ANTIBODIES TO HUMAN IL-4Ra HAVING REDUCED IMMUNOGENICITY AND APPLICATION THEREOF

TECHNICAL FIELD

The present invention relates to the field of genetic engineering, biotechnology and immunology, in particular, to variants of a monoclonal antibody against the alpha chain of a receptor to human interleukin 4 (hIL-4Ra), a pharmaceutical composition containing the antibody, and a use of the antibody for the prevention and treatment of human diseases mediated by the hlL- 4R receptor such as, for example, allergic, autoimmune, infectious and oncological diseases.

BACKGROUND

It is known that some human diseases, in particular, allergic and autoimmune diseases such as, for example, atopic dermatitis, asthma, chronic polyposis rhinosinusitis, eosinophilic esophagitis, as well as the aggravated course of viral infections and some oncological diseases (Simon H.-U. et al., Int. Arch. Allergy Immunol., 2020, 181(8):624-628; Protti M.P. et al., Tissue Antigens, 2014, 83:237-246 and Fridman W.H. et al., Nat. Rev. Cancer, 2012, 12:298-306) are associated with aberrant type 2 immune response (Bertschi N.L. et al., Arch Allergy Immunol, 2021, 182(5):365-380).

One of the most important cytokines that induces type 2 immune response is interleukin 4 (IL-4). It is produced mainly by mast cells, Th2 T lymphocytes, eosinophils and basophils. IL-4 has been shown to promote the differentiation of naive ThO cells into Th2 T lymphocytes (Le Gros G. et al., J. Exp. Med., 1990, 172(3):921-929; Swain S.L. et al., J. Immunol., 1990, 145:3796- 3806). Also, IL-4 shifts the balance between Thl and Th2 towards the Th2, thereby suppressing the type 1 immune response.

An effect from IL-4 on B cells is in the ability to stimulate differentiation of the cells into plasma cells that produce antibodies, as well as in increasing the frequency of antibody isotype switch to IgE. Production of antigen-specific IgE antibodies is the cause of many allergic diseases (Anvari S. et al., Clin. Rev. Allergy. Immunol., 2019, 57(2):244-260).

On the other hand, one of the functions of IL-4 is the polarization of macrophages towards the M2 phenotype. M2 cells are reparative macrophages. They have been shown to secrete immunosuppressive cytokines such as IL- 10 and TGF-R, as well as enzymes that reduce inflammation and promote repair and fibrosis (Braga T.T. et al, Front. Immunol., 2015, 6:602).

Interleukin 13 (IL-13) has similar functions to IL-4. Hypersecretion of IL-13 leads to increased mucus formation in the respiratory tract, inflammation and hypersensitivity in diseases such as asthma (Gour N. and Wills-Karp M., Cytokine, 2015, 75(l):68-78).

IL-4 and IL- 13 act through a common receptor known as IL-4Ra. In a complex with the common gamma chain of interleukin receptors (yC), IL-4Ra forms a receptor that specifically binds to IL-4. In a complex with IL- 13Ra 1, IL-4Ra forms a receptor that is capable of specifically binding IL-4 and IL-13 (Maes T. et aL, Cell Mot. Biol., 2012, 47(3):261-270; Chatila T.A., Trends Mot. Med., 2004, 10(10):493-499).

Blocking the IL-4R receptor through, for example, binding of an antibody to IL-4Ra disrupts the signaling mediated by both types of interleukins. IL-4 and IL- 13 are key type 2 inflammation cytokines that play an important role in the pathogenesis of diseases such as asthma, atopic dermatitis, chronic polyposis rhinosinusitis, and eosinophilic esophagitis.

In clinical studies, it has been shown that the therapeutic effect of monoclonal antibodies against the IL-4Ra subunit is more pronounced as compared with the antibodies against IL-4 or IL-13 interleukins (Cormac S.C., Nat. Biotech., 2018, 36:3-5). This may be due to a lower amount of the antibody against the IL-4Ra subunit that is required to attain the desired therapeutic effect, and a bispecific mode of action of the antibody due to its ability to block the biological function of both IL-4 and IL-13.

It is known that many therapeutic monoclonal antibodies have immunogenicity and can cause the production of responding antibodies by the immune system of a patient, including neutralizing antibodies. Formation of neutralizing antibodies to therapeutic antibodies results in a significant decrease in the effectiveness of the drug. Immunogenicity was shown for many therapeutic antibodies. For example, immunogenicity was shown for the humanized monoclonal antibody against the alpha4-integrin (natalizumab) (Calabresi P.A. et al., Neurology, 2007, 69(14): 1391-1403). If neutralizing antibodies are produced in a significant proportion of patients, it may cause termination of clinical trials of the drug. For example, clinical trial of the monoclonal anti-PCSK9 antibody (bococizumab) has been discontinued despite significant therapeutic benefit. The reason was the high immunogenicity of the drug: 48% of patients produced antibodies to the therapeutic antibody, in 29% of patients the antibodies were neutralizing (Ridker P.M. et al., N. Engl. J. Med., 2017, 376(16): 1517-1526).

The immune response to therapeutic protein can negatively affect not only its therapeutic efficacy but also safety of the patients. The adverse side effects related to immune response may include anaphylaxis, cytokine release syndrome, and cross-reactive neutralization of endogenous proteins involved in critical functions. Consequently, the immunogenicity risk factors should be taken into consideration at the early stages of development of a therapeutic antibody (U.S. Food and Drug Administration (FDA), Immunogenicity Assessment for Therapeutic Protein Products, 2014).

The immunogenicity to a therapeutic antibody can result in hypersensitivity-related reactions. Type 1 hypersensitivity is accompanied by the production of IgE antibodies to the therapeutic antibody. High IgE and IgG titers of antibodies to the therapeutic antibody can lead to significant side effects, including infusion reactions and/or anaphylaxis. The IgE complexes with the therapeutic antibody can activate basophils and mast cells through the Fee receptor, resulting in IgE-mediated anaphylaxis. IgG antibodies can form complexes with a therapeutic antibody. Such immune complexes can cross-bind Fey receptors on neutrophils and release platelet activation factors. Moreover, the resulting large immune complexes, which cannot be cleared, settle in tissues such as the kidneys, synovium, and choroid plexus, resulting in tissue damage and organ failure (Ghaderi D. et aL, Nat. Biotech., 2010, 28:863-867; Matucci A. et al., Expert Rev. Clin. Immunol., 2019, 15: 1263-1271).

The most significant side effects occur when antibodies to a therapeutic drug crossreact with endogenous molecules (U.S. Food and Drug Administration (FDA), Immunogenicity Assessment for Therapeutic Protein Products, 2014; Jawa V. et al., Front. Immunol., 2020, 11 : 1301). In particular, adverse effects may be associated with the formation of anti-idiotype antibodies to the therapeutic antibody, the surface of which mimics the surface of the target antigen. For example, in the case of therapeutic antibodies that block the Spike coronavirus protein, the resulting anti-idiotype antibodies can have a surface similar to that of the Spike protein and, accordingly, bind to the Ace2 protein which is the target of the Spike protein. It is assumed that cases of myocarditis after the introduction of vaccines that cause a response to the Spike protein are associated with the formation of antiidiotype antibodies that interact with Ace2 on myocardial cells (Murphy W.J. and Longo D.L., N. Engl. J. Med., 2022, 386(4):394-396).

Chimeric and humanized therapeutic antibodies can cause the immune response and production of antibodies against therapeutic antibodies, since they contain protein sequences that are foreign to humans. Moreover, it is known that even fully human therapeutic antibodies can also trigger an immune response.

Therapeutic antibodies can be phagocytosed by various cells, including antigen- presenting cells such as dendritic cells (abbreviated as "APC"). APCs can process the engulfed therapeutic antibodies to peptides that are presented on the cell membrane in a complex with the MHC (major histocompatibility complex) class II molecules. Naive CD4+ T cells can recognize the MHC II peptide complex. They are primed, and then proliferate, form clonal effector and memory populations, and direct the immune response against therapeutic antibodies. In particular, follicular CD4+ T lymphocytes select B lymphocytes capable of producing high-affinity antibodies against a therapeutic antibody. CD4+ T cells can direct the production of antibodies against an antigen, for example, against a therapeutic antibody and, in particular, the production of neutralizing antibodies against a therapeutic antibody. A B cell epitope recognized by an antibody against a therapeutic antibody may not match the T cell epitope.

A therapeutic antibody may contain amino acid mutations that modify the original, i.e., the native amino acid sequence (also referred to as the germline sequence, the natural sequence) of the variable domain of the antibody. If such mutations are present in a peptide that is presented on MHC class II molecules, the peptide can be recognized by CD4+ T cells of a patient as a foreign peptide. It results in immunogenicity of the therapeutic antibody. As a result, the adaptive immunity produces high-affinity antibodies against therapeutic antibody, including neutralizing antibodies that block the action of the therapeutic antibody.

The immune response to therapeutic antibodies is highly undesirable, as indicated above. Therefore, in the development of drugs based on therapeutic antibodies, great attention is paid to their immunogenicity. It is desirable that the epitopes of the therapeutic antibody are not presented on MHC class II, so as not to cause an undesirable immune reaction mediated by CD4+ T cells. Therefore, it is important to examine the protein sequence of the antibody for the presence of immunogenic epitopes presented in MHC class II upon the development of therapeutic antibodies. In particular, variable domains of antibodies, for example, regions of hypervariability (so-called "complementarity-determining regions", CDR's) and other parts of the antibody containing mutations as compared with the native sequence of the antibody are subject to an immunogenicity test.

Therapeutic monoclonal antibodies and the preparations based on them having immunogenicity are known. For example, antibodies against IL-4Ra are described that are used for the treatment of a wide range of diseases in which inhibition of IL-4 and IL-13 mediated signal transmission can alleviate the course of a disease (Russian patent No. 2758091 Cl). An antibody to hIL-4Ra, dupilumab, is known (see, for example, a U.S. Patent No. 8,075,887 B2; Blakely K.M. et al, Skin Therapy Lett., 2016, 21(2): 1-5). Dupilumab is a recombinant fully human monoclonal IgG4 antibody that blocks IL-4 and IL- 13 -mediated effects through specific binding to hIL-4Ra. The antibody dupilumab is a component of the drug Dupixent.

A significant drawback of dupilumab is that it is able to induce the formation of antibodies in response to its introduction into the patient's body. Specifically, 5-9% of patients having atopic dermatitis, asthma or chronic polyposis rhinosinusitis who had been receiving Dupixent for 52 weeks have developed antibodies to dupilumab, and 2-4% of patients have developed neutralizing antibodies. The values were even higher for adolescent subjects: approximately 16% of adolescents having atopic dermatitis who had been receiving Dupixent for 16 weeks have developed antibodies to dupilumab, and approximately 5% had neutralizing antibodies (accessdata.fda.gov/drugsatfda_docs/label/2019/761055s0141bl . pdf). The ability of dupilumab to have immunogenicity reduces the effectiveness of the drug based on it for a significant proportion of patients, increases the duration and cost of treatment, and can increase the risk of adverse effects from its use. Therefore, the development of new antibodies against hIL-4R having reduced immunogenicity is an important task in the prevention and treatment of diseases mediated by the hIL-4R.

SUMMARY OF THE INVENTION

An objective technical problem solved by the invention is the provision of novel monoclonal antibodies that specifically bind to the alpha chain of a receptor to human interleukin 4 (hIL-4Ra), and at the same time have reduced immunogenicity as compared with the antibody dupilumab. Another objective technical problem solved by the invention is the provision of novel monoclonal antibodies having the ability to specifically bind to the hIL-4Ra. Other objectives of the invention are to provide the nucleic acids comprising nucleotide sequences encoding the amino acid sequence of the antibodies, a corresponding expression vector and a host cell for the production of the antibodies, a method of production of the host cell, a method of production of the antibodies, a use of the antibodies, and, in particular, a pharmaceutical composition comprising them for the prevention and treatment of diseases that are mediated by the hIL-4R.

The invention broadens the number of monoclonal antibodies that can specifically bind to hIL-4Ra. An antibody according to the invention that can specifically bind to hIL-4Ra has decreased immunogenicity as compared with the antibody dupilumab. Furthermore, an antibody according to the invention that can specifically bind to hIL-4Ra has decreased immunogenicity as compared with the antibody dupilumab, wherein the ability of the antibody of the invention to specifically bind to hIL-4Ra is maintained to the same or almost equal extent as compared with that ability of the dupilumab. Therefore, the immunogenicity of dupilumab can be decreased if an antibody of the invention is provided. Thus, an antibody according to the invention has clear advantages over the antibody dupilumab, in particular: a) an antibody that is provided herein has a reduced immunogenicity as compared with the antibody dupilumab, and b) an antibody that is provided herein has a reduced immunogenicity as compared with the antibody dupilumab, wherein the antibody that is provided has the ability to specifically bind to hIL-4Ra to the same or almost equal extent as compared with that ability of the dupilumab.

The objective technical problems have been solved and advantages of the invention have been achieved by the finding that by modifying the amino acid sequence in complementaritydetermining regions (CDRs) of the light chain and/or the heavy chain of dupilumab in a specific way, it is possible, in particular, to decrease the immunogenicity of the modified antibody as compared with the unmodified antibody. In addition, it was unexpectedly found that the resulting modified antibody having reduced immunogenicity as compared with dupilumab is able to specifically bind to hIL-4Ra, wherein the modified antibody has the ability to specifically bind to hIL-4Ra to the same or almost equal extent as compared with that ability of the dupilumab. In particular, it was found that the immunogenicity of dupilumab can be reduced and an ability of the modified antibody to specifically bind to hIL-4Ra maintained to the same or almost equal extent as compared with that ability of the dupilumab, if the amino acid sequence of, at least, LCDR1, HCDR1, and/or HCDR3 in the dupilumab is modified as described herein.

In view of the foregoing, the present invention provides the following.

In one aspect the present invention provides a monoclonal antibody that specifically binds to a human interleukin 4 receptor alpha chain (hIL-4Ra), comprising: a) LCDR2 and LCDR3 light chain fragments having the amino acid sequences shown in SEQ ID NO: 8 and SEQ ID NO: 10, respectively, and b) HCDR1, HCDR2, and HCDR3 heavy chain fragments having the amino acid sequences shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, wherein the amino acid sequence of a LCDR1 light chain fragment comprises a substitution of at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: 6, wherein said substitution is selected from the group consisting of Y5H, I7N, and a combination thereof.

In a further aspect the present invention provides the antibody as described above, wherein said antibody comprises an amino acid substitution selected from the group consisting of R5S and/or D6S in HCDR1, L5V in HCDR3, and a combination thereof.

In a further aspect the present invention provides the antibody as described above, wherein a light chain of said antibody comprises the amino acid sequence shown in SEQ ID NO: 18 or 20, and a heavy chain of said antibody comprises the amino acid sequence shown in SEQ ID NO: 22 or 24.

In a further aspect the present invention provides the antibody as described above, wherein said antibody is a human antibody, a humanized antibody, or a chimeric antibody.

In a further aspect the present invention provides the antibody as described above, wherein said antibody belongs to a human isotype and is selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, and IgE.

In another aspect the present invention provides a nucleic acid encoding an amino acid sequence of the antibody as described above.

In another aspect the present invention provides an expression vector comprising the nucleic acid as described above.

In another aspect the present invention provides a host cell for producing the antibody as described above, comprising the vector as described above. In a further aspect the present invention provides the host cell as described above, wherein said host cell is a eukaryotic cell or a prokaryotic cell, optionally, said cell is selected from a human cell, a Chinese hamster ovary cell, a fungal cell, a yeast cell, or a bacterial cell.

In another aspect the present invention provides a method for producing the host cell as described above, comprising transforming a eukaryotic cell or a prokaryotic cell with the vector as described above.

In another aspect the present invention provides a method for producing the antibody as described above, comprising: culturing the host cell as described above in a culture medium under the conditions that provide the production of said antibody, and isolating the antibody from the culture medium and/or the host cell.

In another aspect the present invention provides a use of the antibody as described above for the prevention or treatment of a human disease mediated by hIL-4R receptor.

In a further aspect the present invention provides the use as described above, wherein said prevention or treatment of a disease mediated by hIL-4R receptor is attained through inhibiting activity of the hIL-4R receptor.

In a further aspect the present invention provides the use as described above, wherein said disease is mediated by IL-4 and/or IL-13.

In a further aspect the present invention provides the use as described above, wherein said disease is an allergic disease, an autoimmune disease, an infectious disease, or an oncological disease.

In a further aspect the present invention provides the use as described above, wherein said disease is selected from the group consisting of atopic dermatitis, asthma, chronic polyposis rhinosinusitis, and eosinophilic esophagitis.

In yet another aspect the present invention provides a use of the antibody as described above for producing a therapeutic product or a pharmaceutical composition for the prevention or treatment of a human disease mediated by hIL-4R receptor.

In another aspect the present invention provides a pharmaceutical composition for the prevention or treatment of a human disease mediated by hIL-4R receptor, comprising the antibody as described above in an effective amount.

In a further aspect the present invention provides the composition as described above, wherein said composition further comprises a pharmaceutically acceptable carrier, a diluent, and/or an excipient.

In another aspect the present invention provides a method for prevention or treatment of a human disease mediated by hIL-4R receptor, comprising administering the antibody as described above or the composition as described above in an effective amount to a person in need of said prevention or treatment.

In another aspect the present invention provides a kit for the prevention or treatment of a human disease mediated by hIL-4R receptor, comprising, at least, a container containing the composition as described above and, optionally, a syringe, an instruction and/or a case.

Still other objects, features, and attendant advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the embodiments described therein, together with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will now be described in more detail with reference to the exemplary embodiments, given only by way of example, and with reference to the accompanying figures, in which:

Figure 1 shows the results of B -lymphocyte staining with antibodies, where A to D are as follows: A to D are as follows: A - 0.04 pg abD, Alexa594; B - 0.04 pg abD, Alexa647; C - 0.04 pg abD, Alexa594 and 0.04 pg abD, Alexa647; D - 0.04 pg abD, Alexa594 and 0.04 pg abDl, Alexa647.

Figure 2 shows the results of DAUDI cells staining with antibodies AbD and AbD2.

Figure 3 shows the results of competitive binding of antibodies to hIL-4Ra, where A to C are as follows: A - 0.005 pg abD, Alexa647; B - 0.005 pg abD, Alexa647 and 0.005 pg abDl, unlabeled; C - 0.005 pg abD, Alexa647 and 0.005 pg abD2, unlabeled.

Figure 4 shows the results of blocking of the antibody binding to hIL-20 4Ra, where A to H are as follows: A A, E - control; B, F - 0.05 pg abD, Alexa594; C, G - 0.0005 pg abDl, unlabeled, and then 0.05 pg abD, Alexa594; D, H - 0.0005 pg abD2, unlabeled, and then 0.05 pg abD, Alexa594.

Figure 5 shows the results of the analysis of the change of the relative normalized expression of the MRC 1 gene in human PBMC upon stimulation 25 using IL-4.

Figure 6 shows the results of fluocytometric analysis of the amount of divided CFSE ^low CD4+ human T-lymphocytes.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, terms used in the description of the present invention are the terms that are commonly used by a one of ordinary skill in the art, and the meaning of the terms should be understood by the one of ordinary skill in the art.

Unless defined otherwise, the invention can be practiced using methods and techniques that are commonly employed in the field of genetic engineering, biotechnology, immunology, and related fields. The term "antibody" refers to an immunoglobulin molecule or a portion thereof capable of specifically binding to at least one epitope of an antigen through at least one variable domain within the immunoglobulin molecule or a portion thereof. The "antibody" can be a full-length antibody (also referred to as an "intact antibody"), an intact antibody variant, an intact antibody fragment, or an intact antibody fragment variant. Thus, for the sake of brevity and clarity of the present invention, the term "antibody" refers to a full-length antibody, a fragment of the antibody, and a variant thereof.

The antibody can be of any kind, provided that the antibody specifically binds to at least one epitope of the antigen by means of at least one variable domain within the antibody. An antibody fragment or an antibody fragment variant that specifically binds to at least one epitope of an antigen by means of at least one variable domain within the fragment is referred to as an "antigen-binding fragment" or, equivalently, an "antigen-binding fragment of an antibody", or simply an "antibody fragment". As non-limiting examples of the invention, an antibody can be, for example, a full length antibody, a minibody, a diabody, a triabody, a tetrabody, a single chain antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fragment selected from Fab, Fab’, F(ab’)2, Fv, scFv, sc(Fv)2, (SCFV)2, a bispecific sc(Fv)2, a bispecific (scFv)2, as well as covalent antibody conjugates, in particular with one or more residues of one or more carbohydrates, amino sugars, amino acids, organic acids, alcohols, peptides, label molecules, including dye molecules and isotoptic labels, low molecular compounds and/or high molecular compounds.

The term "antibody" according to the invention may be used equivalently to the term "modified antibody", unless other more detailed explanations of the term are given. It is possible because the amino acid sequence of an antibody of the present invention has a high degree of identity as compared with a known antibody. Also, the term "antibody" may be used interchangeably with the term "antibody variant", unless other more detailed explanations of the term are provided. It is possible because the amino acid sequence of a modified antibody of the present invention can be modified in a certain way, for example, in order to obtain variants of the modified antibody. The homology, including identity, of nucleotide and amino acid sequences can be evaluated using known algorithms, for example, BLAST and BLAST 2.0, which are described in Altschul S.F. et al., Nucleic Acids Res., 1977, 25:3389-3402 and Altschul S.F. et al., J. Mot. Biol., 1990, 215:403-410. It is also possible to use the Internet service provided by the National Center for Biotechnology Information (NCBI, .nlm.nih.gov).

Any antibody can be used according to the invention, provided that it is a modified antibody or a variant thereof as explained herein. For example, any antibody can be used according to the invention, provided that it is a modified antibody or a variant thereof as explained herein and is capable of specifically binding to hIL-4Ra.

The antibody can be, in particular, a human antibody, a humanized antibody, or a chimeric antibody.

The antibody can belong to a human isotype, and it can be selected from IgG, IgM, IgA, IgD, and IgE isotypes, some of which in turn may be divided into subclasses such as IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

An antibody comprising two light polypeptide chains (L, from "light") and two heavy polypeptide chains (H, from "heavy"), each chain comprising a variable domain (V, from "variable") and a constant domain (C, from "constant"), is considered to be a full-length antibody. For the purposes of the present invention, the variable domain of the light chain and the heavy chain is designated, respectively, as VL (from "variable light") and VH (from "variable heavy"). The variable domain of the chain contains three complementarity-determining regions, designated as CDR1, CDR2 and CDR3 (from a "complementarity-determining region"). Therefore, the complementarity-determining regions of the light chain and the heavy chain are designated, respectively, as LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3, and, in general, as CDR's. CDR's and amino acid residues within CDR's, VL, VH, L, and H are numbered and positioned using the Kabat nomenclature (Kabat E. et al., 1991, sequence of proteins of immunological interest. US Public Health Services. NIH, Bethesda, MD, Publication no. 91-3242), unless otherwise specified. Amino acid residues are designated as one- or three-letter codes according to conventional nomenclature (Nelson D.L. and Cox M.M., 2000, Lehninger Principles of Biochemistry, 3 ^rd Ed.).

An antibody is able to "specifically bind" to an antigen. The term "specifically bind" can mean the predominant ability of a given antibody to form a complex, that is, to bind to a particular epitope of an antigen as compared to another epitope of the same or a different antigen. The term "specifically binds" also can mean the predominant ability of a given antigen epitope to complex with a desired antibody over other antibodies. For purposes of the present invention, the antibody is capable of specifically binding to hIL-4Ra. An hIL-4Ra can comprise at least one epitope so that an antibody of the present invention is able to specifically bind to the epitope. Thus, as an antigen to the antibody as described herein can be the hlL- 4Ra. Given that the hIL-4Ra can be a common subunit of receptors that belong to different types of receptors, the antibody according to the invention "specifically binds" to any receptor, provided that the receptor contains the hIL-4Ra. For example, an antibody can specifically bind to an IL-4 and/or IL-13 receptor, provided that the receptor comprises the hIL-4Ra.

The ability of an antibody to specifically bind to an antigen can be determined by any known method. The affinity of an antibody for an antigen is referred to as "antibody affinity," or for the simplicity, as "affinity".

The term "monoclonal antibody" refers to an antibody derived from a single copy of a progenitor cell or clone, including, for example, any eukaryotic, prokaryotic, or phage clone of a progenitor cell, or derived from a population of substantially homogeneous antibodies, that is, individual antibodies that form a population of identical antibodies, the individual members of which differ in view of possible point mutations which are naturally acquired that do not affect the properties of the antibodies or affect, but insignificantly as compared with the general antibody population. Methods for producing monoclonal antibodies are known (see, for example, Monoclonal antibody production, Washington (DC): National Academies Press (US), 1999). The antibody of the invention can be prepared using methods which are commonly used to produce monoclonal antibodies, such as, for example, hybridoma technologies, recombinant technologies, phage display technologies, synthetic technologies, or a combination of these, or other technologies which are well known to those skilled in the art.

The term "epitope" can refer to a portion of an antigen molecule that is capable of being recognized and bound to one or more antigen-binding regions of an antibody. This epitope is referred to as a B-cell epitope. A polypeptide, such as, for example, a protein, may contain a B- cell epitope and need not be immunogenic, but it may also be immunogenic. Immunogenicity of a polypeptide is a result of the presence of one or more immunogenic T-cell epitopes in the polypeptide. As a T-cell epitope, a peptide fragment can be that is presented on MHC class II and capable of inducing an immune response of CD4+ T-cells. The immunogenic activity of a polypeptide, including, for example, a protein and, in particular, a therapeutic antibody, can be determined by any known method.

As used herein, the terms "immunogenicity" and "immunogenic activity" used in relation to an antibody or a fragment thereof are equivalent.

The term "immunogenicity" of an antibody refers to the ability of an antibody to elicit an immune response of an organism to the antibody. In the context of the present invention, the immunogenic activity of an antibody refers to such activity in humans. An antibody of the present invention has reduced immunogenic activity as compared with that activity of an unmodified antibody such as, for example, an antibody having L and H chains the amino acid sequences of which are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

An antibody having the amino acid sequences of L and H chains shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively, is referred to as an "unmodified antibody" for clarity. The antibody is also referred to as an "intact antibody" because, as explained above, it is a full-length antibody.

An antibody according to the invention contains at least one CDR, the amino acid sequence of which has been modified as compared with the CDR of the antibody having L and H chains the amino acid sequences of which are shown, respectively, in SEQ ID NO: 2 and SEQ ID NO: 4. One or more CDR's in the L chain and/or H chain of the unmodified antibody having the amino acid sequences of the L chain (SEQ ID NO: 2) and H chain (SEQ ID NO: 4) can be modified as described herein so that to obtain the antibody of the present invention. Therefore, an antibody of the present invention may have one or more CDR's that are modified and one or more CDR's that are not modified as compared with the CDR's of the unmodified antibody. One or more CDR's that can be modified are selected from the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of the unmodified antibody.

An antibody in which the amino acid sequence of at least one CDR has been modified relative to an unmodified antibody is referred to as a "modified antibody" for clarity. Therefore, it is acceptable that a "modified antibody" may, for simplicity, be referred to as an "antibody" as described herein, unless otherwise indicated.

For example, an amino acid sequence of one or more CDR's of the unmodified antibody can be modified as described herein, wherein the one or more CDR's are selected from the group consisting of LCDR1, HCDR1, and HCDR3. One or more changes, that is, mutations, can be introduced independently of each other into one or more amino acid sequences of LCDR 1 (SEQ ID NO: 6), HCDR1 (SEQ ID NO: 12) and HCDR3 (SEQ ID NO: 16) in the unmodified antibody. In particular embodiments, the amino acid sequence of LCDR1 can be modified such that it contains a substitution of an amino acid residue in position 5 and/or 7 in the LCDR1, specifically, a Y5H mutation and/or a I7N mutation; and/or the amino acid sequence of HCDR1 can be modified such that it contains a substitution of an amino acid residue in position 5 and/or 6 in the HCDR1, specifically, a R5S mutation and/or a D6S mutation; and/or the amino acid sequence of HCDR3 can be modified such that it contains a substitution of an amino acid residue in position 5 in the HCDR3, specifically, a L5V mutation. In a specific example, an antibody of the present invention can have one or more CDR's that are modified as compared with the unmodified antibody such as, for example, a LCDR1 having the amino acid sequence QSLLHSIGYNY, QSLLYSNGYNY, or QSLLHSNGYNY; a HCDR1 having the amino acid sequence GFTFSDYA, GFTFRSYA, or GFTSSDYA; and/or a HCDR3 having the amino acid sequence AKDRVSITIRPRYYGLDV.

A nucleotide sequence of LCDR2 and the amino acid sequence encoded by the nucleotide sequence of the modified antibody are identical, respectively, to the nucleotide sequence of LCDR2 and the amino acid sequence of dupilumab, and they are shown in, respectively, in SEQ ID NO: 7 (nucleotide sequence: ttgggttct; designated as “LCDR2, dupilumab, nt”) and SEQ ID NO: 8 (amino acid sequence: LGS; designated as “LCDR2, dupilumab, aa”). As the L chain of dupilumab contains LCDR2, the nucleotide sequence (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) of the dupilumab include, respectively, the nucleotide sequence (SEQ ID NO: 7) and the amino acid sequence (SEQ ID NO: 8) of the LCDR2 of the modified antibody.

The position of amino acid residues in a particular CDR is counted from the N- terminus of that CDR. For the sake of clarity, it is noted that the positions of the amino acid residues tyrosine (Tyr, Y) and isoleucine (He, I) in positions 5 and 7, respectively, in the LCDR1 are identical to the positions of the residues in positions 31 and 33, respectively, in the amino acid sequence of the light chain of dupilumab (SEQ ID NO: 2) and correspond to positions 31 and 33, respectively, in a modified antibody (for example, SEQ ID NO: 18 and SEQ ID NO: 20); the positions of the amino acid residues arginine (Arg, R), aspartic acid (Asp, D) and leucine (Leu, L) in positions 5 and 6, respectively, in the HCDR1 and position 5, respectively, in the HCDR3 are identical to the positions of the residues in positions 30, 31 and 101, respectively, in the amino acid sequence of the heavy chain of dupilumab (SEQ ID NO: 4) and correspond to positions 30, 31 and 101, respectively, in a modified antibody (for example, SEQ ID NO: 22 and SEQ ID NO: 24). A position of other amino acid residues in the CDR’s and outside the CDR’s of an antibody can be determined in a similar way by comparing the amino acid sequence of the selected region in the antibody with the amino acid sequence of the light chain or heavy chain of the dupilumab.

The antibody containing at least one CDR, the amino acid sequence of which has been modified as compared with the CDR of the antibody having L and H chains the amino acid sequences of which are shown, respectively, in SEQ ID NO: 2 and SEQ ID NO: 4 can be modified further so that to contain in a non-CDR region in the L and/or H chain of the antibody one or more mutations. For example, an antibody having a substitution of an amino acid residue in position 5 and/or 6 in the HCDR1, which correspond to the positions 30 and 31 in the amino acid sequence of the heavy chain of dupilumab (SEQ ID NO: 4), can be modified further by introducing a mutation in the position 35 in the amino acid sequence of the heavy chain of the dupilumab. An exemplary mutation is the T35S substitution, in which threonine (Thr, T) in the position 35 in the amino acid sequence of the heavy chain of dupilumab (SEQ ID NO: 4) is replaced to serine (Ser, S).

The kind and number of mutations in the CDR’s can be combined with each other so that to obtain an antibody that differs from the unmodified antibody by at least one CDR. In particular, the modified antibody can have a light chain having the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 20 and/or a heavy chain having the amino acid sequence shown in SEQ ID NO: 22 or SEQ ID NO: 24.

Together with one or more of the above-mentioned mutations in LCDR1, HCDR1, and HCDR3, other one or more mutations, including substitution, deletion, insertion of one or more amino acid residues, can be introduced into the amino acid sequence of a modified antibody independently of each other, provided that the resulting “antibody variant” can be referred to as the modified antibody as explained herein. The one or more mutations can be introduced into any region of the amino acid sequence of the antibody such as, for example, a CDR and/or a non-CDR region in the L and/or the H chain of the antibody. As a result of such modification of the antibody, an antibody variant can be obtained which is, for example, a modified antibody as explained herein and capable of specifically binding to hIL-4Ra. Such modifications can be introduced, for example, into regions of the antibody that do not affect or affect insignificantly the ability of the antibody to specifically bind to hIL-4Ra. Preferably, but not necessarily, the affinity of the antibody variant is the same or higher and/or the immunogenic activity of the antibody variant is the same or lower as compared with an antibody that is modified, for example, with an antibody that is a modified antibody as explained herein and is capable of specifically binding to hIL-4Ra.

Methods for modification of amino acid sequence of proteins, including antibodies, are known (Hsieh P. and Vaisvila R., Methods Mol. Biol., 2013, 978: 173-186). Methods for determining the binding specificity of an antibody to an antigen are known (Bordeaux J. et al., Biotechniques, 2010, 48(3): 197-209). Methods for determining the immunogenic activity of antibodies are also known (U.S. Food and Drug Administration (FDA), Immunogenicity Assessment for Therapeutic Protein Products, 2014; Jawa V. et al., Front. Immunol., 2020, 11 : 1301). Thus, it is also within the skill and experience of the ordinary person skilled in the art to modify the amino acid sequence of an antibody and test the ability of the resulting antibody variant to specifically bind to hIL-4Ra, affinity and immunogenicity of the resulting antibody variant.

The amino acid sequence of an antibody is encoded by its corresponding nucleotide sequence. It is also acceptable that a degenerative nucleotide sequence can also be used as a coding nucleotide sequence, that is possible due to the degeneracy of the genetic code. A nucleotide sequence encoding the amino acid sequence of an antibody may comprise regions that do not encode the amino acid sequence of the antibody, such as introns. In addition, a nucleotide sequence encoding the amino acid sequence of an antibody can be optimized so that to increase its expression level in a host cell of a particular organism (see, for example, Fu H. et al., Sci. Rep., 2020, 10(1): 17617) and/or linked directly or through a nucleotide linker to a nucleotide sequence encoding a leader peptide. Leader peptides (also referred to as signal peptides, localization signals, etc.) are commonly used to increase the secretion efficiency of secreted proteins. A nucleic acid containing a nucleotide sequence encoding an amino acid sequence of an antibody can be expressed in a host cell which belongs to a eukaryotic or a prokaryotic organism. For example, a mammalian cell, a fungal cell, a yeast cell, or a bacterial cell can be used to produce an antibody (see, for example, Frenzel A. et al., Front. Immunol., 2013, 4:217; and ibid.). In particular, the host cell can be an Escherichia coli cell, a Chinese hamster ovary cell (CHO), or a human cell, such as, for example, a cell that is obtained using a recombinant DNA technology and characterized by a high transfection and proteinproducing ability.

A host cell can be transformed with a nucleic acid that is in the expression vector, such that the resulting vector contains the nucleotide sequence encoding the light and heavy polypeptide chains of an antibody simultaneously. Alternatively, nucleic acids encoding the light and heavy polypeptide chains of an antibody can be transformed into a host cell as parts of different expression vectors. The expression vector may be selected according to, for example, the type of a host cell used, and examples of such vectors are known (Li Y.-M. et al., Mol. Biol. Rep., 2018, 45(6):2907-2912; Carter P. et al., 1992, 10: 163-167; etc.). Known techniques, including electroporation, calcium phosphate precipitation, transfection, etc., can be used to transform a cell with a DNA molecule (Sambrook J. et al., Molecular Cloning: A Laboratory Manual (2 ^nd ed.), Cold Spring Harbor, NY, 1989; Current protocols in molecular biology, ed. by M. Ausubel et al., Vol. 1 and 2, John Wiley & Sons, Inc., Media, PA, 1988).

The culturing of a host cell can be performed in a culture medium under conditions that allow production of the antibody. The culture medium, or culture liquid, can be appropriately selected so that the host cell can grow, proliferate and produce the antibody. Under the “production of an antibody” by a host cell is meant accumulation of an antibody in a host cell or accumulation of an antibody in the culture medium, including a combination of them. The conditions and methods for culturing cells that can be used to produce antibodies are known to the one of ordinary skill in the art, and they are described, for example, in Carvalho L.S. et al., Production processes for monoclonal antibodies (in Fermentation Processes, ed. by A. Jozala, IntechOpen, 2017) and Current protocols in cell biology, ed. by Bonifacino J.S. et al., John Wiley & Sons, Inc., Media, PA, 2000.

By a way of example, stable monoclonal antibody producing cell lines can be obtained using a transfection of a CHO-S suspension cell line (Thermo Fisher) with vector constructs containing light and heavy chains of an antibody at an optimal ratio. Clonal lineages having high antibody level (more than 100 mg/L) can be prepared using the ClonePix robotic platform (Molecular Devices). The analysis of the productivity of the selected clones can be carried out using an automated Biomek FX robotics system (Beckman Coulter) and an analytical system Octet RED96 (Pall Life Sciences). Serum-free media that do not contain proteins of animal origin can be used for cultivation of antibody-producing cells. The preparation of an antibody can be carried out in a fermenter having a loading volume of 100 liters.

The antibody can be isolated from a host cell and/or a culture medium in a purified form thereof to a desired degree of purity or as a composition containing an antibody purified to a desired degree of purity and one or more other components. The degree of purity of an antibody may be, but is not limited to, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. A composition containing the antibody may include one or more components that increase stability of the antibody, for example, when the antibody is stored or in a solution for the administration to a patient, including components that increase the bioavailability and/or efficiency of the antibody, and the like. The antibody can be purified to the desired purity using any known one or more methods that are commonly used for purification of proteins, including antibodies. Such methods include affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration, and the so forth. For purification of an antibody by a metal affinity chromatography, a polyhistidine tag consisting of, for example, at least six histidine residues can be introduced at the N- or C-terminus of the amino acid sequence of one or more chains of the antibody.

An antibody of the present invention can be used to obtain a therapeutic product based on the antibody for the prevention or treatment of a disease mediated by a IL-4R receptor, such as, for example, a IL-4 and/or IL- 13 interleukin-mediated disease. A therapeutic product that can be used to prevent or treat a disease in a patient can be prepared using an antibody as described herein as an active agent. Therefore, the antibody can be used for the prevention or treatment in a patient of a disease mediated by over-activation of the IL-4R receptor as compared with a situation when over-activation of the IL-4R receptor in the patient is not observed and, therefore, the prevention or treatment in a patient of a disease mediated by the receptor is not required. A disease mediated by over-activation of the IL-4R receptor includes cases when the disease is mediated by IL-4 and/or IL-13 interleukins, or when the amount of IL-4 and/or IL-13 in a cell and/or a body of the patient is increased as compared with the usual content of such interleukins in the cell and/or the body of the patient, and a clinical picture inherent in the disease mediated by the increased amount in the patient of IL-4 and/or IL- 13 is possible or manifested. As a therapeutic product based on the antibody described herein, there may be a pharmaceutical composition containing the antibody. Therefore, a use of the antibody for the preparation of a therapeutic product based on the antibody can be considered as the use of the antibody for the preparation of a pharmaceutical composition containing it. Examples of the formulation and composition of a pharmaceutical composition containing the antibody are described below.

The term “a disease mediated by hIL-4R receptor”, which is equivalent to the term “a disease mediated by hIL-4R”, means a disease that is directly mediated by over-activation of hIL-4R receptor, and a disease in which the aggravated course of such a disease is or may be due to the over-activation of hIL-4R receptor. An example of a disease that is directly mediated by over-activation of hIL-4R receptor may be an autoimmune and allergic disease. An example of a disease in which the aggravated course of such a disease is or may be due to over-activation of hIL-4R receptor may be an infectious viral disease and an oncological disease.

Prevention and treatment of a disease mediated by hIL-4R receptor can be achieved through inhibiting the activity of the receptor due to the ability of the antibody as described herein to specifically bind to the alpha chain of the receptor (hIL-4Ra). As a result, the activity of the receptor, and therefore a cellular signal mediated by the receptor, is reduced. Since hIL-4R is a receptor, in particular, for IL-4 and IL-13, inhibition of activity of the receptor by means of binding to the antibody results in the decrease of the cellular signal mediated by IL-4 and IL-13.

A “patient” can be a mammal, including, but not limited to, a primate, including a human.

Allergic or autoimmune disease, for example, atopic dermatitis, asthma, chronic polyposis rhinosinusitis, eosinophilic esophagitis and other diseases in the pathogenesis of which the activation of hIL-4R is involved can be the diseases for which measures of prevention or treatment using the antibody can be taken. The antibody can also be used for treating oncological diseases, treating or correcting an immune response to infectious diseases, including viral infectious diseases, in order to suppress a type 2 immune response, including activation and proliferation of Th2 T lymphocytes and/or eosinophils, in favor of the dominance of a type 1 immune response, including activation and proliferation of Thl T lymphocytes, cytotoxic T lymphocytes and natural killers. This is possible because it is known that the dominance of the type 2 immune response over the type 1 immune response, mediated by activation of hIL-4R by IL-4 and/or IL-13, may have a negative effect on i) the efficacy of the anti-tumor immune response in cancers (Protti M.P. et al., Tissue Antigens, 2014, 83:237-246; Fridman W.H. et al., Nat. Rev. Cancer, 2012, 12:298-306) and ii) the efficacy and safety of the immune response in viral infectious diseases (Simon H.-U. et al., Int. Arch. Allergy Immunol., 2020, 181 (8) : 624-628).

The antibody may be administered to a patient or formulated as a pharmaceutical composition for administration to the patient alone or in a combination with another one or more components, such as, for example, a carrier, diluent, excipient, another therapeutic agent as a component of the pharmaceutical composition. The pharmaceutical composition can be prepared, for example, by preparing a citrate buffer (10 mM, pH 6-7) followed by the addition of the remaining components: antibody (50-300 mg/ml), NaCl (50-150 mM), sucrose or trehalose (0.3-0.5% w/v), water for injection up to 1 ml.

The pharmaceutical composition may be formulated in accordance with conventional methods known to those skilled in the art, as set forth, for example, in J.P. Remington and A.R. Gennaro, Remington: the science and practice of pharmacy, 19 ^th ed., Easton, PA: Mack Publishing, 1995.

The antibody can be administered in amounts, doses, and over a period of time that are individually or collectively effective for the prevention or treatment of a disease mediated by hIL-4R. Methods for the selection of the amount of the active substance of a drug, doses, time parameters, and the like are known to the person skilled in the relevant field of technology, or they can be selected experimentally using standard methods, depending, for example, on the nature and/or stage of the disease, a particular patient, individual and/or clinical characteristics of the patient, the desired method, mode and duration of administration of the antibody, in particular, containing its pharmaceutical composition. For example, known administration and storage parameters of a pharmaceutical composition comprising dupilumab as the active substance may be used or adjusted appropriately to administer an antibody of the invention to a patient in order to obtain the desired therapeutic effect (accessdata. fda.gov/drugsatfda_docs/label/2019/761055s0141bl.pdf).

Methods of administering a pharmaceutical composition are not specifically limited and include oral administration, parenteral administration, intranasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, or inhalation administration.

For the prevention or treatment of a disease mediated by hIL-4R receptor, a kit comprising at least a container, for example, a vial, ampoule or syringe ampoule, with a pharmaceutical composition contained therein may be used. If the kit does not contain an ampoule syringe with a pharmaceutical composition, the kit may include an injection syringe in addition to a container with the pharmaceutical composition. The kit may be placed in a case suitable for transport and storage of the pharmaceutical composition, and it may further contain an instruction for a use of the pharmaceutical composition in the form of, for example, a label and/or package insert. For example, the kit may include an ampoule with a pharmaceutical composition, a 1 ml syringe, and an instruction that are packaged in a case made of plastic or a cardboard material.

EXAMPLES

The present invention will be more specifically explained below with reference to the following non-limiting examples.

Example 1. Identification of immunogenic sites in dupilumab.

Identification of potentially immunogenic epitopes in the light chain of the dupilumab antibody (SEQ ID NO: 2), which may be presented in common MHC class II contexts according to the AFND database (The Allele Frequency Net Database, allelefrequencies.net), was carried out using NetMHCIIpan 4.0 software (services. healthtech. dtu.dk/service. php?NetMHCIIpan-4.0). The MHC II contexts considered were, among others, DRBl*03:01, DRBl*07:01, DRBl*13:01, DRB1*13:O2, DRBl*15:01, HLA-DQAl*05:01-DQBl*02:01, HLA-DQAl*03:01-

DQBl*03:02, HLA-DQAl*05:01-DQBl*03:01, HLA-DQAl*03:01-DQBl*03:01, HLA- DQAl*01:01-DQBl*05:01, HLA-DQAl*01:03-DQBl*06:03, HLA-DQA1 *03:01 -

DQBl*06:03, HLA-DQAl*01:02-DQBl*06:03, HLA-DPA1 *01 :03-DPB 1*04:01, HLA- DPAl*01:03-DPBl*02:01, HLA-DPAl*01:03-DPBl*03:01, HLA-DPA1 *01: 03 -DPB 1*04:02 and HLA-DPAl*02:01-DPBl*17:01. The results of the analysis are shown in Table 1.

The analysis showed that the hypermutations in positions 31 and 33 in SEQ ID NO: 2 are located within the peptides that bind to common MHC class II with relatively high affinity (<200 nM), including peptides SSQSLLYSIGYNYLD (DRB1 0701, LLYSIGYNY site, affinity 51.16 nM) and QSLLYSIGYNYLDWY (LYSIGYNYL site, HLA-DPA10103-DPB10401, affinity 48.46 nM, HLA-DPA10103-DPB 10201, affinity 37.77 nM).

The same analysis was performed for the dupilumab heavy chain (SEQ ID NO: 4). The MHC II contexts considered were, among others, DRBl*03:01, DRBl*07:01, DRBl*13:01, DRB1*13:O2, DRBl*15:01, HLA-DQAl*05:01-DQBl*02:01, HLA-DQAl*03:01-

DQBl*03:02, HLA-DQAl*05:01-DQBl*03:01, HLA-DQAl*03:01-DQBl*03:01, HLA- DQAl*01:01-DQBl*05:01, HLA-DQAl*01:03-DQBl*06:03, HLA-DQA1 *03:01 -

DQBl*06:03, HLA-DQAl*01:02-DQBl*06:03, HLA-DPA1 *01 :03-DPB 1*04:01, HLA- DPAl*01:03-DPBl*02:01, HLA-DPAl*01:03-DPBl*03:01, HLA-DPAl*01:03 -DPB 1*04:02 and HLA-DPAl*02:01-DPBl*17:01. The results of the analysis are shown in Table 2.

The analysis showed that the hypermutations in positions 30, 31 and 35 in SEQ ID NO: 4 are located within the peptides that bind to common MHC class II with relatively high affinity (<200 nM), including peptides FRDYAMTWVRQAPGK (DRB1 0701, YAMTWVRQA site, affinity 88.14 nM) and GSGFTFRDYAMTWVR (HLA-DPA10103-DPB10201, FTFRDYAMT site, affinity 106.34 nM).

Thus, the analysis showed that the immunogenicity of dupilumab can be due to mutations in positions 31 and/or 33 as counted in the amino acid sequence of the light chain (SEQ ID NO: 2), and/or mutations in positions 30, 31 and/or 35 as counted in the amino acid sequence of the heavy chain (SEQ ID NO: 4). The analysis also revealed potentially immunogenic peptides that include HCDR3 region of dupilumab that are presented in the MHC II complexes DRB1 0301, DRB1 1301 and DRB1 1302 with relatively high affinity (Table 2).

Example 2. Removal of immunogenic sites in dupilumab.

In order to reduce the immunogenic activity of dupilumab, mutations in the light chain variable domain of dupilumab that distinguish the sequence of the light chain of dupilumab from the native sequence of the light chain of a human antibody were removed. Hereinafter, “mutation removal” refers to the substitution of an amino acid residue at a mutant site in dupilumab for an amino acid residue located at the same site in the amino acid sequence of a human native antibody.

In the first step, the amino acid residue Tyr31 in SEQ ID NO: 2 was replaced with the amino acid residue His31 to provide the substitution Y31H (SEQ ID NO: 18). The amino acid residue Ile33 in SEQ ID NO: 18 was then replaced with the amino acid residue Asn33 to provide the substitution I33N (SEQ ID NO: 20).

Thus, novel light chain amino acid sequences of the antibody were obtained that are different from the light chain amino acid sequence of dupilumab in that the potentially most immunogenic regions in the light chain amino acid sequence of dupilumab were removed.

Also, in order to reduce the immunogenic activity of dupilumab, mutations in the heavy chain variable domain of dupilumab that distinguish the sequence of the heavy chain of dupilumab from the native sequence of the heavy chain of a human antibody were removed. In one embodiment, the amino acid residue LeulOl in SEQ ID NO: 4 was replaced with the amino acid residue VallOl to provide the substitution L101V (SEQ ID NO: 22). In another embodiment, amino acid residues Arg30, Asp31, and Thr35 in SEQ ID NO: 4 were replaced, respectively, with amino acid residues Ser30, Ser31, and Ser35 to provide the corresponding substitutions R30S, D31S, and T35S (SEQ ID NO: 24).

Example 3. Construction of expression vectors.

Synthesis of nucleic acids encoding the light and heavy chains of the control antibody (dupilumab) and modified antibodies was ordered from Cloning Facility, Moscow, Russia.

In order to clone a heavy chain, a polymerase chain reaction (PCR) using primers Pl (SEQ ID NO: 25) and P2 (SEQ ID NO: 26) and a high-precision polymerase Q5 (New England Biolabs, USA) were used for introduction of restriction sites EcoRI and Nhel into the 5’- and 3 ’-terminus of variable domains. The resulting nucleic acids were purified on Qiagen columns (Germany) using a reagent kit (cat. No. 28104) and then cloned into a commercially available pFuse2ss-CHIg- hG4 vector containing constant region of a gene segment of the heavy chain of a human antibody (IgG4) (InvivoGen, USA). The sequences of cloned fragments were confirmed by Sanger sequencing. Site-directed mutagenesis was performed using primers P3 (SEQ ID NO: 27) and P4 (SEQ ID NO: 28). Mutagenesis was carried out by amplification of the vector, the plasmid obtained in the first round of cloning was used as the template. Then, the amplified fragment was treated with Dpnl restrictase and transformed into E. coll cells (strain XL 1 -Blue, CC004M, Evrogen, Russia). The presence of the substitution was confirmed by Sanger sequencing.

In order to clone a light chain, a PCR using primers P5 (SEQ ID NO: 29) and P6 (SEQ ID NO: 30) and the high-precision polymerase Q5 was used for introduction into the 5’- and 3’- terminus of variable domains the regions that are complementary to the vector sequence pFUSE2ss-CLIg-hK (InvivoGen, USA). The purified fragments were annealed to the pFUSE2ss- CLIg-hK vector digested at the EcoRI and TVcoI sites, and the reaction was carried out using Q5 polymerase (CyClone method). E. coll cells were transformed with the resulting mixture. The clones were selected on the next day The sequences of cloned fragments were confirmed by Sanger sequencing.

As the result, plasmids containing the coding sequences for the three variants of light (L) chain and three variants of heavy (H) chain of the antibody were obtained: SEQ ID NO: 2 (L of dupilumab), SEQ ID NOs: 18 and 20 (L of modified antibody) and SEQ ID NO: 4 (H of dupilumab), SEQ ID NOs: 22 and 24 (H of modified antibody).

Example 4. Preparation of recombinant antibodies.

Antibodies were prepared by transfection of plasmids (Example 3) into a suspension of the Expi293F cell line. Expi293 expression system (Gibco, USA) including Expi293F cell culture, Expi293 Expression Medium serum free culture medium and ExpiFectamine transfection reagent were used. Cultivation of cells, transfection, and isolation of antibodies were performed according to the manufacturer’s protocols. Purification of the resulting antibodies was performed using HiTrap Protein A HP antibody purification columns (Cytiva, USA) followed by dialysis against a single sodium phosphate buffer (PBS buffer). Sodium azide, 0.05% (w/v) was added to the aliquots of the antibody solution for long-term storage to prevent bacterial contamination. The concentration of purified antibodies ranged from 0.5 to 5 pg/ml. Purified antibodies were stained using the Alexa Fluor™ 594 Microscale Protein Labeling Kit (Invitrogen, USA). Antibodies were stored in PBS buffer at +4°C in a dark place.

As a result, an antibody abD containing polypeptides having amino acid sequences shown in SEQ ID NOs: 2 and 4 (dupilumab) and modified antibodies abDl containing polypeptides having amino acid sequences shown in SEQ ID NOs: 18 and 22, abD2 containing polypeptides having amino acid sequences shown in SEQ ID NOs: 20 and 22, and abD3 containing polypeptides having amino acid sequences shown in SEQ ID NOs: 20 and 24 were obtained.

Example 5. Confirmation of the specificity of antibodies and determination of their affinity.

White blood cells (peripheral blood mononuclear cells, PBMC) were stained with antibody abD labeled with Alexa594 dye (Figure 1 A) or Alexa647 (Figure IB), a mixture of abD antibodies labeled with Alexa594 or Alexa647 dye (Figure 1C), and a mixture of antibody abD labeled with Alexa594 dye and abDl antibody labeled with Alexa647 dye (Figure ID). All antibodies were taken in the same amount (0.04 pg). Figure 1 shows only gated CD19+ B lymphocytes.

As one can see from the experimental data presented in Figure 1 A-D, the modified antibody abDl stains the same B-lymphocyte subpopulation and at the same intensity as the control antibody abD. Therefore, the modified antibody abDl can specifically bind to the desired target (hIL-4Ra), and the modified antibody abDl has the ability to specifically bind to hIL-4Ra to the same or almost equal extent as compared with that ability of the control antibody abD.

In another experiment, DAUDI cells (Klein E. et al., Cancer Res., 1968, 28: 1310-1330) were stained at an increasing concentration of antibodies abD or abD2 labeled with Alexa594 dye. As one can see from the experimental data presented in Figure 2, the increase of intensity for cells staining curves for the antibodies abD and abD2 are the same within the experimental error bars. Therefore, the modified antibody abD2 has the affinity to hIL-4Ra to the same or almost equal extent as compared with that affinity of the control antibody abD.

Example 6. Competitive binding of antibodies to hIL-4Ra.

PBMC cells were stained with antibody abD labeled with Alexa647 dye (Figure 3A), a mixture of antibody abD labeled with Alexa647 dye and unlabeled antibody abDl (Figure 3B), and a mixture of antibody abD labeled with Alexa647 dye and unlabeled antibody abD2 (Figure 3C). All antibodies were taken in the same amount (0.005 pg). Figure 3 shows only gated CD19+ B lymphocytes.

As one can see from the experimental data presented in Figure 3, the intensity of B- lymphocyte stained with the control antibody abD is significantly reduced in the presence of unlabeled modified antibody abDl or abD2. Therefore, each modified antibody abDl and abD2 is capable of substantially competing with the control antibody abD for the binding site on hIL-4Ra.

Example 7. Blocking the binding of antibodies to hIL-4Ra.

PBMC cells were stained with antibody abD labeled with Alexa647 dye without prior incubation with cells (Figure 4B,F), after pre-incubation of cells in the presence of unlabeled antibody abDl (Figure 4C,G) or unlabeled antibody abD2 (Figure 4D,H). The antibodies abDl and abD2 were taken in an amount 100 times less than the amount of the antibody abD. Figure 4 shows only gated CD3+ T lymphocytes (Figure 4A-D) and CD 19+ B lymphocytes (Figure 4E-H). The unstained control is shown in Figure 4A,E.

As one can see from the experimental data presented in Figure 4, the intensity of B- lymphocyte stained with the control antibody abD is significantly reduced in the case of preincubation of cells in the presence of unlabeled modified antibody abDl or abD2. Therefore, each modified antibody abDl and abD2 is capable of blocking the binding site of the antibody abD on hIL-4Ra.

Example 8. Functional activity of antibodies.

As an example, illustrating the functional activity of the modified antibodies, the activity of antibodies abD (dupilumab) and abD2 (Example 4) was compared. Human PBMC cells derived from a healthy donor were incubated in the presence of IL-4, followed by evaluation of the expression level of the MRC1 gene encoding a mannose receptor C-type 1. It is known that MRC1 is a key factor in the polarization of macrophages according to the phenotype M2, the expression of which increases in response to the activation of IL-4 macrophages (see, for example, Martinez F.O. et al., Annu. Rev. Immunol., 2009, 27(1):451-483). The human HPRT “housekeeping” gene was used as an internal control.

PBMC were isolated from peripheral blood of a healthy donor by means of a density gradient centrifugation using ficoll. 500,000 PBMC cells dissolved in 400 pL of a complete medium using autologous serum were dispensed into each well on 24 well plates. Antibodies abD and abD2 were diluted to a concentration of 25 pg/mL, and 50 pL of the solution were added to each well. The plate was incubated for 60 minutes in a CO2 incubator under standard conditions (37°C, 5% CO2). Then, 50 pl of solution of IL-4 (SCI-STORE, Russia) diluted to a concentration of 100 ng/ml was added to the wells.

Four incubation variants of PBMC were used, in 3 biological replicates each:

1) 3 wells: no addition of IL-4, no addition of antibodies;

2) 3 wells: 50 pl of IL-4 solution, no addition of antibodies;

3) 3 wells: 50 pl of IL-4 solution, with the preliminary addition of 50 pl of antibody abD solution;

4) 3 wells: 50 pl of IL-4 solution, with the preliminary addition of 50 pl of antibody abD2 solution.

Cells were incubated for 2 h, 3 h and 20 h. Cells were removed from the wells by pipetting, centrifuged, and the precipitate was lysed in a RLT buffer (Qiagen). Isolation of total RNA was performed using an RNeasy MinElute reagent kit (Qiagen), including the DNase 1 step (Qiagen), and eluted from the column with sterile RNAse-free water (Qiagen).

The expression level of the MRC1 and HPRT genes was determined using real-time PCR. The PCR template used was the first cDNA strand that was synthesized using Smart Scribe Revertase (Clontech) and a poly-T oligonucleotide using the manufacturer’s protocol (Clontech). The first cDNA strand was purified on AMPure-XP magnetic particles (Beckman Coulter) at a ratio of 1 : 1.5, respectively. PCR was performed using a qPCRmix-HS Sybr reagent kit (Eurogen) on a DT-Prime instrument (DNA technology). PCR program was as follows: 95°C 5 min; 95°C 15 s / 58°C 20 s / 72°C 30 s - 45 cycles. DNA primers P7 (SEQ ID NO: 31) and P8 (SEQ ID NO: 32) for the MRC1 gene, and P9 (SEQ ID NO: 33) and P10 (SEQ ID NO: 34) for the HPRT gene were used. To determine the expression of each gene, three replicates were made for each cDNA sample.

Evaluation of the change in the relative level of expression of the MRC1 gene was carried out using the delta-delta Ct (AACt) method using HPRT as the normalizing gene. The threshold number of cycles (Ct) was averaged over three technical replicates. The difference in the mean values of the threshold cycle for the test gene (A MRCI) between the test and control samples was then calculated, as well as the difference in the mean values of the threshold cycle for HPRT (ACtHPRr) between the test and control samples. PBMC without IL-4 stimulation and without addition of IL-4 blocking antibodies was taken as a control. The difference between A HPRT and ACtMRCi was then calculated. All computations and visualizations of results were performed in R (R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. R-proj ect.org).

The expression level of the MRC1 gene was significantly increased when incubated with IL-4 (Figure 5). Pre-addition of the control antibody abD, which blocks the effects mediated by IL-4 and IL-13, resulted in the decrease of the MRC1 gene expression. In the case of the preliminary addition of the modified antibody abD2, the expression level of the MRC1 gene has decreased to the same extent as in the case of the control antibody. These experimental data show that the efficacy of specific blocking of hIL-4Ra by the modified antibody abD2 and the control antibody (dupilumab) is the same.

Example 9. Comparative analysis of antibody immunogenicity.

As an example, illustrating the immunogenic activity of the modified antibodies, the immunogenic activity of antibodies abD (dupilumab) and abD2 (Example 4) was compared. Immunogenic activity was evaluated by determining the presence of a T cell response to epitopes containing hypermutations within the CDR1 fragment of the light chain of the antibody dupilumab (SEQ ID NO: 2). For this purpose, a method of culturing CFSE (carboxyfluorescein succinimidylester) stained T cells with antigen-specific stimulation in the presence of peptide A (SEQ ID NO: 35) or peptide B (SEQ ID NO: 36) was used, or without the addition of the peptides A and B (negative control), instead of which dimethyl sulfoxide (DMSO) was used. Peptide A is a fragment of the light chain of the control antibody abD (SEQ ID NO: 2). Peptide B is a fragment of the light chain of the modified antibody abD2 (SEQ ID NO: 20).

Cells were cultured for 8 days in three biological replicas, the divided CD4+ CFSE ^low cells were sorted and the T-cell repertoire was analyzed. White blood cells obtained from patients previously treated with the antibody dupilumab as a component of Dupixent were used as the samples for the study, as well as the samples from healthy donors who had not previously received therapy with the antibody. In each replica, 600,000 white blood cells were used as starting material.

The results of fluocytometric analysis of the number of divided CD4+ T lymphocytes for three patients and four healthy donors, two types of antigenic peptide (peptide A or peptide B) and without the peptide (negative control), in three replicates are shown in Figure 6. As one can see from the results of the analysis, for two out of three patients analyzed in the presence of peptide A, an increased number of proliferated CD4+ T lymphocytes was observed relative to replicates cultured in the presence of peptide B, as well as relative to the control replicates. For healthy donors, there was no statistically significant difference between CD4+ T cells dividing in the presence of peptide A and peptide B. Thus, it was shown using the fluocytometric analysis that the modified antibody abD2 containing peptide B has reduced immunogenic activity as compared with the control antibody abD containing peptide A.

Divided CFSE ^low CD4+ T lymphocytes for two patients (conditionally, patients No.l and No.3), for whom the increased number of such cells was observed after cultivation in the presence of peptide A, were sorted using flow cytometry. Isolation of total RNA was performed using an RNeasy MinElute reagent kit (Qiagen), including the DNase 1 step (Qiagen), and eluted from the column with sterile RNAse-free water (Qiagen). The cDNA libraries of T cell receptor beta chains were obtained using Human TCR multiplex kit (MiLaboratories) and sequenced using Illumina MiSeq 150+150 nt. T cell receptor beta chain CDR3 repertoires were extracted using MiXCR software.

Comparative analysis of the obtained repertoires of T-cell receptors showed that the replicas of CD4+ T-lymphocytes of patient No.3 divided in the presence of peptide A are reproducibly enriched with variants of beta chains of T-cell receptors relative to replicas of CD4+ T- lymphocytes divided in the presence of peptide B, as well as relative to the negative control. At the same time, none of the two patients had T cell receptor beta chain variants that were reproducibly enriched in CD4+ T lymphocyte replicates that divided in the presence of peptide B.

Therefore, the analysis of T-cell receptor repertoires has confirmed the results of the fluocytometric analysis, indicating the presence of clonal memory populations of CD4+ T- lymphocytes specific to one or more MHC class II epitopes that are a fragment of peptide A in the blood of patients treated with the antibody dupilumab. The presence of the clonal populations of CD4+ T lymphocytes indicates the involvement of the light chain of the dupilumab in the formation of an antigen-specific humoral response. The absence of a pronounced antigen-specific humoral response to peptide B indicates the reduced immunogenicity of the modified antibody abD2 as compared with the control antibody abD.

While the invention has been described in detail with reference to exemplary embodiments thereof, it will be apparent to one of ordinary skill in the art that various changes can be made and equivalents employed, and such changes and equivalents are within the scope of the invention.

Table 1. Identified MHC class II light chain epitopes of dupilumab.

# NetMHCIIpan version 4.0

# Input of data in FASTA format

# Peptide Length 15

# Prediction type : EL+BA

# Threshold for strongly binding peptides (iDegree) 2%

# Threshold for weakly binding peptides (iDegree) 10%

# Threshold for output filtering (iDegree) 10%

# Allel: DRB1 0301

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

Number of strong bindings: 0 Number of weak bindings: 0

# Allel: DRB1 0701

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

77 TLKISRVEAEDVGFY ISRVEAEDV 149.86 5.78 <=SB

49 PQLLIYLGSNRASGV LIYLGSNRA 56.74 1. 98 <=WB

25 SSQSLLYSIGYNYLD LLYSIGYNY 51.16 1.74 <=WB

73 GTDFTLKISRVEAED FTLKISRVE 762.01 23.35 <=WB

95 QALQTPYTFGQGTKL YTFGQGTKL 296.06 11.12 <=WB

63 VPDRFSGSGSGTDFT FSGSGSGTD 3184.87 53. 61 <=WB

Number of strong bindings: 1 Number of weak bindings: 5

# Allel: DRB1 1301

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

48 SPQLLIYLGSNRASG LLIYLGSNR 37.46 0.79 <=WB

49 PQLLIYLGSNRASGV LIYLGSNRA 29.86 0.48 <=WB

Number of strong bindings: 0 Number of weak bindings: 2

# Allel: DRB1_13O2

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

49 PQLLIYLGSNRASGV LIYLGSNRA 42.46 1.10 <=WB

52 LIYLGSNRASGVPDR LGSNRASGV 77.72 2.34 <=WB

73 GTDFTLKISRVEAED FTLKISRVE 1182.69 28.16 <=WB

75 DFTLKISRVEAEDVG LKISRVEAE 476.45 14.04 <=WB

Number of strong bindings: 0 Number of weak bindings: 4

# Allel: DRB1 1501

Location Peptide Site Affinity (nM) iDegree BA Degree of binding 48 SPQLLIYLGSNRASG LLIYLGSNR 14.47 0.07 <=SB

49 PQLLIYLGSNRASGV LIYLGSNRA 13.45 0.06 <=SB

77 TLKISRVEAEDVGFY ISRVEAEDV 685.54 19.75 <=WB

Number of strong bindings: 2 Number of weak bindings: 1

# Allel: HLA-DQA10501-DQB10201

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

77 TLKISRVEAEDVGFY ISRVEAEDV 157.91 0.37 <=SB

68 SGSGSGTDFTLKISR SGTDFTLKI 3050.93 44.13 <=WB

8 PLSLPVTPGEPASIS VTPGEPASI 5305.00 62.57 <=WB

1 DIVMTQSPLSLPVTP VMTQSPLSL 902.53 12.48 <=WB

Number of strong bindings: 1 Number of weak bindings: 3

# Allel: HLA-DQA10301-DQB10302

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

75 DFTLKISRVEAEDVG LKISRVEAE 686.20 1.76 <=SB

77 TLKISRVEAEDVGFY ISRVEAEDV 889.56 3.36 <=WB

49 PQLLIYLGSNRASGV LIYLGSNRA 4790.32 49.68 <=WB

Number of strong bindings: 1 Number of weak bindings: 2

# Allel: HLA-DQA10501-DQB10301

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

8 PLSLPVTPGEPASIS VTPGEPASI 39.24 0.80 <=SB

77 TLKISRVEAEDVGFY ISRVEAEDV 819.99 21.47 <=WB

14 TPGEPASISCRSSQS PASISCRSS 715.69 19.42 <=WB

Number of strong bindings: 1 Number of weak bindings: 2

# Allel: HLA-DQA10301-DQB10301

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

10 SLPVTPGEPASISCR VTPGEPASI 215.75 2.75 <=WB

77 TLKISRVEAEDVGFY ISRVEAEDV 1290.74 25.37 <=WB

14 TPGEPASISCRSSQS PASISCRSS 997.13 19.81 <=WB

Number of strong bindings: 0 Number of weak bindings: 3

# Allel: HLA-DQA10101-DQB10501

Location Peptide Site Affinity (nM) iDegree BA Degree of binding 48 SPQLLIYLGSNRASG LIYLGSNRA 3243.29 31.29 <=WB

77 TLKISRVEAEDVGFY ISRVEAEDV 2209.17 22.69 <=WB

31 YSIGYNYLDWYLQKS GYNYLDWYL 214.34 0.98 <=WB

34 GYNYLDWYLQKSGQS YLDWYLQKS 983.78 9.81 <=WB

Number of strong bindings: 0 Number of weak bindings: 4

# Allel: HLA-DQA10103-DQB10603

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

57 SNRASGVPDRFSGSG ASGVPDRFS 3941.24 32.44 <=SB

Number of strong bindings: 1 Number of weak bindings: 0

# Allel: HLA-DQA10301-DQB10603

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

48 SPQLLIYLGSNRASG LIYLGSNRA 4087.29 3.39 <=SB

75 DFTLKISRVEAEDVG LKISRVEAE 5169.41 8.55 <=WB

77 TLKISRVEAEDVGFY ISRVEAEDV 5676.52 11.93 <=WB

73 GTDFTLKISRVEAED FTLKISRVE 5632.23 11.61 <=WB

Number of strong bindings: 1 Number of weak bindings: 3

# Allel: HLA-DPA10103-DPB10401

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

27 QSLLYSIGYNYLDWY LYSIGYNYL 48.46 0.63 <=SB

32 SIGYNYLDWYLQKSG YNYLDWYLQ 789.47 11.78 <=WB

Number of strong bindings: 1 Number of weak bindings: 1

# Allel: HLA-DPA10103-DPB10201

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

27 QSLLYSIGYNYLDWY LYSIGYNYL 37.77 0.62 <=WB

32 SIGYNYLDWYLQKSG YNYLDWYLQ 447.69 10.73 <=WB

34 GYNYLDWYLQKSGQS YLDWYLQKS 791.08 17.19 <=WB

73 GTDFTLKISRVEAED FTLKISRVE 2909.94 41.45 <=WB

Number of strong bindings: 0 Number of weak bindings: 4

# Allel: HLA-DPA10103-DPB10301

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

56 GSNRASGVPDRFSGS RASGVPDRF 7808.63 55.19 <=WB Number of strong bindings: 0 Number of weak bindings: 1

# Allel: HLA-DPA10103-DPB10402

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

32 SIGYNYLDWYLQKSG YNYLDWYLQ 1732.36 12.43 <=WB

73 GTDFTLKISRVEAED FTLKISRVE 5829.14 41.19 <=WB

27 QSLLYSIGYNYLDWY LYSIGYNYL 340.83 1.17 <=WB

33 IGYNYLDWYLQKSGQ YLDWYLQKS 2000.57 14.72 <=WB

49 PQLLIYLGSNRASGV LIYLGSNRA 1627.69 11.53 <=WB

Number of strong bindings: 0 Number of weak bindings: 5

# Allel: HLA-DPA10201-DPB11701

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

73 GTDFTLKISRVEAED FTLKISRVE 5691.34 32.64 <=WB

48 SPQLLIYLGSNRASG LIYLGSNRA 2042.54 7.72 <=WB

Number of strong bindings: 0 Number of weak bindings: 2

Table 2. Identified MHC class II heavy chain epitopes of dupilumab.

# NetMHCIIpan version 4.0

# Input of data in PASTA format

# Peptide Length 15

# Prediction type : EL+BA

# Threshold for strongly binding peptides (iDegree) 2%

# Threshold for weakly binding peptides (iDegree) 10

# Threshold for output filtering (iDegree) 10

# Allel: DRB1 0301

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

55 GGNTYYADSVKGRFT YYADSVKGR 355.25 3.67 <=SB

97 AKDRLSITIRPRYYG LSITIRPRY 178.16 1.88 <=WB

83 MNSLRAEDTAVYYCA LRAEDTAVY 291.89 3.07 <=WB

66 GRFTISRDNSKNTLY ISRDNSKNT 430.46 4.43 <=WB

76 KNTLYLQMNSLRAED LYLQMNSLR 677.84 6.72 <=WB

90 DTAVYYCAKDRLSIT VYYCAKDRL 970.05 9.32 <=WB

64 VKGRFT ISRDNSKNT FTISRDNSK 1420.94 13.35 <=WB

Number of strong bindings: 1 Number of weak bindings: 6

# Allel: DRBl_0701

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

29 FRDYAMT VRQAPGK YAMT VRQA 88.14 3.33 <=WB

77 NTLYLQMNSLRAEDT YLQMNSLRA 224.22 8.67 <=WB

42 GKGLEWVSSISGSGG LEWVSSISG 256.12 9.80 <=WB

43 KGLEWVSSISGSGGN WVSSISGSG 284.29 10.74 <=WB

48 VSSISGSGGNTYYAD ISGSGGNTY 772.41 23.57 <=WB

Number of strong bindings: 0 Number of weak bindings: 5

# Allel: DRB1_13O1

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

99 DRLSITIRPRYYGLD ITIRPRYYG 20.23 0.17 <=SB

97 AKDRLSITIRPRYYG LSITIRPRY 22.55 0.23 <=SB

61 ADSVKGRFTISRDNS VKGRFTISR 336.13 13.26 <=SB

76 KNTLYLQMNSLRAED LYLQMNSLR 68.66 2.22 <=WB

Number of strong bindings: 3 Number of weak bindings: 1

# Allel: DRB1 1302

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

98 KDRLSITIRPRYYGL LSITIRPRY 194.59 6.23 <=SB

99 DRLSITIRPRYYGLD ITIRPRYYG 273.93 8.50 <=SB

77 NTLYLQMNSLRAEDT YLQMNSLRA 118.49 3.74 <=WB

56 GNTYYADSVKGRFTI YYADSVKGR 1770.12 36.84 <=WB 65 KGRFTISRDNSKNTL FTISRDNSK 312.02 9.60 <=WB

83 MNSLRAEDT AVY YC A LRAEDTAVY 434.61 12.95 <=WB

48 VSSISGSGGNTYYAD ISGSGGNTY 479.62 14.12 <=WB

Number of strong bindings: 2 Number of weak bindings: 5

# Allel: DRB1_15O1

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

42 GKGLEWVSSISGSGG LEWVSSISG 512.47 15.79 <=WB

76 KNTLYLQMNSLRAED YLQMNSLRA 82.16 2.11 <=WB

Number of strong bindings: 0 Number of weak bindings: 2

# Allel: HLA-DQA10501-DQB10201

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

80 YLQMNSLRAEDTAVY MNSLRAEDT 235.14 1.12 <=WB

55 GGNTYYADSVKGRFT YYADSVKGR 3339.36 47.10 <=WB

53 GSGGNTYYADSVKGR GNTYYADSV 3187.18 45.60 <=WB

Number of strong bindings: 0 Number of weak bindings: 3

# Allel: HLA-DQA10301-DQB10302

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

106 RPRYYGLDVWGQGTT YYGLDVWGQ 4479.76 46.71 <=SB

55 GGNTYYADSVKGRFT YYADSVKGR 5539.10 56.10 <=WB

29 FRDYAMTWVRQAPGK YAMTWVRQA 2139.24 18.91 <=WB

77 NTLYLQMNSLRAEDT YLQMNSLRA 1069.00 5.11 <=WB

43 KGLEWVSSISGSGGN WVSSISGSG 5634.97 56.85 <=WB

53 GSGGNTYYADSVKGR GNTYYADSV 4722.65 49.07 <=WB

80 YLQMNSLRAEDTAVY MNSLRAEDT 1177.31 6.33 <=WB

42 GKGLEWVSSISGSGG LEWVSSISG 5229.10 53.55 <=WB

Number of strong bindings: 1 Number of weak bindings: 7

# Allel: HLA-DQA10501-DQB10301

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

55 GGNTYYADSVKGRFT TYYADSVKG 679.72 18.67 <=WB

44 GLEWVSSISGSGGNT WVSSISGSG 331.72 10.52 <=WB

29 FRDYAMTWVRQAPGK YAMTWVRQA 250.68 8.21 <=WB

80 YLQMNSLRAEDTAVY MNSLRAEDT 562.25 16.12 <=WB

56 GNTYYADSVKGRFTI YYADSVKGR 812.12 21.32 <=WB

90 DTAVYYCAKDRLSIT YYCAKDRLS 1471.10 32.71 <=WB

106 RPRYYGLDVWGQGTT YYGLDVWGQ 1771.00 37.22 <=WB

Number of strong bindings: 0 Number of weak bindings: 7 # Allel: HLA-DQA10301-DQB10301

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

55 GGNTYYADSVKGRFT TYYADSVKG 1141.63 22.54 <=SB

29 FRDYAMTWVRQAPGK YAMTWVRQA 375.19 6.39 <=WB

43 KGLEWVSSISGSGGN WVSSISGSG 395.10 6.85 <=WB

56 GNTYYADSVKGRFTI YYADSVKGR 1274.83 25.07 <=WB

90 DTAVYYCAKDRLSIT YYCAKDRLS 1879.43 35.08 <=WB

106 RPRYYGLDVWGQGTT YYGLDVWGQ 2134.13 38.79 <=WB

50 SISGSGGNTYYADSV GSGGNTYYA 444.01 8.01 <=WB

Number of strong bindings: 1 Number of weak bindings: 6

# Allel: HLA-DQA10101-DQB10501

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

53 GSGGNTYYADSVKGR GNTYYADSV 4777.23 41.61 <=SB

54 SGGNTYYADSVKGRF NTYYADSVK 4305.78 38.61 <=SB

23 AGSGFTFRDYAMTWV GFTFRDYAM 286.57 1.63 <=WB

77 NTLYLQMNSLRAEDT YLQMNSLRA 1434.24 14.94 <=WB

55 GGNTYYADSVKGRFT GNTYYADSV 5214.24 44.20 <=WB

104 TIRPRYYGLDVWGQG PRYYGLDVW 3102.39 30.19 <=WB

Number of strong bindings: 2 Number of weak bindings: 4

# Allel: HLA-DQA10103-DQB10603

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

90 DTAVYYCAKDRLSIT YYCAKDRLS 3824.21 31.41 <=WB

54 SGGNTYYADSVKGRF TYYADSVKG 6010.45 48.67 <=WB

29 FRDYAMTWVRQAPGK YAMTWVRQA 715.01 2.16 <=WB

30 RDYAMTWVRQAPGKG MTWVRQAPG 1201.07 6.01 <=WB

42 GKGLEWVSSISGSGG LEWVSSISG 2789.70 21.65 <=WB

106 RPRYYGLDVWGQGTT YYGLDVWGQ 4516.50 37.26 <=WB

Number of strong bindings: 0 Number of weak bindings: 6

# Allel: HLA-DQA10301-DQB10603

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

76 KNTLYLQMNSLRAED YLQMNSLRA 4119.88 3.50 <=SB

42 GKGLEWVSSISGSGG LEWVSSISG 6952.58 22.27 <=SB

55 GGNTYYADSVKGRFT TYYADSVKG 7091.00 23.46 <=SB

30 RDYAMTWVRQAPGKG MTWVRQAPG 3440.10 1.57 <=WB

29 FRDYAMTWVRQAPGK YAMTWVRQA 2774.50 0.53 <=WB

106 RPRYYGLDVWGQGTT YYGLDVWGQ 6960.40 22.33 <=WB

44 GLEWVSSISGSGGNT WVSSISGSG 7954.97 31.28 <=WB

56 GNTYYADSVKGRFTI YYADSVKGR 7709.16 29.07 <=WB

61 ADSVKGRFTISRDNS VKGRFTISR 10255.04 51.63 <=WB

63 SVKGRFTISRDNSKN FTISRDNSK 8328.68 34.75 <=WB Number of strong bindings: 3 Number of weak bindings: 7

# Allel: HLA-DPA10103-DPB10401

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

24 GSGFTFRDYAMTWVR FTFRDYAMT 752.09 11.29 <=WB

29 FRDYAMTWVRQAPGK YAMTWVRQA 1307.13 17.65 <=WB

Number of strong bindings: 0 Number of weak bindings: 2

# Allel: HLA-DPA10103-DPB10201

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

24 GSGFTFRDYAMTWVR FTFRDYAMT 106.34 2.49 <=SB

29 FRDYAMTWVRQAPGK YAMTWVRQA 580.81 13.41 <=WB

102 SITIRPRYYGLDVWG IRPRYYGLD 2612.45 38.87 <=WB

Number of strong bindings: 1 Number of weak bindings: 2

# Allel: HLA-DPA10103-DPB10301

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

84 NSLRAEDTAVYYCAK LRAEDTAVY 2739.95 23.18 <=WB

97 AKDRLSITIRPRYYG DRLSITIRP 665.27 4.97 <=WB

105 IRPRYYGLDVWGQGT RYYGLDVWG 3901.70 32.11 <=WB

Number of strong bindings: 0 Number of weak bindings: 3

# Allel: HLA-DPA10103-DPB10402

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

24 GSGFTFRDYAMTWVR FTFRDYAMT 1080.77 6.87 <=SB

29 FRDYAMTWVRQAPGK YAMTWVRQA 1399.17 9.57 <=WB

77 NTLYLQMNSLRAEDT YLQMNSLRA 1856.33 13.50 <=WB

106 RPRYYGLDVWGQGTT YGLDVWGQG 4749.86 34.78 <=WB

Number of strong bindings: 1 Number of weak bindings: 3

# Allel: HLA-DPA10201-DPB11701

Location Peptide Site Affinity (nM) iDegree BA Degree of binding

97 AKDRLSITIRPRYYG RLSITIRPR 3300.50 16.44 <=WB

29 FRDYAMTWVRQAPGK YAMTWVRQA 2170.95 8.57 <=WB

Number of strong bindings: 0 Number of weak bindings: 2