Anti-CHIKV antibodies and their use

Abstract

To provide an antibody that neutralizes Chikungunya virus (CHIKV) and is suitable for prophylactic and therapeutic use against CHIKV, and also to provide pharmaceutical compositions comprising the antibody and the use of the antibody for the prevention and treatment of CHIKV disease.SOLUTION: The present invention provides an isolated monoclonal antibody that binds to CHIKV and comprises three heavy chain complementarity determining regions (CDRHs) and three light chain complementarity determining regions (CDRLs), where the CDRHs and the CDRLs have specific amino acid sequences.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an antibody and an antigen-binding fragment of the antibody that specifically bind to and neutralize the chikungunya virus (CHIKV), which can be manipulated to provide prophylactic and therapeutic solutions for the prevention and treatment of CHIKV disease. The present invention also relates to a pharmaceutical composition containing an antibody that neutralizes CHIKV and to the use of the antibody for the prevention and treatment of CHIKV disease. [Background technology]

[0002] CHIKV is a re-emerging, mosquito-borne pathogen. While endemic to Africa, India, and Southeast Asia, CHIKV can spread beyond these regions, infecting millions of people in unpredictable and rapid outbreaks with high rates of attack (Non-Patent Literature 1). A mutation in the CHIKV envelope glycoprotein 1 (E1) allows the virus to be transmitted by Aedes albopictus in addition to Aedes aegypti, leading to a severe epidemic in Réunion, India, and Indonesia in 2005, followed by rapid outbreaks in Italy, France, and China, initially among travelers (Non-Patent Literature 2; Non-Patent Literature 3; Non-Patent Literature 4; Non-Patent Literature 5; Non-Patent Literature 6). Based on the geographical range of Aedes albopictus, the virus is expected to spread to new areas, and Europe and the Americas are now at risk of CHIKV outbreaks.

[0003] CHIKV is an enveloped, positive-strand RNA virus belonging to the genus Alphavirus in the family Togaviridae. It is a member of the Semlik Forest Virus Complex and is closely related to Ross River virus and Onyonnyon virus (ONNV); it can also be called an arbovirus (a virus carried by arthropods) because it is transmitted by arthropods, namely mosquitoes.

[0004] CHIKV enters cells via receptor-mediated internalization and type II membrane fusion events triggered by low pH in early endosomes. CHIKV disease is characterized by acute, post-acute, and chronic polyarthritis / polyarthralgia phases, the latter of which is usually symmetrical, often resulting in incapacitation, and can last for months or even years. Other symptoms such as fever, rash, muscle pain, and / or fatigue may also be present during the acute phase. Recent outbreaks have been associated with atypical and severe clinical forms of CHIKV disease, some of which have been fatal, and appear to be limited to very young and elderly patients with comorbidities.

[0005] Currently, there are no specific preventive or therapeutic measures for CHIKV disease. CHIKV is usually treated with bed rest, fluid therapy, and medications such as simple analgesics and / or nonsteroidal anti-inflammatory drugs (NSAIDs) to alleviate symptoms of fever and pain. While a vaccine candidate against CHIKV was first proposed 45 years ago, many vaccine candidates tested to date have failed to induce protective antibodies or demonstrate significant safety outcomes. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Powers AM,Logue CH,2007,J.Gen.Virol.88:2363-2377 [Non-Patent Document 2] Tsetsarkin KA et al. 2007, PLoS Pathog.3:e201; [Non-Patent Document 3] Schuffenecker I et al. 2006, PLoS Med.3:e263; [Non-Patent Document 4] Wu D, Zhang Y et al. 2013, Virol.J.10:174; [Non-Patent Document 5] Rezza G et al. 2007, Lancet 370:1840-1846; [Non-Patent Document 6] Burt FJ 2012, Lancet 379:662-671 [Overview of the project] [Problems that the invention aims to solve]

[0007] There is still a need for treatments that demonstrate improved therapeutic efficacy against CHIKV, including the use of specific monoclonal antibodies targeting CHIKV. There is a need in the art for CHIKV-neutralizing antibodies suitable for prophylactic and therapeutic use. In particular, such antibodies need to appropriately neutralize different strains of CHIK virus with high target binding affinity, exhibit appropriate pharmacokinetic parameters, have a suitable half-life upon administration, and retain their binding to FcγRIIIa, which is associated with effector function, while enabling efficient production on a large scale. [Means for solving the problem]

[0008] As disclosed in this invention, the inventors of this application were able to select and manipulate specific CHIKV neutralizing antibodies, improve their exposure-related pharmacokinetics, and maintain their binding to FcγRIIIa related to effector function, thereby adapting them to the development of therapeutics for preventing and treating CHIKV disease and addressing the need in the art for effective therapies against CHIKV.

[0009] The antibodies of the present invention have high binding affinity (within the nanomolar concentration range) to different CHIKV strains. Therefore, they exhibit broad and very potent neutralizing activity against different CHIKV strains. Furthermore, the antibodies of the present invention have improved binding to the human FcRn receptor, while simultaneously retaining their binding to FcγRIIIa, thereby increasing their half-life and compatibility, and maintaining their binding to FcγRIIIa, which is associated with effector function.

[0010] In a first aspect, the present invention is an isolated monoclonal antibody that binds to CHIKV and comprises three heavy chain complementarity determining regions (CDRH) and three light chain complementarity determining regions (CDRL), wherein: i. the CDRH have the amino acid sequences of SEQ ID NO: 5, 6 and 7, and the CDRL have the amino acid sequences of SEQ ID NO: 8, GNT and SEQ ID NO: 10, or ii. the CDRH have the amino acid sequences of SEQ ID NO: 11, 12 and 13, and the CDRL have the amino acid sequences of SEQ ID NO: 14, GTS and SEQ ID NO: 16, or iii. the CDRH and CDRLH have amino acid sequences that differ from those of i. or ii. by one or two amino acid substitutions; and the antibody further comprises: iv. an alanine at position 434, or v. alanines at positions at 307, 380 and 434, respectively, or vi. a glutamine at position 250 and a leucine at position 428, respectively, or vii. a leucine at position 428 and a serine at position 434, respectively, or viii. a tyrosine at position 252, a threonine at position 254 and a glutamate at position 256, and further comprises an Fc region comprising at least one residue selected from the group consisting of: where the positions of the amino acids are with respect to the monoclonal antibody given by the EU index.

[0011] In one embodiment, the isolated monoclonal antibody binds to CHIKV and comprises three heavy chain complementarity determining regions (CDRH) and three light chain complementarity determining regions (CDRL), wherein: i. the CDRH have the amino acid sequences of SEQ ID NO: 5, 6 and 7, and the CDRL have the amino acid sequences of SEQ ID NO: 8, GNT and SEQ ID NO: 10, or ii. The CDRH has the amino acid sequences of SEQ ID NOs. 11, 12, and 13, and the CDRL has the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, or iii. The CDRH and CDRLH have amino acid sequences that differ from the sequences in i. or ii. due to one or two amino acid substitutions; And the antibody is: iv. Alanine at position 434, or v. Alanine at positions 307, 380 and 434, respectively, or vi. Glutamine at position 250 and leucine at position 428, respectively, vii. Leucine at position 428 and serine at position 434, respectively, or viii. Tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, respectively. It further comprises an Fc region containing at least one residue selected from the group consisting of; Here, the position of the amino acid is given by the EU index; And antibodies are as follows: ix. CHIKV pE2-E1 target binding dissociation equilibrium constant (K) less than approximately 10 nM D ) is used to join them; x. Human FcRn and Kless approximately 200 nM D Join them together; xi. Human FcγRIII and K D Join them together It possesses one or more properties.

[0012] In another embodiment, the monoclonal antibody comprises three heavy chain complementarity-determining regions (CDRHs) having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, and the antibody comprises three light chain complementarity-determining regions (CDRLs) having the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences.

[0013] In another embodiment, the monoclonal antibody is: - CDRH1 consisting of the sequence of sequence number 5; - CDRH2 consisting of the sequence of sequence number 6; - CDRH3 consisting of the sequence of sequence number 7; - CDRL1 consisting of the sequence of sequence number 8; - CDRL2 consisting of the GNT array; - CDRL3 consisting of the sequence of sequence number 10 Includes.

[0014] In a further embodiment, the monoclonal antibody comprises three heavy chain complementarity-determining regions (CDRHs) having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, or different amino acid sequences obtained by one or two amino acid substitutions from these sequences, and the antibody is Each contains either the amino acid sequence of SEQ ID NO: 14, GTS, and SEQ ID NO: 16, or three light chain complementarity-determining regions (CDRLs) having different amino acid sequences due to one or two amino acid substitutions from these sequences.

[0015] In another embodiment, the monoclonal antibody is: - CDRH1 consisting of the sequence of sequence number 11; - CDRH2 consisting of the sequence of sequence number 12; - CDRH3 consisting of the sequence of sequence number 13; - CDRL1 consisting of the sequence of sequence number 14; - CDRL2 consisting of the GTS sequence; - CDRL3 consisting of the sequence of sequence number 16 Includes.

[0016] In another embodiment, the monoclonal antibody is: i. Leucine at position 428 and serine at position 434, or ii. Tyrosine at position 252, threonine at position 254, and glutamate at position 256, respectively. It includes an Fc region containing a residue selected from the group consisting of the following, Here, the position of the amino acid is given by the EU index.

[0017] In a further embodiment, the monoclonal antibody comprises an Fc region, where the Fc region comprises leucine at position 428 and serine at position 434, and the positions of the amino acids are given by the EU index.

[0018] In another embodiment, the monoclonal antibody comprises a kappa light chain or a lambda light chain.

[0019] In another embodiment, the monoclonal antibody has an Fc region comprising or consisting of sequences having at least 80% identity with SEQ ID NOs. 59, 60, 61, 62, and 63.

[0020] In another embodiment, the monoclonal antibody has a variable region of its heavy chain that includes or consists of a sequence having at least 80% identity with SEQ ID NO: 1.

[0021] In another embodiment, the monoclonal antibody has a variable region of its light chain that includes or consists of a sequence having at least 80% identity with SEQ ID NO: 2.

[0022] In another embodiment, the monoclonal antibody has a heavy chain comprising or consisting of a sequence having at least 80% identity with SEQ ID NO: 31.

[0023] In another embodiment, the monoclonal antibody has a light chain comprising or consisting of a sequence having at least 80% identity with SEQ ID NO: 20.

[0024] In a second embodiment, an isolated monoclonal antibody is bound to CHIKV or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRHs) having amino acid sequences of SEQ ID NOs. 11, 12, and 33, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, and the antibody or its antigen-binding fragment further comprises three light chain complementarity-determining regions (CDRLs) having amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, where: i. The amino acid at position 8 of SEQ ID NO: 33 is not M, and / or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; and / or iii. The amino acid at position 13 in sequence number 33 is not G.

[0025] In one embodiment, a monoclonal antibody or its antigen-binding fragment is: CDRH1 consisting of the sequence of sequence number 11; CDRH2 consists of the sequence at sequence number 12; CDRH3 consisting of the sequence of sequence number 33; CDRL1 consists of the sequence of sequence number 14; CDRL2 consisting of GTS CDRL3 consisting of sequence number 16 Includes; here: i. The amino acid at position 8 of SEQ ID NO: 33 is not M, and / or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; and / or iii. The amino acid at position 13 in sequence number 33 is not G.

[0026] In another embodiment, the monoclonal antibody or its antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRHs) having the amino acid sequences of SEQ ID NOs. 11, 12, and 33, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, and the antibody or its antigen-binding fragment further comprises three light chain complementarity-determining regions (CDRLs) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, wherein: i. The amino acid at position 8 in SEQ ID NO: 33 is not M, or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; or iii. The amino acid at position 13 in sequence number 33 is not G.

[0027] In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 11, 12, and 33, respectively, and the antibody or its antigen-binding fragment further comprises three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, where: i. The amino acid at position 8 in SEQ ID NO: 33 is not M, or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; or iii. The amino acid at position 13 in sequence number 33 is not G.

[0028] In another embodiment, the monoclonal antibody or its antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRHs) having the amino acid sequences of SEQ ID NOs. 11, 12, and 33, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, and the antibody or its antigen-binding fragment further comprises three light chain complementarity-determining regions (CDRLs) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, wherein: i. The amino acid at position 8 in SEQ ID NO: 33 is not M, or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; or iii. The amino acid at position 13 of sequence number 33 is not G, Here, the antibodies are as follows: iv. CHIKV pE2-E1 target has a binding-dissociation equilibrium constant of less than 10 nM (K D ) is used to join them; v. Human FcRn with K200nM D Join them together; vi. Human FcγRIII with K600 nM D Join them together It possesses one or more properties.

[0029] In another embodiment, the monoclonal antibody comprises an amino acid at position 8 of SEQ ID NO: 33, selected from the group consisting of I, L, V, Q, and N.

[0030] In another embodiment, the monoclonal antibody is: - CDRH1 consisting of the sequence of sequence number 11; - CDRH2 consisting of the sequence of sequence number 12; - CDRH3 consisting of the sequence at sequence number 34; - CDRL1 consisting of the sequence of sequence number 14; - CDRL2 consisting of GTS; - CDRL3 consisting of the sequence of sequence number 16 Includes.

[0031] In another embodiment, the monoclonal antibody comprises an amino acid at position 12 of SEQ ID NO: 33, selected from the group consisting of Q, E, S, T, and D.

[0032] In further embodiments, the monoclonal antibody is: - CDRH1 consisting of the sequence of sequence number 11; - CDRH2 consisting of the sequence of sequence number 12; - CDRH3 consisting of the sequence of sequence number 35; - CDRL1 consisting of the sequence of sequence number 14; - CDRL2 consisting of GTS; - CDRL3 consisting of the sequence of sequence number 16 Includes.

[0033] In another embodiment, the monoclonal antibody comprises an amino acid at position 13 of SEQ ID NO: 33, selected from the group consisting of A, S, and T.

[0034] In further embodiments, the monoclonal antibody is: - CDRH1 consisting of the sequence of sequence number 11; - CDRH2 consisting of the sequence of sequence number 12; - CDRH3 consisting of the sequence at sequence number 36; - CDRL1 consisting of the sequence of sequence number 14; - CDRL2 consisting of GTS; - CDRL3 consisting of the sequence of sequence number 16 Includes.

[0035] In another embodiment, the monoclonal antibody has a variable region of its heavy chain that includes or consists of a sequence having at least 80% identity with SEQ ID NO: 57.

[0036] In another embodiment, the monoclonal antibody has a variable region of its light chain that includes or consists of a sequence having at least 80% identity with SEQ ID NO: 4.

[0037] In another embodiment, the monoclonal antibody has a heavy chain comprising or consisting of a sequence having at least 80% identity with SEQ ID NO: 47.

[0038] In another embodiment, the monoclonal antibody has a light chain comprising a sequence having at least 80% identity with SEQ ID NO: 38.

[0039] In another aspect of this second embodiment, the monoclonal antibody is: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively, or v. Tyrosine at position 252, threonine at position 254, and glutamate at position 256, respectively. The Fc region further comprises a residue selected from the group consisting of the following: Here, the position of the amino acid is given by the EU index.

[0040] In another embodiment, the monoclonal antibody is: i. Leucine at position 428 and serine at position 434, or ii. Tyrosine at position 252, threonine at position 254, and glutamate at position 256 It has an Fc region containing a residue selected from the group consisting of the following, Here, the position of the amino acid is given by the EU index.

[0041] In another embodiment, the monoclonal antibody has an Fc region comprising leucine at position 428 and serine at position 434, where the positions of the amino acids are given by the EU index.

[0042] In another embodiment of this aspect of the present invention, the monoclonal antibody comprises a kappa light chain or a lambda light chain.

[0043] In another embodiment, the monoclonal antibody has an Fc region comprising or consisting of sequences having at least 80% identity with SEQ ID NOs. 59, 60, 61, 62, and 63.

[0044] In another embodiment, the monoclonal antibody has a variable region of its heavy chain that includes or consists of a sequence having at least 80% identity with SEQ ID NO: 57.

[0045] In another embodiment, the monoclonal antibody has a variable region of its light chain that includes or consists of a sequence having at least 80% identity with SEQ ID NO: 4.

[0046] In another embodiment, the monoclonal antibody has a heavy chain comprising a sequence having at least 80% identity with SEQ ID NO: 53.

[0047] In another embodiment, the monoclonal antibody has a light chain comprising a sequence having at least 80% identity with SEQ ID NO: 38.

[0048] In a fourth aspect, the present invention relates to a monoclonal antibody for use as a pharmaceutical.

[0049] In another embodiment, the monoclonal is intended for use in the treatment of joint pain associated with CHIKV.

[0050] In another embodiment, a monoclonal antibody is used to prevent CHIKV infection. It belongs to them.

[0051] In a fifth aspect, the present invention relates to a pharmaceutical composition comprising a monoclonal antibody and at least one excipient.

[0052] In a sixth aspect, the present invention relates to a cell line that produces a monoclonal antibody.

[0053] A seventh embodiment is a method for producing a monoclonal antibody, the method comprising: (i) culturing a cell line according to the sixth embodiment; (ii) purifying the produced monoclonal antibody; and optionally (iii) forming the monoclonal antibody into a pharmaceutical composition.

[0054] In an eighth embodiment, the present invention relates to a polynucleotide comprising a sequence encoding an antibody characterized in the present invention or an antigen-binding fragment thereof. In one embodiment, the polynucleotide encodes a polypeptide having at least 80% identity with one of the sequences of SEQ ID NOs: 18, 21, 22, 24, 26, 28, 30, 32, 39, 40, 42, 44, 46, 48, 50, 52, and 54. In one embodiment, the polynucleotide is characterized by having a sequence having at least 80% identity with one of the sequences of SEQ ID NOs: 18, 21, 22, 24, 26, 28, 30, 32, 39, 40, 42, 44, 46, 48, 50, 52, and 54.

[0055] In a ninth embodiment, the present invention relates to a kit comprising at least one antibody characterized in the present invention. In one embodiment, the kit optionally comprises packaging material. [Brief explanation of the drawing]

[0056] [Figure 1]This figure shows the amino acid sequences of the CH1, hinge, CH2, and CH3 regions of the heavy chain of human IgG1; the hinge and CH2 and CH3 regions constitute the Fc region. The amino acid residue numbering follows the EU index described in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th, Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Substituted residues in the Fc region, which are the main focus of this invention, are enclosed in squares. [Figure 2] This figure shows the sequence alignment of the Fc region of IgG1: SEQ ID NOs. 17, 59, 60, 61, 62, and 63. [Figure 3] This figure shows the effect of mutations within CDRH3 of mAb2, designed to eliminate potential deamination and oxidation motifs, on binding to the E2-E1 target. A comparison of results is shown in two sequences for each mutant. [Figure 4-1] This figure shows the effect of substitutions in the Fc region of mAb1 and mAb2 on binding to FcRn. A comparison of the results at pH 6.0 is shown in two sequences for mAb1 (Figure 4A) and mAb2 (Figure 4C) on human FcRn, and for mAb1 (Figure 4B) and mAb2 (Figure 4D) on mouse FcRn. [Figure 4-2] Continuation of Figure 4-1. [Figure 4-3] Continuation of Figure 4-2. [Figure 4-4] Continuation of Figure 4-3. [Figure 5-1] This figure shows the effect of mutations in the Fc region of mAb1 and mAb2 on binding to the E2-E1 target. The results are shown in pairs for the E1-E2 antigens induced from strains LR2006 (Figure 5A) and SL15649 (Figure 5B), respectively. [Figure 5-2] Continuation of Figure 5-1. [Figure 6-1]This figure shows the effect of substitutions in the Fc region of mAb1 and mAb2 on FcγRIIIa binding. The binding results are shown in two sets (Figure 6C for mAb1 and Figure 6D for mAb2) for mAb1 (Figure 6A) and mAb2 (Figure 6B) against the human FcγRIIIa high-affinity receptor (FcγRIIIaV158), and for the human FcγRIIIa low-affinity receptor (FcγRIIIaF158). [Figure 6-2] Continuation of Figure 6-1. [Figure 6-3] Continuation of Figure 6-2. [Figure 6-4] Continuation of Figure 6-3. [Figure 7-1] This figure shows the neutralizing activity of mAb1 and mAb7 using a standard plaque reduction assay. mAb1 and mAb7 inhibit chikungunya virus from all three genotypes, namely the Asian type (Figure 7A), East Central and Southern African type (ESCA) (Figure 7B), and West African type (Figure 7C), with very potent activity. [Figure 7-2] Continuation of Figure 7-1. [Figure 7-3] Continuation of Figure 7-2. [Figure 8] A study on mAb-mediated prevention in mice. This figure shows the effect of mAb1 and mAb7, administered at 250 μg / mouse 2, 7, or 14 days prior to CHIKV infection, on viral titer 3 days after inoculation into the right hind leg of DBA / 1J mice. Viral titer is plotted as the 50% cell culture infectious dose (CCID50) per gram of tissue. [Figure 9-1]This figure shows post-exposure therapy for mAb in mice. Viral titers 5 days (dpi) after single intraperitoneal administration of fixed 250 μg doses of mAb2, 7, 11, and 14 in the right hind leg at 3 dpi (Figure 9A). The effect of dose ranges on viral titers in the right hind leg at 5 dpi for mAb7 and mAb14 after single doses of various doses (10 to 250 μg / mouse) at 3 dpi (Figure 9B). Viral titers are plotted as the 50% cell culture infectious dose per gram of tissue (CCID50). The upper horizontal line represents the mean limit of detection for tissue homogenates. [Figure 9-2] Continuation of Figure 9-1. [Figure 10] This figure shows the pharmacokinetics of mAbs in non-human primates. It compares the pharmacokinetics of mAb1 and mAb7 administered intravenously (IV) at a dose of 2.5 mg / kg to male cynomolgus monkeys (Macaca Fascicularis). [Modes for carrying out the invention]

[0057] definition An antibody is a natural or common antibody in which two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by disulfide bonds. In mammals, antibodies are classified into five major classes or isotypes: IgA, IgD, IgE, IgG, and IgM. They are further classified by the heavy chains they contain: alpha, delta, epsilon, gamma, or mu. These differ in the sequence and number of constant domains, hinge structure, and antibody valency. There are two types of light chains, lambda(l) and kappa(k), of which kappa light chains are more common. Although their protein sequences are relatively different, they share similar structures and functions.

[0058] Five major heavy chain classes (or isotypes) determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. IgG is the most abundant antibody in normal human serum, accounting for 70-85% of the total immunoglobulin pool. It is a monomer with a molecular weight of approximately 150 kDa, is the major antibody in the second immune response, and has the longest half-life of the five immunoglobulin classes. IgG has four human subclasses (IgG1, IgG2, IgG3, and I), each containing a different heavy chain. IgG1 and IgG4 consist of IgG1, IgG4, and IgG4. They are highly homologous, differing mainly in their hinge regions and the degree to which they activate the host immune system. IgG1 and IgG4 contain two interchain disulfide bonds in their hinge regions; IgG2 has four, and IgG3 has eleven.

[0059] The light chain contains two domains or regions: one variable domain (VL) and one constant domain (CL). The heavy chain contains four domains: one variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant domains of the light chain (CL) and heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of immunoglobulin and consists of one light chain and one heavy chain variable region. Antibody specificity lies in the structural complementarity between the antibody binding site and the antigenicity determinant. The antibody binding site is primarily composed of residues from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable groups or framework regions (FRs) affect the entire domain structure and, therefore, the binding site. A "complementarity-determining region or CDR" refers to the amino acid sequence that together determines the binding affinity and specificity of the native Fv region of the original immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, respectively, called CDR1-L, CDR2-L, CDR3-L (for the light chain's complementarity-determining region) or CDRL1, CDRL2, CDRL3 and CDR1-H, CDR2-H, CDR3-H (for the heavy chain's complementarity-determining region) or CDRH1, CDRH2, CDRH3. The antigen-binding site of a typical antibody therefore contains six CDRs, including sets of CDRs from the V regions of both the heavy and light chains.

[0060] The "framework region" (FR) refers to the amino acid sequences interspersed between CDRs, i.e., these portions of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins within a single species. The light and heavy chains of immunoglobulins each have four FRs, called FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively.

[0061] As used herein, the “human framework region” refers to a framework region that is substantially identical (about 85% or more, particularly 90%, 95%, 97%, 99%, or 100%) to the framework region of a naturally occurring human antibody.

[0062] In one embodiment, the definition of CDR / FR in immunoglobulin light or heavy chains should be determined based on the IMGT definition (Lefranc, MP et al., 2003, Dev Comp Immunol. 27(1):55-77; www.imgt.org). The CDR sequences primarily dealt with in this invention are given according to the IMGT nomenclature.

[0063] As used herein, the term “antibody” refers to a normal or full-length antibody (i.e., an antibody containing two heavy chains and two light chains), a single-domain antibody, and fragments of normal and single-domain antibodies. As used herein, the term “antibody” includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and multispecific antibodies (e.g., bispecific and trispecific antibodies). The term “antibody” refers to both antibodies containing a signal peptide (or propeptide, if present), and the mature form obtained by the secretion and proteolytic processing of the chain(s).

[0064] The antibodies or immunoglobulins used herein include relatively recently described "single-domain antibodies," which have a complementarity-determining region that is a single-domain polypeptide. A single-domain antibody is an antibody that lacks a light chain. Examples of single-domain antibodies include heavy-chain antibodies, antibodies that naturally lack a light chain, single-domain antibodies derived from normal four-chain antibodies, and engineered single-domain antibodies. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. Single-domain antibodies can also be naturally occurring single-domain antibodies known as heavy-chain antibodies lacking a light chain. In particular, camelid species, such as camels, dromedaries, llamas, alpacas, and guanacos, naturally produce heavy-chain antibodies lacking a light chain. Heavy-chain antibodies from camelid animals also lack the CH1 domain.

[0065] The variable heavy chains of these single-domain antibodies lacking light chains are known in the art as "VHH" or "nanobodies." Like typical VH domains, VHH contains four FRs and three CDRs. Nanobodies have advantages over typical antibodies: they are approximately 10 times smaller than IgG molecules, and as a result, appropriately folded functional nanobodies can be produced by in vitro expression with high yields. Furthermore, nanobodies are highly stable and resistant to protease action. The properties and production of nanobodies have been reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov;77(1):13-22).

[0066] As used herein, “isolated antibody” refers to an antibody that does not primarily contain other antibodies having different antigen specificities; for example, an isolated antibody that binds to CHIKV, or a fragment thereof, or its antigen-binding fragment, does not primarily contain an antibody that specifically binds to antigens other than CHIKV.

[0067] In this specification, the terms "blocking antibody," "neutralizing antibody," "antibody that neutralizes CHIKV activity," "antibody that exhibits / exerts activity to neutralize CHIKV," "antibody that neutralizes CHIKV," or "anti-CHIKV antibody" refer to antibodies whose binding to CHIKV inhibits at least one of the biological activities of CHIKV. For example, an antibody can neutralize a CHIKV strain by blocking its attachment to cells, thereby preventing infection of those cells by CHIKV.

[0068] As used herein, the terms “monoclonal antibody” or “mAb” refer to an antibody molecule consisting of a single amino acid composition against a specific antigen and should not be interpreted as requiring the production of the antibody by any particular method. Monoclonal antibodies are produced by a single clone of a B cell or hybridoma, but can also be produced by recombination, i.e., protein engineering.

[0069] The term "chimeric antibody," in its broadest sense, refers to an engineered antibody containing one or more regions from one antibody and one or more regions from one or more other antibodies. In particular, a chimeric antibody contains the VH and VL domains of an antibody derived from a non-human animal, conjugated to the CH and CL domains of another antibody, especially a human antibody. Any animal can be used as the non-human animal, such as a mouse, rat, hamster, or rabbit. A chimeric antibody can also mean a multispecific antibody that has specificity for at least two different antigens. In one embodiment, the chimeric antibody has a variable domain of mouse origin and a constant domain of human origin.

[0070] The term "humanized antibody" refers to an antibody that is originally entirely or partially of non-human origin and has been modified by replacing certain amino acids, particularly in the framework regions of the heavy and light chains, in order to avoid or minimize the immune response in humans. The constant domain of a humanized antibody is, in most cases, the human CH and CL domains. In one embodiment, the humanized antibody has a constant domain of human origin.

[0071] A (typical) antibody "fragment" contains a portion of the complete antibody, particularly the antigen-binding or variable region of the complete antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and bispecific and multispecific antibodies formed from antibody fragments. A typical antibody fragment can also be a heavy chain antibody or a single-domain antibody such as VHH.

[0072] The term "Fab" refers to an antibody fragment obtained by treating IgG with the protease papain, in which approximately half of the N-terminal side of the H chain and the entire L chain are linked together via disulfide bonds, resulting in a molecular weight of approximately 50,000 and antigen-binding activity.

[0073] The term "F(ab')2" refers to an antibody fragment obtained by treating IgG with the protease pepsin. This fragment is slightly longer than the Fab fragment, which is linked via a disulfide bond in the hinge region, and has a molecular weight of approximately 100,000 and antigen-binding activity.

[0074] The term "Fab'" refers to an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0075] The “Fc region” or “Fc domain” is defined as the carboxyl terminus of the antibody heavy chain and contains protein sequences common to all immunoglobulins as well as determinants specific to individual different classes of immunoglobulins. For example, the heavy chain of human IgG1 includes the CH1, hinge, CH2, and CH3 regions; the hinge and CH2 and CH3 regions constitute the Fc region. As shown in Figure 1, the numbering of amino acid residues in the Fc region for the purposes of this invention follows the EU index described in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Consequently, the expression “the position of the amino acid is given by the EU index” refers to this numbering of the Fc region shown in Figure 1, as described in Kabat et al., 1991.

[0076] The Fc domain is central to determining the biological function of immunoglobulins, and these biological functions are referred to as “effector functions.” The activity mediated by these Fc domains is mediated by immunological effector cells, including B lymphocytes, natural killer cells, macrophages, basophils, neutrophils, and mast cells, or various complement components. These effector functions include activation of receptors on the surface of the effector cells by binding the Fc domain of an antibody to the aforementioned receptor (or “Fc receptor”) or complement component(s). Antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent cell cytotoxicity (CDC) activities belong to these effector functions and include the binding of the Fc domain to Fc receptors such as FcγRI(CD64), FcγRII, FcγRIII, or complement component(s) such as C1q on effector cells. Among the various human immunoglobulin classes, human IgG1 and IgG3-mediated ADCC are more effective than IgG2 and IgG4. The term "Fc receptor" includes, but is not limited to, FcγRI(CD64), FcγRIIA and FcγRIIB(CD32), FcγRIIIA(CD16a) and FcγRIIIB(CD16b), Fcα receptor (FcαRI or CD89) and Fcε receptor (FcεRI and FcεRII(CD23)). Several amino acid substitutions have been found in the literature. This has been reported to be linked to reduced effector function in different human IgG isotypes (see Table 2 in Strohl 2009, Current Opinion in Biotechnology 20:685-691).

[0077] A single-chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer, typically expressed from a gene fusion containing the genes encoding VH and VL, linked by a peptide-encoding linker. Human scFv fragments can contain CDRs that are preserved in a suitable structure, particularly by using recombination techniques. Bivalent and multivalent antibody fragments can be formed spontaneously by the association of monovalent scFvs or generated by coupling monovalent scFvs with a peptide linker such as bivalent sc(Fv)2. "dsFv" is a disulfide-bonded VH::VL heterodimer. "(dsFv)2" represents two dsFvs coupled by a peptide linker.

[0078] The term "bispecific antibody" or "BsAb" refers to an antibody that combines the antigen-binding sites of two different antibodies within a single molecule. Therefore, a BsAb can bind to two different antigens simultaneously. Genetic engineering is increasingly used to design, modify, and produce antibodies or antibody derivatives using a desired set of binding properties and effector functions, for example, as described in EP2050764A1.

[0079] The term "multispecific antibody" refers to an antibody that combines the antigen-binding sites of two or more antibodies within a single molecule.

[0080] The term "diabody" refers to a small antibody fragment that has sites where two antigens bind, and the fragment contains a variable domain (VH) (VH-VL) of the heavy chain connected to a variable domain (VL) of the light chain on the same polypeptide chain. By using a linker that is too short to allow the two domains to be paired on the same chain, these domains are forced to pair with a complementary domain on another chain, resulting in two antigen-binding sites.

[0081] The term "hybridoma" refers to a cell obtained by cell fusion of B cells produced by immunizing non-human mammals with an antigen and myeloma cells derived from a mouse or the like, and it produces a desired monoclonal antibody having antigen specificity.

[0082] As used herein, "specifically binds to" or "specifically binds with" or "binds to" etc. means that an antibody or its antigen-binding fragment forms a relatively stable complex with an antigen under physiological conditions. Specific binding can be characterized by, at least, an equilibrium dissociation constant (K -8 ) of about 1×10 D M or less (for example, a smaller K D represents a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies have been characterized by their specific binding to CHIKV and / or CHIKV antigens using, for example, surface plasmon resonance, such as BIACORE (trademark).

[0083] As used herein, "CHIKV antigen" means the specific natural antigen of the antibodies described herein, i.e., the protein E2 of CHIKV. It is used, for example, in surface plasmon resonance binding experiments for measuring the binding affinity of anti-CHIKV antibodies described in the materials and methods of this specification in vitro, and also includes recombinant proteins containing the envelope protein E1 of CHIKV and the specific antigen E2 of CHIKV, which are called "pE2-E1 protein" or "pE2-E1 target" or "p62-E1" or "his-tagged CHIKV E2 " or "CHIKV target pE2-E1" or "pE2-E1 antigen of CHIKV".

[0084] As used herein, "acidic environment" means an environment with a pH less than 7; it is understood that, for example, a binding experiment performed at pH 6 provides binding data in an acid environment.

[0085] A sequence "at least 80% identical to the reference sequence" is a sequence that, in its entire length, has sequence identity with the reference sequence of 80% or more, particularly 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0086] The percentage of "sequence identity" can be determined by comparing two sequences that are optimally aligned across a comparison frame, where the optimal alignment of the two sequences means that the portion of the polynucleotide or polypeptide sequence in the comparison frame may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions). The percentage is calculated by determining the number of positions in which identical nucleic acid bases or amino acid residues appear in both sequences to obtain the number of matching positions, dividing the number of matching positions in the comparison frame by the total number of positions, and multiplying the result by 100 to obtain the percentage of sequence identity. The optimal alignment of sequences for comparison is performed by overall pairwise alignment, for example, using the algorithm of Needleman and Wunsch J.Mol.Biol.48:443 (1970). The percentage of sequence identity can be easily determined, for example, using the program Needle with the BLOSUM62 matrix and the following parameters: gap-open=10, gap-extended=0.5.

[0087] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue that has a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. Examples of amino acids with side chains of similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. The conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophan, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine glutamine.

[0088] The terms “antigen-binding moiety” of an antibody, and “antigen-binding fragment” of an antibody, as used herein, include any naturally occurring, enzymatically obtained, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The terms “antigen-binding moiety” of an antibody, or “antibody fragment,” as used herein, refer to one or more fragments of an antibody that retain the ability to bind to CHIKV and / or CHIKV antigen. Such antigen-binding moieties typically include the CDR of an antibody.

[0089] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a type of cytotoxic effect where antibodies secreted and bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) trigger these cytotoxic effectors. ADCC refers to a cytotoxic form of antibody that allows cells to specifically bind to antigen-carrying target cells and subsequently kill those target cells. In other words, ADCC is a cell-mediated immune mechanism in which effector cells of the immune system, primarily natural killer cells, actively lyse target cells to which specific antibodies are bound. ADCC is one of the mechanisms by which antibodies, as part of the humoral immune response, can limit and contain infection. To estimate the ADCC activity of molecules of interest, we also considered in vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337.

[0090] The expression "an amino acid sequence(single or multiple) that differs from sequence X or Y by one or two amino acid substitutions(single or multiple)" means that the sequence differs from sequence X or Y by a maximum of two amino acid substitutions, that is, by just one or two amino acid substitutions.

[0091] For example, the expression "sequence X is different from sequence Y by amino acid substitution Z and optionally one or two additional amino acid substitutions (one or more)" means that the sequence X is: - Amino acid substitution Z only, or - Amino acid substitution Z and one or two amino acid substitutions different from amino acid substitution Z This means that it is different from array Y.

[0092] The expression "sequence number X with one amino acid substitution at position Y" means that the sequence of sequence number X is different from sequence number X due to one amino acid substitution at position Y.

[0093] As used herein, "CHIKV" refers to the chikungunya virus, an enveloped positive-sense RNA virus belonging to the alphavirus genus of the Togaviridae family, as described in the introduction above. CHIKV includes, but is not limited to, different representative infectious strains ("CHIKV strains"), such as the LR2006OPY1[LR] strain with the sequence indicated by NCBI acceptance number DQ443544.2 dated October 24, 2006; the West African genotype, another example being the NI64IbH35 strain with the sequence indicated by NCBI acceptance number HM045786.1 dated December 28, 2010; the Asian genotype, another example being the RSU1 strain with the sequence indicated by NCBI acceptance number HM045797.1 dated December 28, 2010; and the 99659[2014 Caribbean type] strain with the sequence indicated by NCBI acceptance number KJ451624 dated September 11, 2014. Other strains belonging to different genotypes have also been identified; for example, strain S27 has the sequence shown as Q8JUX5 in the UniProtKB / Swiss-Prot reference dated September 16, 2015, with NCBI acceptance number AF369024.2 dated January 14, 2014, as another example; and SL15649 has the sequence shown as GU189061, with NCBI acceptance number GU189061 dated December 14, 2011. Other examples of the referenced CHIKV strains can be found in the Viral Pathogen Database, for their genomes: https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=doQuickTextSearch&decorator=toga&pageTo=1&selectionContext=1476362322448, or for related proteins: https: / / www.viprbrc.org / brc / vipr_protein_search.spg?method=doQuickTextSearch&decorator=toga&pageTo=1&selectionContext=1476362669763 It is available.

[0094] Like other alphavirus genomes, the CHIKV genome has two envelope sugars. The proteins E2 and E1 encode the virus, which are derived from a larger polyprotein precursor (capsid / E3 / E2 / 6K / E1; indicated by NCBI acceptance number NC_004162.2 dated June 27, 2012) and embedded in the viral membrane. Mature virions contain three major structural proteins: a nucleocapsid protein and two glycoproteins, E1 and E2, where E2 functions in cell adhesion and E1 is involved in viral fusion. A third glycoprotein, E3, associates with mature virions in some alphaviruses but not with others, and the 6K protein, a membrane-bound peptide resulting from the cleavage of the polyprotein precursor that releases E2 and E1, is incorporated into the particle at a low level. The organization of alphavirus surface glycoproteins within particles has been clarified using cryo-EM, but the atomic structure of CHIKV glycoproteins has recently been elucidated by X-ray crystallography for both mature particles and immature precursor polyproteins. 240 copies of each of the three glycoproteins (E3 / E2 / E1) combine on the mature virus to form 80 spikes, which constitute an icosahedral protein shell around the viral membrane (Voss JE et al. 2010, Nature 468:709-712). The folding, transport to the surface, and function of these glycoproteins depend on their appropriate interactions with one another. E1 consists of three β-sheet domains designated I, II, and III; E2 contains three immunoglobulin-like domains (A, B, and C, with A at the N-terminus). In the complex, domain B is at the far end of the membrane and in contact with E3, domain C is closest to the viral membrane, and domain A is central (Fox JM et al. 2015, Cell 163:1095-1107 and WO2015010125). Sequences and information for CHIKV E1, E2, and E3 proteins are provided as non-exclusive examples in PDB entries No. 2xFB and 2xFC (latest updated November 24, 2010), and PDB entries No. 3N40, 3N41, 3N42, 3N43, and 3N44 (latest updated December 1, 2010).

[0095] The antibodies primarily used in this invention For therapeutic purposes, it is desirable to produce mAbs that are better suited to the required pharmaceutical properties, particularly by improving their binding to the antigen(s) they target, their stability, pharmacokinetics and pharmacodynamics, and their function.

[0096] The primary objective is that the anti-CHIKV antibodies, based entirely on human parental antibodies, mAb1 and mAb2 respectively, have high binding affinity to different CHIKV strains, particularly to their respective protein E2 (within the nanomolar concentration range). Therefore, they exhibit broad and very potent neutralizing activity against various CHIKV strains.

[0097] mAb1 is: - array: [ka] A heavy chain of variable domains consisting of (the framework region includes CDRH1, CDRH2, and CDRH3); - array: [ka] A variable light chain consisting of (framework regions include CDRL1, CDRL2, and CDRL3) Includes, mAb2 is: - array: [ka] It includes a variable heavy chain domain consisting of CDRH1, CDRH2, and CDRH3; - array: [ka] It includes a variable light chain domain consisting of CDRL1, CDRL2, and CDRL3, and the framework region includes CDRL1, CDRL2, and CDRL3.

[0098] In a first embodiment, the present invention provides mutated antibodies of mAb1 and mAb2 that bind to CHIKV, and which contain at least one amino acid substitution in their Fc domain, thus having improved binding to the FcRn receptor in an acidic environment. Such mutations result in an increased serum half-life of such mutated antibodies when administered to a patient. Examples of such substitutions include, but are not limited to, modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434, where the amino acid positions are given using EU index numbering (Figure 1).

[0099] The inventors confirmed that binding to human and mouse FcRn receptors at pH 6 is enhanced when substitutions selected from the group below are introduced into the Fc region of mAb1 or mAb2 antibodies, as shown in Figure 4 and Example 2: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively, or v. Tyrosine at position 252, threonine at position 254, and glutamate at position 256.

[0100] Furthermore, as described in Example 3 and Figure 5, the inventors have shown that the binding of mAb1 and mAb2 to their CHIKV targets pE2-E1, when introduced into their Fc regions, is unaffected by the above substitutions and enhances binding to human and mouse FcRn. As shown in Example 2 and Figure 4, substitutions of the Fc regions of mAb1 or mAb2 show the strongest binding to human and mouse FcRn when one of the following is introduced into their respective Fc regions: tyrosine at position 252, threonine at position 254 and glutamic acid at position 256, or leucine at position 428 and serine at position 434, respectively.

[0101] As is known in this field, the Fc region is essential for determining the biological function of immunoglobulins, known as "effector function." ADCC is one of the cell-mediated immune mechanisms in which effector cells (primarily natural killer cells) of the immune system lyse target cells to which specific antibodies are bound. Therefore, ADCC is one of the mechanisms for limiting and containing infection. Cell-mediated activity involves the binding of the Fc domain to Fc receptors such as FcγRI(CD64), FcγRII, and FcγRIII on effector cells.

[0102] Since monoclonal antibodies are highly specific to the CHIKV strain and are primarily used for therapeutic purposes, as is the case in this invention, their ability to activate ADCC is an important parameter to measure, as ADCC appears to be related to the activity of anti-CHIKV antibodies in controlling infection. As shown in Example 4 and Figure 6, we have shown that the binding of mAb1 and mAb2 to FcγRIIIa is retained when substitutions selected from the group below are introduced into their respective Fc regions: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively.

[0103] In contrast, binding to FcγRIIIa was reduced when mAb1 and mAb2 were substituted with tyrosine at position 252, threonine at position 254, and glutamate at position 256, respectively.

[0104] Therefore, the inventors have confirmed that antibodies having improved binding to the human FcRn receptor maintain binding to FcγRIIIa, thereby increasing the half-life and compatibility of the antibodies of the present invention while maintaining their effector function.

[0105] Therefore, in a first embodiment, the present invention relates to an isolated monoclonal antibody that conjugates to CHIKV and comprises three heavy chain complementarity-determining regions (CDRH) and three light chain complementarity-determining regions (CDRL), wherein: i. The CDRH has the amino acid sequences of SEQ ID NOs. 5, 6, and 7, and the CDRL has the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, or ii. The CDRH has the amino acid sequences of SEQ ID NOs. 11, 12, and 13, and the CDRL has the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, or iii. The CDRH and CDRLH have different amino acid sequences from the sequence in i. or ii. by one or two amino acid substitutions; The aforementioned antibody is: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively, or v. Tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, respectively. It further comprises an Fc region containing at least one residue selected from the group consisting of, Here, the position of the amino acid is given by the EU index.

[0106] The Fc regions containing such substitutions are shown in Figures 1 and 2 (SEQ ID NO: 17; SEQ ID NOs: 59-63).

[0107] In one embodiment, the anti-CHIKV antibody includes an Fc region containing alanine at position 434. In another embodiment, the anti-CHIKV antibody includes an Fc region containing alanine at positions 307, 380, and 434. In yet another embodiment, the anti-CHIKV antibody includes an Fc region containing glutamine at position 250 and leucine at position 428. In yet another embodiment, the anti-CHIKV antibody includes an Fc region containing leucine at position 428 and serine at position 434. In yet another embodiment, the anti-CHIKV antibody includes an Fc region containing tyrosine at position 252, threonine at position 254, and glutamic acid at position 256.

[0108] The antibodies primarily dealt with in this invention are derived from mAb1 or mAb2 and comprise three heavy chain complementarity-determining regions (CDRH) and three light chain complementarity-determining regions (CDRL), which are: i. The amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, and the amino acid sequences of SEQ ID NOs. 8, GNT, and SEQ ID NOs. 10, or ii. The amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, and the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, or iii. A different amino acid sequence due to one or two amino acid substitutions with the sequence in i. or ii. Each has one of the following:

[0109] In a further embodiment, the antibody comprises a CDR having an amino acid sequence different from the sequences of SEQ ID NOs. 5, 6, and 7, and the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, each by one or two amino acid substitutions, or a CDR having an amino acid sequence different from the sequences of SEQ ID NOs. 11, 12, and 13, and the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, each by one or two amino acid substitutions.

[0110] The amino acid substitutions according to the present invention may be conservative or non-conservative. Examples of conservative substitutions are shown in Table 1 below.

[0111] [Table 1]

[0112] In another embodiment, the antibody comprises three heavy chain complementarity-determining regions (CDRHs) having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, and the antibody comprises three light chain complementarity-determining regions (CDRLs) having the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, and: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively, or v. Tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, respectively. It comprises an Fc region containing at least one residue selected from the group consisting of the following, Here, the position of the amino acid is given by the EU index.

[0113] In another embodiment, the antibody comprises three heavy chain complementarity-determining regions (CDRHs) derived from mAb2 and having amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, and the antibody comprises three light chain complementarity-determining regions (CDRLs) having amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, and: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively, or v. Tyrosine at position 252, threonine at position 254 and position Glutamic acid in position 256, The Fc region includes at least one residue selected from the group consisting of the following: Here, the position of the amino acid is given by the EU index.

[0114] In another embodiment, the antibody comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, respectively, and: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively, or v. Tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, respectively. It comprises an Fc region containing at least one residue selected from the group consisting of the following, Here, the position of the amino acid is given by the EU index.

[0115] In a further embodiment, the deformed antibody (mAb3) comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, respectively, and an Fc region containing alanine at position 434, where the amino acid positions are given according to the EU index.

[0116] In another further embodiment, the deformed antibody (mAb4) comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, respectively, as well as an Fc region containing alanine at positions 307, 380, and 434, respectively, where the amino acid positions are given by the EU index.

[0117] In another further embodiment, the deformed antibody (mAb5) comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, respectively, and an Fc region containing at least glutamine at position 250 and leucine at position 428, respectively, where the positions of the amino acids are given by the EU index.

[0118] In another further embodiment, the deformed antibody (mAb7) comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, respectively, and an Fc region comprising at least leucine at position 428 and serine at position 434, respectively, where the positions of the amino acids are given by the EU index.

[0119] In another further embodiment, the deformed antibody (mAb6) comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, respectively, and at least tyrosine at position 252, position 2 It includes an Fc region containing threonine at position 54 and glutamic acid at position 256, where the positions of the amino acids are given by the EU index.

[0120] In another embodiment, the deformed antibody has three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, and: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively, or v. Tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, respectively. It comprises an Fc region containing at least one residue selected from the group consisting of the following, Here, the position of the amino acid is given by the EU index.

[0121] In a further embodiment, the deformed antibody comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, and also comprises an Fc region containing at least alanine at position 434, the position of which is given according to the EU index.

[0122] In another further embodiment, the deformed antibody comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, and an Fc region containing alanine at positions 307, 380, and 434, respectively, where the amino acid positions are given by the EU index.

[0123] In another further embodiment, the deformed antibody comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, and an Fc region containing at least glutamine at position 250 and leucine at position 428, respectively, where the positions of the amino acids are given by the EU index.

[0124] In another further embodiment, the deformed antibody (mAb8) comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, and an Fc region comprising at least leucine at position 428 and serine at position 434, respectively, where the positions of the amino acids are given by the EU index.

[0125] In another further embodiment, the deformed antibody (mAb9) comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, and an Fc region comprising at least tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, respectively. This includes, where the position of the amino acid is given by the EU index.

[0126] The inventors have shown that the binding of mAb1 and mAb2 to their CHIKV target pE2-E1 is unaffected when they contain an Fc region containing at least one residue as described above, as shown in Example 3 and Figure 5. The inventors have also shown that all of these antibodies containing an Fc region containing at least one residue as described above show enhanced binding to human and mouse FcRn (Example 2 and Figure 4), which has a favorable effect on their respective half-lives and is therefore beneficial for anti-CHIKV therapy; and that the best binding to human and mouse FcRn is shown when any of tyrosine at position 252, threonine at position 254 and glutamic acid at position 256, or leucine at position 428 and serine at position 434, respectively, is introduced into their respective Fc regions.

[0127] Therefore, in a typical embodiment, the deformed antibody comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, and three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, respectively, and an Fc region containing at least leucine at position 428 and serine at position 434, respectively, or an Fc region containing at least tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, respectively, where the positions of the amino acids are given by the EU index.

[0128] Furthermore, as shown in Example 4 and Figure 6, the inventors have shown that the binding of mAb1 and mAb2 to FcγRIIIa is as follows: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively. We showed that the substitutions selected from are retained when introduced into their respective Fc regions.

[0129] In contrast, binding to FcγRIIIa was reduced when mAb1 and mAb2 were substituted with tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, respectively.

[0130] Therefore, the inventors have identified at least one antibody that retains binding to FcγRIIIa while its binding to its target is unaffected, and its binding to the human FcRn receptor is improved, while maintaining their effector functions, and thus, with respect to its increased half-life, is suitable for the development of therapeutics for preventing and treating CHIKV disease.

[0131] In a typical embodiment, the deformed antibody comprises three heavy chain complementarity-determining regions (CDRH) having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively, three light chain complementarity-determining regions (CDRL) having the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, respectively, and an Fc region containing at least leucine at position 428 and serine at position 434, respectively, where the positions of the amino acids are given by the EU index.

[0132] In other words, the antibody primarily dealt with in this invention is an isolated monochloroform that binds to CHIKV and contains three heavy chain complementarity-determining regions (CDRH) and three light chain complementarity-determining regions (CDRL). It can be described as a single antibody, here: i. The CDRH has the amino acid sequences of SEQ ID NOs. 5, 6, and 7, and the CDRL has the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, or ii. The CDRH has the amino acid sequences of SEQ ID NOs. 11, 12, and 13, and the CDRL has the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, or iii. The CDRH and CDRLH have different amino acid sequences from the sequence in i. or ii. by one or two amino acid substitutions; And the antibody is: iv. Alanine at position 434, or v. Alanine at positions 307, 380 and 434, or vi. Glutamine at position 250 and leucine at position 428, or vii. Leucine at position 428 and serine at position 434, or viii. Tyrosine at position 252, threonine at position 254, and glutamate at position 256. It further comprises an Fc region containing at least one residue selected from the group consisting of, Here, the position of the amino acid is given by the EU index; And antibodies are as follows: i. CHIKV pE2-E1 target has a binding-dissociation equilibrium constant (K) of less than approximately 10 nM. D ) is used to join them; ii. Human FcRn with K200 nM D Join them together; iii. Human FcγRIII with K600 nM D Join them together It possesses one or more properties.

[0133] In one embodiment, the antibody according to the present invention has binding-dissociation equilibrium constants (K) of less than approximately 5 nM, 4 nM, 3, 2, 1 nM, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 nM, or less than approximately 0.25 nM, 0.20 nM, 0.15 nM, 0.1 nM for the CHIKV pE2-E1 target. D They are joined together using ).

[0134] In another embodiment, the antibody according to the present invention has a K content of less than about 200 nM, less than about 100 nM, less than about 50 nM, less than 45, 40, 35, 30 nM, or less than about 25 nM, 20, 15, or 10 nM relative to human FcRn. D They are joined together.

[0135] In another embodiment, the antibody according to the present invention has a K content of less than about 600 nM, about 500 nM, 400 nM, less than 300 nM, about 200 nM, 150, 100, or less than 50 nM in human FcγRIII. D They are joined together.

[0136] Binding to the CHIKV pE2-E1 target, human FcRn, and human FcγRIII can be measured, for example, by surface plasmon resonance assay, at 37°C. This assay can be carried out, for example, as described in Examples 1 to 4.

[0137] The “Fc region” according to the present invention may belong to one of the heavy chains of four human subclasses of IgG (IgG1, IgG2, IgG3, and IgG4) that determine the functional activity of the antibody. In one embodiment, the Fc region belongs to the heavy chain of the IgG1 subtype. In another embodiment, the Fc region belongs to the heavy chain of the IgG2 subtype. In another embodiment, the Fc region belongs to the heavy chain of the IgG3 subtype. In another embodiment, the Fc region belongs to the heavy chain of the IgG4 subtype. In another embodiment, the Fc region includes or consists of the sequence of the FC region of IgG1 (SEQ ID NO: 17), excluding the mutations described herein (Figures 1 and 2).

[0138] In one embodiment, the antibody has an Fc region containing or consisting of a sequence having at least 80% identity with SEQ ID NO: 17. In another embodiment, the antibody has an Fc region containing or consisting of a sequence having at least 85% identity with SEQ ID NO: 17. In yet another embodiment, the antibody has an Fc region containing or consisting of a sequence having at least 90% identity with SEQ ID NO: 17. In a further embodiment, the antibody has an Fc region containing or consisting of a sequence having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 17.

[0139] In one embodiment, the antibody has an Fc region containing one or more substitutions described above and in the examples. In another embodiment, the antibody has an Fc region containing or consisting of sequences having at least 80% identity with SEQ ID NOs. 59, 60, 61, 62, and 63.

[0140] Alternatively, or in addition, cysteine ​​residues(s) can be introduced into the Fc region, thereby enabling the formation of interchain disulfide bonds in this region. Homodimer antibodies thus produced can have improved internal migration ability and / or increased complement-mediated cytotoxicity and / or antibody-dependent cytotoxicity (ADCC) (Caron, PC et al., 1992, J Exp Med. 176(4):1191-1195 and Shopes B., 1992, J Immunol. 148(9):2918-2922).

[0141] In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having at least 80% identity with SEQ ID NO: 1. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having at least 85% identity with SEQ ID NO: 1. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having at least 90% identity with SEQ ID NO: 1. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1.

[0142] In another embodiment, the antibody includes a variable region of its light chain containing or comprising sequences having at least 80% identity with SEQ ID NO: 2. In another embodiment, the antibody includes a variable region of its light chain containing or comprising sequences having at least 85% identity with SEQ ID NO: 2. In another embodiment, the antibody includes a variable region of its light chain containing or comprising sequences having at least 90% identity with SEQ ID NO: 2. In another embodiment, the antibody includes a variable region of its light chain containing or comprising sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 2.

[0143] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 19. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 19. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 19. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 19.

[0144] In another embodiment, the antibody light chain contains or consists of a sequence having at least 80% identity with sequence number 20. In another embodiment, the antibody light chain is sequence number In another embodiment, the antibody light chain contains or consists of sequences having at least 85% identity with sequence number 20. In yet another embodiment, the antibody light chain contains or consists of sequences having at least 90% identity with sequence number 20.

[0145] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 23. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 23. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 23. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 23.

[0146] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 25. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 25. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 25. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 25.

[0147] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 27. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 27. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 27. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 27.

[0148] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 29. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 29. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 29. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 29.

[0149] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 31. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 31. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 31. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 31.

[0150] In one embodiment, the antibody is a combination of a heavy chain and a light chain, or a combination of a heavy chain and a light chain encoded by a nucleotide sequence of the sequence listed in Table 2 below.

[0151] [Table 2]

[0152] In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having at least 80% identity with SEQ ID NO: 3. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having at least 85% identity with SEQ ID NO: 3. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having at least 90% identity with SEQ ID NO: 3. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 3.

[0153] In another embodiment, the antibody includes a variable region of its light chain containing or comprising sequences having at least 80% identity with SEQ ID NO: 4. In another embodiment, the antibody includes a variable region of its light chain containing or comprising sequences having at least 85% identity with SEQ ID NO: 4. In another embodiment, the antibody includes a variable region of its light chain containing or comprising sequences having at least 90% identity with SEQ ID NO: 4. In another embodiment, the antibody includes a variable region of its light chain containing or comprising sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 4.

[0154] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 37. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 37. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 37. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 37.

[0155] In another embodiment, the antibody light chain contains or consists of a sequence having at least 80% identity with SEQ ID NO: 38. In another embodiment, the antibody light chain contains or consists of a sequence having at least 85% identity with SEQ ID NO: 38. In one embodiment, the antibody light chain contains or consists of sequences having at least 90% identity with SEQ ID NO: 38. In another embodiment, the antibody light chain contains or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 38.

[0156] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 41. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 41. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 41. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 41.

[0157] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 43. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 43. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 43. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 43.

[0158] In one embodiment, the antibody is a combination of a heavy chain and a light chain, or a combination of a heavy chain and a light chain encoded by a nucleotide sequence of the sequence listed in Table 3 below.

[0159] [Table 3]

[0160] As already mentioned, for therapeutic purposes, it is highly desirable to produce mAbs that are reliable in terms of stability, pharmacokinetics, pharmacodynamics, and their function, while maintaining their binding affinity to their specific targets.

[0161] In a second embodiment, the inventors identified hotspots that can be used to improve homogeneity and mitigate disadvantages in chemical properties, production, and control (CMC). Such analyses focused on undesirable motifs exposed to solvents, such as oxidation, deamination, isomerization, acidic cleavage, glycosylation, and additional free cysteine ​​residues, which can potentially lead to heterogeneity in degradation products or antibody formulations, as confirmed either in silico or experimentally. As an example, deamination of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly prone to deamination when they exist primarily in the Asn-Gly sequence, and less so in other dipeptide sequences such as Asn-Ala. If a mino-regulation site, particularly Asn-Gly, is present in the antibody or polypeptide described herein, it is typically desirable to remove the site by conservative substitution, which involves removing one of the residues involved.

[0162] As shown in Example 1 and Figure 3, the inventors identified at least three amino acids in CDRH3 of mAb2 (SEQ ID NO: 13) because, when used for therapeutic purposes, they may have adverse consequences or give rise to unfavorable motifs based on previous criteria. As shown in Figure 3, the inventors generated at least three variant antibodies of mAb2 that maintain their binding affinity to the CHIKV pE2-E1 antigen by single substitutions at positions 8, 12, and 13, respectively, of SEQ ID NO: 13 in order to suppress undesirable amino acids or motifs. Therefore, the inventors generated variants of mAb2 that, when produced in a bioreactor, have lower impurities associated with the product while maintaining their target affinity, and exhibit higher stability and homogeneity compared to CMC criteria.

[0163] Therefore, in a second embodiment, the present invention relates to an mAb2 variant antibody or its antigen-binding fragment that binds to CHIKV and comprises three heavy chain complementarity-determining regions (CDRHs) having amino acid sequences of SEQ ID NOs. 11, 12, and 33, respectively, or having different amino acid sequences due to one or two amino acid substitutions from these sequences, wherein the antibody or its antigen-binding fragment further comprises three light chain complementarity-determining regions (CDRLs) having amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, or having different amino acid sequences due to one or two amino acid substitutions from these sequences: i. The amino acid at position 8 of SEQ ID NO: 33 is not M, and / or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; and / or iii. The amino acid at position 13 in sequence number 33 is not G.

[0164] In one embodiment, the deformed antibody comprises CDRH having a different amino acid sequence from the sequences of SEQ ID NOs. 11, 12, and 33 by one or two amino acid substitutions, and CDRL having a different amino acid sequence from the sequences of SEQ ID NOs. 14, GTS, and 16 by one or two amino acid substitutions, respectively. "Different amino acid sequence by one or two amino acid substitutions" means, with respect to SEQ ID NOs. 33, which is the primary subject of this invention, an additional amino acid substitution compared to the substitutions envisioned at positions 8, 12, and 13 of SEQ ID NOs. 33.

[0165] Amino acid substitutions can be conservative or non-conservative. Examples of conservative substitutions are shown in Table 1 above.

[0166] In another embodiment, the deformed antibody comprises CDRH3 of SEQ ID NO: 33, where the amino acid at position 8 of SEQ ID NO: 33 is not M, the amino acid at position 12 of SEQ ID NO: 33 is not N, and the amino acid at position 13 of SEQ ID NO: 33 is not G. In another embodiment, the deformed antibody comprises CDRH3 of SEQ ID NO: 33, where the amino acid at position 8 of SEQ ID NO: 33 is M, the amino acid at position 12 of SEQ ID NO: 33 is not N, and the amino acid at position 13 of SEQ ID NO: 33 is not G. In yet another embodiment, the deformed antibody comprises CDRH3 of SEQ ID NO: 33, where the amino acid at position 8 of SEQ ID NO: 33 is not M, the amino acid at position 12 of SEQ ID NO: 33 is N, and the amino acid at position 13 of SEQ ID NO: 33 is not G; CDRH3 of SEQ ID NO: 33, where the amino acid at position 8 of SEQ ID NO: 33 is not M, the amino acid at position 12 of SEQ ID NO: 33 is not N, and the amino acid at position 13 of SEQ ID NO: 33 is G; CDRH3 of SEQ ID NO: 33, where the amino acid at position 8 of SEQ ID NO: 33 is M, the amino acid at position 12 of SEQ ID NO: 33 is N, and the amino acid at position 13 of SEQ ID NO: 33 is not G. 3; CDRH3 of SEQ ID NO: 33, in which the amino acid at position 8 of SEQ ID NO: 33 is M, the amino acid at position 12 of SEQ ID NO: 33 is not N, and the amino acid at position 13 of SEQ ID NO: 33 is G; CDRH3 of SEQ ID NO: 33, in which the amino acid at position 8 of SEQ ID NO: 33 is not M, the amino acid at position 12 of SEQ ID NO: 33 is N, and the amino acid at position 13 of SEQ ID NO: 33, in which the amino acid at position 8 of SEQ ID NO: 33 is not M, the amino acid at position 12 of SEQ ID NO: 33 is N, and the amino acid at position 13 of SEQ ID NO: 33 is not G. In other words, SEQ ID NO: 33 cannot be identical to SEQ ID NO: 13.

[0167] The expression "the amino acid at position X is not M" means that the amino acid at position X may be any amino acid other than M. Similarly, the expression "the amino acid at position X is not N" means that the amino acid at position X may be any amino acid other than N. Similarly, the expression "the amino acid at position X is not G" means that the amino acid at position X may be any amino acid other than G. As a non-limiting example, a deformed antibody containing CDRH3 of SEQ ID NO: 33, where the amino acid at position 8 of SEQ ID NO: 33 is not M, may contain any amino acid at position 8 selected from the group consisting of A, G, V, L, I, F, W, Y, S, T, N, Q, C, D, E, K, R, and H. As another non-limiting example, a deformed antibody containing CDRH3 of SEQ ID NO: 33, where the amino acid at position 12 of SEQ ID NO: 33 is not N, may contain any amino acid at position 12 selected from the group consisting of A, G, V, L, I, F, W, Y, S, T, M, Q, C, D, E, K, R, and H. As another non-limiting example, a deformed antibody containing CDRH3 of SEQ ID NO: 33, in which the amino acid at position 13 of SEQ ID NO: 33 is not G, may contain any amino acid at position 13 selected from the group consisting of A, N, V, L, I, F, W, Y, S, T, M, Q, C, D, E, K, R, and H.

[0168] In another embodiment, the deformed antibody or its antigen-binding fragment is: - CDRH1 consisting of the sequence of sequence number 11; - CDRH2 consisting of the sequence of sequence number 12; - CDRH3 consisting of the sequence of sequence number 33; - CDRL1 consisting of the sequence of sequence number 14; - CDRL2 consisting of GTS; - CDRL3 consisting of the sequence of sequence number 16 This includes, and here: i. The amino acid at position 8 of SEQ ID NO: 33 is not M, and / or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; and / or iii. The amino acid at position 13 in sequence number 33 is not G.

[0169] In another embodiment, the deformed antibody or its antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRHs) having the amino acid sequences of SEQ ID NOs. 11, 12, and 33, respectively, or having different amino acid sequences due to one or two amino acid substitutions from these sequences, and further comprises three light chain complementarity-determining regions (CDRLs) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, or having different amino acid sequences due to one or two amino acid substitutions from these sequences, wherein: i. The amino acid at position 8 in SEQ ID NO: 33 is not M, or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; or iii. The amino acid at position 13 in sequence number 33 is not G.

[0170] In another embodiment, the deformed antibody or its antigen-binding fragment has three heavy chain complementarity-determining regions (CDRHs) having the amino acid sequences of SEQ ID NOs. 11, 12, and 33, respectively. It comprises, and further comprises three light chain complementarity determining regions (CDRLs) having the amino acid sequences of SEQ ID NO: 14, GTS, and SEQ ID NO: 16, respectively, where: i. The amino acid at position 8 in SEQ ID NO: 33 is not M, or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; or iii. The amino acid at position 13 in sequence number 33 is not G.

[0171] In other words, as shown in Example 1 and Figure 3, the present inventors have included three heavy chain complementarity-determining regions (CDRHs) having the amino acid sequences of SEQ ID NOs. 11, 12, and 33, respectively, or different amino acid sequences obtained by one or two amino acid substitutions from these sequences, and further including three light chain complementarity-determining regions (CDRLs) having the amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, or different amino acid sequences obtained by one or two amino acid substitutions from these sequences, where: i. The amino acid at position 8 in SEQ ID NO: 33 is not M, or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; or iii. The amino acid at position 13 of sequence number 33 is not G, The antibodies are as follows: i. CHIKV pE2-E1 target has a binding-dissociation equilibrium constant (K) of less than approximately 10 nM. D ) is used to join them; ii. Human FcRn with K200 nM D Join them together; iii. Human FcγRIII with K600 nM D Join them together Antibodies or their antigen-binding fragments possessing one or more of these properties were identified.

[0172] In one embodiment, the antibody binds to the CHIKV pE2-E1 target with binding-dissociation equilibrium constants (K) of less than approximately 5 nM, 4 nM, 3, 2, 1 nM, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 nM, or less than approximately 0.25 nM, 0.20 nM, 0.15 nM, 0.1 nM. D They are joined together using ).

[0173] In another embodiment, the antibody has a K content of less than approximately 200 nM, less than approximately 100 nM, less than approximately 50 nM, less than 45, 40, 35, 30 nM, or less than approximately 25 nM, 20, 15, or 10 nM to human FcRn. D They are joined together.

[0174] In another embodiment, the antibody according to the invention binds to human FcγRIII with a K of less than about 600 nM, about 500 nM, 400 nM, less than about 300 nM, about 200 nM, 150, 100 or less than 50 nM D to bind.

[0175] Binding to the CHIKV pE2-E1 target, human FcRn and human FcγRIII can be measured, for example, by surface plasmon resonance assay, for example at 37°C. This assay can be performed, for example, as described in Examples 1 to 4.

[0176] In another embodiment, the engineered antibody or antigen-binding fragment thereof comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 33, and the amino acid at position 8 of SEQ ID NO: 33 is selected from the group consisting of I, L, V, Q and N.

[0177] In another embodiment, the engineered antibody (mAb10) or antigen-binding fragment thereof is: - CDRH1 consisting of the sequence of SEQ ID NO: 11; - CDRH2 consisting of the sequence of SEQ ID NO: 12; - CDRH3 consisting of the sequence of SEQ ID NO: 34; - CDRL1 consisting of the sequence of SEQ ID NO: 14; - CDRL2 consisting of GTS; - CDRL3 consisting of the sequence of SEQ ID NO: 16 including.

[0178] In another embodiment, the engineered antibody or antigen-binding fragment thereof comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 33, and the amino acid at position 12 of SEQ ID NO: 33 is selected from the group consisting of Q, E, S, T and D.

[0179] In another embodiment, the engineered antibody (mAb11) or antigen-binding fragment thereof is: - CDRH1 consisting of the sequence of SEQ ID NO: 11; - CDRH2 consisting of the sequence of SEQ ID NO: 12; - CDRH3 consisting of the sequence of sequence number 35; - CDRL1 consisting of the sequence of sequence number 14; - CDRL2 consisting of GTS; - CDRL3 consisting of the sequence of sequence number 16 Includes.

[0180] In another embodiment, a deformed antibody or its antigen-binding fragment comprises CDRH3 having the amino acid sequence of SEQ ID NO: 33, wherein the amino acid at position 13 of SEQ ID NO: 33 is selected from the group consisting of A, S, and T.

[0181] In another embodiment, the deformed antibody (mAb12) or its antigen-binding fragment is: - CDRH1 consisting of the sequence of sequence number 11; - CDRH2 consisting of the sequence of sequence number 12; - CDRH3 consisting of the sequence at sequence number 36; - CDRL1 consisting of the sequence of sequence number 14; - CDRL2 consisting of GTS; - CDRL3 consisting of the sequence of sequence number 16 Includes.

[0182] In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having at least 80% identity with SEQ ID NO: 56. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having at least 85% identity with SEQ ID NO: 56. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having at least 90% identity with SEQ ID NO: 56. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 56.

[0183] In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising a sequence having at least 80% identity with SEQ ID NO: 57. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising a sequence having at least 85% identity with SEQ ID NO: 57. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising a sequence having at least 90% identity with SEQ ID NO: 57. In another embodiment, the antibody includes a variable region of its heavy chain containing or comprising a sequence having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 57.

[0184] In another embodiment, the antibody includes a variable region of its heavy chain that contains or comprises a sequence having at least 80% identity with SEQ ID NO: 58. In another embodiment, the antibody includes a variable region of its heavy chain that contains or comprises a sequence having at least 85% identity with SEQ ID NO: 58. In another embodiment, the antibody has at least SEQ ID NO: 58 In another embodiment, the antibody includes a variable region of its heavy chain that contains or comprises sequences having 90% identity with SEQ ID NO: 58, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 58.

[0185] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 45. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 45. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 45. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 45.

[0186] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 47. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 47. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 47. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 47.

[0187] In another embodiment, the antibody heavy chain includes or consists of a sequence having at least 80% identity with SEQ ID NO: 49, or a sequence encoded by a nucleotide sequence having at least 80% identity with SEQ ID NO: 50. In another embodiment, the antibody heavy chain includes or consists of a sequence having at least 85% identity with SEQ ID NO: 49. In another embodiment, the antibody heavy chain includes or consists of a sequence having at least 90% identity with SEQ ID NO: 49. In another embodiment, the antibody heavy chain includes or consists of a sequence having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 49.

[0188] In one embodiment, the antibody is a combination of a heavy chain and a light chain encoded by a nucleotide sequence of the sequence listed in Table 4 below.

[0189] [Table 4]

[0190] In a third embodiment, the inventors combined the above beneficial embodiments. On the one hand, they are substituted within CDRH3 to suppress amino acids or motifs that are not advantageous based on the criteria of chemical properties, manufacturing, and control (CMC) disadvantages. On the other hand, variants of mAb2 were generated that have improved binding to the FcRn receptor in an acidic environment while retaining binding to FcγRIIIa associated with effector function, because they contain at least one amino acid substitution in their Fc domain.

[0191] As described in Example 2 and FIGS. 4C and 4D, the inventors have shown that variants of mAb2 in which substitutions within CDRH3 were introduced are in the following group: i. leucine at position 428 and serine at position 434, respectively, or ii. tyrosine at position 252, threonine at position 254, and glutamate at position 256 show enhanced binding to the human and mouse FcRn receptors at pH 6 when the selected substitutions are introduced into their Fc regions.

[0192] Furthermore, as described in Example 3 and FIG. 5, the inventors confirmed that for antibodies stacking the substitutions described above within their CDRH3 and their Fc regions, their binding to the CHIKV target pE2-E1 is not affected.

[0193] As shown in Example 4 and FIGS. 6B and 6D, the inventors also confirmed that for such antibodies, at least, when leucine at position 428 and serine at position 434 are introduced into their Fc regions, binding to FcγRIIIa is retained. In contrast, FcγRIIIa binding was weakened when such antibodies were substituted with tyrosine at position 252, threonine at position 254, and glutamate at position 256, respectively.

[0194] Therefore, the inventors have confirmed the beneficial effects of substitutions introduced within the respective Fc regions of antibodies according to the present invention that suppress unfavorable amino acids or motifs based on the criteria of disadvantages in chemical properties, manufacturing, and control (CMC).

[0195] Therefore, in this third embodiment, the present invention relates to a modified antibody of mAb2 or its antigen-binding fragment that binds to CHIKV and comprises three heavy chain complementarity-determining regions (CDRHs) having amino acid sequences of SEQ ID NOs. 11, 12, and 33, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, wherein the antibody or its antigen-binding fragment further comprises three light chain complementarity-determining regions (CDRLs) having amino acid sequences of SEQ ID NOs. 14, GTS, and 16, respectively, or different amino acid sequences due to one or two amino acid substitutions from these sequences, wherein: i. The amino acid at position 8 of SEQ ID NO: 33 is not M, and / or ii. The amino acid at position 12 of SEQ ID NO: 33 is not N; and / or iii. The amino acid at position 13 of sequence number 33 is not G; The aforementioned antibody is at least: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively, or v. Tyrosine at position 252, threonine at position 254, and glutamate at position 256 From the group consisting of, each includes an Fc region containing a selected mutation, Here, the position of the amino acid is given by the EU index.

[0196] In a third embodiment, any antibody comprising CDRH of the second embodiment, i.e., SEQ ID NO: 33, may contain any mutation in the Fc region described in the first embodiment, and the antibody is: i. Alanine at position 434, or ii. Alanine at positions 307, 380 and 434, respectively, or iii. Glutamine at position 250 and leucine at position 428, respectively, or iv. Leucine at position 428 and serine at position 434, respectively, or v. Tyrosine at position 252, threonine at position 254, and glutamate at position 256, respectively. It comprises an Fc region having at least one residue selected from the group consisting of the following, Here, it is understood that the position of the amino acid is given by the EU index.

[0197] In another embodiment, the deformed antibody (mAb13) or its antigen-binding fragment is: - CDRH1 consisting of the sequence of sequence number 11; - CDRH2 consisting of the sequence of sequence number 12; - CDRH3 consisting of the sequence of sequence number 35; - CDRL1 consisting of the sequence of sequence number 14; - CDRL2 consisting of GTS; - CDRL3 consisting of the sequence of sequence number 16; Furthermore, it includes an Fc region comprising at least tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, where the positions of the amino acids are given by the EU index.

[0198] In another embodiment, the deformed antibody (mAb14) or its antigen-binding fragment is: - CDRH1 consisting of the sequence of sequence number 11; - CDRH2 consisting of the sequence of sequence number 12; - CDRH3 consisting of the sequence of sequence number 35; - CDRL1 consisting of the sequence of sequence number 14; - CDRL2 consisting of GTS; - CDRL3 consisting of the sequence of sequence number 16; Furthermore, it includes an Fc region comprising at least leucine at position 428 and serine at position 434, respectively, where the positions of the amino acids are given by the EU index.

[0199] In another embodiment, the antibody heavy chain includes or consists of sequences having at least 80% identity with SEQ ID NO: 51. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 85% identity with SEQ ID NO: 51. In another embodiment, the antibody heavy chain includes or consists of sequences having at least 90% identity with SEQ ID NO: 51. In another embodiment, the antibody heavy chain includes or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 51.

[0200] In another embodiment, the antibody heavy chain contains or consists of a sequence having at least 80% identity with SEQ ID NO: 53. In another embodiment, the antibody heavy chain contains or consists of a sequence having at least 85% identity with SEQ ID NO: 53. In one embodiment, the antibody heavy chain contains or consists of sequences having at least 90% identity with SEQ ID NO: 53. In another embodiment, the antibody heavy chain contains or consists of sequences having 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 53.

[0201] In one embodiment, the antibody according to this particular aspect is a combination of a heavy chain and a light chain encoded by a nucleotide sequence of the sequence listed in Table 5 below.

[0202] [Table 5]

[0203] Nucleic acids, vectors, and recombinant host cells A further object of the present invention is to relate to nucleic acid sequences comprising or comprising polypeptides, heavy chains, light chains, or sequences encoding fragments thereof, which include or comprise antibodies as defined in the present invention or described herein.

[0204] Typically, the nucleic acids are DNA or RNA molecules, which can be contained in any suitable vector, such as plasmids, cosmids, episomes, artificial chromosomes, phage or viral vectors, etc.

[0205] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle through which a DNA or RNA sequence (e.g., an exogenous gene) can be introduced into a host cell, thereby transforming the host and promoting the expression (e.g., transcription and translation) of the introduced sequence.

[0206] Therefore, a further object of the present invention is primarily concerned with vectors containing nucleic acids as described herein.

[0207] Such vectors include regulatory elements, such as promoters, enhancers, and terminators, and when administered to a subject, they can induce or direct the expression of the polypeptide. Examples of promoters and enhancers used in expression vectors for animal cells include enhancers and promoters for human cytomegalovirus (Nelson, J., 1996 J. Virology 70:32073986), early promoters and enhancers for SV40 (Mizukami, T. and Itoh, S. et al., 1987, J Biochem. 101(5):1307-1310), LTR promoters and enhancers for Moloney's mouse leukemia virus (Kuwana Y. et al., 1987, Biochem Biophys Res Commun. 149:960-968), promoters (Mason, JO et al., 1985, Cell 41:479-487), and enhancers for the H chain of immunoglobulins (Gillies, SD et al., 1983, Cell 33:717-728), etc.

[0208] Any expression vector for animal cells can be used, as long as it can insert and express the gene encoding the C region of the human antibody. Examples of suitable vectors include pAGE107 (Miyaji, H. et al., 1990, Cytotechnology 3(2):133-140), pAGE103 (Mizukami, T. and Itoh, S. et al., 1987, J Biochem. 101(5):1307-1310), pHSG274 (Brady, G. et al., 1984, Gene 27(2):223-232), pKCr (O'Hare, K. et al., 1981, Proc Natl Acad Sci USA. 78(3):1527-1531), pSG1beta d2-4- (Miyaji, H. et al., 1990, Cytotechnology 4:173-180), etc.

[0209] Other examples of plasmids include replication plasmids containing the origin of replicated pCEP5, or integrative plasmids such as pUC, pcDNA, and pBR.

[0210] Other examples of viral vectors include vectors for adenoviruses, retroviruses, herpesviruses, and AAVs. Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection using helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, and others. Detailed protocols for producing such recombinant viruses with replication defects can be found, for example, in WO95 / 14785, WO96 / 22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056 and WO94 / 19478.

[0211] In one embodiment, the present invention relates to a polynucleotide having at least 80% identity with one of the sequences selected from the group consisting of SEQ ID NOs: 18, 21, 22, 24, 26, 28, 30, 32, 39, 40, 42, 44, 46, 48, 50, 52, and 54. In another embodiment, the present invention relates to a polynucleotide having at least 85% identity with one of the sequences selected from the group consisting of SEQ ID NOs: 18, 21, 22, 24, 26, 28, 30, 32, 39, 40, 42, 44, 46, 48, 50, 52, and 54. In yet another embodiment, the present invention relates to a polynucleotide having at least 90% identity with one of the sequences selected from the group consisting of SEQ ID NOs: 18, 21, 22, 24, 26, 28, 30, 32, 39, 40, 42, 44, 46, 48, 50, 52, and 54. In another embodiment, the present invention relates to a polynucleotide having at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with one of the sequences selected from the group consisting of SEQ ID NOs: 18, 21, 22, 24, 26, 28, 30, 32, 39, 40, 42, 44, 46, 48, 50, 52, and 54. In another embodiment, the present invention relates to a polynucleotide encoding one of the heavy chains or one of the light chains or both of the heavy and light chains of the antibody described in the present invention, i.e., mAb3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In another embodiment, the present invention relates to a polynucleotide comprising a sequence encoding the antibody or its antigen-binding fragment, which is the main subject of the present invention.

[0212] A further object primarily addressed in this invention relates to cells transfected, infected, or transformed with nucleic acids and / or vectors described herein.

[0213] Therefore, the present invention relates to a cell line that produces one of the antibodies described herein.

[0214] The term "transformation" refers to the process by which a host cell expresses an introduced gene or sequence, thereby producing a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. This refers to the introduction of "foreign" (i.e., external) genes, DNA, or RNA sequences into a host cell, which will then produce them. The host cell that receives and expresses the introduced DNA or RNA is "transformed."

[0215] The nucleic acids primarily used in this invention can be used to produce recombinant anti-CHIKV antibodies from a suitable expression system. The term "expression system" refers, for example, to a host cell and a compatible vector under conditions suitable for expressing a protein encoded by foreign DNA that has been introduced into the host cell via a vector.

[0216] Common expression systems include Escherichia coli (E. coli) host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, and plant cells). Specific examples include Escherichia coli, yeasts of the genera Kluyveromyces or Saccharomyces, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, HEK293 cells, etc.), and primary or established mammalian cell cultures (e.g., those produced from lymphoblasts, fibroblasts, germ cells, epithelial cells, nerve cells, adipocytes, etc.). Examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells with a defect in the dihydrofolate reductase gene (hereafter referred to as the "DHFR gene") (Urlaub, G. et al., 1980, Proc Natl Acad Sci USA. 77(7):4216-4220), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereafter referred to as "YB2 / 0 cells"). YB2 / 0 cells are interesting because the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in these cells.

[0217] In particular, regarding the expression of humanized antibodies, expression vectors are either of the type in which the genes encoding the antibody's heavy chain and light chain are in separate vectors, or of the type in which both genes are in the same vector (tandem type). Tandem-type humanized antibody expression vectors are generally used due to their ease of construction, ease of introduction into animal cells, and balance between the expression levels of the antibody's H and L chains in animal cells (Shitara, K. et al., 1994, J Immunol Methods. Jan. 3:167(1-2):271-8). Examples of tandem-type humanized antibody expression vectors include pKANTEX93 (WO97 / 10354) and pEE18.

[0218] The present invention also relates to a method for producing recombinant host cells expressing antibodies according to the present invention, the method comprising the steps of: (i) introducing the recombinant nucleic acid or vector described above into competent host cells in vitro or ex vivo; (ii) culturing the resulting recombinant host cells in vitro or ex vivo; and (iii) optionally selecting cells that express and / or secrete the antibodies.

[0219] Such recombinant host cells can be used for the production of the anti-CHIKV antibodies described herein.

[0220] Accordingly, the present invention relates to a method for producing a monoclonal antibody according to the present invention, the method comprising (i) culturing the cell line described above; (ii) purifying the produced monoclonal antibody; and optionally (iii) forming the monoclonal antibody into a pharmaceutical composition.

[0221] Method for producing anti-CHIKV antibodies The anti-CHIKV antibodies primarily dealt with in this invention are produced by any technique known in the art, such as, but not limited to, any chemical, biological, genetic, or enzymatic technique, either alone or in combination.

[0222] If the amino acid sequence of a desired sequence is known, those skilled in the art can readily produce the antibody or immunoglobulin chain by standard techniques for polypeptide production. For example, they can be synthesized using well-known solid-phase methods, particularly using commercially available peptide synthesizers (such as those made by Applied Biosystems, Foster City, California), according to the manufacturer's instructions. Alternatively, antibodies and immunoglobulin chains can be synthesized by recombinant DNA techniques well known in the art. For example, these fragments can be obtained as DNA expression products after the incorporation of the DNA sequence encoding the desired (poly)peptide into an expression vector and its introduction into a suitable eukaryotic or prokaryotic host expressing the desired polypeptide, from which the fragments can then be isolated using well-known techniques.

[0223] In particular, the present invention further relates to a method for producing an antibody, comprising the steps of (i) culturing host cells transformed according to the present invention; (ii) expressing the antibody or polypeptide; and (iii) recovering the expressed antibody or polypeptide.

[0224] In other words, the present invention is: (i) A step of providing cells that express an anti-CHIKV antibody; (ii) The step of culturing the cells; (iii) the step of purifying the antibody; and (iv) Depending on the case, the step of forming the antibody into a pharmaceutical composition. This relates to a method for producing antibodies, including [the specified method].

[0225] Methods for producing humanized or chimeric antibodies include conventional recombinant DNA and gene transfection techniques well known in the art (see Morrison, SL and Oi, VT, 1984, Annu Rev Immunol 2:239-256 and Patent Documents US5,202,238; and US5,204,244).

[0226] In certain embodiments, the chimeric antibodies of the present invention can be produced by obtaining nucleic acid sequences encoding the VL and VH domains of a mouse, as previously described, constructing a chimeric antibody expression vector, inserting them into an expression vector for animal cells having genes encoding the CH and CL domains of a human antibody, and expressing the encoding sequences by introducing the expression vector into animal cells.

[0227] The antibodies primarily used in this invention are appropriately separated from the culture medium by conventional immunoglobulin purification procedures, such as affinity chromatography of protein A, ceramic hydroxyapatite chromatography, mixed-mode chromatography, and size-exclusion chromatography.

[0228] Fab can be obtained by treating an antibody that specifically reacts with CHIKV with a protease such as papain. Fab is then obtained by inserting the DNA sequences encoding both strands of the antibody Fab into a vector for expression in prokaryotes or eukaryotes. It can also be produced by introducing the vector into prokaryotic or eukaryotic cells (appropriate) to express Fab.

[0229] F(ab')2 can be obtained by treating an antibody that specifically reacts with CHIKV with a protease and pepsin. F(ab')2 can also be produced by attaching Fab' via a thioether bond or a disulfide bond, as described below.

[0230] Fab' can be obtained by treating F(ab')2, which reacts specifically with CHIKV, with a reducing agent such as dithiothreitol. Fab' can also be produced by inserting the DNA sequence encoding the Fab' chain of an antibody into a vector for expression in prokaryotes or eukaryotes, introducing the vector into a prokaryotic or eukaryotic cell (appropriate), and then performing expression.

[0231] scFv can be produced by constructing DNA encoding the scFv fragment using the CDR or VH and VL domain sequences, inserting that DNA into a prokaryotic or eukaryotic expression vector, and then introducing that expression vector into a prokaryotic or eukaryotic cell (appropriate) to express scFv. To generate humanized scFv fragments, a well-known technique called CDR grafting can be used, which involves selecting complementarity-determining regions (CDRs) according to the present invention and grafting them onto a framework of a human scFv fragment with a known three-dimensional structure (see, for example, W098 / 45322;WO87 / 02671;US5,859,205;US5,585,089;US4,816,567;EP0173494).

[0232] A single-chain antibody or VHH against CHIKV can be obtained, for example, by a method comprising the steps of (a) immunizing a mammal belonging to the Camelidae family with CHIKV or fragments thereof to induce antibodies against CHIKV (and especially heavy-chain antibodies); (b) obtaining a biological sample containing heavy-chain antibody sequences and / or VHH sequences against CHIKV from the thus immunized Camelidae; and (c) recovering (e.g., isolating) the heavy-chain antibody sequences and / or VHH sequences against CHIKV from the biological sample. A suitable single-chain antibody or VHH can also be obtained by screening a library containing heavy-chain antibody sequences and / or VHH sequences of heavy-chain antibody sequences and / or VHH sequences that compete for binding to pE2-E1 of CHIKV, as an example, but not limited to.

[0233] Modification of the anti-CHIKV antibody of the present invention A further object of the present invention is to encompass functionally conserved variants of the antibodies described herein.

[0234] For example, a certain amino acid can be substituted in a protein structure by another amino acid without any noticeable loss of activity. Since the ability and properties of proteins to interact determine their biological functional activity, a certain amino acid substitution can occur in the protein sequence and, needless to say, in the DNA coding sequence that encodes that sequence, and yet a protein with similar properties can be obtained. Therefore, it is considered that various changes can be made to antibody sequences, or the corresponding DNA sequences that encode the antibodies, without any noticeable loss of their binding activity.

[0235] Some amino acids have a similar hydropathic index or score to other amino acids. This technology allows for the substitution of amino acids to produce proteins with similar biological activity, that is, to obtain proteins that are still biologically functionally equivalent. It is known in the field that, in order to identify all amino acids that can be substituted in an antibody without significant loss of binding to the antigen, well-established techniques such as alanine scanning can be used. Such residues can be qualitatively classified as neutral because they do not participate in antigen binding or the maintenance of the antibody structure. One or more of these neutral positions can be substituted by alanine or another amino acid without altering the major properties of the antibody.

[0236] Therefore, as outlined above, amino acid substitutions are generally based on the relative similarity of the side-chain substituents of amino acids, such as their hydrophobicity, hydrophilicity, charge, size, etc. Several typical substitutions that take these properties into account are well known to those skilled in the art: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0237] Amino acid modification of other types of antibodies can be useful in altering the antibody's original glycosylation pattern by deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody. The presence of either the tripeptide sequence asparagine X-serine or asparagine X-threonine, where X is any amino acid other than proline, gives rise to potential glycosylation sites. Addition or deletion of glycosylation sites to an antibody is usually achieved by modifying the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites).

[0238] Another type of covalent modification involves chemically or enzymatically coupling glycosides to antibodies. These procedures are advantageous in that they do not require the production of antibodies in a host cell capable of glycosylation for N- or O-linked glycosylation. Depending on the coupling mode used, sugars(s) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as the free radical of cysteine, (d) free hydroxyl groups such as the free radicals of serine, threonine, or hydroxyproline, (e) aromatic residues such as phenylalanine or tyrosine residues, or (f) amide groups of glutamine. For example, such a method is described in WO87 / 05330.

[0239] The removal of any carbohydrate moiety present in an antibody can be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This process causes cleavage of most or all sugars except linked sugars (N-acetylglucosamine or N-acetylgalactosamine), while the antibody remains intact. Chemical deglycosylation has been described by Sojahr, H. et al. (1987, Arch Biochem Biophys. 259(1):52-57) and Edge, AS et al. (1981, Anal Biochem. 118(1):131-137). Enzymatic cleavage of carbohydrate moieties in antibodies can be achieved using various endo and exoglycosidases described by Thotakura, NR et al. (1987, Methods Enzymol 138:350-359).

[0240] Another type of covalent modification of antibodies involves linking the antibody to one of various non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner described in U.S. Patents 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0241] Pharmaceutical composition The anti-CHIKV antibodies primarily used in this invention can also be combined with pharmaceutically acceptable excipients and, optionally, with a sustained-release matrix such as a biodegradable polymer to form therapeutic compositions.

[0242] Therefore, the present invention also relates to a pharmaceutical composition comprising the anti-CHIKV antibody of the present invention and a pharmaceutically acceptable carrier.

[0243] This invention also relates to antibodies according to the present invention for use as pharmaceuticals.

[0244] "Pharmacologically" or "pharmaceutically acceptable" means molecular entities and compositions that, when administered appropriately to mammals, particularly humans, do not produce harmful allergic or other adverse reactions. Pharmaceutically acceptable carriers or excipients include non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or any type of dosage form aid.

[0245] The form of the pharmaceutical composition, the route of administration, the dosage, and the administration plan naturally depend on the condition being treated, the severity of the disease, the patient's age, weight, and sex, etc.

[0246] Pharmaceutical compositions can be formulated into dosage forms for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration.

[0247] In particular, pharmaceutical compositions contain vehicles that are pharmaceutically acceptable for injectable dosage forms. These may be isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts), or lyophilized compositions that, in particular as may be added, enable the formation of injectable solutions.

[0248] The dose used for administration can be adapted as a function of various parameters, particularly as a function of the mode of administration used, the pathology involved, or the desired duration of treatment.

[0249] To manufacture pharmaceutical compositions, an effective amount of antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0250] In one embodiment, the present invention relates to a pharmaceutical composition comprising a preventive or therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to CHIKV as described herein, and a pharmaceutically acceptable carrier.

[0251] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to be readily injectable. It must be stable under manufacturing and storage conditions and stored in a manner that prevents contamination by microorganisms such as bacteria and fungi.

[0252] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or a suitable mixture thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants, stabilizers, cryoprotectants, or antioxidants. Prevention of biological activity can be achieved with antimicrobial and antifungal agents. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride.

[0253] Sterile injectable solutions are prepared by incorporating the required amount of active compound in a suitable solvent, along with several other components listed above, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other components as needed from those described above. In the case of sterile powders for preparing sterile injectable solutions, common manufacturing methods include vacuum drying and freeze-drying techniques, producing powders of the active ingredients plus any additional desired components from those solutions that have been previously sterilized and filtered.

[0254] Once formulated, the solution will be administered in a manner suitable for the dosage form and in a therapeutically effective amount. The formulation can be easily administered in various forms of administration, such as the injectable solution types described above, but drug-releasing capsules and the like can also be used.

[0255] For parenteral administration in aqueous solutions, for example, the solution should be appropriately buffered if necessary, and the diluent should first be isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, those skilled in the art will know of sterile aqueous media that can be used in light of this disclosure. For example, a single dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of a large volume of subcutaneous injection fluid or infused into the proposed site of infusion (see, e.g., Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038, 1570-1580). Some variation in the dose will inevitably occur depending on the condition of the subject being treated. The person responsible for administration will determine an appropriate dose for the individual subject in any given event.

[0256] The antibody may be formulated into a dosage form within a therapeutic mixture and may contain approximately 0.01 to 100 milligrams or close to that amount per dose.

[0257] In one embodiment, a single or multiple doses of the anti-CHIKV antibody described herein may be administered to a subject over a specified time course.

[0258] Methods according to this embodiment include sequentially administering multiple doses of antibody against CHIKV to a subject. As used herein, “sequentially administer” means that each dose of antibody against CHIKV is administered to the subject at different times, for example, on different days at predetermined intervals (e.g., hours, days, weeks, or months). The present invention includes a method comprising sequentially administering to a patient one initial dose of antibody against CHIKV, followed by one or more secondary doses of antibody against CHIKV, and optionally one or more tertiary doses of antibody against CHIKV.

[0259] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to the sequence of antibody administrations against CHIKV in chronological order. Thus, the “initial dose” is the dose administered at the start of the treatment regimen (also referred to as the “baseline dose”); the “secondary dose” is the dose administered after the initial dose; and the “tertiary dose” is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of antibody against CHIKV, but generally they may differ from one another in terms of the frequency of administration. However, in some embodiments, the amounts of antibody against CHIKV contained in the initial, secondary, and / or tertiary doses may change from one another during the course of treatment (e.g., adjusted up or down as appropriate). In some embodiments, two or more doses (e.g., 2, 3, 4, or 5) may be used. The dose is administered as the "charged dose" at the start of the treatment administration plan, followed by doses administered at reduced frequency (e.g., the "maintenance dose").

[0260] Therapeutic methods and uses In another aspect, the present invention provides a method for preventing CHIKV infection in patients in need, or for treating patients suffering from CHIKV infection, or for alleviating at least one symptom or complication associated with CHIKV infection, the method comprising administering to a patient in need one or more antibodies or antigen-binding fragments thereof as described herein, or a pharmaceutical composition comprising one or more anti-CHIKV antibodies or fragments thereof, which are the primary focus of the present invention, to prevent CHIKV infection or reduce, alleviate, or decrease the severity and / or duration of at least one symptom or complication associated with the infection.

[0261] In some embodiments, the method alleviates symptoms associated with CHIKV infection.

[0262] In some embodiments, the method alleviates symptoms associated with acute, post-acute, or chronic polyarthritis / polyarthralgia / CHIKV-associated joint pain, fever, rash, muscle pain, and / or fatigue. In one embodiment, the method relieves pain associated with CHIKV infection in a subject. In one embodiment, the antibody is used to treat / relieve symptoms associated with CHIKV infection and can cross-react with and treat symptoms associated with other alphavirus infections. In one embodiment, the antibody is used to treat / relieve acute, post-acute, and chronic polyarthritis / polyarthralgia phases associated with CHIKV infection.

[0263] Other examples of such alphaviruses include, but are not limited to, Onyonnyon (ONNV), Ross River (RRV), Bammer-Forrest (BFV), Western Equine Encephalitis (WEEV), Semlyki-Forrest (SFV), Sindvis (SINV), Eastern Equine Encephalitis (EEEV), and Venezuelan Equine Encephalitis (VEEV). Symptoms treated or relieved may include, but are not limited to, pain and fever.

[0264] In another embodiment, a method is provided for treating a subject infected with chikungunya virus or reducing the likelihood of infection of a subject at risk of contracting chikungunya virus, comprising delivering to the subject an antibody or antigen-binding fragment according to the present invention, which contains the CDR sequence of an antibody listed in Tables 2 to 5, respectively.

[0265] In another embodiment, a method is provided for treating a subject infected with chikungunya virus or reducing the likelihood of infection in a subject at risk of contracting chikungunya virus, comprising delivering antibodies that bind to CHIKV, as listed in Tables 2 to 5, to the subject. In another embodiment, one, two, or more antibodies from those listed in Tables 2 to 5 may be combined. In another embodiment, the antibodies may encode a deformed antibody containing heavy and light chains having a variable sequence having 70%, 80%, 90%, or 95% identity with one of the variable sequences of the antibodies listed in Tables 2 to 5. The antibody fragment may be a recombinant ScFv (single-chain variable fragment) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. The antibody may be an IgG and / or a chimeric antibody. The antibody or antibody fragment may be administered prior to or after infection. Delivery may include administration of the antibody or antibody fragment, or genetic delivery using a vector encoding an RNA or DNA sequence or an antibody or antibody fragment.

[0266] As mentioned above, the method of the present invention involves administering to a subject in need thereto a CHIKV infection / symptom prevention or treatment one antibody selected from the group consisting of mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb11, mAb12, mAb13, and mAb14, as listed in Tables 2, 3, 4, and 5 as described herein.

[0267] As mentioned above, the method of the present invention involves administering to a subject in need of an antibody selected from the group consisting of mAb6, mAb7, mAb8, mAb9, mAb13, and mAb14 described herein, in order to prevent or treat infection / symptoms caused by CHIKV. In another embodiment, the method of the present invention involves administering to a subject in need of an antibody selected from the group consisting of mAb7 and mAb14, which is the primary focus of the present invention, in order to prevent or treat infection / symptoms caused by CHIKV.

[0268] In another embodiment, the present invention relates to a monoclonal antibody described herein for use as a pharmaceutical. In one embodiment, the present invention relates to a monoclonal antibody described herein for use in the treatment of CHIKV infection. In one embodiment, the present invention relates to a monoclonal antibody for use in the treatment of arthralgia associated with CHIKV. In one embodiment, the present invention relates to a monoclonal antibody for use in the treatment of acute, post-acute and chronic polyarthritis / polyarthralgia associated with CHIKV infection. In one embodiment, the present invention relates to a monoclonal antibody for use in the treatment of arthralgia, fever, rash, muscle pain, and / or fatigue associated with acute, post-acute or chronic polyarthritis / polyarthralgia / CHIKV. In one embodiment, the present invention relates to a monoclonal antibody for use to alleviate pain in subjects associated with CHIKV infection. In another embodiment, the present invention relates to a monoclonal antibody for use in the prevention of CHIKV infection. In another embodiment, the present invention relates to monoclonal antibodies selected from the group consisting of mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb11, mAb12, mAb13, and mAb14, listed in Tables 2, 3, 4, and 5 above, for use in the treatment of infections and symptoms listed above. In another embodiment, the present invention relates to monoclonal antibodies selected from the group consisting of mAb6, mAb7, mAb8, mAb9, mAb13, and mAb14, listed in Tables 2, 3, and 5, for use in the treatment of infections and symptoms listed above. In another embodiment, the present invention relates to monoclonal antibodies selected from mAb7 and mAb14, listed in Tables 2 and 5, for use in the treatment of infections and symptoms listed above.

[0269] In another embodiment, the monoclonal anti-CHIKV antibody or its antigen-binding fragment of the present invention may also be used in combination with one or more additional therapeutic agents to prevent or treat CHIKV infection or related symptoms. As used herein, the expression “in combination with” means that the additional therapeutic agent is administered before, after, or concurrently with the pharmaceutical composition comprising the anti-CHIKV antibody, which is the primary subject of the present invention. The term “in combination with” also includes sequential or simultaneous administration of the anti-CHIKV antibody and the second therapeutic agent.

[0270] For example, when administered "before" the pharmaceutical composition containing the anti-CHIKV antibody, the additional therapeutic agent can be administered approximately 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, or 10 minutes before the administration of the pharmaceutical composition containing the anti-CHIKV antibody. When administered "after" the pharmaceutical composition containing the anti-CHIKV antibody, the additional therapeutic agent can be administered approximately 10 minutes, 15 minutes, 30 minutes, 1 hour, or 2 hours before the administration of the pharmaceutical composition containing the anti-CHIKV antibody. It can be administered approximately 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours later. Administration “simultaneously” with or together with a pharmaceutical composition containing anti-CHIKV antibody means that the additional therapeutic agent is administered to the subject in a different dosage form within 5 minutes (before, after, or simultaneously) of the administration of the pharmaceutical composition containing anti-CHIKV antibody, or as a single combination dosage form containing both the additional therapeutic agent and the anti-CHIKV antibody.

[0271] Combination therapies may include the anti-CHIKV antibodies described herein, and any additional therapeutic agents that may be advantageous when combined with the above antibodies or with biologically active fragments thereof.

[0272] For example, a second or third therapeutic agent may be used to help alleviate symptoms associated with CHIKV infection, including but not limited to acute, post-acute, or chronic polyarthritis, polyarthralgia / CHIKV-associated joint pain, fever, rash, muscle pain, and / or fatigue. For example, a second or third therapeutic agent may be used to help alleviate pain associated with CHIKV infection.

[0273] Use for diagnosis In another embodiment, the monoclonal antibodies or antigen-binding fragments primarily dealt with in the present invention are used to detect the presence or absence of CHIKV antigen in a sample. In one embodiment, the antibodies described herein are used as components of an assay comprising the steps of contacting a sample with the antibody or antigen-binding fragment described herein, and detecting the binding of the monoclonal antibody or antigen-binding fragment to CHIKV antigen, where detection of binding indicates the presence of CHIKV antigen, or the absence of detection of binding to CHIKV antigen indicates the absence of CHIKV antigen.

[0274] In particular, the monoclonal antibodies or antigen-binding fragments described herein are used as components of both therapeutic agents and diagnostic assays.

[0275] In one embodiment, the antibody is intended for use in vitro or ex vivo. For example, CHIKV can be detected in vitro or ex vivo in a biological sample obtained from a subject using the anti-CHIKV antibody described herein.

[0276] This invention is: i. Contact the target biological sample with an anti-CHIKV antibody, and in particular, under conditions sufficient for the antibody to form a complex with the biological sample. ii. Measuring the level of antibodies bound to the biological sample, iii. Detecting the presence of CHIKV infection by comparing the measured level of bound antibodies with a control; an increased level of bound antibodies compared to a control indicates CHIKV infection. The present invention further relates to an in vitro or ex vivo method for detecting the presence of CHIKV infection in a subject, comprising the steps of:

[0277] The present invention also relates to an in vitro or ex vivo method for determining the susceptibility of a patient infected with CHIKV to a therapeutic agent targeting CHIKV, particularly to an anti-CHIKV antibody or its antigen-binding fragment described herein, the method being: i. Contacting a biological sample from a patient infected with CHIKV with an anti-CHIKV antibody or its antigen-binding fragment, particularly under conditions sufficient for the antibody to form a complex with the biological sample. ii. Measuring the level of antibodies bound to the biological sample, iii. Comparing the measured level of the antibody bound to the biological sample with the level of the antibody bound to the control. The steps include, Here, an increased level of antibody bound to the biological sample compared to a control indicates a patient who is susceptible to a CHIKV-targeting therapeutic agent.

[0278] In the above method, the control may be a reference value determined to be a representative value of antibody binding levels in a normal, uninfected biological sample of the same type, or in a normal biological sample of the same type.

[0279] This invention is: i. A biological sample of a subject being treated for CHIKV infection is brought into contact with the antibody or its antigen-binding fragment described herein, in particular, under conditions sufficient for the antibody to form a complex with the biological sample. ii. Measuring the level of antibodies bound to the biological sample, iii. Compare the measured level of the bound antibody with the level of the antibody bound to the control. Further relating here to methods, either in vitro or ex vivo, for monitoring the effectiveness of treatment for CHIKV infection, including steps comprising the following: The reduced level of antibodies bound to the biological sample compared to the control indicates the effectiveness of the treatment for CHIKV infection.

[0280] In the above method, an increased level of antibody bound to the biological sample compared to the control indicates ineffectiveness of the treatment of the CHIKV infection.

[0281] The aforementioned controls are, in particular, biological samples of the same type as the biological samples subjected to analysis, but obtained from the subjects prior to the course of treatment for CHIKV infection.

[0282] In one embodiment, the anti-CHIKV antibody or its antigen-binding fragment (e.g., E2-binding fragment) described herein may also be labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule, or any other label known in the art that provides a signal (either directly or indirectly).

[0283] With respect to the antibodies according to the present invention, the term “labeled” as used herein is intended to include direct labeling of antibodies by coupling (i.e., physically linking) a polypeptide with a detectable substance, such as a radioactive reagent or a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), or indocyanine (Cy5)), as well as indirect labeling by reactivity using a detectable substance for the polypeptide.

[0284] The "sample" or "biological sample" that can be used in the CHIKV diagnostic assay according to the present invention includes any tissue or body fluid sample that can be obtained from a patient in a normal or pathological state.

[0285] Biological specimens include, but are not limited to, blood and other fluid specimens of biological origin, biopsy specimens or tissue cultures or cells derived therefrom, and solid tissue specimens such as their offspring. Therefore, biological specimens encompass clinical specimens, cells in culture, cell supernatants, cell lysates, serum, plasma, body fluids, and tissue specimens.

[0286] kit The present invention also provides a kit containing at least one anti-CHIKV antibody or antigen-binding fragment. The kit containing the antibody or antigen-binding fragment finds applications in the detection of CHIKV or in therapeutic or diagnostic assays. The kit may contain polypeptides or antibodies coupled to a solid support, such as a tissue culture plate or beads (e.g., Sepharose beads). A kit containing antibodies for in vitro detection and quantification of CHIKV, for example, by ELISA or Western blotting, can be provided. Such antibodies useful for detection may be provided labeled with fluorescence or radiolabeling, etc.

[0287] In one embodiment, the present invention relates to a kit comprising at least one antibody primarily dealt with in the present invention, and optionally a packaging material, and optionally a label or packaging insert contained within the packaging material indicating that the antibody primarily dealt with in the present invention is effective in preventing / treating CHIKV infection or symptoms associated with CHIKV infection.

[0288] A brief explanation of arrays Sequence ID 1 shows the VH sequence of the "mAb1" antibody. Sequence ID 2 shows the VL sequence of the "mAb1" antibody. Sequence ID 3 shows the VH sequence of the "mAb2" antibody. Sequence ID 4 shows the VL sequence of the "mAb2" antibody. Sequence IDs 5-7 show the sequences of CDR1H, CDR2H, and CDR3H of the "mAb1" antibody. Sequence ID 8 shows the CDR1L sequence of the "mAb1" antibody. Sequence ID 9 shows the sequence of the recombinant target "His-tagged CHIKV E2 LR2006" for recombinant pE2-E1. Sequence ID No. 10 shows the CDR3L sequence of the "mAb1" antibody. Sequence IDs 11-13 show the sequences of CDR1H, CDR2H, and CDR3H of the "mAb2" antibody. Sequence ID 14 shows the CDR1L sequence of the "mAb2" antibody. Sequence ID 15 shows the sequence of the recombinant pE2-E1 recombinant target "His-tagged CHIKV E2 SL15649". Sequence ID 16 shows the CDR3L sequence of the "mAb2" antibody. Sequence ID 17 is the sequence of the Fc region of IgG1 without substitution, which is the main focus of this invention and is shown in Figure 1. Sequence ID 18 shows the nucleic acid sequence of the Fc region of IgG1. Sequence ID 19 shows the HC sequence of the "mAb1" antibody. Sequence ID 20 shows the LC sequence of the "mAb1" antibody. Sequence ID 21 shows the nucleic acid sequence of the HC of the "mAb1" antibody. Sequence ID 22 shows the nucleic acid sequence of the LC of the "mAb1" antibody. Sequence ID 23 shows the HC sequence of the "mAb3" antibody. Sequence ID 24 shows the nucleic acid sequence of the HC of the "mAb3" antibody. Sequence ID 25 shows the HC sequence of the "mAb4" antibody. Sequence ID 26 shows the nucleic acid sequence of the HC of the "mAb4" antibody. Sequence ID 27 shows the HC sequence of the "mAb5" antibody. Sequence ID 28 shows the nucleic acid sequence of the HC of the "mAb5" antibody. Sequence ID 29 shows the HC sequence of the "mAb6" antibody. Sequence ID 30 shows the nucleic acid sequence of the HC of the "mAb6" antibody. Sequence ID 31 shows the HC sequence of the "mAb7" antibody. Sequence ID 32 shows the nucleic acid sequence of the HC of the "mAb7" antibody. Sequence ID 33 shows the consensus sequence of CDRH3 in the "mAb2" antibody. Sequence ID 34 shows the CDRH3 sequence of the "mAb10" antibody. Sequence ID 35 shows the CDRH3 sequence of the "mAb11" antibody. Sequence ID 36 shows the CDRH3 sequence of the "mAb12" antibody. Sequence ID 37 shows the HC sequence of the "mAb2" antibody. Sequence ID 38 shows the LC sequence of the "mAb2" antibody. Sequence ID 39 shows the nucleic acid sequence of the HC of the "mAb2" antibody. Sequence ID 40 shows the nucleic acid sequence of the LC of the "mAb2" antibody. Sequence ID 41 shows the HC sequence of the "mAb8" antibody. Sequence ID 42 shows the nucleic acid sequence of the HC of the "mAb8" antibody. Sequence ID 43 shows the HC sequence of the "mAb9" antibody. Sequence ID 44 shows the nucleic acid sequence of the HC of the "mAb9" antibody. Sequence ID 45 shows the HC sequence of the "mAb10" antibody. Sequence ID 46 shows the nucleic acid sequence of the HC of the "mAb10" antibody. Sequence ID 47 shows the HC sequence of the "mAb11" antibody. Sequence ID 48 shows the nucleic acid sequence of the HC of the "mAb11" antibody. Sequence ID 49 shows the HC sequence of the "mAb12" antibody. Sequence ID 50 shows the nucleic acid sequence of the HC of the "mAb12" antibody. Sequence ID 51 shows the HC sequence of the "mAb13" antibody. Sequence ID 52 shows the nucleic acid sequence of the HC of the "mAb13" antibody. Sequence ID 53 shows the HC sequence of the "mAb14" antibody. Sequence ID 54 shows the nucleic acid sequence of the HC of the "mAb14" antibody. Sequence ID 55 shows the sequence of the constant region of IgG1, as shown in Figure 1. Sequence ID 56 shows the VH sequence of the "mAb10" antibody. Sequence ID 57 shows the VH sequence of the "mAb11" antibody. Sequence ID 58 shows the VH sequence of the "mAb12" antibody. Sequence ID 59 shows the sequence of the Fc region of IgG1 having alanine at position 434 according to the present invention, as shown in Figure 2. Sequence ID 60 shows the sequence of the Fc region of IgG1 having alanine at positions 307, 380, and 434, respectively, according to the present invention, as shown in Figure 2. Sequence ID 61 shows the sequence of the Fc region of IgG1 according to the present invention, which has glutamine at position 250 and leucine at position 428, as shown in Figure 2. Sequence ID 62 shows the sequence of the Fc region of IgG1 according to the present invention, as shown in Figure 2, with tyrosine at position 252, threonine at position 254, and glutamic acid at position 256. Sequence ID 63 shows the sequence of the Fc region of IgG1 having leucine at position 428 and serine at position 434, according to the present invention, as shown in Figure 2. [Examples]

[0289] material and method Analysis and manipulation of monoclonal antibodies: The amino acid sequences of anti-CHIKV antibodies were analyzed using an antibody inspector (an in-house developed antibody sequence analysis tool) coupled with a 3D model / structural analysis tool (Biovia Discovery Studio Suite) to screen for potential problems and disadvantages for development. Disadvantage analysis focused on motifs undesirable to solvent exposure, such as oxidation, deamination, isomerization, acid cleavage, glycosylation, and additional free Cys. All solvent exposure disadvantages were prioritized based on their location (CDR, variable domain framework, constant domain). Mutations were suggested to minimize the disadvantages found in the sequences.

[0290] Optimized antibody generation: Codon-optimized gene fragments were synthesized and cloned in mammalian expression vectors. Transfection was performed using the Expi293F expression system (Thermo Fisher Scientific) according to the manufacturer's protocol. The harvested samples in conditioned media were purified by passing them through a protein A column, and the eluted fractions were buffered and prepared in Gibco PBS at pH 7.4.

[0291] Binding analysis: Antigen binding, FcRn binding to human and mouse, FcγIIIa binding The binding of recombinant CHIKV E2 antigen was measured by surface plasmon resonance using a Biacore T200 instrument. Anti-tetraHis antibody (Qiagen) was immobilized to saturation levels on CM5 series S sensor chips using a standard amine coupling procedure provided by Biacore. Recombinant CHIKV E2 antigen constructs, i.e., His-tagged CHIKV E2 LR2006 (SEQ ID NO: 9) and His-tagged CHIKV E2 SL15649 (SEQ ID NO: 15) recombinant protein constructs, were based on Voss et al., 2010 (Voss JE et al. 2010, Nature 468:709-712) and were transiently expressed and purified in HEK293 cells (Pal et al., 2013, PloS Pathog 9, e1003312; Smith et al., 2015, Cell Host & Microbe 18:86-95). Essentially, these constructs are designed as the signal peptide-E3_E2-(G4S)4-E1-His8, but in the mature form, the signal peptide and E3 are removed by cleavage. His-tagged CHIKV E2 LR2006 and His-tagged CHIKV E2 SL15649 recombinant antigens were diluted in HBS-EP+ electrophoresis buffer and injected for 30 seconds to achieve capture levels between 10 and 30 RU. Test antibodies were sequentially diluted 3-fold from 30 nM to 1.1 nM. Low-affinity binders were sequentially diluted from 900 nM. Each antibody was injected in two decans for 3 minutes at a flow rate of 65 μL / min onto the captured antigen and control surface, and dissociated for 5 or 15 minutes. The surface was regenerated with glycine at pH 1.5. Reaction rate constants were calculated using a 1:1 binding model with Biacore T200 evaluation software.

[0292] To measure binding to FcRn, recombinant human or mouse FcRn was directly immobilized onto the tip of a CM5 Series S sensor using an amine chemical reaction, achieving surface densities of 1700 RU and 800 RU, respectively. Test antibodies were diluted to 200 and 50 nM in 50 mM sodium phosphate, 150 mM NaCl, and 0.05% surfactant P20 at pH 6.0 or pH 7.4. The diluted samples were injected for 3 minutes, followed by dissociation in a double-stream at 10 μL / min of buffer for 5 minutes. The surface was regenerated in borate buffer at pH 8.5.

[0293] Binding to FcγRIIIa was measured using a Biacore 3000 instrument. Anti-HPC4 antibody was immobilized to a saturation level on a CM5 chip by amine coupling. Two polymorphs of recombinant human FcγRIIIa were compared analytically (Val158 and Phe158). Recombinant human HPC4-tagged FcγRIIIa-V158 and FcγRIII-F158 were diluted in HBS-P+ buffer containing 2 mM CaCl2 and injected into Fc2 or Fc4 at 10 μL / min for 30 seconds, respectively, to achieve capture levels of 10–40 RU. Samples were diluted to 900, 300, and 100 nM and injected for 2 minutes, followed by dissociation in two strands at 30 μL / min in buffer for 3 minutes. The surface was regenerated at 20 μL / min for 3 minutes using 10 mM EDTA in HBS-EP+ buffer.

[0294] result [Examples]

[0295] Antigen binding of mAb2 with substitution in CDRH3 The results are shown in Figure 3. The following were introduced to eliminate potential deamination and oxidation motifs. The effect of the mutation in CDRH3 of mAb2 was measured by its binding to the pE2-E1 antigen of CHIKV derived from the LR2006 strain of CHIKV. Binding was: Measurements were taken for mAb2, and its derived variants mAb10, mAb11, and mAb12, which contain isoleucine at position 8 of its CDRH3, glutamine at position 12 of its CDRH3, and alanine at position 13 of its CDRH3, respectively, as well as for the variant containing alanine at position 8 of its CDRH3, and variants containing different combinations of the two substitutions listed above.

[0296] Among the seven mutants created by eliminating potential deamination and oxidation motifs, only mAb11 (mAb2 N108Q), which contained glutamine at position 12 of CDRH3, retained target binding affinity equivalent to the parent mAb. Note that mAb10 (mAb2 M104I), which contained isoleucine at position 8 of CDRH3, and mAb12 (mAb2 G109A), which contained alanine at position 13 of CDRH3, showed intermediate profiles; the double mutants lost their target binding affinity, as did the variant (mAb2 M104A), which contained alanine at position 8 of CDRH3. [Examples]

[0297] FcRn binding The results are shown in Figure 4. Binding to human FcRn (Figures 4A and 4C) and binding to mouse (Figures 4B and 4D): - In those Fc regions, mAb1, mAb3, mAb4, mAb5, mAb6, and mAb7 contain alanine at position 434, alanine at positions 307, 380, and 434 respectively, glutamine at position 250 and leucine at position 428, serine at position 428 and 434 respectively, and tyrosine at position 252, threonine at position 254 and glutamic acid at position 256, respectively. - In their Fc regions, mAb2, mAb8, mAb9, mAb11, mAb13, and mAb14 each contain tyrosine at position 252, threonine at position 254, glutamic acid at position 256, leucine at position 428, and serine at position 434, respectively, and glutamine at position 12 of their CDRH3, tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, as well as glutamine at position 12 of their CDRH3, leucine at position 428, and serine at position 434, and glutamine at position 12 of their CDRH3, respectively. The measurement was performed at pH 6.0.

[0298] All mutants showed increased FcRn binding affinity, which is expected to result in an extended half-life, and therefore a favorable effect on their usefulness in anti-CHIKV therapy. Mutants containing leucine at position 428 and serine at position 434, respectively, and tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, respectively, showed the strongest binding compared to the other mutants. [Examples]

[0299] Effect of substitution in the Fc region on target binding of mAb1 and mAb2 The results are shown in Figure 5. The effect of substitution in the Fc region was measured for mAb1 and mAb2 by their binding to the pE2-E1 antigen of CHIKV derived from CHIKV strains LR2006 (Figure 5A) and SL15649 (Figure 5B), respectively. Binding was: - mAb1, mAb3, mAb4, mAb contain alanine at position 434, alanine at positions 307, 380, and 434 respectively, glutamine at position 250 and leucine at position 428, leucine at position 428 and serine at position 434 respectively, and tyrosine at position 252, threonine at position 254 and glutamic acid at position 256, respectively. 5, mAb6 and mAb7; - mAb2, mAb8, mAb9, mAb11, mAb13, and mAb14 each contain tyrosine at position 252, threonine at position 254, glutamic acid at position 256, leucine at position 428, and serine at position 434, respectively, glutamine at position 12 of its CDRH3, tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, as well as glutamine at position 12 of its CDRH3, leucine at position 428, and serine at position 434, and glutamine at position 12 of its CDRH3. Each of these was measured.

[0300] Substitutions in the Fc region that enhanced the binding of mAb1 and mAb2 to FcRn did not affect the binding of the target CHIKV pE2-E1 antigen. [Examples]

[0301] FcγRIIIa binding The results are shown in Figure 6. The effects of substitutions in the Fc region of mAb1 and mAb2 were measured by binding to FcγRIIIa, respectively. FCγRIIa(CD16a) is expressed by NK cells and macrophages and can induce antibody-dependent cell-mediated cytotoxicity (ADCC) and macrophage-mediated cytokine release.

[0302] The binding results are shown for mAb1 (Figure 6A) and mAb2 (Figure 6B) in the human FcγRIIIa high-affinity receptor (FcγRIIIaV158), and for mAb1 (Figure 6C) and mAb2 (Figure 6D) in the human FcγRIIIa low-affinity receptor (FcγRIIIaF158).

[0303] Join: - mAb1, mAb3, mAb4, mAb5, mAb6, and mAb7, each containing alanine at position 434, alanine at positions 307, 380, and 434 respectively, glutamine at position 250 and leucine at position 428, leucine at position 428 and serine at position 434 respectively, and tyrosine at position 252, threonine at position 254 and glutamic acid at position 256, respectively; - mAb2, mAb8, mAb9, mAb11, mAb13, and mAb14 contain tyrosine at position 252, threonine at position 254, glutamic acid at position 256, leucine at position 428, and serine at position 434, respectively, glutamine at position 12 of its CDRH3, tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, as well as glutamine at position 12 of its CDRH3, leucine at position 428, serine at position 434, and glutamine at position 12 of its CDRH3. Each of these was measured.

[0304] Among the substitutions in the Fc region of mAb1 and mAb2 that enhanced binding to FcRn, tyrosine at position 252, threonine at position 254, and glutamate at position 256 led to decreased binding affinity to FcγRIIIa and may have had a negative impact on cell-mediated effector function and anti-CHIKV therapy compared to mAb1 or mAb2 without Fc region substitution. Other mutations, such as alanine at position 434, alanine at positions 307, 380, and 434, glutamine at position 250 and leucine at position 428, leucine at position 428, and serine at position 434, respectively, retained binding affinity to FcγRIIIa.

[0305] Alanine at position 434, alanine at positions 307, 380 and 434 respectively, or glutamine at position 250 and leucine at position 428, or leucine at position 428 mAb1 and mAb2, which contain serine at position 434 and respectively, but do not contain tyrosine at position 252, threonine at position 254, or glutamic acid at position 256, retained FcγRIIIa binding linked to the effector function.

[0306] Therefore, mAb1 or mAb2 containing leucine at position 428 and serine at position 434 exhibits the best FcRn binding while simultaneously retaining FcγRIIIa binding, which is linked to the effector function.

[0307] [Table 6]

[0308] [Table 7] [Examples]

[0309] Neutralizing activity of mAbs using a standard plaque reduction assay mAb1, mAb7, and mAb CTR (anti-lysozyme rhlgG1 control antibody) were tested in vitro against three different CHIKV protozoan strains (Caribbean strain, Reunion (LR), and strain 37997) representing three CHIKV lineages (Asian, East Central and Southern Africa (ESCA), and West Africa).

[0310] A predetermined amount of virus was mixed with an equal volume of antibody diluted with PBS or diluent. The mixture was incubated at 37°C for 2 hours. The mixture was then added to a confluent monolayer of Vero cells in a 6-well plate. After 2 hours of incubation, the plate was overlaid with 5% CMC in DMEM. 48 hours after injection, the plate was fixed with formalin and then stained with methylene blue. Plaques were counted and EC was measured. 50 To determine this, we analyzed the data using Prism-Graph Pad.

[0311] The results are shown in Table 8 and Figures 7A to 7C below. mAb1 and mAb7, as shown in Figures 7A, 7B, and 7C respectively, exhibited very potent activity against all three genotypes of CHIKV strains from Asia, East Central and Southern Africa (ESCA), and West Africa (EC). 50 Inhibition was achieved at values ​​<10 ng / mL (and <1 ng / mL for mAb7). Anti-lysozyme antibodies (CTR of mAbs) were used as a nonspecific negative control.

[0312] [Table 8] [Examples]

[0313] In vivo protection studies in mice In vivo studies using the DBA1 / J mouse model were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at the National Institute of Allergy and Infection Diseases (NIAID). Infection experiments were conducted at an A-BSL3 facility with the approval of the NIAID Animal Studies Committee.

[0314] 0.05 ml of CHIKV-LR2006 was inoculated subcutaneously into the plantar of the right hind foot, towards the ankle, in 7-8 week old DBA / 1J mice. For therapeutic studies, 10 5.5 CCID 50 Three days after CHIKV infection, 0.1 ml of CHIKV-LR2006 was administered subcutaneously to the sole of the foot, followed by a single dose of recombinant human IgG anti-CHIKV mAb administered intraperitoneally in individual, specialized doses. Viral titers at the injection site were monitored five days after infection. For preventive studies, 105.5 CCID 50 Recombinant human IgG anti-CHIKVmAb was administered by intraperitoneal injection 2, 7, or 14 days prior to subcutaneous infection of the sole of the foot using 0.1 ml of CHIKV-LR2006. Viral titers at the injection site were monitored 3 days after infection.

[0315] In vivo prevention with mAbs Two, seven, or fourteen days prior to subcutaneous injection of CHIKV-LR2006, mice were given a single dose of 250 μg (approximately 12.5 mg / kg) of recombinant hIgG anti-CHIKV mAb (mAb1, mAb7) or a control anti-isotype lysozyme mAb (mAb1). CTR All mice treated with the isotype control mAb were inoculated in the right hind leg (CCID). 50 High viral titers were observed 3 days after the introduction of mAb7 / g tissue (Figure 8). Pretreatment with both mAb7 and mAb1 at different time points before infection (-2 to -14 days) completely protected DBA1 / J mice from the virus loaded at the injection site, as the viral levels in their respective right hind legs were nearly equivalent to those of uninfected mice from the SHAM-PBS group.

[0316] Post-exposure in vivo mAb therapy 7-8 week old DBA / 1J mice were given 0.05 ml of 10 oz subcutaneously in the plantar area of ​​the right hind foot, towards the ankle. 5.5 CCID 50 0.1 ml of CHIKV-LR was administered. A single 250 μg dose of each mAb (mAb1, mAb2, mAb7, mAb11, or mAb14) was administered intraperitoneally 3 days after CHIKV infection. Viral titers at the injection site were monitored 5 days after infection. All tested mAbs showed the same titer to neutralize the viral load in tissues in the mouse model of CHIKV infection (Figure 9A). mAb7 and mAb14 were further characterized in dose titration studies. For this purpose, DBA1 / J mice were injected subcutaneously into the plantar surface of the right hind paw toward the ankle with 0.05 ml of 10 5.5 CCID 500.1 ml of CHIKV-LR2006 was inoculated. mAb7 or mAb14 was administered by a single intraperitoneal injection at 3 dpi at doses of 10, 25, 50, 100, and 250 μg (approximately 0.5, 1, 2.5, 5, and 12.5 mg / kg). The primary result was a 5 dpi viral titer at the viral challenge site in the hind limbs. mAb7 reduced the viral titer in the joints of CHIKV-infected DBA / 1J mice in a dose-dependent manner (Figure 9B). Significant reductions were observed at doses from 50 to 250 μg, with the best effect achieved at the highest dose. mAb14 significantly reduced viral titer at any dose, but no dose-effect was observed under the test conditions. [Examples]

[0317] Pharmacokinetics of mAbs in non-human primates mAb1 and mAb7 were administered to male cynomolgus monkeys (Macaca Fascicularis) at a dose of 2.5 mg / kg via intravenous (IV) bolus. Animals receiving mAb1 and mAb7 were different (two separate studies). Plasma samples were assayed using an extrapolated Elisa bioanalysis method with an antibody against human IgG1 to recognize mAb1 and mAb7 expressed by rabbit immunization. Pharmacokinetic comparisons were performed using the terminal half-life (i.e., t) for mAb7. 1 / 2 This showed an increase in the time required for the concentration or amount of the drug in the body to decrease by exactly half, which was 22.7 days for mAb1 and 25.5 days for mAb7, and 25.5 days for mAb7, respectively (Figure 10).

Claims

1. A pharmaceutical composition for use in the treatment or prevention of chikungunya virus (CHIKV) infection in a subject requiring such treatment, comprising a prophylactic or therapeutically effective amount of isolated human IgG monoclonal antibody, wherein the isolated human IgG monoclonal antibody is conjugated to CHIKV and comprises three heavy chain complementarity-determining regions (CDRH) and three light chain complementarity-determining regions (CDRL), i. The CDRH has the amino acid sequences of SEQ ID NOs. 5, 6, and 7, and the CDRL has the amino acid sequences of SEQ ID NOs. 8, GNT, and 10, or ii. The CDRH has the amino acid sequences of SEQ ID NOs: 11, 12, and 13, and the CDRL has the amino acid sequences of SEQ ID NOs: 14, GTS, and 16. The aforementioned monoclonal antibody further, iii. Alanine at position 434, or iv. Alanine at positions 307, 380 and 434, or v. Glutamine at position 250 and leucine at position 428, or vi. Leucine at position 428 and serine at position 434, or vii. Tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, It comprises an Fc region containing at least one residue selected from the group consisting of the following, The positions of the aforementioned amino acids are given by the EU index. The aforementioned prevention includes reducing the likelihood of the subject contracting CHIKV infection. The treatment comprises reducing, alleviating, or decreasing the severity or duration of at least one symptom or complication associated with CHIKV infection, according to the pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, comprising at least one excipient.

3. The isolated monoclonal antibody has the following properties: i. CHIKV binds to the pE2-E1 target with a binding-dissociation equilibrium constant (KD) of less than approximately 10 nM; ii. Binds to human FcRn with a KD of less than approximately 200 nM; and iii. Binds to human FcγRIII with a KD ratio of less than approximately 600 nM. The pharmaceutical composition according to claim 1, comprising one or more of the above.

4. The pharmaceutical composition according to claim 1, wherein CDRH has the amino acid sequences of SEQ ID NOs. 5, 6, and 7, and CDRL has the amino acid sequences of SEQ ID NOs. 8, GNT, and 10.

5. The pharmaceutical composition according to claim 1, wherein CDRH has the amino acid sequences of SEQ ID NOs: 11, 12, and 13, and CDRL has the amino acid sequences of SEQ ID NOs: 14, GTS, and 16.

6. The Fc region of the antibody is: Leucine at position 428 and serine at position 434, or Tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, Includes residues selected from, The pharmaceutical composition according to claim 1, wherein the position of the amino acid is given by the EU index.

7. The pharmaceutical composition according to claim 1, wherein the Fc region of the antibody comprises leucine at position 428 and serine at position 434, and the positions of the amino acids are given by the EU index.

8. The pharmaceutical composition according to claim 1, wherein the Fc region of the antibody includes or consists of a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 62, and 63.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein at least one symptom or complication associated with the CHIKV infection is pain.

10. The pharmaceutical composition according to any one of claims 1 to 8, wherein at least one symptom or complication associated with CHIKV infection is selected from the group consisting of fever, rash, muscle pain, and fatigue.

11. The pharmaceutical composition according to any one of claims 1 to 8, wherein the treatment comprises reducing the severity or duration of a condition associated with CHIKV infection.

12. The pharmaceutical composition according to claim 11, wherein the medical condition associated with CHIKV infection includes joint pain associated with CHIKV.

13. The pharmaceutical composition according to claim 12, wherein the joint pain associated with CHIKV includes polyarthritis and / or polyarthralgia.

14. The pharmaceutical composition according to claim 13, wherein the polyarthritis and / or polyarthritis of the subject are those in the acute phase.

15. The pharmaceutical composition according to claim 13, wherein the polyarthritis and / or polyarthritis of the subject are those in the post-acute phase.

16. The polyarthritis and / or polyarthritis of the subject is chronic, The pharmaceutical composition described in item 13.

17. The pharmaceutical composition according to any one of claims 1 to 8, wherein the treatment further comprises treating symptoms associated with an alphavirus infection other than CHIKV infection.

18. The pharmaceutical composition according to claim 17, wherein the alphavirus infection other than CHIKV infection is selected from the group consisting of Onyonnyon (ONNV) infection, Ross River (RRV) infection, Bermer Forrest (BFV) infection, Western equine encephalitis (WEEV) infection, Semlyki ​​Forrest (SFV) infection, Sindvis (SINV) infection, Eastern equine encephalitis (EEEV) infection, and Venezuelan equine encephalitis (VEEV) infection.

19. The pharmaceutical composition according to any one of claims 1 to 8, wherein the isolated monoclonal antibody is formulated for parenteral administration.

20. The pharmaceutical composition according to any one of claims 1 to 8, wherein the isolated monoclonal antibody is formulated for administration in combination with an additional therapeutic agent.

21. The pharmaceutical composition according to any one of claims 1 to 8, wherein the Fc region comprises or consists of the amino acid sequence of SEQ ID NO:

63.

22. The pharmaceutical composition according to any one of claims 1 to 4 or 6 to 8, wherein the isolated monoclonal antibody comprises a heavy chain including a variable region comprising or consisting of the amino acid sequence of SEQ ID NO:

1.

23. The pharmaceutical composition according to any one of claims 1 to 4 or 6 to 8, wherein the isolated monoclonal antibody comprises a light chain having a variable region comprising or consisting of the amino acid sequence of SEQ ID NO:

2.

24. The pharmaceutical composition according to any one of claims 1 to 4 or 6 to 8, wherein the isolated monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:

31.

25. The pharmaceutical composition according to any one of claims 1 to 4 or 6 to 8, wherein the isolated monoclonal antibody comprises a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:

20.

26. The pharmaceutical composition according to any one of claims 1 to 4 or 6 to 8, wherein the isolated monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 31 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:

20.

27. ​​The pharmaceutical composition according to any one of claims 1 to 3, 5 or 6 to 8, wherein the isolated monoclonal antibody comprises a heavy chain including a variable region comprising or consisting of the amino acid sequence of SEQ ID NO:

3.

28. The pharmaceutical composition according to any one of claims 1 to 3, 5 or 6 to 8, wherein the isolated monoclonal antibody comprises a light chain having a variable region having or comprising the amino acid sequence of SEQ ID NO:

4.

29. The pharmaceutical composition according to any one of claims 1 to 3, 5 or 6 to 8, wherein the isolated monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:

43.

30. The isolated monoclonal antibody comprises or comprises the amino acid sequence of SEQ ID NO:

38. A pharmaceutical composition according to any one of claims 1 to 3, 5, or 6 to 8, comprising a light chain.

31. The pharmaceutical composition according to any one of claims 1 to 3, 5 or 6 to 8, wherein the isolated monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 43 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:

38.

32. The pharmaceutical composition according to any one of claims 1 to 8, wherein the antibody is formulated for administration in a single dose or a series of sequential doses.

33. The pharmaceutical composition according to any one of claims 1 to 8, wherein at least one symptom or complication associated with the CHIKV infection is fatigue.

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