B-lactamase-inhibiting antibody and use thereof in the treatment and / or prevention of infections caused by b-lactamase-producing bacteria

Abstract

The present invention relates to antibodies that inhibit B-lactamases, for example class A B-lactamase from Mycobacterium abscessus, as well as related compositions, and uses of same for and / or preventing infections caused by B-lactamase-producing bacteria.

Description

[0001] DESCRIPTION

[0002] B-lactamase inhibitor antibody and use for the treatment and / or prevention of infections caused by bacteria that produce B-lactamases

[0003] The present invention belongs to the field of Biotechnology and Biomedicine, and relates to a class A p-lactamase inhibitor antibody, and its use in the treatment of infections caused by p-lactamase-producing bacteria in a subject.

[0004] BACKGROUND OF THE INVENTION

[0005] The global emergence of drug-resistant microorganisms is one of the most urgent health threats to be addressed. The overuse of antibiotics in healthcare, agriculture, and livestock farming worldwide has dramatically accelerated the emergence of nosocomial infections, primarily affecting people with compromised immune systems. Although more than 1,200,000 people die from this cause each year, this number is expected to rise to more than 10 million in the coming years. Experts say that even common infections will no longer be treatable with conventional antibiotics in the near future.

[0006] The expression of β-lactamase enzymes is one of the mechanisms of resistance of pathogenic bacteria to antibiotics. β-Lactamases are enzymes capable of degrading β-lactam antibiotics, therefore, these enzymes constitute an interesting target for the development of new therapeutic strategies against pathogenic bacteria (Bush, K. (2018). Antimicrobial agents and chemotherapy, 62(10), e01076-18).

[0007] The development of antibodies capable of inhibiting beta-lactamases is a promising alternative to treatment with current antibiotics, and various developments based on this strategy are found in the state of the art.

[0008] WO2020254861A1 describes the use of antibodies and antigen-binding fragments to neutralize β-lactamases and treat infections by antibiotic-resistant bacteria. The TEM-1 protein, a class A β-lactamase expressed by Gram-negative bacteria, was used as an antigen in obtaining antibodies for the treatment of bacterial infections. The publication “Conrath, KE, et al., (2001). Antimicrobial agents and chemotherapy, 45(10), 2807-2812” describes the use of the TEM-1 and Bell proteins, type A and B β-lactamases respectively, for obtaining single-domain antibodies that inhibit β-lactamase. The TEM-1 β-lactamase is expressed in Gram-negative bacteria while the Bell β-lactamase is expressed in Gram-positive bacteria.

[0009] In the publication “Cawez, F., et al, (2023). Antimicrobial Agents and Chemotherapy, 67(4), e01499-22” three soluble single-domain fragments derived from the unique variable region of camelid heavy chain (VHH) antibodies are disclosed, which are inhibitors of the CMY-2 protein, a class C p-lactamase produced by Gram-negative bacteria isolated from veterinary and human environments.

[0010] On the other hand, in the publication “Sohier, JS, et al. ,(2013). Biochemical Journal, 450(3), 477-486” a single domain antibody capable of inhibiting class B p-Lactamasase VIM-4, expressed in Gram-negative bacteria, was obtained.

[0011] However, the strategies described in the prior art are limited to antibodies that inhibit class A, B, and C β-lactamases from Gram-negative bacteria or class B β-lactamases from Gram-positive bacteria. Therefore, there is a need to generate new strategies that allow the inhibition of class A β-lactamases from Gram-positive bacteria.

[0012] DESCRIPTION OF THE INVENTION

[0013] The present inventors have obtained antibodies capable of binding and inhibiting class A P-lactamase enzymes from Gram-positive bacteria, with an inhibition constant of less than 10 pM, which indicates an inhibition capacity at low concentrations (Table 2).

[0014] Thus, in a first aspect, the present invention relates to an antibody that inhibits class A β-lactamase from Gram-positive bacteria or a fragment thereof, hereinafter “antibody or fragment of the invention”, characterized in that said antibody or fragment thereof is an inhibitor of a β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO. 21.

[0015] In a preferred embodiment of the antibody or fragment of the invention, said antibody or fragment thereof comprises an inhibition constant (K¡) of less than 10 pM, more preferably it comprises a K¡ of between 0.01 pM and 10 pM, between 0.01 pM and 8 pM, between 0.01 pM and 7 pM, between 0.01 pM and 6 pM, between 0.5 pM and 6 pM or between 1 pM and 6 pM, against a β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21 , preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO. 21.

[0016] In the present invention, the polypeptide or β-lactamase comprising an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 21, corresponds to a functionally equivalent variant to a polypeptide or β-lactamase comprising the amino acid sequence SEQ ID NO. 21.

[0017] In another even more preferred embodiment of the antibody or fragment of the invention, said fragment or antibody comprises a K¡ of between 2.2 pM and 5.5 pM against a β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO. 21.

[0018] The inhibition capacity of the antibody or fragment of the invention entails that said antibody or fragment is specific for the β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO. 21.

[0019] The amino acid sequence SEQ ID NO. 21 comprises amino acids 31 to 263 of M. abscessus class A β-lactamase (NCBI reference for M. abscessus class A β-lactamase: WP_005091054.1). The amino acid sequence SEQ ID NO. 21 corresponds to a fragment of M. abscessus class A β-lactamase comprising the catalytic center without the 30 amino acids at the N-terminal end of said enzyme, making it a functional fragment.

[0020] Secuencia de aminoácidos del fragmento de p-lactamasa de clase A de M. abscessus, SEQ ID NO. 21 :

[0021] APDELASLEKDFGGRIGVYALDTGSGDTVGHRADERFLMCSTVKTFIVSAILRRRLSE PGLLDQRIQYTQSDVLEWAPITSQHVSTGMTVSELCDATLRYSDNTGANLLITQLGGP KETEKFVRSLGDNVTRMDRTEVQLNIPDGDLDTSTPQQLVANLRRLVLDEGLDSRGR DLLTDWLKRNTTGDQSIRAAVPAGWTVADKTGGGFKGETNDIAVIWPPGRAPIVMAV LTVPEDPTSTKGKPTIAAATRIVLRAFGA

[0022] The term “antibody,” as used herein, refers to a polypeptide or protein comprising at least one “immunoglobulin variable domain” sequence, which refers to the structural unit of an immunoglobulin capable of binding to an antigen.As is known to those skilled in the art, an immunoglobulin variable domain consists of a polypeptide comprising four framework regions (FRs) referred to as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", respectively; said framework regions being interrupted by three "complementary determining regions" or "CDRs" referred to as "complementary determining region" or "CDR1"; "complementary determining region 2" or "CDR2"; and "complementary determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain may be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.CDRs are short amino acid sequences, typically between 3 and 20 amino acids, which mediate the specific binding of an antibody to an antigen. Therefore, the immunoglobulin variable domain(s) confer antigen specificity to an antibody by providing the antigen-binding site. In the present invention, the term “immunoglobulin variable domain” may also be referred to as “antigen-binding variable region” or “antigen-binding domain.”

[0023] The antibody of the present invention may be a complete antibody. Complete antibodies, for example from humans or mice, comprise at least two heavy (H) polypeptide chains linked by disulfide bonds, and in the case of antibodies of human origin also comprise two light (L) polypeptide chains, each linked to a heavy chain by disulfide bonds. Each heavy chain has a variable domain or region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable domain or region (VL) at one end and a constant region at the other; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Examples of complete antibodies include, but are not limited to, IgG or IgY antibodies.

[0024] In the present invention, the antibody may be an antibody composed solely of two heavy chains, each chain comprising an immunoglobulin variable domain. Such antibodies are referred to as “heavy chain antibodies (HCAbs).” HCAbs are naturally produced by camelids, where examples of camelids include, but are not limited to, camels, llamas, dromedaries, or alpacas.

[0025] Thus, in a preferred embodiment, the antibody or fragment of the invention is human, mouse, rat, camelid, bird, a chimeric antibody or a humanized antibody.

[0026] The term "humanized antibody" refers to an antibody that has been engineered to comprise one or more human framework regions in the variable region along with non-human (e.g., mouse or synthetic) complementarity determining regions (CDRs) of the heavy and / or light chain. In some embodiments, a humanized antibody comprises sequences that are fully human except for the CDR regions. Humanized antibodies are typically less immunogenic to humans than non-humanized antibodies, thereby offering therapeutic advantages in certain situations.

[0027] The term "chimeric antibody" refers to an antibody that has been engineered to comprise at least one human constant region.

[0028] In another preferred embodiment of the antibody or fragment of the invention, the antibody is selected from the list consisting of IgY, IgG1, IgG2, IgG3, IgG4 and a heavy chain antibody.

[0029] As is known to those skilled in the art, antibodies can be monospecific or multispecific. In the present invention, the term "monospecific antibody" refers to an antibody capable of binding a single antigen, while the term "multispecific antibody" refers to an antibody capable of binding more than one antigen. Multispecific antibodies comprise at least two distinct immunoglobulin variable domains, each of which recognizes a distinct antigen (each domain is specific for a distinct epitope). The antibody of the present invention comprises at least one immunoglobulin variable domain that specifically binds to the polypeptide comprising the amino acid sequence with at least 80% identity with SEQ ID NO: 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO. 21.

[0030] Thus, in another preferred embodiment, the antibody or fragment of the invention is monospecific or multispecific; preferably, the antibody or multispecific fragment is bispecific.

[0031] In another preferred embodiment of the antibody or fragment of the invention, the antibody is monoclonal. The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible vagrant antibodies, for example, containing naturally occurring mutations or arising during the production of a monoclonal antibody preparation, said vagrants generally being present in minor amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant of an antigen.

[0032] The term "antibody fragment" as used herein refers to a functional portion of a whole antibody, wherein said functional portion preserves antigen-binding capacity. In the present invention, the antibody fragment comprises at least one immunoglobulin variable domain. Examples of antibody fragments include, but are not limited to, a single domain antibody (sdAb, whether VL, VH, or a VHH domain), IgG1 heavy chain antibody (HCIgGI), IgG2 heavy chain antibody (HCIgG2), IgG3 heavy chain antibody (HCIgG3), IgG4 heavy chain antibody (HCIgG4), Fab, F(ab')2, Fv fragments, disulfide-linked Fv fragments (sdFv), an Fd fragment, or scFv fragments.

[0033] Thus in a preferred embodiment of the antibody or fragment of the invention, the antibody fragment is selected from the list consisting of a single domain antibody (sdAb, either VL, VH or a VHH domain of camelid antibodies), an IgG1 heavy chain antibody (HCIgGI), an IgG2 heavy chain antibody (HCIgG2), an IgG3 heavy chain antibody (HCIgG3), an IgG4 heavy chain antibody (HCIgG4), a Fab fragment, an F(ab')2 fragment, an Fv fragment, Fv fragments linked by a disulfide bridge (sdFv), an Fd fragment and an scFv fragment.

[0034] Antibody fragments have advantages over whole antibodies, since they are smaller in size and therefore more stable, they are soluble and can bind more easily to the epitope of the antigen to which they bind.

[0035] Thus, in a preferred embodiment of the antibody or fragment of the invention, the antibody is a single domain antibody (sdAb).

[0036] In the present invention, the terms “single domain antibody,” “nanobody,” “dAb,” or “sdAb,” used interchangeably, refer to a polypeptide comprising, preferably consisting of, a single immunoglobulin variable domain that specifically binds to an antigen. The single domain antibody may be a VH single domain antibody, a VL single domain antibody, or a VHH single domain antibody.

[0037] In the present invention, the terms “VHH single domain antibody”, “VHH nanobodies”, “VHH sdAb” or “VHH”, used interchangeably, refer to a polypeptide consisting of a single camelid immunoglobulin variable domain or a polypeptide consisting of a single humanized camelid immunoglobulin variable domain (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)).

[0038] The use of VHHs in the field of biomedicine, either alone or as part of a larger polypeptide, offers a number of significant advantages over the use of conventional VH and VL domains, scFvs or conventional antibody fragments (such as Fab- or F(ab')2-fragments):

[0039] Only a single domain is required to bind an antigen with high affinity and selectivity, so there is no need to have two separate domains present, nor to ensure that these two domains are present in the correct conformation and spatial configuration (i.e., by using specially designed linkers, as with scFvs);

[0040] VHH domains can be expressed from a single gene and do not require folding or post-translational modifications;

[0041] VHH domains can be easily designed in multivalent and multispecific (formed) formats;

[0042] VHH domains are highly soluble and do not tend to aggregate;

[0043] VHH domains are highly stable to heat, pH, proteases and other denaturing agents or conditions and can therefore be prepared, stored or transported without the need for refrigeration equipment, thereby saving costs, time and the environment;

[0044] VHH domains are easy and relatively inexpensive to prepare, even at the scale required for their production;

[0045] The VHH domains are relatively small (approximately 15 kDa, or 10 times smaller than a conventional IgG) compared to conventional 4-chain antibodies and their antigen-binding fragments, and therefore show high tissue penetration and can be administered in higher doses than such conventional 4-chain antibodies and their antigen-binding fragments;

[0046] VHH domains can display so-called cavity-binding properties (especially due to their extended CDR3 loop, compared to conventional VH domains) and can therefore also access targets and epitopes not accessible to conventional 4-chain antibodies and their antigen-binding fragments.

[0047] Camelid-derived VHH domains may be "humanized" by substituting one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more of the amino acid residues found at the corresponding position(s) in a VH domain of a conventional 4-chain antibody from a human (also referred to as "sequence optimization", and in addition to humanization, sequence optimization also encompasses other sequence modifications by one or more mutations to provide improved VHH characteristics, such as the removal of potential sites for post-translational modification). A humanized VHH domain may contain one or more fully human framework region sequences.

[0048] Thus, in a more preferred embodiment, the antibody of the invention is a VHH single domain antibody or a humanized VHH single domain antibody.

[0049] In another preferred embodiment, the antibody or fragment of the invention is an isolated antibody or fragment, where the term “isolated” refers to a portion isolated from its natural environment, for example, a single antibody fragment that is substantially free of other antibodies, antibody fragments and / or substantially free of cells or cellular materials.

[0050] According to the inventors' results, the antibody of the invention is capable of acting as a β-lactamase inhibitor (Table 2). The term "β-lactamases" refers to those enzymes, expressed by Gram-positive or Gram-negative bacteria, capable of metabolizing and inactivating a β-lactam antibiotic. β-Lactamases can inactivate β-lactam antibiotics by hydrolyzing the β-lactam ring of said antibiotics. β-Lactamases are mainly classified into three classes depending on their activity: class A, B and C. Class A β-lactamases comprise a serine residue in their catalytic center and exhibit pelikinylase activity (examples of class A β-lactamases include, but are not limited to, enzymes of the SHV, CTX-M or KPC subclasses).Class B β-lactamases are metallo-β-lactamases that require Zinc as a cofactor and exhibit cephalosporinase activity (examples of class B β-lactamases include, but are not limited to, enzymes from subclasses VI M or Bell). Class C β-lactamases comprise a serine residue in their catalytic center and exhibit cephalosporinase activity (examples of class C β-lactamases include, but are not limited to, enzymes from subclasses CYM, ACT, DHA, FOX, or MIR) (Bush, K. (2018). Antimicrobial agents and chemotherapy, 62(10), e01076-18).

[0051] The term "inhibitor", and its grammatical derivations, refer in the present invention to the ability of an agent (e.g. the antibody of the invention) to block, partially block, interfere, decrease, reduce or deactivate a biological molecule (e.g. a β-lactamase enzyme), pathway or mechanism of action, thus in the present invention the term inhibitor refers to the ability of the antibody or fragment of the invention to inhibit a β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO: 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably with an inhibition constant of less than 10 M.

[0052] Surprisingly, the present inventors have obtained antibodies with inhibitory activity against β-lactamase, specifically the β-lactamase enzyme comprising the sequence SEQ ID NO. 21. The enzyme comprising SEQ ID NO. 21 corresponds to a class A β-lactamase enzyme encoded by the Blamab gene of the bacterium Mycobacterium abcessus (NCBI reference of the Blamab gene of M. abscessus: NG_088376.1 ; NCBI reference of the protein: WP_005091054.1). M. abcessus is a Gram-positive bacterium, commonly known by the deposit number ATCC19977, it can cause lung infections, skin infections, nervous system infections, bacteremias and eye infections. SEQ ID NO. 21 preserves the amino acids of the catalytic site with respect to other Gram-positive class A β-lactamase sequences (Fig. 1 ).

[0053] The B-lactamase enzyme Blamab from Mycobacterium abcessus preserves the amino acids of the catalytic site together with other B-lactamase enzymes from other bacterial species, as indicated in Example 1 of the present invention. Therefore, the B-lactamase enzyme Blamab is a suitable target for the generation of inhibitory antibodies capable of acting against B-lactamases that share the structural characteristics of the amino acids involved in their catalytic action. In view of the experiments provided by the present inventors, the antibodies of the invention were capable of inhibiting other B-lactamases from bacteria other than Mycobacterium abcessus (Example 4).

[0054] The term "identity," as used herein, refers to the proportion of identical amino acids between two peptides or proteins being compared. Sequence comparison methods are known in the art and include, but are not limited to, the BLASTP or BLASTN programs, ClustalW, and FASTA. It can be assumed that peptides or proteins with identity percentages of at least 80% will maintain the same properties as the β-lactamase comprising the sequence SEQ ID NO. 21.

[0055] The antibodies obtained by the inventors presented a low inhibition constant (less than 10 pM) against the polypeptide comprising the amino acid sequence SEQ ID NO. 21, so they are capable of acting as β-lactamase inhibitors at very low concentrations.

[0056] In the present invention, the term “inhibition constant” or “K¡” is widely known to those skilled in the art and relates to the capacity of an enzyme to metabolize a substrate in the presence of an inhibitor (in the present invention, the inhibitor is the antibody of the invention). In the present invention, the term inhibition constant (K¡) has the meaning / interpretation of the person skilled in the art in view of common general knowledge. The inhibition constant can be calculated by methods known to those skilled in the art, including, but not limited to, linearization methods such as the Lineweaver-Burk method, the Eadie-Hofstee method, the Hanes method, or the Scatchard method (Paolo Ascenzi, Maria Grazia Ascenzi, Gino Amiconi).Enzyme competitive inhibition, graphical determination of Ki and presentation of data in comparative studies, Biochemical Education, Volume 15, Issue 3, 1987, Pages 134-135, ISSN 0307- 4412). The kinetic parameters of maximum velocity (Vmax) and the Michaelis constant (Km) can be obtained by methods known to the person skilled in the art such as, but not limited to, the nitrocefin assay (Kumar P, et al. Antimicrob Agents Chemother. 2017 Sep 22;61(10):e00866-17).

[0057] In the present invention, the inhibition constant (of the antibody or fragment of the invention) is obtained by the Lineweaver-Burk method, and where the kinetic parameters (maximum velocity, Vmax, and the Michaelis constant, Km, of the antibody or fragment of the invention) are obtained by a nitrocefin assay; preferably, the Vmax, Km and the inhibition constant of the antibody or fragment of the invention are obtained according to example 3 of the present invention.

[0058] In another preferred embodiment, the antibody or fragment of the invention comprises: a) a CDR1 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 1: GTIFVYPY, a CDR2 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 2: EFVAGIGQGATTYY, and a CDR3 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 3: AYRYRDDVRRYGIDRYHT; b) a CDR1 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 4: GTIFYYPY, a CDR2 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 5: EFVAGIDYGSTTYY, and a CDR3 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 6: VVYLVTRKGASQQDEIYS; c) a CDR1 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 7: GTIFAQDY, a CDR2 comprising, preferably consisting of, the amino acid sequence SEQ ID NO.8: EFVASIAYGTITYY, and a CDR3 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 9: VYPTPVTDYI; d) a CDR1 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 10: GNISDLGT, a CDR2 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 11: EFVAGIGTGSNTYY, and a CDR3 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 12: VIAGRSYWIYFY; or e) a CDR1 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 13: GTISAVSG, a CDR2 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 14: EFVASINRGSTTNY, and a CDR3 comprising, preferably consisting of, the amino acid sequence SEQ ID NO. 15: ALHGTGRVHV.

[0059] In another more preferred embodiment, the antibody or fragment of the invention comprises: a) a CDR1 comprising the amino acid sequence SEQ ID NO. 1, a CDR2 comprising the amino acid sequence SEQ ID NO. 2 and a CDR3 comprising the amino acid sequence SEQ ID NO. 3, wherein the antibody comprises a K¡ of between 2.3 pM and 2.4 pM against the β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO. 21 ; b) a CDR1 comprising the amino acid sequence SEQ ID NO. 4, a CDR2 comprising the amino acid sequence SEQ ID NO. 5 and a CDR3 comprising the amino acid sequence SEQ ID NO.6, wherein the antibody comprises a K¡ of between 2.5 pM and 2.8 pM against the β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO. 21; c) a CDR1 comprising the amino acid sequence SEQ ID NO. 7, a CDR2 comprising the amino acid sequence SEQ ID NO. 8 and a CDR3 comprising the amino acid sequence SEQ ID NO. 9, wherein the antibody comprises a K¡ of between 4.9 pM and 5.5 pM against the β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO. 21; d) a CDR1 comprising the amino acid sequence SEQ ID NO.10, a CDR2 comprising the amino acid sequence SEQ ID NO. 11 and a CDR3 comprising the amino acid sequence SEQ ID NO. 12, wherein the antibody comprises a K¡ of between 3.7 pM and 3.9 pM against the β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21 , preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO. 21 ; or e) a CDR1 comprising the amino acid sequence SEQ ID NO. 13, a CDR2 comprising the amino acid sequence SEQ ID NO. 14 and a CDR3 comprising the amino acid sequence SEQ ID NO. 15, wherein the antibody comprises a K¡ of between 4.4 pM and 5.4 pM against the β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO.21 , preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO. 21 ; where the K¡ (inhibition constant) is obtained by the Lineweaver-Burk method, and where the kinetic parameters (Vmax and Km) are obtained by a colorimetric assay with nitrocefin; preferably, the Vmax, Km and the inhibition constant are obtained according to example 3 of the present invention.

[0060] In another preferred embodiment of the antibody or fragment of the invention, the complementarity determining regions CDR1, CDR2 and CDR3 correspond to complementary complementarity regions of the immunoglobulin variable domain regions of the heavy chain, which is why they are called CDRH1, CDRH2 and CDRH3 respectively.

[0061] In another even more preferred embodiment, the antibody of the invention comprises, preferably consists of, the amino acid sequence SEQ ID NO. 16, the amino acid sequence SEQ ID NO. 17, the amino acid sequence SEQ ID NO. 18, the amino acid sequence SEQ ID NO. 19 or the amino acid sequence SEQ ID NO. 20.

[0062] In the present invention, antibodies of sequence SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20 are referenced as antibody B2, B3, B4, B5 and B6, respectively.

[0063] Amino acid sequence of the β-lactamase inhibitor antibody B2, SEQ ID NO. 16:

[0064] MQVQLQESGGGLVQAGGSLRLSCAASGTIFVYPYMGWYRQAPGKEREFVAGIGQG ATTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYRYRDDVRRYGIDR YHTYWGQGTQVTVSS

[0065] The antibody of SEQ ID NO. 16 comprises CDR1, CDR2 and CDR3 of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO.3, respectively.

[0066] Amino acid sequence of the β-lactamase inhibitor antibody B3, SEQ ID NO. 17:

[0067] MQVQLQESGGGLVQAGGSLRLSCAASGTIFYYPYMGWYRQAPGKEREFVAGIDYGS TTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAWYLVTRKGASQQDEIY SYWGQGTQVTVSS

[0068] The antibody of SEQ ID NO. 17 comprises CDR1, CDR2 and CDR3 of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0069] Amino acid sequence of the β-lactamase inhibitor antibody B4, SEQ ID NO. 18:

[0070] MQVQLQESGGGLVQAGGSLRLSCAASGTIFAQDYMGWYRQAPGKEREFVASIAYGT ITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVYPTPVTDYIYWGQGTQ VTVSS

[0071] The antibody of SEQ ID NO. 18 comprises CDR1, CDR2 and CDR3 of SEQ ID NO.

[0072] 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

[0073] Amino acid sequence of the β-lactamase inhibitor antibody B5, SEQ ID NO. 19:

[0074] MQVQLQESGGGLVQAGGSLRLSCAASGNISDLGTMGWYRQAPGKEREFVAGIGTG SNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVIAGRSYWIYFYYWG QGTQVTVSS

[0075] The antibody of SEQ ID NO. 19 comprises CDR1, CDR2 and CDR3 of SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, respectively.

[0076] Amino acid sequence of the β-lactamase inhibitor antibody B6, SEQ ID NO. 20:

[0077] MQVQLQESGGGLVQAGGSLRLSCAASGTISAVSGMGWYRQAPGKEREFVASINRGS TTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAALHGTGRVHVYWGQG TQVTVSS

[0078] The antibody of SEQ ID NO. 20 comprises CDR1, CDR2 and CDR3 of SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15, respectively.

[0079] Antibodies can be covalently linked to molecules of interest such as therapeutic agents or detectable agents, thus forming conjugates that allow the molecules of interest to be specifically directed to a target such as a cell expressing the antigen to which the antibody or fragment thereof binds.

[0080] Thus, in another preferred embodiment of the antibody or fragment of the invention, the antibody or fragment is linked to a therapeutic agent or a detectable marker. In the present invention, the term "therapeutic agent" refers to a compound or group of compounds administered to a subject for therapeutic, preventive, or veterinary purposes.

[0081] In a more preferred embodiment of the antibody or fragment of the invention, the therapeutic agent is an antibacterial agent. The term “antibacterial agent” as used herein refers to a compound capable of inhibiting, reducing, or preventing the growth of bacteria, capable of inhibiting or reducing the ability of a bacterium to cause infection in a subject, or capable of inhibiting or reducing the ability of a bacterium to multiply or remain infective in the environment. The term “antibacterial agent” also refers to compounds capable of decreasing the infectivity or virulence of bacteria. Examples of antibacterial agents include, but are not limited to, antimicrobial peptides, defensins, antibiotics, bacteriocins, and endolysins.

[0082] In the present invention, the terms “detectable label” or “detectable agent,” used interchangeably, refer to a compound or compounds capable of emitting a detectable signal indicating the presence of a desired molecule, for example, the antigen recognized by the antibody to which said label or detectable agent is bound. Examples of a detectable label include, but are not limited to, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a radioisotope, a prosthetic group, a positron-emitting metal, and a non-radioactive paramagnetic metal ion. The examples described in this paragraph are within the scope of the present invention.

[0083] The detectable marker signal can be determined by any of the methods known to those skilled in the art, which include, without limitation, fluorescence spectroscopy techniques, computed tomography (CT), magnetic resonance imaging (MRI), optical imaging, single photon emission computed tomography (SPECT), positron emission tomography (PET), X-ray imaging, gamma ray imaging or the like.

[0084] The therapeutic agent or detectable marker is linked to the antibody or fragment of the invention by covalent bonds, preferably by the functional groups (e.g. -OH, -NH2, -SH) of the side chains of the amino acids of said antibody or fragment.

[0085] In another preferred embodiment, the antibody or fragment of the invention is immobilized on a solid support, a lipid particle, a nanoparticle, on the surface of a virus or on the surface of a cell.

[0086] As used herein, the term "solid support" refers to any solid or stationary material to which reagents such as antibodies, antibody fragments, or antigens are attached. For example, solid supports include, but are not limited to, plastic, glass, microtiter plate wells, microscope slides, coverslips, lateral flow assay strips, cell culture flasks, or gels.

[0087] In the present invention, the term “lipid particle” refers to a particle composed primarily of lipids, for example, liposomes or micelles.

[0088] In the present invention, the term “nanoparticle” means a particle having a size within the nanometric scale, such as metallic nanoparticles of gold, silver or copper, silica nanoparticles or polymeric nanoparticles.

[0089] The antibody or fragment of the invention may be immobilized bound to proteins or lipids on the surface of a virus or cell. The cell on which the antibody or fragment of the invention may be immobilized may be either a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., Saccharomyces cerevisiae, a non-human animal cell, or a human cell). Thus, the antibody or fragment of the invention may be bound to or form part of a display system in yeast, phages, or bacteria; or in animal or human cells, such as CAR-T cells.

[0090] The antibody or fragment of the invention may be immobilized by, without limitation, electrostatic interactions, covalent binding (e.g., peptide bond or avidin-biotin binding), adsorption, or the like.

[0091] In another aspect, the present invention relates to a pharmaceutical composition, hereinafter the "pharmaceutical composition of the invention," which comprises the antibody or fragment of the invention. In the present invention, the term "pharmaceutical composition" refers to any substance used for the diagnosis, prevention, alleviation, treatment, or cure of a disease in humans or animals. The pharmaceutical composition of the invention can be used either alone or in combination with other pharmaceutical compositions. In a preferred embodiment, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier or excipient.

[0092] In a preferred embodiment, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable excipient and / or carrier.

[0093] The term "excipient" refers to a substance that aids in the absorption of the pharmaceutical composition comprising the antibody of the invention, stabilizes it, or assists in its preparation by giving it a specific consistency, shape, flavor, or any other functional characteristic. Thus, excipients could have the function of keeping the ingredients together, such as starches, sugars, or cellulose, sweetening function, coloring function, protective function, such as isolating it from air and / or moisture, filling function of a tablet, capsule, or any other form of presentation, such as dibasic calcium phosphate, or disintegrating function to facilitate the dissolution of the components and their absorption, without excluding other types of excipients not mentioned in this paragraph.

[0094] A "carrier" refers to those substances, or combinations of substances, known in the pharmaceutical industry, used in the preparation of pharmaceutical dosage forms and includes, but is not limited to, solids, liquids, solvents, or surfactants. The carrier may be an inert substance or one that acts analogously to any of the compounds of the present invention and whose function is to facilitate the incorporation of the drug, as well as other compounds, allow for improved dosage and administration, or give consistency and shape to the pharmaceutical composition. When the form of presentation is liquid, the carrier is the diluent.

[0095] The term “pharmacologically acceptable” (or “pharmacologically acceptable”) refers to a compound that is permitted and evaluated so that it does not cause harm to the organisms to which it is administered.

[0096] The pharmaceutical composition of this invention can be administered by any route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the chosen route of administration. In a preferred embodiment, the pharmaceutical composition of the invention can be formulated in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, creams, microspheres or aerosols. According to an even more preferred embodiment of the present invention, the pharmaceutical composition is presented in a form suitable for oral, intravenous, intramuscular, intraarterial, intravenous, vaginal, intradermal, subcutaneous, topical, ophthalmic or inhalation administration.

[0097] The form adapted for oral administration refers to a physical state that allows for oral administration. Such a form adapted for oral administration is selected from the list that includes, but is not limited to, drops, syrup, herbal tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granules, cachet, pill, tablet, lozenge, troche, or lyophilized tablet.

[0098] The form adapted for topical administration refers to a physical state that allows for topical administration. Such a form adapted for topical administration is selected from the list that includes, but is not limited to, paste, cream, gel, or ointment.

[0099] A form adapted for parenteral administration refers to a physical state that allows for injectable administration, i.e., preferably in a liquid state. Parenteral administration can be carried out by intramuscular, intraarterial, intravenous, ophthalmic, intradermal, or subcutaneous routes, but is not limited to these types of parenteral administration routes.

[0100] The form adapted for administration by inhalation refers to a physical state that can allow its administration by nasal or oral inhalation, preferably in the form of powder, nebulized or aerosol.

[0101] Another possibility is that the pharmaceutical composition is presented in a form adapted to sublingual, intrathecal, bronchial, lymphatic, rectal or transdermal administration.

[0102] In another aspect, the present invention relates to the antibody, fragment or pharmaceutical composition of the invention for use as a medicament.

[0103] The term “drug” refers to any substance or combination of substances that is presented as having properties for the treatment or prevention of diseases in a subject, or that can be used in a subject or administered to a subject for the purpose of restoring, correcting or modifying physiological functions by exerting a pharmacological, immunological or metabolic action, or of establishing a medical diagnosis.

[0104] In another aspect, the present invention relates to the antibody, fragment or pharmaceutical composition of the invention for use in the treatment and / or prevention of an infection caused by a β-lactamase-producing bacterium in a subject, hereinafter “antibacterial use of the invention”.

[0105] In the present invention, the term "β-lactamase-producing bacteria" refers to a bacterium that expresses β-lactamase and is therefore capable of metabolizing β-lactam antibiotics, thereby presenting resistance to said antibiotics. As is known to those skilled in the art, examples of bacteria that produce β-lactamase include, but are not limited to, pathogenic bacteria belonging to the genera Mycobacterium, Pseudomona, Klebsiella, Listeria, Staphylococcus, Enterococcus, Streptococcus, Clostridium, Bacillus, or Corynebacterium.

[0106] In the present invention, the term “treatment” refers to combating the effects caused as a consequence of a disease or pathological condition of interest in a subject (preferably a mammal, and more preferably a human) including:

[0107] (i) inhibit the disease or pathological condition, that is, stop its development;

[0108] (i) alleviate the disease or pathological condition, that is, cause the regression of the disease or pathological condition or its symptomatology;

[0109] (iii) stabilize the disease or pathological condition.

[0110] In the present invention, the term “prevention” refers to avoiding the occurrence of the disease, that is, preventing the disease or pathological condition from occurring in a subject (preferably a mammal, and more preferably a human), in particular, when said subject has a predisposition for the pathological condition.

[0111] In a preferred embodiment of the antimicrobial use of the invention, the antibody, fragment or pharmaceutical composition of the invention is administered to the subject in a therapeutically effective amount. In the present invention, the expressions “therapeutically effective dose” or “therapeutically effective amount”, used interchangeably herein, refer to that amount or dose of the antibody, fragment or pharmaceutical composition of the invention that when administered to a subject, preferably a mammal, and more preferably a human, is sufficient to produce treatment and / or prevention of an infection caused by a β-lactamase-producing bacteria in a subject.The therapeutically effective amount will vary, for example, depending on the metabolic stability and duration of action of the extracts; the age, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the combination with other drugs; and the severity of the particular disease or pathological condition of said subject. It is routine practice for one skilled in the art, for example, a medical specialist, to determine the therapeutically effective dose for each subject, preferably a human, based on his or her own knowledge and the subject's physiological conditions or variables.

[0112] The term “subject,” as understood in the present invention, refers to a human or a non-human animal, where examples of non-human animals include, but are not limited to, dogs, cats, birds, cows, pigs, goats, sheep, fish, reptiles, non-human primates, mice, or rats. Thus, in a preferred embodiment of the antimicrobial use of the invention, the subject is selected from a human or a non-human animal.

[0113] In a more preferred embodiment of the antimicrobial use of the invention, where the bacteria is a bacteria that produces a class A β-lactamase.

[0114] In another preferred embodiment of the antimicrobial use of the invention, the bacteria is Gram positive.

[0115] In an even more preferred embodiment of the antimicrobial use of the invention, the bacteria is selected from the list consisting of Mycobacterium abscessus, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Mycobacterium leprae, Klebsiella pneumoniae Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Clostridium botulinum, Clostridium novyi, Clostridium septicum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium mangenotii, Bacillus anthracis, and Corynebacterium diphtheriae.

[0116] In a preferred embodiment of the antimicrobial use of the invention, the antibody, fragment or pharmaceutical composition of the invention is administered to the subject by a route of administration selected from the list consisting of oral, intravenous, intramuscular, intraarterial, intravenous, intradermal, vaginal, subcutaneous, topical, ophthalmic and inhalation.

[0117] In an even more preferred embodiment of the antimicrobial use of the invention, the infection is selected from the list consisting of pulmonary infection, urinary infection, skin infection, nervous system infection, bacteremia, septicemia, ocular infection, and sexually transmitted infection.

[0118] In another aspect, the present invention relates to a polynucleotide encoding the antibody or fragment of the invention, hereinafter “the polynucleotide of the invention”.

[0119] The terms "nucleic acid" or "polynucleotide," used interchangeably, refer to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides, or any substrate that can be incorporated into a DNA or RNA polymer by a polymerase or synthetic reaction.

[0120] The term "encode" refers to the genetic code that determines how a sequence of nucleotides is translated into a polypeptide or protein. The order of nucleotides in a sequence determines the order of amino acids throughout a polypeptide or protein.

[0121] In another aspect, the present invention relates to an expression vector, hereinafter “the expression vector of the invention” comprising the polynucleotide of the invention.

[0122] A vector is a nucleic acid molecule used to transfer genetic material into a cell. Aside from genetic material, a vector may also contain various functional elements, including transcription control elements such as promoters or operators, transcription factor-binding regions or enhancers, and control elements for initiating and terminating translation. Vectors include, but are not limited to, plasmids, cosmids, viruses, phages, recombinant expression cassettes, and transposons. Some vectors are capable of autonomous replication or division once introduced into the host cell, such as bacterial vectors with a bacterial origin of replication or mammalian episomal vectors. Other vectors can integrate into the host cell's genome and thus replicate along with the cell's genome. An expression vector is one capable of directing the expression of genes to which it has been operably linked.An expression vector is used for the translation and transcription of a gene of interest, typically controlled by a promoter. A promoter is a nucleotide sequence that controls the translation of the gene of interest. The promoter is operably linked to the gene of interest. “Operably linked” refers to the functional relationship and location of the promoter sequence with respect to the gene of interest; for example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, an operably linked promoter is contiguous with the sequence of interest. However, an enhancer does not have to be contiguous with the sequence of interest to control its expression.

[0123] In another aspect, the present invention relates to a host cell comprising the polynucleotide of the invention or the expression vector of the invention. The cell may be prokaryotic, for example, but not limited to, a bacterium such as E. coli, which is used to produce the antibody or fragment of the invention. The cell may be eukaryotic, such as, but not limited to, a yeast or an insect cell, which is used to produce the antibody or fragment of the invention.

[0124] In another aspect, the present invention relates to a polypeptide consisting of the amino acid sequence SEQ ID NO. 21.

[0125] In another aspect, the present invention relates to the in vitro use of a polypeptide comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21 , preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO. 21 , for obtaining antibodies or fragments thereof, hereinafter "use for obtaining the invention". Preferably, the use for obtaining the invention is directed to obtaining antibodies, or fragments thereof, which specifically bind to β-lactamases. In the method of obtaining the invention, the polypeptide comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO.21 , is used as an antigen to obtain antibodies or fragments thereof.

[0126] In the present invention, the term "antigen" refers to a molecule comprising an epitope to which an antibody, or fragment thereof, specifically binds via at least one immunoglobulin variable domain. Thus, in the method of obtaining said antigen according to the invention, said antigen allows for the selection of antibodies, or fragments thereof, specific for β-lactamases, preferably Gram-positive class A β-lactamases.

[0127] According to the common knowledge of the person skilled in the art, molecules of a peptide nature, such as the polypeptide comprising the amino acid sequence SEQ ID NO. 21, can be used as antigens for obtaining specific antibodies by in vitro methods that include, without limitation, protein display systems in viruses (phage display or phage surface display), protein display systems in prokaryotes (bacterial display or bacterial surface display), for example E. coli, or protein display systems in eukaryotes, for example in yeast (yeast display or yeast surface display) (Valldorf, Bernhard, et al., Biological Chemistry, vol. 403, no. 5-6, 2022, pp. 455-477).

[0128] In a preferred embodiment of the use of obtaining the invention, the antibodies or fragments thereof are from human, mouse, rat, camelid, bird, chimeric antibodies or humanized antibodies.

[0129] In another preferred embodiment of the method of obtaining the invention, the antibodies are selected from the list consisting of IgY, IgG1, IgG2, IgG3, IgG4 and heavy chain antibodies (HCAb).

[0130] In another preferred embodiment of the use of obtaining the invention, the antibodies are monoclonal.

[0131] In another preferred embodiment of the method of obtaining the invention, the antibody fragments are selected from the list consisting of single domain antibodies (sdAb, either VL, VH or a VHH domain of camelid antibodies), IgG1 heavy chain antibodies (HCIgGI), IgG2 heavy chain antibodies (HCIgG2), IgG3 heavy chain antibodies (HCIgG3), IgG4 heavy chain antibodies (HCIgG4), Fab fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv fragments (sdFv), Fd fragments and scFv fragments.

[0132] In a preferred embodiment of the method of obtaining the invention, the antibodies are single domain antibodies.

[0133] In a more preferred embodiment of the method of obtaining the invention, the antibodies are VHH single domain antibodies or humanized VHH single domain antibodies.

[0134] In another aspect, the present invention relates to an in vitro method for obtaining antibodies or antibody fragments against β-lactamase, hereinafter "method for obtaining the invention", where said method comprises: a) providing a library of nucleic acids encoding antibodies or fragments thereof, where said antibodies or fragments comprise at least one immunoglobulin variable domain; b) introducing the library from step a) into a surface display system selected from the list consisting of a phage surface display system, a prokaryotic surface display system and a eukaryotic surface display system; c) contacting the display system from step b) with the antigen consisting of a polypeptide comprising an amino acid sequence with at least 80% identity with SEQ ID NO: 21;and d) selecting antibodies, or fragments thereof, that specifically bind to the antigen from step b).;

[0135] The expression "antibodies or antibody fragments against β-lactamase" in the method of obtaining the invention refers to the fact that the antibodies or fragments thereof are specific for β-lactamase, preferably a β-lactamase comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO. 21.

[0136] In the present invention, the term "nucleic acid library" refers to a set of nucleic acids or polynucleotides, preferably deoxyribonucleic acid (DNA), where the DNA is genomic DNA or complementary DNA (cDNA, synthetic DNA that has been transcribed from a specific mRNA by a reaction using the enzyme reverse transcriptase). As is known to the person skilled in the art, the nucleic acid library for obtaining antibodies or fragments thereof in vitro may come from a source of cells such as immunized B cells, non-immunized B cells, or may be synthetically generated. In the case of the source of immunized B cells, these cells may come from a subject (for example, a human or a non-human mammal, preferably a camelid) which has been immunized with an antigen of interest (for example, a polypeptide comprising the amino acid sequence SEQ ID NO.21) for which antibodies, or fragments thereof, that specifically bind to said antigen are to be obtained. Once the B cells have been isolated from the immunized subject, the nucleic acids are extracted and amplified using specific primers for the genomic regions that code for the antibodies or fragments thereof (for example, the genome sequences that code for the variable domains of antibodies). On the other hand, nucleic acid libraries for the in vitro production of antibodies, or fragments thereof, can be obtained from B cells that come from a non-immunized subject ("naive" libraries).In this case, nucleic acids are extracted and amplified using primers specific to the genomic regions that code for the antibodies or fragments thereof, and during amplification, variability is introduced into the CDR regions using directed mutation techniques. Furthermore, nucleic acid libraries for the in vitro production of antibodies or fragments thereof can be generated synthetically, for example, by assembling known and synthetic FR sequences, and where the CDRs are varied using directed mutation techniques. Directed mutation methodologies that can be used in the optimization of libraries for obtaining antibodies or fragments thereof include, but are not limited to, CRISPR-Cas or error-prone polymerase chain reaction (EPR) (Valldorf, Bernhard, et al., Biological Chemistry, vol. 403, no. 5-6, 2022, pp. 455-477).

[0137] Thus, in a preferred embodiment of the method of the invention, the library of step a) is obtained from B cells isolated from a non-immunized subject, by assembly of synthetic FRs sequences with mutated CDRs in a targeted manner, or from B cells of a subject immunized with a polypeptide comprising an amino acid sequence with at least 80% identity with SEQ ID NO: 21, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; where the immunized or non-immunized subject is selected from a human or a non-human mammal, preferably where the selected one is the list consisting of a camelid, a mouse or a rat.

[0138] After the provision of the library from step a) of the method of the invention, it is introduced into a display system selected from the list consisting of a phage display system, a prokaryotic display system and a eukaryotic display system, according to step b).

[0139] In the present invention, the terms “display system” or “surface display system”, used interchangeably, refer to viruses or cells that express polypeptides of interest on their surface, for which the nucleic acid encoding the polypeptide of interest is introduced into the surface display system, which comprises translating said nucleic acid. The nucleic acid encoding the polypeptide of interest can be introduced into it as part of a vector such as a plasmid. The introduction of the nucleic acid molecules of the library in step b) is carried out by known techniques such as, but not limited to, electroporation, exposure to calcium phosphate or viral transfection in the case of cellular display systems.Thus, the display system after step b) of the method of the present invention expresses and presents on its surface the antibodies or fragments thereof encoded by the nucleic acids of the library of step a).

[0140] In a preferred embodiment of the method of obtaining the invention, the eukaryotic surface display system is a mammalian cell surface display system or a yeast surface display system (yeast display, yeast surface display or YSD).

[0141] After step b) of the method for obtaining the invention, the exposure system is contacted with the antigen in step c) and the antibodies, or fragments thereof, (expressed on the surface of the exposure system) that bind specifically to said antigen are selected.

[0142] In aspect c) of the method of obtaining the invention, the antigen may be bound to a solid support, such as, but not limited to, a gel, glass, plastic or gold nanoparticles.

[0143] Once the display system has been contacted with the antigen in step c), the selection step d) is carried out, preferably by means of a washing operation under conditions in which the binding between the antibodies or fragments thereof exposed on the surface of the display system and the antigen is maintained, while the weakest bonds are eliminated. Methods for selecting antibodies that specifically bind to an antigen are known to the person skilled in the art, and include, but are not limited to, immunological techniques such as MACS (magnetic activated cell sorting), FACS (fluorescent activated cell sorting), or ELISA (enzyme linked immunosorbent assay).

[0144] In another preferred embodiment of the method of obtaining the invention, steps c) and d) are repeated at least once.

[0145] Following the selection in step d), the nucleic acids contained in the phages, prokaryotic cells, or prokaryotic cells of the display system are isolated, and following isolation, sequence analysis of said nucleic acids is carried out, thereby determining the nucleic acid sequence(s) encoding the antibodies, or fragments thereof, capable of specifically binding to the antigen. The sequence analysis can be carried out by known methods such as, but not limited to, Sanger sequencing or next-generation sequencing. The nucleic acids encoding the antibodies or fragments thereof that have been selected in the method for obtaining the selected antibodies are then cloned and expressed in a host cell.

[0146] In a preferred embodiment of the method of obtaining the invention, the antibodies or fragments thereof are from human, mouse, rat, camelid, bird, chimeric antibodies or humanized antibodies.

[0147] In another preferred embodiment of the method of obtaining the invention, the antibodies are selected from the list consisting of IgY, IgG1, IgG2, IgG3, and heavy chain antibodies (HCAb).

[0148] In another preferred embodiment of the method of obtaining the invention, the antibodies are monoclonal.

[0149] In another preferred embodiment of the method of obtaining the invention, the antibody fragments are selected from the list consisting of single domain antibodies (sdAb, either VL, VH or a VHH domain of camelid antibodies), IgG1 heavy chain antibodies (HCIgGI), IgG2 heavy chain antibodies (HCIgG2), IgG3 heavy chain antibodies (HCIgG3), IgG4 heavy chain antibodies (HCIgG4), Fab fragments, F(ab')2 fragments, Fv fragments, Fv fragments linked by a disulfide bridge (sdFv), Fd fragments and scFv fragments.

[0150] In a preferred embodiment of the method of obtaining the invention, the antibodies are single domain antibodies.

[0151] In a more preferred embodiment of the method of obtaining the invention, the antibodies are VHH single domain antibodies or humanized VHH single domain antibodies.

[0152] In another preferred embodiment of the method of obtaining the invention, in step c) of the invention polypeptide comprising an amino acid sequence with at least 81%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO. 21.

[0153] The terms “antibody”, “immunoglobulin variable domain”, “heavy chain antibody (HCAb)”, “humanized antibody”, “chimeric antibody”, “antibody fragment”, “single domain antibody”, “β-lactamase” and “inhibitor” have been defined and explained in previous paragraphs in the antibody and fragment aspect of the invention, and both said definitions and their preferred embodiments are applicable to the use and method aspects of the invention. In the present invention, ranges of values ​​such as the inhibition constant refer not only to values ​​within said range, but also include values ​​at the upper and lower limits of said ranges.

[0154] DESCRIPTION OF THE FIGURES

[0155] Fig. 1. Multiple alignment of different Gram-positive class A β-lactamases. Arrows indicate residues that are especially important in the structural and functional characteristics of the enzyme; S70 and E166 of the catalytic site, and residues R164 and D179 contained in the Q loop. MA: amino acid sequence of the Mycobacterium abscessus antigenic protein SEQ ID NO. 21. MT: amino acid sequence of Mycobacterium tuberculosis class A β-lactamase, SEQ ID NO. 35 (NCBI Reference Sequence: WP_057351649.1). LM: amino acid sequence of Listeria monocytogenes class A β-lactamase, SEQ ID NO. 36 (NCBI Reference Sequence: WP_036094553.1). SA: amino acid sequence of Staphylococcus aureus class A β-lactamase, SEQ ID NO. 37 (NCBI Reference Sequence: WP_149508696.1). SP: Class A p-Lactamase amino acid sequence from Streptococcus pneumoniae, SEQ ID NO. 38 (GenBank: CGG65551.1).CD: Clostridium difficile class A p-lactamase amino acid sequence, SEQ ID NO. 39 (GenBank: HBH2479975.1). BA: Bacillus anthracis class A p-lactamase amino acid sequence, SEQ ID NO. 40 (GenBank: PES83414.1).

[0156] Fig. 2. Comparison of the class A β-lactamase of M. abscessus encoded by the Blamab gene, with the KPC-2 protein of Klebsiella pneumoniae. A) Solved structure resulting from the crystallization of KPC-2 (SEQ ID NO. 32) B) Model of class A β-lactamase of M. abscessus generated by the artificial intelligence tool Alphafold.

[0157] Fig. 3. Alignment of the amino acid sequences of the class A β-lactamase from M. abscessus (SEQ ID NO. 21) and the KPC-2 β-lactamase from Pseudomonas aeruginosa (SEQ ID NO. 32).

[0158] EXAMPLES

[0159] The invention will now be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention. Example 1. Design and process for obtaining class A β-lactamase.

[0160] The antigen used to obtain antibodies against β-lactamase is a fragment of the protein encoded by the Blamab gene of Mycobacterium abscessus, SEQ ID NO. 21 (NCBI reference of the Blamab gene of M. abscessus: NG_088376.1; NCBI reference of the protein encoded by the Blamab gene of M. abscessus: WP_005091054.1). The protein encoded by the Blamab gene of Mycobacterium abscessus has class A β-lactamase activity and consists of the amino acid sequence SEQ ID NO. 31 (NCBI reference of the protein encoded by the Blamab gene of M. abscessus: WP_005091054.1).

[0161] Complete amino acid sequence of Mycobacterium abscessus β-lactamase, SEQ ID NO. 31:

[0162] MISRRALLVGGVSAVGWAAGCSRNGNRRPAPDELASLEKDFGGRIGVYALDTGSGD TVGHRADERFLMCSTVKTFIVSAILRRRLSEPGLLDQRIQYTQSDVLEWAPITSQHVST GMTVSELCDATLRYSDNTGANLLITQLGGPKETEKFVRSLGDNVTRMDRTEVQLNIPD GDLDTSTPQQLVANLRRLVLDEGLDSRGRDLLTDWLKRNTTGDQSIRAAVPAGWTVA DKTGGGFKGETNDIAVIWPPGRAPIVMAVLTVPEDPTSTKGKPTIAAAT RIVLRAFGA

[0163] Amino acids 1 to 30 of the amino acid sequence SEQ ID NO. 31 correspond to the sequence of which is processed and immediately eliminated in the periplasm and is not part of the protein released into the periplasmic space.

[0164] Class A β-lactamases are a very large family of proteins in Gram-positive bacteria. The antigenic protein was chosen based on its high homology with other β-lactamases that exist in Gram-positive bacteria and are associated with serious infectious diseases, where this homology was determined by a multiple alignment of various Gram-positive β-lactamases against the peptide of SEQ ID NO. 21. The alignment was carried out with the NCBI BLASTp tool, and the amino acid sequences SEQ ID NO. 21, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39 and SEQ ID NO. 40 were included. High homology is demonstrated in the amino acids of the catalytic site between the sequence SEQ ID NO. 21 with the rest of the amino acid sequences of Gram-positive class A p-lactamases, and its essential residues S70 and E166, in addition to R164 and D179 contained in the Q loop and characteristic of this family (Fig.1).

[0165] Antigenic protein of Mycobacterium abscessus SEQ ID NO. 21 .

[0166] Amino acid sequence of Mycobacterium tuberculosis class A p-lactamase, SEQ ID NO. 35 (NCBI Reference Sequence: WP_057351649.1):

[0167] MRNRGFGRRELLVAMAMLVSVTGCARHASGARPASTTLPAGADLADRFAEL ERRYDARLGVYVPATGTTAAIEYRADERFAFCSTFKAPLVAAVLHQNPLTHLD KLITYTSDDIRSISPVAQQHVQTGMTIGQLCDAAIRYSDGTAANLLLADLGGPG GGTAAFTGYLRSLGDTVSRLDAEEPELNRDPPGDERDTTTPHAIALVLQQLVL GNALPPDKRALLTDWMARNTTGAKRIRAGFPADWKVIDKTGTGDYGRANDIA WWSPTGVPYVVAVMSDRAGGGYDAEPREALLAEAATCVAGVLA

[0168] Amino acid sequence of Listeria monocytogenes class A p-lactamase SEQ ID NO. 36 (NCBI Reference Sequence: WP_036094553.1):

[0169] MRMRKILLGLTVIFAVAGLVACGAPDKNAAATKKEETKQETENAQVKAALEKL EKKYGATLGVYGMDMGGKQTISFNAERFAAYASTFKAIAGGILLKNLTDEQLN KRFTFSKEDLVDHSPITEKHVDSGMTMKELINASMTYSDNTAANLQLQLGGP KGFEKELVKIGDKTMKPVRFEPELNDAVPGDIRDTTTPEAMAKTFAYLLTEGN LPADRLAYFKQTLIDNTTGATLIRAGVPAGYIVGDRTGAGSYGTRNAIAMIYPK DKDTKPLVWVIYSKKTGKDDEYNDQLIADAAKVLSDYYSL

[0170] Amino acid sequence of p-Lactamase class A from Staphylococcus aureus SEQ ID NO: 37 (NCBI Reference Sequence: WP_149508696.1):

[0171] VKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDA GQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLL TTIGGPKELTAFLHNMGDHVTRLDWEPELNEAIPNDERDTTMPVAMATTLRK LLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGI IAALGPDGKPSRVWIYTTGSQATMDERQIAE

[0172] Amino acid sequence of Streptococcus pneumoniae class A p-Lactamase SEQ ID NO. 38 (GenBank: CGG65551.1): MYVLNKFFTYKKIVPWLLSCVTLIGCSNSNTPSELPKQTKQIKQIKQENTRHD SFAKLEKEYNAKLGIYALDTGTNQTVAYHPNDRFAFASTSKSLAAGALLRQNSI EALDERITYTREDLSNYNPITEKHVDTGMTLKELADASVRYSDSTAHNLILKKL GGPSAFEKILRETGDTVTKSERFEPELNEVHPGETRDTSTPEAIAKTLQSFTLG SALPTEKRELLVDWMKRNTTGDKLIRAGVPKGWEVADKTGAGSYGTRNDIAII WPPNKPKPIVLAILSNHDKEDAEYDDKLIADATKVVLDTLKVTNKN

[0173] Amino acid sequence of Clostridium Difficile class A p-Lactamase SEQ ID NO. 39 (GenBank: HBH2479975.1):

[0174] MKKLIFLIAIALVLSACNSNSSHAKELNDLEKKYNAHIGVYALDTKSGKEVKFNS DKRFAYASTSKAI NSAI LLEQVPYN KKI HIN KDDI VAYSPI LEKYVGKDITLKE LIEASMAYSDNTANNKIIKEIGGIKKVKQRLKELGDKVTNPVRYEIELNYYSPKS KKDTSTPAAFGKTLNKLIANGKLSKENKKFLLDLMLNNKSGDTLIKDGVSKDCK VADKSGQAITYASRNDVAFVYPKGQSEPIVLVIFTNKDNKSDKPNDKLISETAK SVMKEF

[0175] Amino acid sequence of p-Lactamase class A from Bacillus anthracis SEQ ID NO. 40 (GenBank: PES83414.1):

[0176] MIVLNKFFNMLPYKKIVPVLFLSCATLIGCSNSNTQSEPPQQQENTVNHSFA KLEKEFDAKLGIYALLDTNQTVTYQSDERFAYASTKALAVGVLLQKKSIEDL NQRILYTREDLVNYNPITEKYVDTGMTLKELADASLRYSDNQLQLQLGGLG SEFKKSLREIGDTVTNPERFEPELNEVHPGDTHDTSTPKALATSLQAFALGDIL STEKRNLLIDWMKRNTTGDKLIRAGVPGGWEVADKTGSGSYGTRNDIAIIWPP NKPPIVLAILSNQGKEDAKYDDKLIAEATKIVLHALKTPNKQ

[0177] The 3D structure of the protein expressed by Blamab gene from Mycobacterium abscessus (SEQ ID NO. 21) (Fig. 2b) was generated using the artificial intelligence tool Alphafold (Jumper, J.,et al. 2021. Nature, 596(78), 583; et al. 2022. Nucleic acids research, 50(D1), D439-D444).

[0178] KPC-2 family proteins are phylogenetically closest to Blamab (Brandt C, et al. 2017. Sci Rep. Feb 24; 7:43232.) and are also found in Gram-negative bacteria such as Klebsiella pneumoniae or Pseudomonas aeruginosa. Both the KPC-2 protein from Klebsiella pneumoniae (Uniprot: Q9F663) and the KPC-2 protein from Pseudomonas aeruginosa consist of the amino acid sequence SEQ ID NO. 32:

[0179] MSLYRRLVLLSCLSWPLAGFSATALTNLVAEPFAKLEQDFGGSIGVYAMDTGSGATVS YRAEERFPLCSSFKGFLAAAVLARSQQQAGLLDTPIRYGKNALVPWSPISEKYLTTGM TVAELSAAAVQYSDNAAANLLLKELGGPAGLTAFMRSIGDTTFRLDRWELELNSAIPG DARDTSSPRAVTESLQKLTLGSALAAPQRQQFVDWLKGNTTGNHRIRAAVPADWAV GDKTGTCGVYGTANDYAVVWPTGRAPIVLAVYTRAPNKDDKHSEAVIAAAARLALEG LGVNGQ

[0180] More than 50% of people who die in hospital with acquired pneumonia die if their infection is caused by a strain that produces KPC-2. The 3D model of the protein encoded by the Blamab gene of M. abscessus was found to contain the structural characteristics of KPC-2 proteins (Fig. 2a), such as: the three helices, the Q loop, the hinge region and the loop between helices a3 and a4 (Fig. 2b) (Galdadas I, et al., 2018. Sci Rep. Aug 27;8(1):12916). Furthermore, the BLASTp alignment of the sequences SEQ ID NO. 21 and SEQ ID NO. 32 shows a high level of conservation, especially in the residues essential for enzymatic activity: S70, E166, R164 and D179 (Fig.3).

[0181] In the design of the DNA construct for the expression of the protein encoded by the Blamab gene of M. abscessus to which a TEV protease recognition zone, a DYKDDDDK tag and a histidine tail were added in the C-terminal zone that would allow its purification (SEQ ID NO. 33 nucleotide sequence encoding the protein encoded by the modified Blamab gene of M. abscessus). The construct was flanked with BamHI restriction sites that allowed "in frame" cloning within the expression vector pET26b. The sequence was optimized for production in E. coli. K12 using EMBOSS Backtranseq (Madeira F, Pearce M, Tivey ARN, et al. 2022. Nucleic Acids Research Jul;50(W1):W276-W279).

[0182] Nucleotide sequence of synthetic construct for the expression of the protein fragment encoded by the modified Blamab gene of M. abscessus, SEQ ID NO. 33:

[0183] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAG

[0184] CCGGCGATGGCCATGGATATCGGAATTAATTCGGATCCGGCGCCGGATGAACTG

[0185] GCGAGCCTGGAAAAAGATTTTGGCGGCCGCATTGGCGTGTATGCGCTGGATACC GGCAGCGGCGATACCGTGGGCCATCGCGCGGATGAACGCTTTCTGATGTGCAGC ACCGTGAAAACCTTTATTGTGAGCGCGATTCTGCGCCGCCGCCTGAGCGAACCG GGCCTGCTGGATCAGCGCATTCAGTATACCCAGAGCGATGTGCTGGAATGGGCG CCGATTACCAGCCAGCATGTGAGCACCGGCATGACCGTGAGCGAACTGTGCGAT GCGACCCTGCGCTATAGCGATAACACCGGCGCGAACCTGCTGATTACCCAGCTG GGCGGCCCGAAAGAAACCGAAAAATTTGTGCGCAGCCTGGGCGATAACGTGACC CGCATGGATCGCACCGAAGTGCAGCTGAACATTCCGGATGGCGATCTGGATACC AGCACCCCGCAGCAGCTGGTGGCGAACCTGCGCCGCCTGGTGCTGGATGAAGG CCTGGATAGCCGCGGCCGCGATCTGCTGACCGATTGGCTGAAACGCAACACCAC CGGCGATCAGAGCATTCGCGCGGCGGTGCCGGCGGGCTGGACCGTGGCGGATA AAACCGGCGGCGGCTTTAAAGGCGAAACCAACGATATTGCGGTGATTTGGCCGC CGGGCCGCGCGCCGATTGTGATGGCGGTGCTGACCGTGCCGGAAGATCCGACC AGCACCAAAGGCAAACCGACCATTGCGGCGGCGACCCGCATTGTGCTGCGCGCG TTTGGCGCGGAAAACCTGTATTTTCAGAGCGATTATAAAGATGATGATGATAAAGA GGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCA CCACCACCACTGA

[0186] The protein or polypeptide encoded by the nucleotide sequence SEQ ID NO. 33 consists of the amino acid sequence SEQ ID NO. 34, which comprises the amino acid sequence SEQ ID NO. 21. That is, SEQ ID NO. 34 comprises the class A β-lactamase fragment of M. abscessus (SEQ ID NO. 21) that was used as an antigen for obtaining antibodies (hereinafter antigenic protein). The protein with the amino acid sequence SEQ ID NO. 34 comprises the amino acid sequence SEQ ID NO. 21, and at its C-terminal end comprises a C-terminal region, a TEV protease recognition region SEQ ID NO. 42: ENLYFQS, a DYKDDDDK tag (SEQ ID NO. 43), and a HHHHHH histidine tail (SEQ ID NO. 25) (underlined in SEQ ID NO. 34), for its purification.

[0187] Amino acid sequence of the peptide comprising fragment of the protein encoded by the M. abscessus Blamab gene (SEQ ID NO. 21) + TEV protease recognition region, a DYKDDDDK tag and a HHHHHH histidine tail, SEQ ID NO. 34:

[0188] MKYLLPTAAAGLLLLAAQPAMAMDIGINSDPAPDELASLEKDFGGRIGVYALDTGSGD

[0189] TVGHRADERFLMCSTVKTFIVSAILRRRLSEPGLLDQRIQYTQSDVLEWAPITSQHVST

[0190] GMTVSELCDATLRYSDNTGANLLITQLGGPKETEKFVRSLGDNVTRMDRTEVQLNIPD GDLDTSTPQLVANLRRLVLDEGLDSRGRDLLTDWLKRNTTGDQSIRAAVPAGWTVA DKTGGGFKGETNDIAVIWPPGRAPHIVMAVLTVPSTACTATKIVLGRAPH NLYFQSDYKDDDDKEDPNSSSVDKLAAALEHHHHHH

[0191] The synthetic DNA (SEQ ID NO. 33) was cloned into the pET26b vector for periplasmic expression in E. coli. Once the protein of SEQ ID NO. 34 is expressed, the fragment corresponding to amino acids 1 to 30 of said sequence is immediately eliminated in the periplasm and is not part of the protein released into the periplasmic space that is subsequently used as an antigen in the process of obtaining antibodies. After cell lysis of induced E. coli cultures, the protein was purified by immobilized metal ion affinity chromatography. The concentration was calculated by Bradford, and the integrity was checked by denaturing PAGE gels and subsequent western blot analysis. Finally, nitrocefin assays were performed to verify the enzymatic activity of Blamab, which resulted in a Km = 45.3 ± 2.5 pM. The Km reflects the activity of the enzyme, which was found to be in the range of other β-lactamases described in the literature.

[0192] Example 2. Process for obtaining class A B-lactamase inhibitor antibodies.

[0193] Antibodies were obtained by selective selection from a naive library of sdAbs displayed on the surface of yeast. The library was obtained by assembly of synthetic sequences where the FRs are fixed and the CDRs contained increased variability by error-prone PCR. Since the FR sequences were known and fixed, it was possible to determine the position and sequence of the CDRs. FR1, FR2, FR3 and FR4 of the sdAbs in the synthesized library comprised the amino acid sequences SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46 and SEQ ID NO. 47, respectively.

[0194] Amino acid sequences of FR1, SEQ ID NO. 44:

[0195] QVQLQESGGGLVQAGGSLRLSCAAS

[0196] Amino acid sequence of FR2, SEQ ID NO. 45:

[0197] MGWYRQAPGKER Amino acid sequence of FR3, SEQ ID NO. 46:

[0198] ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA

[0199] Amino acid sequence of FR4, SEQ ID NO. 47:

[0200] YWGQGTQVTVSS

[0201] The original collection contained approximately 10 A Nine combinations of sdAbs were generated by error-prone POR. The conditions for introducing random mutations into DNA were achieved by adding 2 pM of 8-oxo-dGTP and 2 pM of dPTP to the POR reaction. These two nucleotide analogs are introduced during the DNA extension step, and in a second conventional POR, they are replaced by natural nucleotides, thus creating a new sequence. The new constructs were transformed by electroporation into S. cerevisiae cells. A library with an approximate variability of 10 A 9 sdAbs. The library was amplified and stored at -80°C for later use.

[0202] For the selection process, the antigenic protein obtained in Example 1 (which comprises the amino acid sequence SEQ ID NO. 21) was incubated with yeast cells expressing the sdAbs library and subsequently stained with anti-His and anti-DYKDDDDK antibodies linked to a fluorophore. This makes it possible to trap cells expressing antibodies that recognize the antigenic protein. The process must be repeated in at least four rounds of selection by MACS (Magnetic-activated cell sorting) and another round of selection by FACS (Fluorescence-activated Cell Sorting). This process is carried out as many times as necessary until an increase in the population of antigen-binding cells is observed, as verified by flow cytometry.

[0203] Once a collection containing approximately 200 antibodies was selected by FACS, ELISA assays were performed on all variants in replicates. The method is similar to a sandwich ELISA, except that it works with whole cells. The selected clones are grown on the FACS in 96-well U-shaped plates. Subsequently, surface protein expression is induced and incubated with a concentration of 20 pg / mL of antigenic protein for 1 hour at 4°C. For immunological detection, anti-DYKDDDDK antibodies that recognize the antigenic protein were used, and an anti-Rabbit conjugated to alkaline phosphatase was used as the detection antibody. The 10 sdAbs with the highest antigen-binding capacity were selected and sequenced, cloned into the periplasmic expression vector pET26b for expression in E. coli, and subsequently purified by chromatography.Of the 10 selected sdAbs, 2 had identical sequences, and the protein could not be obtained from one, possibly due to its toxicity. Finally, 7 sdAbs were selected to study their affinity and inhibition capacity. The seven sdAbs obtained were named B1, B2, B3, B4, B5, B6, and B7, and presented the CDRs according to Table 1.

[0204] Table 1. Single domain antibodies against β-lactamase

[0205] Amino acid sequence of antibody B1, SEQ ID NO. 29:

[0206] MQVQLQESGGGLVQAGGSLRLSCAASGNIFTVSSMGWYRQAPGKEREFVAAISDGA TTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYASRVGLTVYWGQGT QVTVSS

[0207] Amino acid sequence of antibody B7 SEQ ID NO. 30:

[0208] MQVQLQESGGGLVQAGGSLRLSCAASGTISKAPVMGWYRQAPGKEREFVAGIASGS NTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYVHIRRSGYIALTYWG QGTQVTVSS

[0209] Example 3. Class A B-lactamase inhibitor antibody activity assays.

[0210] The inhibitory capacity of β-lactamase was determined using nitrocefin ionometric assays. This type of assay provides quantitative data that can be used for enzymological studies. The inhibition constant of antibodies B1 to B7 against the β-lactamase comprising the sequence SEQ ID NO. 21 was calculated.

[0211] 10 pM of β-lactamase was added with nitrocefin in a reaction mixture volume of 100 pl at room temperature in a PBS, phosphate buffered saline pH 6.5 solution. The hydrolysis of nitrocefin was monitored using a microplate reader. The concentration of hydrolyzed nitrocefin (c) was calculated from its absorbance (A), molar extinction coefficient (E) (20,500) and path length (I) (0.5 cm) values ​​using Beer’s law: A = sel.

[0212] To evaluate the inhibition of nitrocefin hydrolysis in the presence of antibodies B1 to B7, the antibodies were added, in separate reaction mixtures at a final concentration of 10 pM, to a reaction mixture containing nitrocefin, with the sample without antibodies being used as a control.

[0213] To calculate Vmax and Km, the experiment was carried out with different concentrations of nitrocefin (40pM, 60pM, 80pM, 100pM, 200pM) and 1 / v (initial velocity) was represented against 1 / [c] (nitrocefin concentration) in a Lineweaver-Bu rk diagram.

[0214] Once Vmax and Km were known, K¡ was cleared from the formula K¡ = Km [l] / (Km , apparent - Km ) where Km, apparent corresponds to the Km with inhibitor, and where “[I]” corresponds to the amount of antibody. Thus, sdAbs B2-B6 presented inhibition constants (K¡) between 2.4 and 5.2 pM calculated using the Lineweaver-Bu rk method, lower than those of B1 and B7 (in the case of B7 it does not present inhibition) which means that they are capable of inhibiting the enzymatic activity of β-lactamases at very low concentrations (Table 2). Table 2. Values ​​of Vmax, Km and K¡ obtained in the inhibition assays of antibodies B1-B7 against M. abscessus β-lactamase, with and without antibody. In addition, the affinity of antibodies B2, B5, B7 and a non-β-lactamase specific control antibody (antibody D1.1) for the peptide comprising the amino acid sequence SEQ ID NO. 21 was determined by ELISA assays. All antibodies were added at the same concentration. It was observed that antibodies B2, B5 and B7 had a higher affinity than the control D1.1 in a comparative assay (Table 3). A higher absorbance means that there is a greater amount of bound antibody. Antibody B7 has a higher affinity, but according to the data in Table 2, it does not produce inhibition, so it does not indicate that antibody B7 binds to the catalytic center.

[0215] Table 3. Absorbance values ​​at 405 nm of the ELISA affinity assay

[0216] Amino acid sequence of the control antibody D1.1, SEQ ID NO. 41:

[0217] QVQLQESGGGLVQAGGSLRLSCAASGTISYSYSMGWYRQAPGKDREFVATIAAGGS TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVRYQPQPHPRVYHYYW GQGTQVTVSS

[0218] Example 4. Blamab and VIM-2 B-lactamase inhibitory antibody activity assays

[0219] An in vivo assay was performed to test the inhibition capacity of four β-lactamases from three resistant microorganisms: BlaMab from Mycobacterium abscessus, KPC-2 (UNIPROT Q9F663) and OXA48 (UNIPROT Q6XEC0) from Klebsiella pneumoniae, and VIM-2 (UNIPROT B8QIQ9) from Pseudomonas aeruginosa. The assay was performed using Escherichia coli strain BL21 (DE3) cells transformed with the pET26b(+) expression vector together with the gene sequence of each β-lactamase. Cells correctly transformed with the vector and the insert were selected using a kanamycin resistance gene (KanR) present in the vector. Subsequently, the presence and orientation of the insert was verified using POR and sequencing. Furthermore, the pET26b(+) vector presents an IPTG-inducible promoter to activate the translation of the corresponding p-lactamase.The plasmid also has an export signal, pelB, which directs the β-lactamase to the peñplasmic space of the bacteria in order to perform the inhibition assay with Ndis (nanobodies, single domain antibodies). To obtain Table 4 with the enzymatic constants (Km, Vmax, Kmi and Ki) of Blamab-2, KPC-2, VIM-2 and OXA-48 with B2 and B5, a quantitative assay was performed using a compound analogous to cephalosporins: nitrocefin. This compound turns from yellow to red when hydrolyzed by β-lactamase. This color change is quantifiable by measuring the absorbance at 490 nm. In this way, the penetrance of the antibodies in the bacteria through the outer membrane has been studied; as well as the inhibitory effect of B2 and B5 on the activity of BlaMab, KPC-2, OXA48 and VIM-2. It has been shown that B2 and B5 are able to penetrate the outer membrane and inhibit both the p-lactamase of the gram-positive bacterium M.abscessus, for which they are specific, as well as the p-lactamases of gram-negative bacteria such as KPC-2 and OXA48 of klebsiella pneumoniae, and VIM-2, of Pseudomonas aeruginosa (Table 4).

[0220] Table 4. Enzyme constant results (Km, Vmax, Kmi and K¡) of Blamab-2, KPC-2, VIM-2 and OXA-48 with B2 and B5

Claims

CLAIMS 1. A class A β-lactamase inhibitor antibody from Gram-positive bacteria, or a fragment thereof, characterized in that said antibody or fragment thereof comprises an inhibition constant (K¡) of less than 10 pM against a β-lactamase comprising the amino acid sequence SEQ ID NO.

21.

2. Antibody or fragment thereof according to claim 1, wherein said antibody is from a human, mouse, rat, camelid, bird, a chimeric antibody or a humanized antibody.

3. Antibody or fragment thereof according to any one of claims 1 or 2. 2, where said antibody is selected from the list consisting of IgY, lgG1, lgG2, lgG3 and HCAb.

4. Antibody or fragment thereof according to any one of claims 1 to 3. 3, where said antibody is a monospecific or multispecific antibody.

5. Antibody or fragment thereof according to any one of claims 1 to 3. 4, where the antibody is a monoclonal antibody.

6. Antibody or fragment thereof according to any one of claims 1 to 3. 5, wherein the antibody fragment is selected from the list consisting of a single domain antibody (sdAb), an IgG1 heavy chain antibody (HCIgGI), an IgG2 heavy chain antibody (HCIgG2), an IgG3 heavy chain antibody (HCIgG3), an IgG4 heavy chain antibody (HCIgG4), a Fab fragment, an F(ab')2 fragment, an Fv fragment, disulfide-linked Fv fragments (sdFv), an Fd fragment, and a scFv fragment.

7. Antibody or fragment thereof according to any one of claims 1 to 3. 6, where the antibody is a single domain antibody.

8. Antibody or fragment thereof according to any one of claims 1 to 2. 7, wherein said antibody or fragment comprises: a) a CDR1 consisting of the amino acid sequence SEQ ID NO. 1, a CDR2 consisting of the amino acid sequence SEQ ID NO. 2 and a CDR3 consisting of the amino acid sequence SEQ ID NO. 3; b) a CDR1 consisting of the amino acid sequence SEQ ID NO. 4, a CDR2 consisting of the amino acid sequence SEQ ID NO. 5 and a CDR3 consisting of the amino acid sequence SEQ ID NO. 6; c) a CDR1 consisting of the amino acid sequence SEQ ID NO. 7, a CDR2 consisting of the amino acid sequence SEQ ID NO. 8 and a CDR3 consisting of the amino acid sequence SEQ ID NO. 9; d) a CDR1 consisting of the amino acid sequence SEQ ID NO. 10, a CDR2 consisting of the amino acid sequence SEQ ID NO. 11 and a CDR3 consisting of the amino acid sequence SEQ ID NO. 12; or e) a CDR1 consisting of the amino acid sequence SEQ ID NO. 13, a CDR2 consisting of the amino acid sequence SEQ ID NO. 14 and a CDR3 consisting of the amino acid sequence SEQ ID NO.

15.

9. Antibody or fragment thereof according to any one of claims 1 to 8, wherein the antibody comprises the sequence SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19 or SEQ ID NO.

20.

10. Antibody or fragment thereof according to any one of claims 1 to 9, wherein the antibody or fragment is linked to a therapeutic agent or a detectable marker.

11. Antibody or fragment thereof according to claim 10, wherein the therapeutic agent is an antibacterial agent.

12. Antibody or fragment thereof according to claim 10, wherein the detectable label is selected from the list consisting of a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a radioisotope, a prosthetic group, a positron-emitting metal and a non-radioactive paramagnetic metal ion.

13. Antibody or fragment thereof according to any one of claims 1 to 12, wherein the antibody or fragment thereof is immobilized on a solid support, a lipid particle, a nanoparticle, on the surface of a virus or on the surface of a cell.

14. Pharmaceutical composition comprising the antibody or fragment according to any one of claims 1 to 13.

15. Pharmaceutical composition according to claim 14, wherein said composition comprises a pharmaceutically acceptable excipient and / or vehicle.

16. Antibody according to any one of claims 1 to 13, or pharmaceutical composition according to any one of claims 14 or 15, for use as a medicament.

17. Antibody or fragment according to any one of claims 1 to 13, or pharmaceutical composition according to any one of claims 14 or 15, for use in the treatment and / or prevention of an infection caused by a β-lactamase-producing bacterium in a subject.

18. Antibody, fragment or pharmaceutical composition for use according to claim 17, wherein the bacteria produces a class A β-lactamase.

19. Antibody, fragment or pharmaceutical composition for use according to any one of claims 17 or 18, wherein the bacteria is Gram positive.

20. Antibody, fragment or pharmaceutical composition for use according to any one of claims 17 to 19, wherein the bacterium is selected from the list consisting of Mycobacterium abscessus, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Mycobacterium leprae, Klebsiella pneumoniae, Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Clostridium botulinum, Clostridium novyi, Clostridium septicum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium mangenotii, Bacillus anthracis, and Corynebacterium diphtheriae 21. Antibody, fragment or pharmaceutical composition for use according to any one of claims 17 to 20, wherein the route of administration is selected from the list consisting of oral, intravenous, intramuscular, intramuscular, intraarterial, intravenous, vaginal, intradermal, subcutaneous, topical, ophthalmic and inhalation route.

22. Antibody, fragment or pharmaceutical composition for use according to any one of claims 17 to 21, wherein the infection is selected from the list consisting of pulmonary infection, urinary infection, skin infection, nervous system infection, bacteremia, septicemia, ocular infection and sexually transmitted infection.

23. A polynucleotide encoding the antibody or fragment according to any one of claims 1 to 13.

24. An expression vector comprising the polynucleotide according to claim 23.

25. A host cell comprising the polynucleotide according to claim 23 or the expression vector according to claim 24.

26. A polypeptide consisting of the amino acid sequence SEQ ID NO.

21.

27. In vitro use of a polypeptide comprising an amino acid sequence with at least 80% identity with SEQ ID NO. 21, preferably consisting of the amino acid sequence SEQ ID NO. 21, for obtaining antibodies or fragments thereof.

28. Use according to claim 27, wherein the antibodies, or fragments thereof, are from human, mouse, rat, camelid, bird, chimeric antibodies or humanized antibodies.

29. Use according to any one of claims 27 or 28, wherein the antibodies are selected from the list consisting of IgY, IgG1, IgG2, IgG3, IgG4 and HCAb.

30. Use according to any one of claims 27 to 29, wherein the antibodies are monoclonal.

31. Use according to any one of claims 27 to 30, wherein the antibody fragments are selected from the list consisting of single domain antibodies (sdAb), IgG1 heavy chain antibodies (HCIgGI), IgG2 heavy chain antibodies (HCIgG2), IgG3 heavy chain antibodies (HCIgG3), IgG4 heavy chain antibodies (HCIgG4), Fab fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv fragments (sdFv), Fd fragments and scFv fragments.

32. Use according to any one of claims 27 to 31 wherein the antibodies are single domain antibodies.

33. Use according to any one of claims 27 to 32, wherein the antibodies are VHH single domain antibodies or humanized VHH single domain antibodies.

34. An in vitro method for obtaining an antibody or antibody fragment against β-lactamase, said method comprising: a) providing a library of nucleic acids encoding antibodies or fragments thereof, said antibodies or fragments comprising at least one immunoglobulin variable domain; b) introducing the library from step a) into a surface display system selected from the list consisting of a phage surface display system, a prokaryotic surface display system, and a eukaryotic surface display system; c) contacting the display system from step b) with an antigen consisting of a polypeptide comprising an amino acid sequence with at least 80% identity to SEQ ID NO: 21, preferably consisting of the amino acid sequence SEQ ID NO. 21; and d) selecting antibodies, or fragments thereof, that specifically bind to the antigen from step c).

35. Method according to claim 34, wherein the antibodies, or fragments thereof, are from human, mouse, rat, camelid, bird, chimeric antibodies or humanized antibodies.

36. Method according to any one of claims 34 or 35, wherein the antibodies are selected from the list consisting of IgY, IgG1, IgG2, IgG3, IgG4 and HCAb.

37. Method according to any one of claims 34 to 36, wherein the antibodies are monoclonal.

38. The method of any one of claims 34 to 37, wherein the antibody fragments are selected from the list consisting of single domain antibodies (sdAb), IgG1 heavy chain antibodies (HCIgGI), IgG2 heavy chain antibodies (HCIgG2), IgG3 heavy chain antibodies (HCIgG3), IgG4 heavy chain antibodies (HCIgG4), Fab fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv fragments (sdFv), Fd fragments and scFv fragments.

39. Method according to any one of claims 34 to 38, wherein the antibodies are single domain antibodies.

40. Method according to any one of claims 34 to 39, wherein the antibodies are VHH single domain antibodies or humanized VHH single domain antibodies.