were seeded one cell per 96-well plate and grown for two weeks. B cell culture media containing monoclonal rabbit IgG antibodies were screened by direct ELISA using hIgG4, hIgG1, and cynoIgG. Based on the ELISA hIgG1 / cynoIgG and hIgG4 / cynoIgG signal values, 17 clones possessing both hIgG1 and hIgG4 OD450 > 0.9 and CynoIgG OD450 < 0.2 were selected (Table 1), and their antibody coding sequences were amplified by PCR.

[0065] [Table 1]

[0066] Of these 17 clones, PCR products for both the heavy and light chains were obtained from 11 of them. The heavy and light chain PCR products from each clone were combined in a 1:1 ratio and used to directly transfect HEK293F cells. The cell culture medium of the transfected HEK293F cells was further confirmed by the same ELISA screen assay. Table 2 shows the binding of six rabbit recombinant antibody clones that specifically bind to whole human IgG (hIgG) and Fab but not to cynoIgG, indicated by the signal ratio of hIgG1 or hIgG4 binding to cynomolgus monkey IgG (cynoIgG) determined by ELISA assay. These six clones exhibited excellent hIgG4 / cynoIgG and hIgG1 / cynoIgG ELISA signal ratios ranging from 4.94 to 14.99.

[0067] [Table 2]

[0068] These six clones were further analyzed by Biacore for direct binding to hIgG1, hIgG4, hFab, and cynoIgG, as shown in Figure 1. All six clones showed varying levels of binding to hIgG1, hIgG4, and hFab, but not to cynoIgG. These data indicate that these clones are specific to human IgG and Fab, but not to cynoIgG. [Examples]

[0069] Sequence of rabbit anti-hIgG antibody To determine the characteristics of the six clones, heavy and light chain PCR products were subjected to DNA sequencing. The putative amino acid sequences of the variable regions of the heavy and light chains were aligned using EMBL-EBI web-based Clustal Omega (Sievers et al., Mol Syst Biol. (2011) 7:539). As shown in Figures 2A and 2B, all six clones possessed unique amino acid sequences for both the heavy and light chains. A phylogenetic tree based on Clustal Omega was constructed to visualize relative distances (Figure 2C). In particular, clone 16F5 diverged significantly from the other five clones in both the heavy and light chains, while clone 11F9 was the next clone to diverge in the heavy chain but did not diverge in the light chain.

[0070] Table 3 shows the nucleotide sequences encoding antibodies 2C5, 9E6, 11F9, 11G5, 16F5, and 19B1 (sequence: SEQ ID NO).

[0071] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0072] Table 4 shows the putative amino acid sequences of the variable domains of antibodies 2C5, 9E6, 11F9, 11G5, 16F5, and 19B1. The complementarity-determining regions (CDRs) are shown in bold and underlined.

[0073] [Table 4]

[0074] Table 5 shows the heavy and light chain constant region amino acid sequences (CH and CL, respectively) of the six antibodies 2C5, 9E6, 11F9, 11G5, 16F5, and 19B1.

[0075] [Table 5]

[0076] Table 6 shows the amino acid sequences of the heavy chain CDRs (HCDRs) and light chain CDRs (LCDRs) of antibodies 2C5, 9E6, 11F9, 11G5, 16F5, and 19B1, where CDRs are defined according to the Kabat numbering system. The sequence number is shown in parentheses.

[0077] [Table 6]

[0078] Table 7 shows the sequence number information for antibodies 2C5, 9E6, 11F9, 11G5, 16F5, and 19B1. Unless otherwise indicated by "nt" (nucleotide), all sequences in the table are amino acid sequences.

[0079] [Table 7] [Examples]

[0080] Binding affinity and epitopes of rabbit anti-hIgG antibodies Amplified PCR products containing heavy and light chain antibody coding sequences were cloned into expression vectors and used for transient transfection and antibody purification. Mouse anti-rabbit mAbs were directly immobilized onto CM5 chips using amine coupling (Chu et al., Sci Rep. (2015) 4:7360). Purified rabbit mAb clones were then injected, followed by 1:2 serial dilutions of hFab, hIgG1, hIgG4, and cynoIgG ranging from 80 to 0 nM.

[0081] As illustrated in Table 8, all clones exhibited sub-nanomolecular strong binding affinity (KD) to hIgG1, hIgG4, and hFab, but not to cynoIgG. However, under the same conditions, the control commercially available clone MCA5748G (Bio-rad, Hercules, CA) was able to bind to hIgG1 and hIgG4, but not to hFab and cynoIgG.

[0082] [Table 8]

[0083] To determine whether the six rabbit anti-hIgG clones possess different epitopes from the only commercially available clone, MCA5748G, MCA5748G, 19B1, and 11F9 clones, as well as a mouse anti-hIgG mAb (assay control), were biotinylated. These biotinylated antibodies were then injected into different flow cells of streptavidin chips to reach a range of 500–600 RU, followed by the flow of 1:2 serially diluted hIgG1 antibodies ranging from 80–0 nM in the presence of 240 nM of a different rabbit mAb clone, which was a competitor. As shown in Figures 3A and 3B, when MCA5748G was used as the capture antibody on the chip surface, competition was observed only with clone MAC5748G itself, indicating that this commercially available mouse anti-hIgG clone MAC5748G possesses different epitopes from all six rabbit anti-hIgG clones disclosed herein.

[0084] When rabbit anti-hIgG clone 19B1 was used as the capture antibody on the chip surface, competition was observed with clones 11G5, 19B1, 2C5, and 9E6. Therefore, the epitopes of these four clones were identical but different from those of clones 11F9 and 16F5. Furthermore, when clone 11F9 was captured on the chip surface, competition was observed only with clone 11F9 itself, not with clone 16F5, and not with any of the other four clones that share the same epitope. Therefore, clone 16F5 also had a different epitope from clone 11F9. These results indicate that clones 16F5 and 11F9 each have their own unique epitopes, while clones 11G5 and 19B1 We showed that 2C5 and 9E6 share the same epitope. Paratopes within the CDR determine the antibody-binding epitope, with the heavy chain playing a dominant role, and the epitope classification of the six rabbit anti-hIgG clones correlates well with phylogenetic relationships (Figure 2C).

[0085] Additional Biacore experiments demonstrated that all six rabbit anti-hIgG clones were capable of binding to each of the ten different human IgG molecules and two Fab molecules tested. The ten antibodies included those containing human IgG1 and IgG4. Since these rabbit anti-hIgG mAb clones were generated using Fab as an immunogen, all of them should bind to the Fab region of IgG. Because different human IgG subtypes (IgG1, IgG2, IgG3, and IgG4) are classified by the Fc sequence, which is not part of the Fab region, all six rabbit anti-hIgG antibody clones should be able to recognize all subtypes of human IgG. This would be a significant advantage if these rabbit anti-hIgG mAbs were used in preclinical trials and / or development to detect different subtypes of therapeutic human IgG molecules (e.g., IgG1, IgG2, and IgG4).

[0086] In summary, amino acid sequence alignment demonstrated that all six clones obtained herein are unique and diverse. They bound to three different epitope groups (16F5; 11F9; and 11G5 / 19B1 / 2C5 / 9E6), exhibiting superior binding affinity and targeting different epitopes than the only commercially available mouse-derived anti-hIgG antibody clone, MCA5748G. Clones with different binding epitopes can be used to develop pairwise human IgG detection assays, such as "sandwich" ELISAs and Gyrolab® assays. [Examples]

[0087] Specific detection of human IgG using rabbit antibodies in the Gyrolab® assay. To test whether rabbit anti-hIgG mAbs could detect human IgG molecules in the presence of monkey serum, biotinylated clone 16F6 was initially used as the capture reagent. Gyrolab® xPlore from Gyros Protein Technologies (Uppsala Sweden), Bioaffy 1000nL CD, Rexxip A, and Rexxip F buffers were used in all experiments. The biotinylated capture antibody was diluted to 0.1–0.2 μg / μL with Rexxip A buffer and then Bioaffy The streptavidin bead column was run through the microstructure of the CD. Standard curves and quality control (QC) samples were prepared by spiking hIgG4 or hFab within the indicated ranges in Gyrolab® Rexxip A buffer mixed with 0%, 4%, 10%, and 25% cynomolgus monkey serum. Using human IgG4 antibody hIgG4, standard curves were prepared in 1:4 dilutions in the assay matrix in the range of 1200–0.3 ng / mL.

[0088] As shown in Figure 4A, no matrix effects were observed in the range of 1200–0.3 ng / mL at cynomolgus monkey serum concentrations of 0–4%. When the assay matrix contained 10% and 25% cynomolgus monkey serum, some slight background signal was observed in the lower half of the standard curve. Therefore, the assay matrix containing 4% cynomolgus monkey serum was further tested with three QCs (750, 40, and 0.6 ng / mL hIgG4) using the same standard curve. As shown in Figures 4B and 4C, typical standard curves were obtained, with two of the three QCs satisfying both <20% bias and <20% CV, although the higher QC was only 0.9% off the bias cutoff (20.9%) and its CV was typical (4.56%).

[0089] The compatibility of other rabbit anti-hIgG clones used as capture reagents in the Gyrolab® assay was further tested. This included the commercially available mouse anti-hIgG mAb MCA574. Similar to the 8G clone, all six rabbit anti-hIgG clones were biotinylated and used at similar concentrations in the Gyrolab® assay. Gyrolab® Comprehensive PK Assay Capture Reagent was used as a control. As shown in Figures 5A and 5B, when the entire antibody molecule hIgG4 was used as the standard (same range 1200–0.3 ng / mL), all capture reagents produced concentration-dependent curves, with the highest background observed with the commercially available clone MCA5748G. However, when the Fab antibody molecule hFab was used as the standard (same range 1200–0.3 ng / mL), all six rabbit anti-hIgG capture reagents similarly produced concentration-dependent curves. In contrast, the commercially available mouse anti-hIgG clone MAC5748G and the control Gyrolab® capture reagent were unable to produce concentration-dependent curves. [Examples]

[0090] Dynamic range of rabbit mAb clones in Gyrolab® assays One advantage of the Gyrolab™ assay is that it exhibits a significantly larger dynamic range compared to surface-based assay platforms such as ELISA and MSD (Fraley et al., Bioanalysis (2015) 5:1765-74). Using human whole antibody hIgG4 again, linear 6-point assay standards were prepared at 1:5 dilutions in the range of 5000–0.32 ng / mL, as shown in Table 9.

[0091] [Table 9]

[0092] Similar Gyrolab® assay procedures were performed, and the data were subsequently extracted and compared. At the upper limit (5000 ng / mL), Gyrolab® capture and rabbit mAb clones 9E6, 2C5, and 19B1 all had acceptable bias (<20) and signal-to-noise ratios greater than 100. The three rabbit anti-hIgG clones showed better signal-to-noise ratios than Gyrolab® capture. At the high endpoint of 5000 ng / mL, clone 11G5 had an acceptable mean bias, but its signal-to-noise ratio was low (63.3%), which was due to a relatively high blank signal level (7.4). For the same reason, the commercially available mAb clone MCA5748 also had a very poor signal-to-noise ratio at the upper limit (24.0).

[0093] These data suggest that clones 9E6, 2C5, and 19B1 exhibit similar dynamic ranges with superior signal-to-noise ratios compared to Gyrolab® capture reagents. The dynamic range of detection by clones 9E6, 2C5, and 19B1 was significantly better than that of the commercially available mouse anti-hIgG clone MCA5748G. Clones 16F5 and 11F9 exhibited an average bias of <20 in the range of 0–1000 ng / mL. It worked well at %, but not at the 5000 ng / mL data point. This may be due to the fact that these two clones possess different binding epitopes than the other four clones.

[0094] Therefore, in the initial Gyrolab® assay, when clone 16F5 was used as the capture reagent, a concentration-dependent assay curve was generated with assay matrices containing up to 25% cynomolgus monkey serum. The assay performed well in the range of 0.30–1200 ng / mL with an assay matrix containing 4% cynomolgus monkey serum. In the extended Gyrolab® evaluation assay, all six rabbit mAb clones demonstrated their ability to function as capture reagents for the detection of both whole human IgG and Fab molecules. However, two control capture reagents, Gyros capture reagent and the commercially available clone MCA5748G, could only detect whole human IgG and not Fab molecules.

[0095] As summarized in Table 10, we developed six rabbit anti-human IgG mAb clones that could bind to both whole human IgG molecules and Fab molecules without binding to cynomolgus monkey IgG. These six clones belonged to three different epitope groups and differed from the commercially available clone MCA5748G. Each of these clones was tested for detection of human IgG and Fab in the Gyrolab® assay, and three of them exhibited a larger dynamic range and signal-to-noise ratio.

[0096] [Table 10] [Examples]

[0097] Use of rabbit anti-human IgG monoclonal antibody clones as comprehensive positive controls for PandA assays Rabbit polyclonal anti-drug antibodies (pAbs) are commonly used as a control for detecting anti-drug antibodies (ADAs). However, pAb generation is time-consuming and requires repeated use of experimental animals. Furthermore, pAbs often exhibit low sensitivity as a control. This example describes the use of two recombinant rabbit anti-human IgG antibodies in a PandA assay. This paper describes a study comparing mAbs, 2C5 and 11G5, with a commercially available mouse anti-Fc mAb (JDC-10; Southern Biotech catalog number 9040-01) as a positive control.

[0098] 2C5 and 11G5 were diluted with JDC-10 in monkey plasma pools at concentrations of 5 μg / mL, 1 μg / mL, 0.75 μg / mL, 0.5 μg / mL, and 0 μg / mL, respectively.

[0099] Plasma samples containing diluted antibodies were first diluted 1:5 in assay buffer (300 mM acetate, 2% BSA) containing an excess of the drug (human IgG4 isotype monoclonal antibody; 10-50 μg / mL). The samples were incubated in a polypropylene plate at 450 rpm at 37°C for 1 hour to form a complex between the drug and the added antibody in the sample. Subsequently, 3% PEG in borate (pH 8.0) was added to each sample, and the samples were incubated overnight at 2-8°C. The final concentration of PEG buffer in each sample was 1.5%.

[0100] The following day, the plate was centrifuged at 4000 rpm for 20 minutes to precipitate the complex in the pellet. The pellet was resuspended in 1.5% PEG in borate (pH 8.0) and centrifuged a second time at 4000 rpm for 20 minutes. The washing cycle was repeated three times. After the final centrifugation, each sample was suspended in 100 μL of 300 mM acetic acid and further diluted to 1:10 (20 μL sample + 180 μL acetic acid) to a final sample dilution of 1:50. The diluted samples were replicated and added to the wells of an MSD High Bind plate at a rate of 25 μL per well, and incubated at 24°C for 1 hour with shaking at 450 rpm.

[0101] After incubation, the plates were washed with 1x plate washing buffer and blocked at 24°C for 1 hour with 3% milk in PBS while shaking. The plates were then washed, and 100 ng / mL sulfo-TAG-Drug was added to the sample and incubated at 24°C for 1 hour with shaking. After the final incubation, the plates were washed with 0.05% Tween in PBS. 2x reading buffer T was then added, and the plates were read using a Sector PR2400. In each sample, the electrochemiluminescence (ECL) signal was proportional to the anti-drug antibody.

[0102] As shown in Figure 6A and Table 11, the two rabbit anti-hIgG clones 2C5 and 11G5 demonstrated a greater dynamic range and sensitivity compared to the commercially available anti-Fc mAb clone JDC-10.

[0103] [Table 11]

[0104] Clones 2C5 and 11G5 also served as control groups and were tested in human, rat, monkey, and mouse plasma and serum matrices. The data show that both 2C5 (Figure 6B) and 11G5 (Figure 6C) could distinguish between human, monkey, mouse, and rat serum, with 2C5 being more sensitive than 11G5 (Table 12). In the human matrix, background was increased in both clones, as expected.

[0105] [Table 12]

[0106] The results above demonstrate that recombinant rabbit anti-human IgG mAbs serve as excellent comprehensive positive controls for preclinical assays, particularly in PandA formats (e.g., monkey, rat, and mouse matrices). These antibodies functioned well in both plasma and serum matrices. The dynamic range and assay sensitivity of rabbit IgG mAbs were improved.

Claims

1. An isolated monoclonal antibody or its antigen-binding moiety that specifically binds to human IgG, The antibody or portion thereof Sequence IDs 27-32, Sequence IDs 33-38, Sequence IDs 39-44, Sequence IDs 45-50, Sequence IDs 51-56 or Sequence IDs 57-62 The monoclonal antibody or its antigen-binding moiety, comprising heavy chain complementarity-determining regions (CDRs) 1-3 and light chain CDRs 1-3, respectively.

2. The antibody or portion thereof Sequence IDs 13 and 19, Sequence IDs 14 and 20, Sequence IDs 15 and 21, Sequence IDs 16 and 22, Sequence IDs 17 and 23, or Sequence IDs 18 and 24 The monoclonal antibody or antigen-binding moiety according to claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), respectively.

3. The monoclonal antibody according to claim 1 or 2, wherein the antibody is a rabbit IgG antibody.

4. A monoclonal antibody according to any one of claims 1 to 3, comprising the heavy chain constant region amino acid sequence of SEQ ID NO: 25 and / or the light chain constant region amino sequence of SEQ ID NO:

26.

5. Includes or does not include the leader array. Sequence IDs 63 and 69, Sequence IDs 64 and 70, Sequence IDs 65 and 71, Sequence IDs 66 and 72, Sequence IDs 67 and 73, or Sequence IDs 68 and 74 The monoclonal antibody according to claim 1, comprising a heavy chain and a light chain having the respective amino acid sequences.

6. A monoclonal antibody or antigen-binding moiety according to any one of claims 1 to 5, further comprising a detectable label.

7. A composition or kit comprising a monoclonal antibody or antigen-binding moiety according to any one of claims 1 to 6 in an aqueous buffer solution.

8. An isolated nucleic acid molecule encoding the heavy chain, the light chain, or both of the monoclonal antibody or antigen-binding moiety according to any one of claims 1 to 6.

9. Includes sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and / or 12 The nucleic acid molecule according to claim 8.

10. Sequence IDs 1 and 7, Sequence IDs 2 and 8, Sequence IDs 3 and 9, Sequence IDs 4 and 10, Sequence IDs 5 and 11, or Sequence IDs 6 and 12 The nucleic acid molecule according to claim 9, comprising:

11. An expression construct comprising a nucleic acid molecule according to any one of claims 8 to 10.

12. A host cell comprising a nucleotide sequence encoding the heavy chain and light chain of a monoclonal antibody or antigen-binding moiety according to any one of claims 1 to 6.

13. The host cell according to claim 12, wherein the host cell is a mammalian cell.

14. A method for producing an antibody or an antigen-binding moiety thereof, comprising the steps of: culturing host cells according to claim 13 under conditions that enable the expression of the heavy chain and light chain of the antibody or a portion thereof; and isolating the antibody or the portion thereof from the cultured cells or the supernatant of a cell culture.

15. A method for detecting human IgG or a fragment thereof in a sample, comprising the step of contacting the sample with one or more monoclonal antibodies or antigen-binding moieties described in any one of claims 1 to 6.

16. The method according to claim 15, wherein the sample is obtained from an animal administered with an antibody or fragment containing the human IgG constant region.

17. The method according to claim 15 or 16, wherein the human IgG steady-state region is the human IgG1, IgG2, IgG3, or IgG4 steady-state region.

18. The method according to claim 15 or 16, wherein the animal is administered a Fab or F(ab')2 fragment of an antibody containing a human IgG1, IgG2, IgG3, or IgG4 constant region.

19. The method according to any one of claims 15 to 18, wherein the sample is a tissue sample, and optionally a blood, serum, or plasma sample.

20. The method according to any one of claims 15 to 19, wherein the animal is a non-human primate, and optionally a crab-eating macaque or a rhesus macaque.

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