Monovalent binding molecule composition for binding to enzyme and use thereof

Abstract

Provided are a monovalent binding molecule composition for binding to an enzyme and use thereof, relating to the field of biotechnology. The monovalent binding molecule composition for binding to an enzyme comprises at least two types of monovalent binding molecules. The composition alleviates the problem that blocked enzymes tend to form multimers.

Description

Monovalent binding molecular compositions for binding enzymes and their applications

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 2024118129519, filed on December 10, 2024, entitled "Monovalent Binding Molecular Composition for Binding Enzymes and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biotechnology, and in particular to monovalent binding molecular compositions for binding enzymes and their applications. Background Technology

[0004] The following statements are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0005] Antibody-blocked hot-start enzymes require monoclonal antibodies against specific polymerases. After the monoclonal antibody binds to the polymerase, it forms a complex that effectively blocks the polymerase activity at room temperature, preventing it from exhibiting polymerization activity at low or normal temperatures. At high temperatures, this complex dissociates, releasing the active polymerase for PCR amplification. This effectively avoids primer dimer formation, reduces the amplification of nonspecific products, and prolongs the long-term stability of the polymerase.

[0006] Taking Taq polymerase antibody as an example, it is a monoclonal antibody targeting Taq DNA polymerase, primarily used to improve the specificity and sensitivity of PCR (polymerase chain reaction). Taq polymerase antibody enhances PCR amplification specificity by inhibiting enzyme activity, mainly in two aspects: First, Taq polymerase antibody can bind to Taq DNA polymerase at low temperatures, thereby inhibiting its polymerization activity. This inhibition effectively prevents non-specific primer annealing and primer dimer formation, reducing non-specific amplification. Second, the antibody-blocked Taq DNA polymerase has a hot-start effect; in the initial stage of the PCR reaction, the Taq polymerase antibody is inactivated during the high-temperature denaturation step, releasing the activity of Taq DNA polymerase. This "hot-start" mechanism ensures that the polymerase does not participate in non-specific reactions before the reaction begins, thus improving amplification specificity. In PCR reactions, Taq polymerase antibody can significantly improve the detection sensitivity of low-abundance genes and the amplification uniformity of complex templates, ensuring effective binding of primers to the correct template during the reaction and improving amplification efficiency. Therefore, Taq polymerase antibodies significantly improve the performance of PCR technology through their unique inhibition mechanism and hot-start characteristics, enabling them to be widely used in many fields such as pathogen detection, genetic disease diagnosis and forensic identification, especially in PCR experiments that require high specificity and high sensitivity.

[0007] The development of Taq polymerase antibodies is a systematic process involving multiple steps such as antigen selection, immunization, cell fusion, antibody screening, and purification. While continuous optimization of these steps has led to the production of highly specific and sensitive Taq polymerase antibodies, greatly promoting the development and application of PCR technology, antibody-modified enzymes still exhibit batch-to-batch variations, storage stability issues, and less-than-expected enzyme performance release due to the introduction of antibody modification.

[0008] In view of this, this disclosure is hereby made. Summary of the Invention

[0009] Taking Taq polymerase as an example, this application found that in practical applications, a full-length bivalent antibody can theoretically bind to two Taq polymerases. However, during double antibody blocking, the two antibodies easily ligate multiple Taq polymerases to form polymers, as shown in Figures 1 and 2. The formation of polymers reduces the stability of the antibody-blocking enzyme, which is detrimental to enzyme storage.

[0010] In specific products, when at least two antibodies are used for blocking, multiple polymers can be generated. It's impossible to guarantee that the degree of polymerization of polymers formed in each batch is consistent, often leading to batch-to-batch variations—significant differences between different batches—which severely impacts product quality stability. Polymer formation also makes antibody-blocked enzymes difficult to store, posing significant challenges to preservation and transportation. Furthermore, polymers significantly increase the molecular weight of individual molecules, making them prone to sedimentation. This defect can result in uneven polymer distribution during use, affecting the performance of the product. There are also issues with blocking performance, which relate to whether the polymerase can effectively function in specific application scenarios. Therefore, how to maintain the blocking ability of antibodies against enzymes while mitigating at least one of the above-mentioned defects remains a problem to be solved.

[0011] The purpose of this disclosure is to provide a monovalent binding molecule composition for binding enzymes and its application, wherein the monovalent binding molecule contained therein can reduce the formation of polymers by modified enzymes, thereby alleviating at least one of the defects in the prior art.

[0012] To solve the above-mentioned technical problems, the present disclosure adopts the following technical solution:

[0013] In a first aspect, a monovalent binding molecular composition is provided, comprising at least two monovalent binding molecules, which can block at least two activities of the same enzyme.

[0014] In a second aspect, a method for modifying an enzyme is provided, the method comprising mixing an enzyme with the monovalent binding molecular composition described in the first aspect.

[0015] Thirdly, a modified enzyme is provided, the modified enzyme comprising the monovalent binding molecular composition and enzyme described in the first aspect.

[0016] Fourthly, a kit is provided comprising the monovalent binding molecular composition described in the first aspect, or the modified enzyme described in the third aspect.

[0017] Fifthly, the use of the monovalent binding molecular composition described in the first aspect, or the enzyme modification method described in the second aspect, or the modified enzyme described in the third aspect, or the kit described in the fourth aspect, in the synthesis of polynucleotides or the preparation of products for the synthesis of polynucleotides is provided.

[0018] In a sixth aspect, a hot-start method for polynucleotide synthesis is provided, the method comprising using an enzyme modified as described in the third aspect to catalyze a polynucleotide synthesis reaction.

[0019] In a seventh aspect, a kit for polynucleotide synthesis is provided, comprising: (Ⅰ) and (Ⅱ);

[0020] (I) The monovalent binding molecular composition and enzyme described in the first aspect, or the modified enzyme described in the third aspect;

[0021] (II) Reagents for polynucleotide synthesis reaction. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this disclosure and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 is a schematic diagram illustrating the principle that the enzyme did not form a multimer when two monovalent antibodies were used to block it.

[0024] Figure 2 is a schematic diagram illustrating the principle of forming polymers using two divalent antibodies blocking enzymes.

[0025] Figure 3 is an SDS-PAGE electrophoresis image of the recombinant antibody prepared in Example 3. Lane 02 is TAQ-6A7, lane 03 is TAQ-11D2, lane 04 is TAQ-6A7TB1, lane 05 is TAQ-11D2TB1, and lanes 01 and 06 are markers.

[0026] Figure 4 shows the fluorescence curves of three batches of antibody blocking enzymes prepared by blocking Taq naked enzyme with full-length antibodies (TAQ-6A7 and TAQ-11D2) in Example 4 during the exonuclease activity blocking performance test.

[0027] Figure 5 shows the fluorescence curves of three batches of antibody blocking enzymes prepared by blocking Taq naked enzymes with Fab (TAQ-6A7TB1 and TAQ-11D2TB1) in Example 4, in the detection of their exonuclease activity blocking performance.

[0028] Figure 6 shows the solubility curves of three batches of antibody blocking enzymes prepared by blocking Taq naked enzyme with full-length antibodies (TAQ-6A7 and TAQ-11D2) in the polymerase activity blocking assay in Example 4.

[0029] Figure 7 shows the solubility curves of three batches of antibody blocking enzymes prepared by blocking Taq naked enzymes with Fab (TAQ-6A7TB1 and TAQ-11D2TB1) in the polymer activity blocking assay in Example 4.

[0030] Figure 8 shows the chromatogram of the full-length antibody blocking enzyme in Example 4 characterized by SEC-HPLC;

[0031] Figure 9 is a chromatogram of Fab blocked in Example 4 and characterized by SEC-HPLC;

[0032] Figure 10 shows the DLS pattern of the first batch of full-length antibody blocking enzyme in Example 4;

[0033] Figure 11 shows the DLS pattern of the second batch of full-length antibody blocking enzyme in Example 4;

[0034] Figure 12 shows the DLS pattern of the third batch of full-length antibody blocking enzyme in Example 4;

[0035] Figure 13 shows the DLS pattern of the first batch of Fab blocking enzyme in Example 4;

[0036] Figure 14 shows the DLS pattern of the second batch of Fab blocking enzyme in Example 4;

[0037] Figure 15 shows the DLS pattern of the third batch of Fab blocking enzyme in Example 4;

[0038] Figure 16 shows the fluorescence curves of the antibody blocking enzymes prepared by blocking Taq naked enzyme with full-length antibodies (TAQ-6J12 and TAQ-10C13) in Example 8 in the detection of their exonuclease activity blocking performance.

[0039] Figure 17 shows the fluorescence curves of the antibody blocking enzymes prepared by blocking Taq naked enzymes with Fab antibodies (TAQ-6J12TB1 and TAQ-10C13TB1) in Example 8 during the detection of their exonuclease activity blocking performance.

[0040] Figure 18 shows the fluorescence curves of the antibody blocking enzymes prepared by blocking Taq naked enzyme with full-length antibodies (TAQ-6J12 and TAQ-10C13) in Example 8 during the detection of their blocking performance in polymer activity.

[0041] Figure 19 shows the fluorescence curves of the antibody blocking enzymes prepared by blocking Taq naked enzymes with Fab antibodies (TAQ-6J12TB1 and TAQ-10C13TB1) in Example 8 during the detection of their blocking performance in polymer activity.

[0042] Figure 20 shows the chromatogram of the full-length antibody blocking enzyme in Example 8 characterized by SEC-HPLC;

[0043] Figure 21 is a chromatogram of the Fab antibody blocking enzyme in Example 8 characterized by SEC-HPLC;

[0044] Figure 22 is a chromatogram of the T1 blocking enzyme in Example 8 characterized by SEC-HPLC;

[0045] Figure 23 is a chromatogram of the T2 blocking enzyme in Example 8 characterized by SEC-HPLC;

[0046] Figure 24 shows the DLS pattern of the full-length antibody blocking enzyme in Example 8;

[0047] Figure 25 shows the DLS pattern of the Fab antibody blocking enzyme in Example 8;

[0048] Figure 26 shows the qPCR amplification of the full-length antibody (TAQ-6A7 and TAQ-11D2) blocking enzyme in Example 9;

[0049] Figure 27 shows the qPCR amplification of the Fab (TAQ-6A7TB1 and TAQ-11D2TB1) blocking enzymes in Example 9;

[0050] Figure 28 shows the amplification NTC of the full-length antibody enzyme and Fab antibody enzyme in Example 9. Detailed Implementation

[0051] The technical solutions of this disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0052] In this article, "a," "two," "multiple," "one," "two," and "more than" are used to describe the category or quantity of substances referred to. Their intended meaning can be determined by the context. For example, "at least two" means the presence of two, three, or more; "at least two monovalently bound molecules" means the presence of two, three, or more monovalently bound molecules, each with a different amino acid sequence composition; "at least two activities" means two, three, or more activities; "the same enzyme" refers to enzymes with the same or substantially the same amino acid sequence.

[0053] In this article, “each…independently selected”, “…independently selected respectively”, and “…independently selected” are interchangeable and should all be interpreted broadly. They refer to the range or options that each member of a set of variables or components can choose independently, that is, the choice of each variable or component is independent and is not affected by the choice of other variables or components.

[0054] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0055] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0056] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0057] In this paper, a "domain" refers to a specific region within a molecule that folds into a relatively independent structural unit in three-dimensional space, possessing specific functions and stability. A domain typically consists of one or more continuous portions of a polypeptide chain. These portions may not be continuous in the amino acid sequence of a protein, but they are close to each other in the three-dimensional structure, folding into a relatively independent structural unit. Generally, a domain is responsible for a single functional property and, in many cases, can be added to, removed from, or transferred to other molecules without losing the function of the rest of the molecule and / or the domain itself.

[0058] In this article, the terms “binding,” “specific binding,” or “specifically binding” refer to a non-random binding reaction between two molecules or a blocking effect between two molecules, such as the reaction between an antibody and an antigen, or, for example, the blocking reaction or modification of an enzyme by an antibody.

[0059] In this document, the term "antibody" includes any immunoglobulin capable of binding to a specific antigen. The term "antibody" is used in the broadest sense to encompass a wide range of antibody structures, including but not limited to monoclonal / polyclonal antibodies, monospecific / multispecific antibodies, full-length antibodies, nanobodies, and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Typically, a natural, complete antibody contains two heavy (H) chains and two light (L) chains. Based on the presence or absence of α, δ, ε, γ, and μ heavy chains, antibodies can be classified into five main categories or isotypes: IgA, IgD, IgE, IgG, and IgM. Several major antibody categories can also be subdivided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain), etc. Each heavy chain consists of a variable region (heavy chain variable region, VH) and first, second, third, and fourth (optionally) constant regions (CH1, CH2, CH3, and CH4, respectively). Mammalian light chains can be divided into λ or κ, and each light chain consists of a variable region (light chain variable region, VL) and a constant region (CL).

[0060] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing antigens. The heavy chain variable region can be called "VH," and the light chain variable region can be called "VL." The variable region contains antigen-binding sites. Both the heavy and light chain variable regions consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The extent of the backbone region and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia. Any CDR determination method well-known in the art, including combinations of methods, can identify CDRs of variable domains. CDRs in each chain are held together closely by FRs to form variable regions. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by linking the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0061] The CDR boundaries of antibodies or their antigen-binding fragments described herein can be defined or identified according to the definitions of IMGT, Kabat, Chothia, AbM, and Contact. CDRs defined in other ways acceptable in the art are also within the scope of protection of this disclosure (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains. Dev. Comp. Immunol. 29, 185-203, (2005); RM MacCallum et al., Antibody–antigen interactions: contact analysis and binding site topography J. Mol. Biol. (1996); Martin, ACR Protein sequence and structure analysis of antibody variable domains (Book chapter). In Antibody engineering lab manual Eds. Duebel, S. and Kontermann, R. (2001); Marie-Paule Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Developmental and Comparative Immunology 27 (2003) 55–77).

[0062] In this document, "monovalent binding molecule" refers to any molecule that has only one antigen-binding site and can bind only one antigen epitope. Monovalent binding molecules can include antibodies, antigen-binding fragments of antibodies, CDR graft molecules, or conjugates thereof as defined in this disclosure, or aptamers, including peptide aptamers or nucleic acid aptamers. In some embodiments, the antigen-binding portion of a monovalent binding molecule can be a variable domain (variable region) of an antibody or a variant thereof, such as a nanobody (VHH), a heavy chain variable region (VH) and / or a light chain variable region (VL) of an antibody, or a variant thereof. In some embodiments, there can be two monovalent antibodies, two monovalent aptamers, or a combination of one monovalent antibody and one monovalent aptamer. Different monovalent binding molecules can be identified by differences in their amino acid sequences, especially by differences in the amino acid sequences at their antigen-binding sites, for example, different CDR sequences of monovalent antibodies. In this document, a monovalent antibody refers to any antibody or antigen-binding fragment with only one antigen-binding site; common monovalent antibodies include Fab, Fab', scFV, Fv, or VHH, but are not limited to these.

[0063] In this paper, the term "single-variable domain (VD)" refers to the smallest functional unit constituting the antigen-binding site of an antibody molecule. A single-variable domain can form a variable domain (which can be a heavy-chain or light-chain domain, including VH, VHH, or VL domains) that serves as a functional antigen-binding site without interacting with other variable domains (e.g., in the absence of the required VH / VL interaction between the VH and VL domains of a conventional tetrachain monoclonal antibody). Examples of "single-variable domains" include nanobodies, shark-derived single-domain antibody IgNARs, (single-domain) antibodies (such as dAbs™) that are VH domains or derived from VH domains, and (single-domain) antibodies (such as dAbs™) that are VL domains or derived from VL domains. Immunoglobulin single-variable domains based on and / or derived from heavy-chain variable domains (such as VH or VHH domains) are often optional. A specific example of a single-variable domain is "VHH" as defined below.

[0064] In this paper, the terms "nanobody" and "VHH" refer to single-domain antibodies obtained by cloning heavy chain antibodies that have lost their light chains (e.g., derived from camels or sharks), which are the smallest functional antigen-binding fragments. Nanobodies are characterized by their small molecular weight, high stability, good solubility, ease of expression, and low immunogenicity.

[0065] It should be noted that, in order to distinguish the variable region of heavy chain antibodies that have lost the light chain from the variable region of the heavy chain in a typical four-chain antibody (two heavy (H) chains and two light (L) chains), the variable region of heavy chain antibodies that have lost the light chain is denoted as VHH, and the variable region of the heavy chain in a four-chain antibody is denoted as VH.

[0066] In this document, the term "antigen-binding fragment" refers to a substance containing all or part of the antibody's CDR (Cellular Dependent Ratio), lacking at least some amino acids present in the full-length chain but still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with parental antibodies for binding to the same epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, and single-chain antibody molecules (scFv). Antigen-binding fragments can be prepared through recombinant expression or enzymatic digestion.

[0067] In this paper, the term "Fab" in antibody refers to a portion of an antibody composed of a single light chain (including variable and constant regions) and a single heavy chain whose variable region and first constant region (CH1) are linked by disulfide bonds. Fd refers to a fragment in the heavy chain composed of the variable region and first constant region (CH1) of the heavy chain. An "Fv fragment" is composed of the variable region of a single light chain and / or the variable region of a single heavy chain. A "single-chain Fv antibody" or "scFv" refers to an antibody fragment formed by the direct interconnection of the variable regions of the light chain and the heavy chain, or by linkage through peptide linker sequences. The "smallest recognition unit of an antibody" refers to a single CDR structure containing only the variable region; although the smallest recognition unit has a small molecular weight and low affinity, it possesses the ability to bind to the antigen.

[0068] In this article, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common natural amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0069] In this document, the term "epitope" or "antigenic epitope" refers to any antigenic determinant on an antigen that is bound to the complementary site of an antibody. An antigenic determinant is typically a specific chemical group with a defined composition and structure. Epitopes can be linear (i.e., continuous) or conformational (i.e., consisting of spaced-apart amino acid residues, discontinuous). An epitope defines the minimum binding site of an antibody and is therefore a specific target for the antibody or its antigen-binding fragment. Epitopes can be determined by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structural assays (e.g., nuclear magnetic resonance spectroscopy).

[0070] In this document, the terms "specific recognition," "selective binding," "selective binding," and "specific binding," or similar expressions, refer to the binding of an antigen-binding molecule to a pre-defined epitope on an antigen. Typically, antigen-binding molecules are expressed in quantities of approximately less than 10⁻⁵ M, such as approximately less than 10⁻⁵ M, 10⁻⁶ M, 10⁻⁶ M, etc. - 7 M, 10 -8 M, 10 -9 M or 10 -10M or smaller Kd values ​​are required for binding. The Kd value of an antibody can be determined using methods well-established in the art. Other standard assays for evaluating the binding ability of ligands, such as antibodies, to targets are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry analyses.

[0071] In this document, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, it may optionally encode either the sense or antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0072] In this article, "polymerization-active domain" refers to the domain in an enzyme that is responsible for catalyzing the polymerization reaction of deoxyribonucleotides and / or ribonucleotides to form new nucleic acid chains.

[0073] In this document, "exonuclease active domain" refers to a domain in an enzyme that can sequentially cleave deoxyribonucleotides and / or ribonucleotides from the ends of a nucleic acid chain. In some embodiments, the exonuclease active domain bound by a monovalent binding molecule is an exonuclease active domain having 5'→3' exonuclease activity or 3'→5' exonuclease activity.

[0074] In this article, "blocking" refers to the binding of an enzyme's domain to a binding molecule, resulting in a temporary reduction or disappearance of the enzyme's activity. For example, a blocked enzyme is one that loses all or part of its activity compared to an unblocked enzyme.

[0075] In this document, "activity" refers to the catalytic function of an enzyme. Typically, an enzyme possesses one or more activities. Common activities of molecular enzymes include polymerization activity (e.g., DNA polymerization, RNA polymerization), exonuclease activity (e.g., 5'→3' exonuclease activity, 3'→5' exonuclease activity), and RNase H activity. The generation or inactivation of activity usually originates from the action of specific structural domains of the enzyme. For example, as demonstrated in the examples herein, the compositions of this application can block at least two activities (e.g., exonuclease activity and polymerization activity).

[0076] In this document, the enzyme in any embodiment includes a wild-type, mutant, or modified enzyme. Wild-type refers to an enzyme having a natural amino acid sequence; mutant enzyme refers to an enzyme obtained by mutating one or more sites in the amino acid sequence of a wild-type enzyme.

[0077] In this article, "thermally stable enzyme" refers to an enzyme that is relatively stable to heat. Thermostable enzymes can tolerate high-temperature environments, typically above 50°C, such as maintaining activity at 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or even higher temperatures. Blocked enzymes may lose all or part of their activity at suitable temperatures, but can recover or partially recover their activity at a heat-start temperature.

[0078] Firstly, this paper provides a monovalently bound molecular composition.

[0079] In some embodiments, the monovalent binding molecular composition comprises at least two monovalent binding molecules, the composition being capable of blocking at least two activities of the same enzyme. For example, as demonstrated in the examples herein, two monovalent binding molecules can block at least two activities (exoclease activity and polymerization activity) of the same enzyme. In some embodiments, the composition can block at least polymerization activity and / or exoclease activity.

[0080] In some embodiments, the monovalent binding molecular composition comprises at least two monovalent binding molecules, the composition being capable of binding at least two different domains of the same enzyme.

[0081] In some embodiments, each monovalent binding molecule in the monovalent binding molecular composition binds only one antigenic epitope, avoiding the formation of polymers during enzyme binding.

[0082] In some embodiments, each enzyme domain is independently selected from a polymerization active domain, an exonuclease active domain, a palm domain, a finger domain, a thumb domain, a linker domain, an N-terminal domain, a reverse transcription active domain, or an RNase H domain.

[0083] In some embodiments, the exolytic active domain has 5'→3' exolytic activity or 3'→5' exolytic activity.

[0084] In some embodiments, the enzyme includes a thermostable enzyme.

[0085] In some embodiments, the enzyme includes polymerase or reverse transcriptase.

[0086] In some embodiments, the enzyme includes a thermostable DNA polymerase or an RNA reverse transcriptase.

[0087] In some embodiments, the thermostable DNA polymerase includes, but is not limited to, Taq DNA polymerase, Pfu DNA polymerase, Tth DNA polymerase, Bst DNA polymerase, KOD DNA polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, 9°N-30 DNA polymerase, Phusion DNA polymerase, or Bca DNA polymerase.

[0088] In some embodiments, the thermostable DNA polymerase is Taq DNA polymerase, which is also referred to herein as Taq enzyme.

[0089] In some embodiments, the thermostable RNA reverse transcriptase includes MMLV reverse transcriptase, AMV reverse transcriptase, HIV-1 reverse transcriptase, and SIV reverse transcriptase.

[0090] In some embodiments, the monovalent binding molecular composition comprises at least two monovalent binding molecules, at least one monovalent binding molecule specifically binding a polymerization active domain, and / or, at least one monovalent binding molecule specifically binding an exotropic active domain.

[0091] In some embodiments, the monovalent binding molecular composition comprises two or more monovalent binding molecules that specifically bind polymeric active domains; different monovalent binding molecules that specifically bind polymeric active domains may specifically bind different antigenic epitopes of polymeric active domains; or specific binding polymeric active domains may be specific binding polymeric active domains to antigenic epitopes but have different antigen-binding domains, for example, different CDR sequences or different molecular structures; or specific binding polymeric active domains may be specific binding polymeric active domains to antigen epitopes and have the same antigen-binding domains, but other regions may differ, such as constant regions or backbone regions.

[0092] In some embodiments, the monovalent binding molecular composition comprises two or more monovalent binding molecules that specifically bind exonuclease active domains. Different monovalent binding molecules that specifically bind exonuclease active domains may specifically bind to different antigenic epitopes of the exonuclease active domains; or they may specifically bind to the same antigenic epitopes of the exonuclease active domains but have different antigen-binding domains, for example, different CDR sequences or different molecular structures; or they may specifically bind to the same antigenic epitopes of the exonuclease active domains and have the same antigen-binding domains, but differ in other regions, such as constant regions or backbone regions.

[0093] In some embodiments, the monovalent binding molecular composition comprises two monovalent binding molecules, one of which specifically binds to a polymeric active domain and the other of which specifically binds to an exonucleotide active domain.

[0094] In some embodiments, the monovalent binding molecule does not contain part or all of the antibody constant region.

[0095] In some embodiments, the monovalent binding molecule does not contain at least one of the antibody's CH1, CH2, CH3, and CH4.

[0096] In some embodiments, the monovalent binding molecule does not contain at least one of the antibody's CH2, CH3, and CH4.

[0097] In some embodiments, the monovalent binding molecule does not contain the hinge region of the antibody.

[0098] In some embodiments, the monovalently bound molecules are independently selected from Fab, Fab', scFV, Fv, or VHH.

[0099] In some embodiments, the monovalently bound molecules are not linked together. It is understood that the monovalently bound molecules are independent of each other, without any linker to fuse them; for example, there are no linking peptides between the monovalently bound molecules.

[0100] In some implementations, at least one monovalently bound molecule is Fab or VHH.

[0101] In some embodiments, the monovalent binding molecular composition comprises two monovalent binding molecules, one of which is a Fab or VHH that specifically binds to a polymeric active domain, and the other of which is a Fab or VHH that specifically binds to an exotropic active domain.

[0102] In some embodiments, the monovalent binding molecular composition comprises two monovalent binding molecules, one of which is a Fab that specifically binds to a polymeric active domain, and the other of which is a Fab that specifically binds to an exocleent active domain.

[0103] In some embodiments, the monovalent binding molecular composition comprises two monovalent binding molecules, one of which is a VHH that specifically binds to the polymeric active domain, and the other of which is a VHH that specifically binds to the exocleent active domain.

[0104] In some embodiments, the binding molecule can be any molecule capable of binding to the enzyme, such as any antibody capable of binding to different domains of the enzyme. The sequences of these antibodies are not limited, as long as they are modified into a monovalent antibody form to implement this disclosure. For example, antibodies binding to Taq enzymes can be selected from the following antibodies and modified into a monovalent antibody form.

[0105] The Taq enzyme antibody can be selected from any antibody in patent CN202510840492.3 or PCT / CN2025 / 102588.

[0106] In some embodiments, the monovalent binding molecule includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the heavy chain variable region of the heavy chain sequence shown in SEQ ID NO. 5 or 7; and the light chain variable region is selected from the light chain variable region of the light chain sequence shown in SEQ ID NO. 6 or 8.

[0107] In some embodiments, the antibody may block polymerization activity. In some embodiments, the antibody may block exonuclease activity. In some aspects, some antibodies may block both polymerization and exonuclease activity.

[0108] In some embodiments, the heavy chain variable region of the monovalently bound molecule is selected from SEQ ID NO.1 or 3; the light chain variable region of the monovalently bound molecule is selected from SEQ ID NO.2 or 4.

[0109] In some implementations, the constant region of the full-length antibody is selected from the constant region of any species.

[0110] In some embodiments, the monovalently bound molecule comprises three CDRs of the heavy chain and three CDRs of the light chain; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 5 or 7, and the amino acid sequence of the light chain is shown in SEQ ID NO: 6 or 8.

[0111] In some embodiments, the monovalently bound molecule comprises three CDRs of the heavy chain variable region and three CDRs of the light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 or 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2 or 4.

[0112] In some embodiments, the composition does not contain bivalent or multivalent binding molecules capable of blocking the same enzyme. For example, as described in the examples herein, bivalent or multivalent binding molecules can induce aggregate formation.

[0113] The first aspect provides a monovalent binding molecule composition for binding enzymes, in which the monovalent binding molecule binds only one antigenic epitope, avoiding the aggregation and formation of polymers after modification of enzymes by multivalent binding molecules (such as conventional bivalent tetrachain antibodies). This monovalent binding molecule composition significantly reduces polymer formation during use, improving the stability of antibody-blocking enzymes. Enzymes modified with this monovalent binding molecule composition exhibit better stability, with a significantly reduced amount of precipitation after repeated freeze-thaw cycles.

[0114] Secondly, this paper also provides a method for modifying enzymes.

[0115] In some embodiments, the method of modifying the enzyme includes mixing the monovalent binding molecular composition described in the first aspect with the enzyme.

[0116] In some embodiments, the method of modifying the enzyme includes binding the same enzyme using the monovalent binding molecular composition described in the first aspect.

[0117] In some embodiments, the enzyme includes a polymerase; in other embodiments, the enzyme is a DNA polymerase, such as Taq DNA polymerase.

[0118] In some embodiments, the method of modifying the enzyme includes using the monovalent binding molecular composition described in the first aspect to bind at least two different domains of the same enzyme.

[0119] In an optional embodiment, the modification method includes contacting the enzyme with the monovalently bound molecular composition.

[0120] In an optional embodiment, the modification method includes mixing the enzyme with the monovalently bound molecular composition and reacting for at least 1 hour.

[0121] In an optional embodiment, the enzyme includes Taq polymerase, and the monovalent binding molecules in the monovalent binding molecule composition are all Fab, and the molar concentration ratio of Taq polymerase to each Fab is independently (1-2):(0.5-2).

[0122] Thirdly, this document provides a modified enzyme comprising the monovalent binding molecular composition described in the first aspect and an enzyme defined in any embodiment of the first aspect. In some embodiments, the enzyme comprises a polymerase; in some embodiments, the enzyme is a DNA polymerase, such as Taq DNA polymerase.

[0123] In some respects, compared to using at least two full-length antibodies to modify the enzyme, the enzyme modified in this disclosure has at least one or more of the following properties: improved sedimentation and aggregation, batch-to-batch variation, stability, and amplification efficiency.

[0124] Fourthly, this document provides a kit comprising the monovalent binding molecular composition described in the first aspect, or the modified enzyme described in the third aspect.

[0125] In some embodiments, the enzyme includes a polymerase; in other embodiments, the enzyme is a DNA polymerase, such as Taq DNA polymerase.

[0126] In some embodiments, the kit further comprises reagents for the modification reaction, including but not limited to one or more of buffer components, salts, stabilizers, and surfactants.

[0127] The kits can be in any form that can carry the material, including but not limited to boxes, cards, tubes, strips, etc.

[0128] Fifthly, this document provides the application of the monovalent binding molecular composition described in the first aspect, or the enzyme modification method described in the second aspect, or the modified enzyme described in the third aspect, or the kit described in the fourth aspect in the synthesis of polynucleotides or the preparation of products containing polynucleotides.

[0129] Polynucleotide synthesis is the foundation of nucleic acid amplification and its applications, such as nucleic acid detection and library construction.

[0130] In some embodiments, the nucleic acid detection includes one or more of reverse transcription-based RNA detection, isothermal amplification-based nucleic acid detection, and polymerase chain reaction-based nucleic acid detection.

[0131] In some embodiments, the library amplification is applied to one or more of whole-genome sequencing, whole-exome sequencing, transcriptome sequencing, targeted sequencing, or epigenetic sequencing.

[0132] Sixthly, this article provides a hot-start method for polynucleotide synthesis, the method comprising using an enzyme modified as described in the third aspect to catalyze a polynucleotide synthesis reaction.

[0133] Seventhly, this article provides a kit for polynucleotide synthesis, comprising: (I) and (II):

[0134] (I) The monovalent binding molecular composition and enzyme described in the first aspect, or the modified enzyme described in the third aspect;

[0135] (II) Reagents for polynucleotide synthesis reaction.

[0136] In some embodiments, the polynucleotide synthesis reaction reagents include, but are not limited to, one or more of the following: buffer components, salts, surfactants, preservatives, dATP, dCTP, dGTP, dTTP, dATP, other enzymes not modified by the monovalently bound molecular composition, solvents, primers, probes, positive controls, negative controls, blank controls, quality control materials, standards, and solid-phase carriers. The other enzymes not modified by the monovalently bound molecular composition serve other functions and include, but are not limited to, one or more of the following: UNG enzymes, UDG enzymes, recombinases, single-stranded DNA-binding proteins, and strand displacement DNA polymerases.

[0137] In some embodiments, the polynucleotide synthesis described in the fourth, fifth, and seventh aspects each independently includes one or more of the following: reverse transcription to synthesize DNA, isothermal amplification to synthesize DNA (e.g., loop-mediated isothermal amplification (LAMP), cross-primer amplification (PCA), strand substitution amplification (SDA), rolling circle amplification (RCA), or recombinase polymerase amplification (RPA), etc.), and polymerase chain reaction (PCR) to synthesize DNA (e.g., conventional PCR, fluorescent PCR (qPCR), or digital PCR (dPCR), etc.).

[0138] In some embodiments, any of the enzymes used for modification by the binding molecular composition in the second to seventh aspects is an enzyme as defined in any embodiment of the first aspect.

[0139] In some embodiments, the polynucleotide synthesis includes polymerase chain reaction (PCR) to synthesize DNA.

[0140] In some embodiments, the enzyme in the polynucleotide synthesis reaction system includes the modified Taq polymerase described in the third aspect, wherein the modification sites of the modified Taq polymerase are a polymerization active domain and an exonuclease active domain.

[0141] In some embodiments, this document also provides a nucleic acid molecule that independently encodes various monovalent binding molecules in the monovalent binding molecule composition as described in the first aspect, or simultaneously encodes various monovalent binding molecules in the monovalent binding molecule composition as described in the first aspect. For example, independent encoding means that a first nucleic acid molecule encodes a first monovalent binding molecule, a second nucleic acid molecule encodes a second monovalent binding molecule, and so on; simultaneous encoding means that the nucleic acid molecule simultaneously encodes a first monovalent binding molecule and a second monovalent binding molecule, or more.

[0142] In some embodiments, the nucleic acid molecule may be a combination of one or more nucleic acid molecules.

[0143] In some embodiments, recombinant vectors are also provided herein. The recombinant vectors independently contain nucleic acids of various monovalent binding molecules in the monovalent binding molecular composition as described in the first aspect, or contain nucleic acids simultaneously encoding various monovalent binding molecules in the monovalent binding molecular composition as described in the first aspect. For example, independently containing means that the first recombinant vector contains a first nucleic acid molecule, the second recombinant vector contains a second nucleic acid molecule, and so on; simultaneously containing means that the recombinant vector simultaneously contains a first nucleic acid molecule and a second nucleic acid molecule, or more.

[0144] In some embodiments, the recombinant vector can be a combination of one or more vectors. For example, the coding sequence of the light chain variable region and the coding sequence of the heavy chain variable region Fd segment of the monovalent binding molecule can be located in the same recombinant vector or in two separate recombinant vectors, preferably in two separate recombinant vectors.

[0145] In some embodiments, when ligating the aforementioned nucleic acid molecules to a vector, the nucleic acid molecules can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the aforementioned nucleic acid molecules. The vector may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. The vector may also include components that facilitate its entry into cells, including but not limited to viral particles, liposomes, or protein coats. The vector can be an expression vector or a cloning vector. The expression vector can be derived directly from the vector itself or it can be exogenous, i.e., not derived from the vector itself. Of course, the aforementioned nucleic acid molecules and control elements only need to be operatively linked. In this document, "operatively linked" means linking a foreign gene to the vector so that the control elements within the vector, such as transcription control sequences and translation control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene.

[0146] In some implementations, the aforementioned recombinant vector is a plasmid expression vector.

[0147] In some embodiments, recombinant cells are also provided herein. The recombinant cells independently contain nucleic acids of various monovalently binding molecules in the monovalently binding molecular composition as described in the first aspect, or simultaneously contain nucleic acids of various monovalently binding molecules in the monovalently binding molecular composition as described in the first aspect. For example, independently containing means that the first recombinant cell contains a first nucleic acid molecule, the second recombinant cell contains a second nucleic acid molecule, and so on; simultaneously containing means that the recombinant cell simultaneously contains a first nucleic acid molecule and a second nucleic acid molecule, or more.

[0148] In some embodiments, the aforementioned recombinant cells can be prokaryotic cells, eukaryotic cells, or bacteriophages. The aforementioned prokaryotic cells include, but are not limited to, *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*. The aforementioned eukaryotic cells include fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*; insect cells such as *Ardisia crenata*; plant cells such as tobacco; mammalian cells such as BHK cells, CHO cells, COS cells, NSO cells, 293 series cells, HepG2, HEK293 cell lines, Huh7 cells, and myeloma cells, but do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0149] This article also provides a method for preparing the monovalently bound molecular composition as described in the first aspect, which includes: culturing the aforementioned recombinant cells.

[0150] In some embodiments, the preparation method expresses each monovalently bound molecule independently or simultaneously.

[0151] The present disclosure is illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present disclosure in any way.

[0152] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially or obtained from Feipeng Biotechnology.

[0153] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0154] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.C. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0155] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0156] Example 1: Construction of full-length mouse IgG antibody expression plasmids TAQ-6A7 and TAQ-11D2

[0157] 1.1 Preparation of the variable region gene of TAQ-6A7 and TAQ-11D2 antibodies

[0158] mRNA was extracted from hybridoma cell lines secreting TAQ-6A7 and TAQ-11D2 monoclonal antibodies, respectively. The DNA products were obtained by RT-PCR and inserted into the pMD-18T vector. The cells were then transformed into DH5α competent cells. After bacterial growth, four positive clones of the heavy chain and four positive clones of the light chain genes were sent to a gene sequencing company for sequencing.

[0159] 1.2 Sequence analysis of the variable region genes of TAQ-6A7 and TAQ-11D2 antibodies

[0160] The gene sequences obtained from the sequencing were analyzed in the KABAT antibody database, and the VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct.

[0161] 1.3 Construction of recombinant antibody expression plasmid

[0162] pcDNA TM 3.4 The vector is the constructed recombinant antibody eukaryotic expression vector, which was modified to introduce polyclonal restriction enzyme sites, and will be referred to as the 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers for TAQ-6A7 and TAQ-11D2 antibodies were designed, with restriction endonuclease sites and protective bases at both ends, respectively. The TAQ-6A7 gene, TAQ-11D2 gene and the constant region gene of the corresponding subtype were amplified by overlap PCR to amplify the light chain gene fragment and the heavy chain gene fragment of the correct length.

[0163] The heavy chain and light chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was also double-digested with restriction endonucleases. After purification and recovery of the fragments and vector, the heavy chain gene and light chain gene were respectively ligated into the 3.4A expression vector to obtain recombinant expression plasmids of the heavy chain and light chain, respectively.

[0164] The TAQ-6A7 antibody used in this embodiment is described in Chinese Patent Publication No. CN115785276A (Application No. CN202211017298.8, entitled "An Antibody Against Taq DNA Polymerase and Its Application"), and is incorporated herein by reference. The antibody may contain a group of binding proteins of the

[0062] segment in CN115785276A; the antibody may also contain a group of binding proteins of the

[0110] segment, i.e., the amino acid sequence of the heavy chain variable region of the TAQ-6A7 antibody is SEQ ID NO:1, and the amino acid sequence of the light chain variable region is SEQ ID NO:2.

[0165] The TAQ-11D2 antibody used in this embodiment is described in Chinese Patent Publication No. CN115819601A (Application No. CN202211029351.6, entitled "An Antibody Against Taq DNA Polymerase and Its Application"), and is incorporated herein by reference. The antibody may comprise any group of binding proteins having segments

[0064] to

[0067] of CN115819601A, which have similar properties; the antibody may also be a group of binding proteins having segment

[0115] . Specifically, the amino acid sequence of the heavy chain variable region of the TAQ-11D2 antibody is selected from SEQ ID NO:3, and the amino acid sequence of the light chain variable region is SEQ ID NO:4.

[0166] The full-length Taq-6A7 antibody can bind to Taq enzyme to block exonuclease activity; the full-length TAQ-11D2 antibody can bind to Taq enzyme to block polymerization activity.

[0167] Example 2: Construction of TAQ-6A7TB1 and TAQ-11D2TB1 Fab expression plasmids

[0168] Based on the sequencing results of the variable region gene of the antibody in pMD-18T constructed in Example 1, VL and VH gene-specific primers for TAQ-6A7 and TAQ-11D2 antibodies were designed, with restriction endonuclease sites and protective bases at both ends, respectively. The TAQ-6A7 and TAQ-11D2 genes and the corresponding subtype constant region genes were amplified by overlap PCR, and the heavy chain gene was retained only up to the Fd segment. Finally, the correct length of the light chain gene fragment and the correct length of the heavy chain gene Fd fragment were amplified.

[0169] The Fd fragment of the heavy chain gene and the light chain gene fragment were double-digested with restriction endonucleases, and the 3.4A vector was also double-digested with restriction endonucleases. After purification and recovery of the fragments and vector, the Fd fragment of the heavy chain gene and the light chain gene were ligated into the 3.4A expression vector, respectively, to obtain recombinant expression plasmids of heavy chain Fd and light chain, namely TAQ-6A7TB1 Fab expression plasmid and TAQ-11D2TB1 Fab expression plasmid.

[0170] Example 3: Sample preparation of TAQ-6A7, TAQ-11D2 full-length mouse IgG antibodies and TAQ-6A7TB1, TAQ-11D2TB1 Fab.

[0171] HEK293 cells were revived early and passaged to a 200 mL volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cells / mL, cell viability >95%; centrifuge to wash cells, rehydrate with culture medium, and adjust cell density to 2.9 × 10⁶ cells / mL. 6Cells / mL were used as cell dilution buffers. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA (expressing TAQ-6A7 full-length mouse IgG antibody, TAQ-11D2 full-length mouse IgG antibody, TAQ-6A7TB1 Fab, and TAQ-11D2TB1 Fab, respectively) dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein G affinity chromatography column. The SDS-PAGE image is shown in Figure 3.

[0172] Example 4: Application of Taq-6A7TB1 and Taq-11D2TB1 Fabs

[0173] 4.1 Preparation of antibody blocking enzyme

[0174] Prepare 100 mL of Taq dilution solution: 50 mM Tris, 150 mM NaCl, 0.1 mM EDTA, 0.05% Tween 20, pH 8.0; add the sample according to Tables 2 and 3 in sequence, mix well, and then let it stand at 4℃ for more than 1 hour.

[0175] Table 2. System for preparing full-length antibody blocking enzymes

[0176] Table 3. System for preparing Fab blocking enzyme

[0177] 4.2 Performance testing of antibody blocking enzyme

[0178] (1) Method for detecting the blocking performance of the exonuclease activity: A hairpin structure GB-hairpin was designed, which has an extended single-strand structure at the 5' end; a probe Taq-HP was designed and bound to the extended single strand at the 5' end of GB-hairpin. The 5' end of the probe was modified with FAM, and the 3' end was modified with Q quencher. The reaction system was prepared according to Table 4. Finally, the sample to be tested was added, and the test was performed on a qPCR instrument. The specific program was 37℃ for 30s × 90 cycles, and the fluorescence was collected from FAM. Two control groups were set up: a naked enzyme group and an enzyme-free group NEC. If the fluorescence curve of the test group was close to that of NEC without any curling, it was judged that the blocking performance of the antibody was good.

[0179] Table 4. Preparation of Exocleolytic Activity Detection System

[0180] (2) Polymer activity blocking detection method: Design a pair of cross-linking primers (cross-linking primers 1 and 2) that can be used as templates for each other for extension. Prepare the reaction system according to Table 5, incubate at 37℃ for 1 h, add EDTA to a final concentration of 2 mM to terminate the reaction, and then measure the melting curve on the instrument. The degree of adhesion between the test sample and NEC (enzyme-free group) is used to determine whether the antibody blocking is qualified.

[0181] Table 5. Polymer Activity Blocking Detection System

[0182] (3) Based on the specific activity of Taq polymerase, the prepared antibody blocking enzyme was diluted to prepare a 5 U / μl working solution. Three batches of antibodies were selected to block the same batch of Taq polymerase. The Taq polymerase and the two antibodies were blocked at a molar ratio of 1:1:1, and the blocking efficiency of the antibody blocking enzyme was detected.

[0183] In the blocking results of exonuclease activity (Figures 4 and 5), the fluorescence signal of the naked Taq polymerase was very high. The Taq antibody blocking enzymes prepared with full-length antibodies (TAQ-6A7 and TAQ-11D2) and Fab (TAQ-6A7TB1 and TAQ-11D2TB1) showed fluorescence signals that overlapped with the enzyme-free group (NEC) without any spikes, indicating that both full-length antibodies and Fab could completely block the exonuclease activity of Taq polymerase, and the blocking performance of the three batches showed little difference between batches. In the blocking results of polymerization activity (Figures 6 and 7), both full-length antibodies and Fab could block the polymerization activity of Taq polymerase. In the first two batches blocked with full-length antibodies, the fluorescence signal overlapped with the enzyme-free group (NEC) without any spikes, but the signal of the third batch was slightly higher than that of the enzyme-free group (NEC), indicating that the blocking performance of the three batches was unstable and there were batch-to-batch differences. However, when Fab blocked the Taq polymerase polymerization activity, the fluorescence signals of all three batches overlapped with the enzyme-free group (NEC) without any spikes, indicating that the blocking performance of the three batches showed little difference between batches. Therefore, truncated antibodies have a greater advantage in blocking Taq polymerase activity, not only with blocking ability comparable to full-length antibodies, but also with smaller batch-to-batch differences in blocking performance.

[0184] 4.3 SEC-HPLC characterization of full-length antibody and antibody-blocking enzyme prepared by Fab

[0185] The three batches of antibody blocking enzymes mentioned above were analyzed by SEC-HPLC. The results of Taq polymerase blocking with full-length antibody are shown in Figure 8: the sample peaks were very mixed, with a large number of polymer peaks (>450kDa) in addition to one Taq polymerase peak and two full-length antibody peaks (approximately 400kDa), and the proportion of each component varied greatly among the three batches; the results of Taq polymerase blocking with Fab are shown in Figure 9: the sample peaks were relatively uniform, mainly consisting of one Taq polymerase and two Fab peaks (approximately 200kDa), with no polymeric form, and the peaks of the three batches were uniform, with good consistency between batches.

[0186] 4.4 Dynamic light scattering (DLS) characterization of full-length antibodies and Fab-prepared antibody blocking enzymes.

[0187] Dynamic light scattering (DLS) was performed using the three batches of antibody blocking enzymes described above. The results of Taq polymerase blocking with the full-length antibody are shown in Figures 10-12: the average particle size is approximately 10.2 nm; the results of Taq polymerase blocking with Fab are shown in Figures 13-15: the average particle size is approximately 5.4 nm. The antibody blocking enzyme prepared from the full-length antibody tends to form large aggregates, has a higher sedimentation coefficient, and the proportions of each component differ significantly among the three batches, especially in the volume curves (the upper curve is the intensity curve) of each figure. In contrast, the antibody blocking enzyme prepared with Fab does not form aggregates, has a lower sedimentation coefficient, and the products from all three batches show a single peak, indicating good batch-to-batch consistency.

[0188] Example 5: Construction of full-length rabbit IgG antibody expression plasmids TAQ-6J12 and TAQ-10C13

[0189] pcDNA TM 3.4 The vector is the constructed recombinant antibody eukaryotic expression vector, which was modified to introduce polyclonal restriction enzyme sites, and will be referred to as the 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers for TAQ-6J12 antibody and TAQ-10C13 antibody were designed, with restriction endonuclease sites and protective bases at both ends, respectively. The TAQ-6J12 gene, TAQ-10C13 gene and the constant region gene of the corresponding subtype were amplified by overlap PCR, and the correct length of light chain gene fragment and the correct length of heavy chain gene fragment were obtained.

[0190] The heavy chain and light chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was also double-digested with restriction endonucleases. After purification and recovery of the fragments and vector, the heavy chain gene and light chain gene were respectively ligated into the 3.4A expression vector to obtain recombinant expression plasmids of the heavy chain and light chain, respectively.

[0191] Among them, the heavy chain amino acid sequence of TAQ-6J12 is SEQ ID NO:5; the light chain amino acid sequence is SEQ ID NO:6; the heavy chain amino acid sequence of TAQ-10C13 is SEQ ID NO:7; and the light chain amino acid sequence is SEQ ID NO:8.

[0192] Example 6: Construction of Taq-6J12TB1 and Taq-10C13TB1 Fab expression plasmids

[0193] VL and VH gene-specific primers for TAQ-6J12 and TAQ-10C13 antibodies were designed, with restriction endonuclease sites and protective bases at both ends, respectively. The TAQ-6J12 gene, TAQ-10C13 gene, and the constant region gene of the corresponding subtype were amplified by overlap PCR, and the heavy chain gene was retained only up to the Fd segment. Finally, the correct length of the light chain gene fragment and the correct length of the heavy chain gene Fd fragment were amplified.

[0194] The Fd fragment of the heavy chain gene and the light chain gene fragment were double-digested with restriction endonucleases, and the 3.4A vector was also double-digested with restriction endonucleases. After purification and recovery of the fragments and vector, the Fd fragment of the heavy chain gene and the light chain gene were ligated into the 3.4A expression vector, respectively, to obtain recombinant expression plasmids of heavy chain Fd and light chain, namely Taq-6J12TB1Fab expression plasmid and Taq-10C13TB1Fab expression plasmid.

[0195] Example 7: Sample preparation of TAQ-6J12 / TAQ-10C13 full-length rabbit IgG antibody and Taq-6J12TB1 / Taq-10C13TB1 Fab

[0196] HEK293 cells were revived early and passaged to a 200 mL volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cells / mL, cell viability >95%; centrifuge to wash cells, rehydrate with culture medium, and adjust cell density to 2.9 × 10⁶ cells / mL. 6Cells / mL were used as cell dilution buffers. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA (expressing TAQ-6J12 full-length rabbit IgG antibody, TAQ-10C13 full-length rabbit IgG antibody, Taq-6J12TB1 Fab, and Taq-10C13TB1 Fab, respectively) dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein G affinity chromatography column.

[0197] Example 8: Applications of Taq-6J12TB1 Fab and Taq-10C13TB1 Fab

[0198] 8.1 Preparation of antibody blocking enzyme

[0199] Prepare 100 mL of Taq dilution solution: 50 mM Tris, 150 mM NaCl, 0.1 mM EDTA, 0.05% Tween 20, pH 8.0; add the sample according to Tables 6 and 7, mix well, and then let it stand at 4 °C for more than 1 h.

[0200] Table 6. System for preparing full-length antibody blocking enzymes

[0201] Table 7. System for preparing Fab-blocking enzyme

[0202] 8.2 Performance testing of antibody blocking enzyme

[0203] (1) Method for detecting the blocking performance of exonuclease activity: A hairpin structure GB-hairpin was designed, which has an extended single-strand structure at the 5' end; a probe Taq-HP was designed and bound to the extended single strand at the 5' end of GB-hairpin. The 5' end of the probe was modified with FAM, and the 3' end was modified with Q quenching. The reaction system was prepared according to Table 8. Finally, the sample to be tested was added, and the test was performed on a qPCR instrument. The specific program was 37℃ for 30s × 90 cycles, and the fluorescence was collected from FAM. Two control groups were set up: a naked enzyme group and an enzyme-free group NEC. If the fluorescence curve of the test group was close to that of NEC without any curling, it was judged that the blocking performance of the antibody was good and could be used as a Taq antibody.

[0204] Table 8. Preparation of Exocutaneous Activity Detection System

[0205] (2) Polymer activity blocking detection method: Design a pair of cross-linking primers (cross-linking primers 1 and 2) that can be used as templates for each other for extension. Prepare the reaction system according to Table 9, incubate at 37℃ for 1 h, add EDTA to a final concentration of 2 mM to terminate the reaction, and then measure the melting curve on the instrument. The degree of adhesion between the sample to be tested and NEC (enzyme-free group) is used to determine whether the blocking of the antibody is qualified.

[0206] Table 9. Detection System for Polymer Activity Blocking

[0207] (3) Based on the specific activity of Taq polymerase, the prepared antibody blocking enzyme was diluted to prepare a 5 U / μL working solution. Taq polymerase was used to block the two antibodies at a molar ratio of 1:1:1, and the blocking efficiency of the antibody blocking enzyme was detected.

[0208] In the exonuclease activity assays (Figures 16 and 17), the fluorescence signal of the naked Taq polymerase was very high. The fluorescence signals of the Taq antibody blocking enzymes prepared with full-length antibodies (TAQ-6J12 and TAQ-10C13) and Fab (TAQ-6J12TB1 and TAQ-10C13TB1) overlapped with the signal of the enzyme-free group (NEC) without any overshoot, indicating that both the full-length antibodies and Fab could completely block the exonuclease activity of Taq polymerase. In the polymerization activity assays (Figures 18 and 19), the fluorescence signals of the Taq antibody blocking enzymes prepared with full-length antibodies and Fab overlapped with the signal of the enzyme-free group (NEC) without any overshoot, indicating that both the full-length antibodies and Fab could completely block the polymerization activity of Taq polymerase.

[0209] 8.3 The full-length antibody and the antibody blocking enzyme prepared by Fab were characterized by SEC-HPLC.

[0210] SEC-HPLC analysis was performed using full-length antibodies (TAQ-6J12 and TAQ-10C13) and Fab blocking enzymes (TAQ-6J12TB1 and TAQ-10C13TB1), respectively. The results of Taq polymerase blocking with full-length antibodies are shown in Figure 20: the sample peaks were mixed, with a large number of polymer peaks (>450 kDa) in addition to the peak of one Taq polymerase binding to two full-length antibodies. The results of Taq polymerase blocking with Fab are shown in Figure 21: the sample peaks were relatively uniform, mainly consisting of one Taq polymerase and two Fab peaks (approximately 200 kDa), with no polymeric form. This indicates that the Fab blocking enzyme can prevent the formation of polymers.

[0211] SEC-HPLC analysis was performed using T1 (TAQ-6J12 and TAQ-10C13TB1) and T2 (TAQ-6J12TB1 and TAQ-10C131) blocking enzymes, respectively, with an enzyme-to-antibody modification ratio of 1:1:1. The results of T1 blocking of Taq polymerase are shown in Figure 22, and the results of T2 blocking of Taq polymerase are shown in Figure 23. Both results showed the presence of one Taq polymerase peak binding to one full-length antibody and one Fab antibody peak (approximately 300 kDa), as well as numerous multimeric peaks (>450 kDa), indicating that the presence of bivalent antibodies leads to multimer formation.

[0212] 8.4 Dynamic light scattering (DLS) characterization was performed on the full-length antibody and the antibody blocking enzyme prepared by Fab.

[0213] Dynamic light scattering (DLS) was performed using full-length antibodies (TAQ-6J12 and TAQ-10C13) and Fab (TAQ-6J12TB1 and TAQ-10C13TB1) blocking enzymes, respectively. The results of Taq polymerase blocking with full-length antibodies are shown in Figure 24: the average particle size is approximately 62 nm; the results of Taq polymerase blocking with Fab are shown in Figure 25: the average particle size is approximately 33 nm. The overall results are consistent with those of SEC-HPLC. The blocking enzymes prepared with full-length antibodies tend to form large aggregates with a higher sedimentation coefficient, especially in the volume curves (the upper curve is the intensity curve) of each figure; while the blocking enzymes prepared with Fab do not form aggregates and have a lower sedimentation coefficient.

[0214] Example 9: Application and testing in Taq-6A7TB1 and Taq-11D2TB1 Fab qPCR systems.

[0215] Refer to Tables 2 and 3 to prepare full-length antibody blocking enzymes and Fab antibody blocking enzymes with different modification ratios. Different enzyme and double antibody modification ratios were set as 1:3:3, 1:4:4, and 1:5:5, respectively. After mixing, the enzymes were allowed to stand at 4°C for 1 hour and then diluted to 25 U / μL for later use.

[0216] A 50 μL qPCR reaction system was prepared, mainly consisting of 1.2 μL of 25 U / μL blocking enzyme, 10 μL of template (10³–10⁶ copies / μL), 2 μL of a primer capable of amplifying the target fragment in the template, 25 μL of 2× buffer mix, 1.4 μL of Taq polymerase-free mix, and ddH₂O to make up the difference. Two replicates were set up for each template concentration, and eight replicates were set up for the template-free group (NTC). After preparation, the system was mixed, centrifuged, and then run on the qPCR machine. The program was: 50℃, 2 min; 94℃, 5 min; (94℃, 15 s; 62℃, 30 s for fluorescence collection) × 45 cycles. After the reaction, the product precipitation was observed and the amplification data were analyzed.

[0217] The FAM channel detection results are shown in Figures 26-28. qPCR data show that, under the same modification ratio, whether amplifying positive samples or NTC, the full-length antibody blocking enzyme exhibits amplification curve jitter and baseline instability, while the Fab antibody blocking enzyme shows a normal amplification curve. This indicates that the amplification performance of the Fab antibody blocking enzyme is superior to that of the full-length antibody blocking enzyme. Furthermore, visual observation also revealed that the amplification product precipitation of the Fab antibody blocking enzyme was significantly less than that of the full-length antibody blocking enzyme.

[0218] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this disclosure. Industrial applicability

[0219] This disclosure provides a monovalent binding molecular composition for binding enzymes and its applications, relating to the field of biotechnology. The monovalent binding molecular composition for binding enzymes comprises at least two monovalent binding molecules, and this composition alleviates the problem of blocked enzymes readily forming polymers.

Claims

1. A monovalently bound molecular composition, characterized in that, The composition contains at least two monovalent binding molecules and can block at least two activities of the same enzyme.

2. The monovalently bound molecular composition according to claim 1, characterized in that, The enzyme includes polymerase; Optionally, the enzyme includes a DNA polymerase, such as Taq DNA polymerase.

3. The monovalently bound molecular composition according to claim 1 or 2, characterized in that, The composition can at least block polymerization activity and / or exotropy activity.

4. The monovalently bound molecular composition according to any one of claims 1 to 3, characterized in that, The monovalent binding molecule is a monovalent antibody; Optionally, the monovalently bound molecules are independently selected from Fab, Fab', scFV, Fv, or VHH.

5. The monovalently bound molecular composition according to any one of claims 1 to 4, characterized in that, The monovalently bound molecule comprises three CDRs of the heavy chain and three CDRs of the light chain; the amino acid sequence of the heavy chain is shown in SEQ ID NO:5 or 7, and the amino acid sequence of the light chain is shown in SEQ ID NO:6 or 8. Optionally, the monovalently bound molecule comprises three CDRs of the heavy chain variable region and three CDRs of the light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1 or 3, and the amino acid sequence of the light chain variable region is shown in either SEQ ID NO:2 or 4.

6. The monovalently bound molecular composition according to any one of claims 1 to 5, characterized in that, The composition does not contain bivalent or multivalent binding molecules that can block the same enzyme.

7. A method for modifying an enzyme, characterized in that, This includes mixing the monovalent binding molecular composition according to any one of claims 1 to 6 with an enzyme.

8. The modification method according to claim 7, wherein the enzyme comprises a polymerase; Optionally, the enzyme is a DNA polymerase, such as Taq DNA polymerase.

9. A modified enzyme, characterized in that, The composition comprises the monovalent binding molecular composition and enzyme as described in any one of claims 1 to 6.

10. The modified enzyme according to claim 9, wherein the enzyme is a polymerase; Optionally, the enzyme is a DNA polymerase, such as Taq DNA polymerase.

11. A reagent kit, characterized in that, The enzyme comprises a monovalent binding molecular composition according to any one of claims 1 to 6, or a modified enzyme according to any one of claims 9 to 10.

12. The use of the monovalent binding molecular composition according to any one of claims 1 to 6, or the enzyme modification method according to any one of claims 7 to 8, or the modified enzyme according to any one of claims 9 to 10, or the kit according to claim 11 in the synthesis of polynucleotides or the preparation of products for the synthesis of polynucleotides.

13. A hot-start method for polynucleotide synthesis, characterized in that, This includes using an enzyme modified according to any one of claims 9-10 to catalyze a polynucleotide synthesis reaction.

14. A kit for polynucleotide synthesis, characterized in that, Includes: (Ⅰ) and (Ⅱ) (I) The monovalent binding molecular composition and enzyme according to any one of claims 1 to 6, or the modified enzyme according to any one of claims 9 to 10; (II) Reagents for polynucleotide synthesis reaction.

Images