IL-4R-co antibody variable domain

Abstract

The present invention relates to antibody variable domains that specifically bind to IL-4R and to multispecific antibodies, said multispecific antibodies comprising one or two of said antibody variable domains and at least one further binding domain that specifically binds to a target different to IL-4R. The present invention further relates to a nucleic acid or two nucleic acids encoding said antibody variable domains or said multispecific antibodies, a vector comprising said nucleic acid or said nucleic acids, one or more host cells comprising said nucleic acid or nucleic acids or said vector or vectors, and to methods for producing said antibody variable domains or said multispecific antibodies. The present invention further relates to pharmaceutical compositions comprising said antibody variable domains or said multispecific antibodies and methods of use thereof.

Description

[Technical Field]

[0001] The present invention relates to an antibody-variable domain that specifically binds to IL-4R and a multispecific antibody, wherein the multispecific antibody comprises one or two of the antibody-variable domains and at least one further binding domain that specifically binds to a target different from IL-4R. The present invention further relates to one or two nucleic acids encoding the antibody-variable domain or the multispecific antibody, a vector comprising the nucleic acid or the plurality of nucleic acids, one or more host cells comprising the nucleic acid or the plurality of nucleic acids or the one or more vector, and a method for producing the antibody-variable domain or the multispecific antibody. Furthermore, the present invention relates to a pharmaceutical composition comprising the antibody-variable domain or the multispecific antibody and a method for using the same. [Background technology]

[0002] The interleukin-4 receptor IL-4Rα (also referred to herein as IL-4R or IL4R) is a type I receptor. It binds to interleukin-4 (IL-4) by direct interaction with the gamma chain (γc) of the common cytokine receptor, or by forming a receptor complex with type II receptors, particularly the type II receptor IL-13Rα1. This IL-4R / IL-13Rα1 receptor complex binds to interleukin-4 (IL-4) and interleukin-13 (IL-13) to regulate IgE antibody production in B cells. It is further known that the binding of IL-4 to IL-4R activates macrophages, promotes the differentiation of type II helper T cells (Th2 cells), and induces Th2-mediated inflammation. IL-4 and / or IL-13 bind to IL-4Rα / γ c When bound to and / or IL-4R / IL-13Rα1, it activates Janus kinase (JAK) 1, JAK2, signaling molecules, and transcription activators (STAT) 1, STAT3, STAT6, and STAT dimerization, thereby inducing transcription of specific genes.

[0003] IL-4R signaling has been shown to be associated with many human diseases, such as inflammatory and autoimmune diseases including atopic dermatitis, prurigo nodule, nasal polyposis, chronic urticaria, asthma, and chronic obstructive pulmonary disease.

[0004] Several monoclonal antibodies that reduce or block IL4R-mediated signaling by binding to the cytokine IL-4 and / or IL-13 or IL-4R have been described in the prior art relating to the treatment of such diseases.

[0005] For example, dupilumab (Dupixent®), developed by Regeneron Pharmaceuticals and Sanofi Genzyme, antagonizes IL-4R by binding to its alpha subunit and was approved by the U.S. Food and Drug Administration (FDA) in 2017 for the treatment of moderate to severe atopic dermatitis and chronic sinusitis with nasal polyps (CRSwNP). Furthermore, it is also being evaluated for the treatment of persistent asthma in adults and adolescents. In 2018, dupilumab also received FDA approval for asthma.

[0006] Furthermore, International Publication No. 2014 / 039461 describes an anti-IL4R antagonist antibody for use in the treatment of atopic dermatitis.

[0007] Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by severe itching (i.e., intense itching) and scaly, dry, eczematous lesions. Severe cases can lead to significant psychological problems, marked sleep deprivation, and a reduced quality of life, resulting in high socioeconomic costs. AD often begins in childhood, before the age of five, and can persist into adulthood.

[0008] The pathophysiology of Alzheimer's disease (AD) is influenced by a complex interaction between immunoglobulin E (IgE)-mediated sensitization, the immune system, and environmental factors. Major skin defects may be immune disorders that lead to IgE-mediated sensitization and may involve epithelial barrier dysfunction, which is the result of both genetic mutations and local inflammation.

[0009] However, the anti-IL-4R therapies described above have significant limitations. In many allergic, inflammatory, and autoimmune diseases, even those associated with imbalances in IL-4R signaling, their effectiveness is limited and / or their response rates are low to moderate. One explanation for the suboptimal performance of currently available anti-IL-4R therapies may be that imbalanced IL-4R signaling is not the sole cause of these diseases. Therefore, other signaling pathways also need to be addressed to improve the response rates and / or effectiveness of these therapies. Separately, many of these types of diseases are accompanied by destructive symptoms that also need to be treated, such as itching. Anti-IL-4R therapies do not directly address itching. However, itching is a common symptom in skin-related inflammatory and autoimmune diseases, such as in Alzheimer's disease (AD). Itchy scratching generally promotes inflammation and exacerbates disease-related symptoms. Therefore, there is a critical need for further IL-4R-based treatment options for patients with such allergic, inflammatory, and autoimmune diseases.

[0010] More specifically, it is desirable to have stable and potent anti-IL-4R antibody components readily available that can be easily incorporated into multispecific antibody formats that further disrupt other signaling pathways. These anti-IL-4R components must possess high potency in inhibiting IL-4 and / or IL-13 mediated signaling. Furthermore, these components must possess excellent biophysical properties, particularly high stability, to facilitate their efficient incorporation into multispecific antibodies suitable for drug development.

[0011] In particular, the anti-IL-4R component, i.e., the antibody-binding domain, must exhibit the following minimum characteristics: - This is a monovalent dissociation constant (K) of less than 200 pM, as measured by surface plasmon resonance (SPR). D ) must bind to human IL-4R, - This is cross-reactive with cynomolgus monkey (Macaca fascicularis (Cynomolgus)) IL-4R, and in particular, monovalent K below 5 nM when measured by surface plasmon resonance (SPR). D It must bind to IL-4R in cynomolgus monkeys. - In particular, when prepared in a 50 mM phosphate-citrate buffer solution at pH 6.4 containing 150 mM NaCl, the melting temperature (Tm) measured by differential scanning fluorescence (DSF) must be 63°C or higher.

[0012] Furthermore, when the anti-IL-4R component was measured in HEK-Blue cells using a stat-6 reporter gene assay, the IC50 was less than 5 ng / ml. 50 It neutralizes human IL-4-induced signaling and IC10 is less than 10 ng / ml. 50 It is desirable that the drug has the ability to neutralize human IL-13-induced signaling. Further desirable characteristics are listed in item 7 below.

[0013] While methods for identifying anti-IL-4R antibody variable domains that satisfy one or two of the above criteria are known in the prior art, identifying such antibody variable domains that satisfy all of these criteria, as well as most or all of the criteria mentioned in item 7 below, is difficult and the results are unpredictable. [Overview of the project]

[0014] The object of the present invention is to provide a novel antibody variable domain that specifically binds to IL-4R, can efficiently reduce or eliminate IL-4 and IL-13-mediated activity, and at the same time is highly stable and can be easily incorporated into a multispecific antibody format.

[0015] The inventors have surprisingly discovered that scFv based on the monoclonal rabbit antibody clone 44-34-C10 binds to human IL-4R and cynomolgus monkey IL-4R with high affinity, potently blocks IL-4R signaling, and exhibits excellent stability. This antibody clone is one of a limited number of monoclonal rabbit antibody clones obtained from an extensive immunization campaign that have been confirmed to bind to IL4R with high affinity. In particular, scFv derived from clone 44-34-C10 exhibits a lower pM range dissociation constant (K D ) binds to human IL-4R and has a K level far below 1 nM D It binds to cynomolgus monkey IL-4R, and IC 50 These compounds can neutralize IL-4 and IL-13 inducible signaling at concentrations of less than 1.5 ng / ml and less than 2.5 ng / ml, respectively, have a melting temperature (Tm) of 63°C or higher as measured by DSF, and do not significantly decrease in protein and monomer content even after storage at 4°C for 4 weeks at a concentration of 10 mg / ml. This is particularly surprising considering that none of the scFv produced from other clones exhibit good pharmacological activity and high stability at the same time. Optimizing the pharmacological activity of these scFv almost without exception results in a decrease in stability, and vice versa.

[0016] Therefore, scFv based on clone 44-34-C10 is a suitable component that can be easily incorporated into multispecific antibody formats such as the Morrison format.

[0017] Accordingly, in the first embodiment, the present invention relates to an antibody variable domain that specifically binds to IL-4R and includes the following: a) A VH chain having a sequence selected from SEQ ID NOs: 4, 5, 6, 7, 8, and 9, and b) A VL chain having a sequence selected from sequence numbers 14, 15, 16, 17, and 18.

[0018] In a second embodiment, the present invention is a) One or two antibody variable domains as defined herein, b) The present invention relates to a multispecific antibody comprising at least one binding domain that specifically binds to a target different from IL-4R.

[0019] In a third embodiment, the present invention relates to one or two nucleic acids encoding an antibody variable domain or a multispecific antibody of the present invention.

[0020] In a fourth embodiment, the present invention relates to one vector or two vectors comprising the nucleic acid or two nucleic acids of the present invention.

[0021] In a fifth embodiment, the present invention relates to one host cell or more host cells containing the vector or two vectors of the present invention.

[0022] In a sixth aspect, the present invention relates to a method for producing an antibody variable domain or a multispecific antibody of the present invention, comprising the steps of (i) providing a nucleic acid or two nucleic acids of the present invention, or a vector or two vectors of the present invention, expressing the nucleic acid or the two nucleic acids, or the vector or multiple vectors, and collecting the antibody variable domain or the multispecific antibody from the expression system, or (ii) providing one or more host cells of the present invention, culturing the host cell or the multiple host cells, and collecting the antibody variable domain or the multispecific antibody from the cell culture.

[0023] In a seventh aspect, the present invention relates to a pharmaceutical composition comprising an antibody variable domain or a multispecific antibody of the present invention and a pharmaceutically acceptable carrier.

[0024] In the eighth embodiment, the present invention relates to an antibody variable domain or a multispecific antibody for use as a pharmaceutical.

[0025] In a ninth aspect, the present invention relates to an antibody-variable domain or multispecific antibody of the present invention for use in the treatment of diseases, particularly human diseases, more particularly human diseases selected from allergic diseases, inflammatory diseases, and autoimmune diseases, particularly inflammatory diseases and autoimmune diseases.

[0026] In a tenth aspect, the present invention relates to a method for treating a disease, particularly a human disease, more particularly an allergic disease, an inflammatory disease, and an autoimmune disease, a human disease selected from inflammatory diseases and autoimmune diseases, the method comprising the step of providing the antibody variable domain or multispecific antibody of the present invention to a patient in need thereof.

[0027] The aspects, advantageous features, and preferred embodiments of the present invention, summarized in the following sections, each individually or in combination, further contribute to solving the objectives of the present invention. 1. Antibody variable domains that specifically bind to IL-4R, including the following: a) Variable heavy chain (VH), Here, the variable heavy chain includes the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4 from the N-terminus to the C-terminus, where each HFW represents a heavy chain framework region, and each HCDR represents a heavy chain complementarity determination region, and here The HCDR1 has the sequence of sequence number 1, The HCDR2 has the sequence of sequence number 2, and The HCDR3 has the sequence of sequence number 3, b) Variable light chain (VL), Here, the variable light chain includes the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4 from the N-terminus to the C-terminus, where each LFW represents a light chain framework region, and each LCDR represents a light chain complementarity determination region, and here The aforementioned LCDR1 has the sequence of sequence number 10, The LCDR2 has an array selected from sequence number 11 or 12, and The LCDR3 has the sequence of sequence number 13. 2. The antibody variable domain according to item 1, wherein the variable heavy chain is a VH3 chain and / or the variable light chain is a Vκ1 light chain. 3. The antibody variable domain according to item 1, wherein - the variable heavy chain is a VH3 chain, - LFW1, LFW2, and LFW3 belong to the Vκ1 light chain subtype, and - LFW4 has a human Vλ sequence selected from SEQ ID NOs: 22 - 29. 4. The antibody variable domain according to any one of items 1 - 3, wherein the antibody variable domain is selected from Fab, Fv, scFv, dsFv, and scAB, preferably from Fab, Fv, scFv, and dsFv, particularly from Fab, scFv, and dsFv. 5. The antibody variable domain according to any one of items 1 - 4, wherein the antibody variable domain comprises: a) A VH chain having a sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to any one of the amino acid sequences selected from SEQ ID NOs: 4, 5, 6, 7, 8, and 9, and b) A VL chain having a sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to any one of the amino acid sequences selected from SEQ ID NOs: 14, 15, 16, 17, and 18. 6. The antibody variable domain according to any one of items 1 - 5, wherein the antibody variable domain blocks the binding of IL4 and IL13 to IL4R. 7. The antibody variable domain according to any one of items 1 - 5, wherein when the antibody variable domain is in the scFv format, it exhibits at least two of the following characteristics a. - f.: a. When measured by surface plasmon resonance (SPR), it binds to human IL - 4R with a monovalent dissociation constant (K D ) of 0.1 - 200 pM, particularly 0.1 - 100 pM, particularly 0.1 - 50 pM of K D and b. Cross-reactivity with cynomolgus monkey (Macaca fascicularis) IL-4R is observed, and in particular, when measured by SPR, cynomolgus monkey IL-4R shows 1 pM to 5 nM, especially 1 pM to 3 nM, and especially 1 pM to 2 nM of monovalent potassium. D Combined, c. Marmoset (Callithrix jacchus) shows cross-reactivity with IL-4R, and in particular, when measured by SPR, marmoset IL-4R contains 1 pM to 5 nM, especially 1 pM to 3 nM, and especially 1 pM to 2 nM of monovalent potassium. D Combined, d. In HEK-Blue cells, measurement using the stat-6 reporter gene assay revealed that human IL-4-induced signaling was reduced to 0.01-5 ng / ml IC50. 50 And especially ICs of 0.01~3 ng / ml 50 And in particular, ICs with a concentration of 0.01 to 1.5 ng / ml 50 Neutralize with, In e.HEK-Blue cells, the stat-6 reporter gene assay measured human IL-13-induced signaling, with IC50 ranging from 0.01 to 10 ng / ml. 50 And especially ICs of 0.01~5 ng / ml 50 And especially ICs with concentrations of 0.01 to 2.5 ng / ml 50 Neutralize with, f. When measured by competitive ELISA, the binding of human IL-4 to human IL-4R was measured in IC50 at concentrations of 0.01 to 10 ng / ml. 50 And especially ICs of 0.01~5 ng / ml 50 And especially ICs of 0.01~2 ng / ml 50 inhibited by, Furthermore, if the antibody variable domain is in scFv format, it further exhibits at least one of the following features g.~j.: g. When measured by differential scanning fluorescence (DSF), it has a melting temperature (Tm) of at least 63°C, particularly at least 65°C, particularly at least 67°C, particularly at least 69°C, particularly at least 70°C, particularly at least 71°C, and particularly the scFv here is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl. h. When the initial concentration of scFv is 10 mg / ml, the loss of monomer content after storage at 4°C for 4 weeks is less than 5%, and in particular, here in particular, the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl. i. When the initial concentration of scFv is 10 mg / ml, the loss of protein content after storage at 40°C for 4 weeks is less than 5%, particularly less than 4%, particularly less than 3%, particularly less than 2%, particularly less than 1%, and in particular, here the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl. j. When the initial concentration of the scFv is 10 mg / ml, the loss of monomer content after 5 freeze-thaw cycles is less than 2%, particularly less than 1%, and in particular, the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl. 8. If the antibody variable domain is in scFv format, the antibody variable domain described in item 7, exhibiting at least features a., b., and g. 9. The antibody variable domain described in item 7, wherein, if the antibody variable domain is in scFv format, it exhibits at least features a., b., d., e., g., and h., and in particular exhibits at least features a., b., d., e., f., g., h., and i. 10. If the antibody variable domain is in scFv format, the antibody variable domain described in item 7 exhibits all the features of a. to j. 11. Antibody variable domains listed in any one of items 1-10, including the following: a) A VH chain having a sequence selected from SEQ ID NOs: 4, 5, 6, 7, 8, and 9, and b) A VL chain having a sequence selected from sequence numbers 14, 15, 16, 17, and 18. 12. Antibody variable domains listed in any one of items 1-11, including the following: a) A VH chain having the sequence of SEQ ID NO: 4 and a VL chain having the sequence of SEQ ID NO: 14, or b) A VH chain having the sequence of SEQ ID NO: 4 and a VL chain having the sequence of SEQ ID NO: 17, or c) A VH chain having the sequence of SEQ ID NO: 4 and a VL chain having the sequence of SEQ ID NO: 18, or d) A VH chain having the sequence of SEQ ID NO: 5 and a VL chain having the sequence of SEQ ID NO: 15, or e) A VH chain having the sequence of SEQ ID NO: 6 and a VL chain having the sequence of SEQ ID NO: 16, or f) A VH chain having the sequence of SEQ ID NO: 7 and a VL chain having the sequence of SEQ ID NO: 14, or g) A VH chain having the sequence of SEQ ID NO: 8 and a VL chain having the sequence of SEQ ID NO: 18, or h) A VH chain having the sequence of sequence number 9 and a VL chain having the sequence of sequence number 14. 13. An antibody variable domain described in any one of items 1 to 12, which is an scFv antibody having a sequence selected from sequence numbers 33 to 39. 14. Multispecific antibodies including the following: a) One or two antibody variable domains defined in any one of items 1-13; b) At least one binding domain that specifically binds to a target different from IL-4R. 15. A multispecific antibody as described in item 14, which does not contain the immunoglobulin Fc region. 16. A multispecific antibody as described in item 15, wherein the multispecific antibody is selected from the group consisting of tandem scDb (Tandab), linear dimer scDb (LD-scDb), cyclic dimer scDb (CD-scDb), tandem tri-scFv, tribody (Fab-(scFv)2), Fab-Fv2, triabody, scDb-scFv, tetrabody, di-diabody, tandem-di-scFv, and MATCH. 17. A multispecific antibody as described in item 15, which does not contain the CH1 and / or CL region. 18. The multispecific antibody described in item 17, wherein the antibody is in scDb-scFv, triabody, tetrabody, or MATCH format, and in particular, the multispecific antibody is in MATCH or scDb-scFv format, and more specifically, the multispecific antibody is in MATCH format, more specifically, MATCH3 or MATCH4 format. 19. A multispecific antibody as described in item 14, containing an immunoglobulin Fc region. 20. The multispecific antibody according to item 19, wherein the immunoglobulin Fc region is selected from IgG subclasses, particularly IgG1 and IgG4, and especially IgG4. 21. A multispecific antibody as described in item 20, wherein the format of the multispecific antibody is selected from a bivalent bispecific IgG format, a trivalent bispecific IgG format, and a tetravalent bispecific IgG format. More specifically, the format of the multispecific antibody is selected from KiH-based IgG; DVD-Ig; CODV-IgG and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)), and more specifically from DVD-Ig and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)). The above antibody. 22. The multispecific antibody according to item 21, wherein the format of the multispecific antibody is selected from Morrison-H format and Morrison-L format. 23. The multispecific antibody according to any one of items 14 to 22, wherein the multispecific antibody comprises two antibody-variable domains defined in any one of items 1 to 13 and two binding domains that specifically bind to a second target different from IL-4R. 24. One or two nucleic acids encoding one antibody variable domain from items 1-13, or one multispecific antibody from items 14-23. 25. One vector or two vectors containing the nucleic acids of item 24 or the two nucleic acids. 26. One host cell or more host cells containing the vector described in item 25 or the two vectors. 27. A method for producing an antibody variable domain described in any one of items 1 to 13 or a multispecific antibody described in items 14 to 23, comprising: (i) providing the nucleic acid or the two nucleic acids described in item 24, or the vector or the two vectors described in item 25, expressing the nucleic acid or the two nucleic acids, or the vector or the two vectors, and collecting the antibody variable domain or the multispecific antibody from the expression system; or (ii) providing one or more host cells described in item 26, culturing the host cell or the multiple host cells, and collecting the antibody variable domain or the multispecific antibody from the cell culture. 28. A pharmaceutical composition comprising an antibody variable domain described in any one of items 1 to 13 or a multispecific antibody described in any one of items 14 to 23, and a pharmaceutically acceptable salt carrier. 29. An antibody variable domain as described in any one of items 1 to 13, or a multispecific antibody as described in any one of items 14 to 23, for use as a pharmaceutical. 30. Antibody variable domains as described in items 1-13 or any one of items 14-23, for use in the treatment of diseases, particularly human diseases, more specifically allergic diseases, inflammatory diseases, and autoimmune diseases, particularly selected from inflammatory diseases and autoimmune diseases. 31. The antibody variable domain or multispecific antibody for use as described in item 30, wherein the disease is selected from atopic dermatitis, acute allergic contact dermatitis, chronic spontaneous urticaria, bullous pemphigoid, alopecia areata, dermatomyositis, nodular prurigo, psoriasis, and atopic asthma, and in particular the disease is atopic dermatitis. 32. A method for treating a disease, particularly a human disease, more specifically, a human disease selected from allergic diseases, inflammatory diseases, and autoimmune diseases, particularly from inflammatory diseases and autoimmune diseases, the method comprising the step of administering an antibody variable domain described in items 1 to 13, or a multispecific antibody described in any one of items 14 to 23, to a patient in need thereof. 33. The method according to item 32, wherein the disease is selected from atopic dermatitis, acute allergic contact dermatitis, chronic spontaneous urticaria, bullous pemphigoid, alopecia areata, dermatomyositis, nodular prurigo, psoriasis, and atopic asthma, and in particular the disease is atopic dermatitis. [Brief explanation of the drawing]

[0028] [Figure 1] Figure 1 shows the efficacy of the highest-ranked scFv in neutralizing IL-4-induced signaling in the stat-6 reporter gene assay. Compared to dupilumab, the highest-ranked scFvs, (A) PRO1515 (44-32-F04 sc01) and PRO1517 (4434-C10 sc01), (B) PRO1524 (44-03-A03-sc04), (C) PRO1535 (4434-C10 sc04), and (D) PRO1552 (44-18-C11-sc08), showed IC50 values ​​equivalent to dupilumab for neutralizing IL-4-induced signaling.

[0029] [Figure 2] Figure 2 shows the efficacy of the best-ranked scFv for neutralizing IL-13-induced signaling in the stat-6 reporter gene assay. Compared to dupilumab, the top-ranked scFvs, (A) PRO1515 (44-32-F04 sc01) and PRO1517 (44-34-C10 sc01), (B) PRO1524 (44-03-A03-sc04), (C) PRO1535 (44-34-C10 sc04), and (D) PRO1552 (44-18-C11-sc08), showed IC50 values ​​comparable to dupilumab for neutralizing IL-13-induced signaling.

[0030] [Figure 3]Figure 3 shows the efficacy of optimized anti-IL-4R scFv PRO1898 and PRO1899 in blocking human IL-4-induced signaling (A) and human IL-13-induced signaling (B) of IL-4R in a Stat-6 reporter gene assay in HEK-Blue cells. The efficacy of the analyzed molecules is compared to dupilumab.

[0031] [Figure 4-1] Figure 4 shows the efficacy of (A) PRO1515, (B) PRO1533, (C) PRO1517 and PRO1535, and (D) PRO1538, which are scFvs that inhibit the interaction between IL-4 and human IL-4R. PRO1515 (44-32-F04 sc01), PRO1517 (44-34-C10 sc01), PRO1533 (44-32-F04-sc04), and PRO1535 (44-34-C10 sc04) showed IC50 values ​​equivalent to dupilumab. [Figure 4-2] Figure 4 shows the efficacy of (A) PRO1515, (B) PRO1533, (C) PRO1517 and PRO1535, and (D) PRO1538, which are scFvs that inhibit the interaction between IL-4 and human IL-4R. PRO1515 (44-32-F04 sc01), PRO1517 (44-34-C10 sc01), PRO1533 (44-32-F04-sc04), and PRO1535 (44-34-C10 sc04) showed IC50 values ​​equivalent to dupilumab.

[0032] [Figure 5] Figure 5 shows the efficacy of PRO1898(A) and PRO1899(B), optimized anti-IL-4R scFvs that inhibit the interaction between human IL-4 and human IL-4R, as evaluated by competitive ELISA. [Modes for carrying out the invention]

[0033] Detailed description of the invention Known therapeutic anti-IL-4R antibodies are often limited in efficacy and low to moderate response rates in patients with allergic, inflammatory, and autoimmune diseases, even when these diseases are associated with imbalances in IL-4R signaling. These treatments also do not directly address the destructive symptoms often associated with these diseases, such as itching. Therefore, there is a great need for additional IL-4R-based therapeutic options for patients living with the aforementioned allergic, inflammatory, and autoimmune diseases.

[0034] The present invention provides a novel anti-IL-4R antibody variable domain comprising specific VL and VH chains. The variable domain is based on the monoclonal rabbit antibody clone 44-34-C10. This clone was selected from a limited number of rabbit monoclonal antibodies identified in a broad immunization campaign and found to bind to IL-4R with high affinity. The scFv derived from the monoclonal rabbit antibody clone 44-34-C10 has a lower pM range dissociation constant (K D ) binds to human IL-4R, with a K content of less than 1 nM. D It binds to cynomolgus monkey IL-4R, and IC 50 The concentrations of these substances are less than 1.5 ng / ml and less than 2.5 ng / ml, respectively, and they can neutralize IL-4 and IL-13-induced signaling. The melting temperature (Tm) measured by DSF is 63°C or higher, and they can be stored at a concentration of 10 mg / ml at 4°C for 4 weeks without a significant decrease in protein or monomer content.

[0035] To the best of our knowledge, no prior art possesses an anti-IL-4R antibody variable domain with such advantageous properties.

[0036] The antibody-variable domain of the present invention could be successfully incorporated into a multispecific antibody format targeting IL-31. These anti-IL-4R × IL-31 multispecific antibodies bind to IL-4R with high affinity and potently inhibit IL-4R-mediated signaling, while exhibiting highly advantageous biophysical properties, particularly excellent preparation and storage stability, at antibody concentrations far exceeding 100 mg / ml. This demonstrates that the antibody-variable domain of the present invention also provides advantageous biological and biophysical properties when incorporated into a multispecific antibody format.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to whom this invention relates.

[0038] The terms “comprising” and “including” are used herein in an unrestricted and non-restrictive sense unless otherwise specified. Therefore, with respect to such latter embodiments, the term “comprising” includes the narrower term “consisting of.”

[0039] In the context of this description of the present invention (particularly in the context of the claims), the terms “a,” “an,” “the,” and similar references should be interpreted as encompassing both singular and plural forms unless otherwise indicated herein or unless clearly inconsistent with the context. For example, the term “cell” includes multiple cells and mixtures thereof. Where the plural form is used for compounds, salts, etc., it is considered to mean a single compound, salt, etc.

[0040] In one embodiment, the present invention a) A VH chain having a sequence selected from SEQ ID NOs: 4, 5, 6, 7, 8, and 9, and b) The present invention relates to an antibody variable domain that specifically binds to IL-4R, comprising a VL chain having a sequence selected from SEQ ID NOs: 14, 15, 16, 17, and 18.

[0041] As used herein, terms such as “antibody” include the entire antibody or a single chain thereof; and any antigen-binding variable domain (i.e., “antigen-binding portion”) or a single chain thereof; and molecules containing antibody CDR, VH region, or VL region (including, but not limited to, multispecific antibodies). Naturally occurring “whole antibodies” are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) adjacent to more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors, including the first component (C1q) of the classical complement system.

[0042] As used herein, the term “antibody variable domain” refers to one or more portions of an intact antibody that have the ability to specifically bind to a given antigen (e.g., IL-4R). This may be an intact antibody or any antigen-binding fragment of its single chain (i.e., “antigen-binding portion”), and a molecule containing the CDR, VH region, or VL region of the antibody. Specifically, in the case of the multispecific antibody of the present invention, the term “antibody variable domain” as used herein refers to a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; an Fv fragment (Fv) consisting of the VL and VH domains of a single arm of the antibody; a disulfide-stabilized Fv fragment (dsFv); a single-chain Fv fragment (scFv); and an additional light chain constant domain (C) fused thereto. LThis refers to a single-chain Fv fragment (scAB) having ). Preferably, the antibody variable domain of the present invention is selected from a Fab fragment, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), and an scFv fragment. More preferably, the antibody variable domain of the present invention is selected from a Fab fragment, a disulfide-stabilized Fv fragment (dsFv), and an scFv fragment. In certain embodiments, the antibody variable domain of the present invention is a Fab fragment. In other certain embodiments, the antibody variable domain of the present invention is a single-chain Fv fragment (scFv). In other certain embodiments, the VL domain and VH domain of the scFv fragment are stabilized by interdomain disulfide bonds, in particular the VH domain containing a single cysteine ​​residue at position 51 (AHo numbering), and the VL domain containing a single cysteine ​​residue at position 141 (AHo numbering).

[0043] The term “complementarity-determining region” (“CDR”) is used in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)) (“IMGT” numbering scheme); and Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670. This refers to an amino acid sequence with a boundary determined using one of many well-known schemes, including the scheme described by "AHo" numbering. For example, in the classical format, Kabat numbers the CDR amino acid residues of the heavy chain variable domain (VH) as 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) as 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia numbers the CDR amino acids of VH as 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues of VL as 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Combining the CDR definitions of both Kabat and Chothia, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL.In IMGT, the CDR amino acid residues of VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), while the CDR amino acid residues of VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering based on "Kabat"). In IMGT, the CDR of an antibody can be determined using the program IMGT / DomainGapAlign.

[0044] In the context of this invention, unless otherwise specified, the numbering system proposed by Honegger & Pluckthun ("AHo") is used (Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670). In particular, the following residues are defined as CDRs according to the AHo numbering scheme: LCDR1 (also called CDR-L1): L24~L42; LCDR2 (also called CDR-L2): L58~L72; LCDR3 (also called CDR-L3): L107~L138; HCDR1 (also called CDR-H1): H27~H42; HCDR2 (also called CDR-H2): H57~H76; HCDR3 (also called CDR-H3): H108~H138. To clarify, the numbering system by Honegger & Pluckthun takes into account the length diversity found in naturally occurring antibodies, particularly in the CDR subfamily, both the different VH and VL subfamilies, providing gaps in the sequence. Therefore, in a particular antibody variable domain, not all amino acid residues typically occupy positions 1 through 149.

[0045] As used herein, the term “binding specificity” refers to the ability of an individual antibody to react with one antigenic determinant but not with a different antigenic determinant. As used herein, the terms “specifically bind to” or “specific to” refer to a measurable and reproducible interaction, such as binding of an antibody to a target, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target more readily and / or for a longer duration with higher affinity, binding strength, than an antibody that binds to other targets. In the most common format (unless a defined reference is mentioned), “specific binding” refers to the ability of an antibody to distinguish a molecule unrelated to the target of interest, as measured, for example, according to specificity assay methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) assays, and peptide scans. For example, a standard ELISA assay can be performed. Scoring can be performed by standard colorimetric assays (e.g., secondary antibodies using horseradish peroxidase, and tetramethylbenzidine using hydrogen peroxide). Reactions in a particular well are scored by optical density at, for example, 450 nm. A typical background (= negative reaction) is approximately 0.1 OD. A typical positive reaction is approximately 1 OD. This means that the ratio of positive to negative scores can be more than 10-fold. In further examples, SPR assays can be performed where a difference of at least 10-fold, and especially at least 100-fold, between the background and the signal indicates specific binding. Typically, the determination of binding specificity is performed using a set of about 3-5 unrelated molecules, such as milk powder or transferrin, rather than a single reference molecule.

[0046] In a further embodiment, the present invention relates to a multispecific antibody comprising: a) One or two antibody variable domains as defined herein; b) At least one binding domain that specifically binds to a target different from IL-4R, in particular, wherein the at least one binding domain is hSA-BD and / or IL31-BD.

[0047] In certain embodiments, the multispecific antibody of the present invention does not contain an immunoglobulin Fc region.

[0048] In these specific embodiments, the multispecific antibody is preferably in a format selected from the group consisting of tandem scDb (Tandab), linear dimer scDb (LD-scDb), cyclic dimer scDb (CD-scDb), tandem tri-scFv, tribody (Fab-(scFv)2), Fab-Fv2, triabody, scDb-scFv, tetrabody, di-diabody, tandem-di-scFv, and MATCH (as described in International Publication No. 2016 / 0202457; Egan T., et al., MABS 9 (2017) 68-84). In particular, the multispecific antibody of the present invention is in MATCH format. More specifically, the multispecific antibody of the present invention is in MATCH3 or MATCH4 format.

[0049] As used herein, the terms “immunoglobulin Fc region” or “Fc region” are used to define the C-terminal region of the immunoglobulin heavy chain, i.e., the CH2 and CH3 domains of the heavy chain constant region. The term “Fc region” includes native sequence Fc regions and variant Fc regions, i.e., Fc regions that have been engineered to exhibit specific desired properties, such as modified Fc receptor binding function and / or reduced or suppressed Fab arm replacement. An example of such engineered Fc regions is the knob-into-hole (KiH) technique (see, for example, Ridgway et al., Protein Eng. 9:617-21 (1996) and Spiess et al., J Biol Chem. 288(37):26583-93 (2013)). Natural sequence Fc regions include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In particular, FcR is the natural human FcR sequence, which binds to IgG antibodies (gamma receptors) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors (including allele variants and alternative splice forms of these receptors), the FcγRII receptor, which includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), these of which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 5:203-234 (1997)).FcRs are outlined in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capet et al, Immunomethods 4: 25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those to be identified in the future, are included in the term “FcR” as used herein. The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is responsible for the transport of maternal IgG to the fetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994). Methods for measuring binding to FcRn are publicly known (e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem. TJI (8): 6213-6 (2004); International Publication No. 2004 / 92219 (Hinton et al)). In vivo binding to FcRn and the serum half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice, transfected human cell lines expressing human FcRn, or primates administered with polypeptides containing variant Fc regions. International Publication No. 2004 / 42072 (Presta) describes antibody variants that improve or reduce binding to FcR. See, for example, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0050] In other specific embodiments, the multispecific antibody of the present invention includes an immunoglobulin Fc region.

[0051] Furthermore, in certain embodiments, the multispecific antibody of the present invention includes an IgG region.

[0052] As used herein, the term “IgG region” refers to the heavy and light chains of immunoglobulin G, i.e., the Fc region as defined above, and the Fab region consisting of the VL, VH, CL, and CH1 domains. The term “IgG region” includes naturally occurring IgG regions, such as human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4, as well as engineered IgG regions exhibiting specific desired properties, such as those defined above for the Fc region.

[0053] In certain embodiments, the immunoglobulin Fc region contained in the multispecific antibody of the present invention is selected from the Fc region of the IgG subclass, particularly from the Fc region of the IgG subclasses IgG1 and IgG4, and especially from the Fc region of IgG4.

[0054] As used herein, terms such as “binding domain,” “antigen-binding fragment,” and “antigen-binding moiety” refer to one or more parts of an intact antibody that retain the ability to specifically bind to a particular antigen. The antigen-binding function of an antibody is performed by fragments of the intact antibody. Specifically, in the case of the multispecific antibody of the present invention, terms such as “binding domain,” “antigen-binding fragment,” and “antigen-binding moiety” as used herein refer to the Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; the Fv fragment consisting of the VL and VH domains of a single arm of the antibody; the disulfide-stabilized Fv fragment (dsFv); and the single-chain Fv fragment (scFV). Preferably, the binding domain of the multispecific antibody of the present invention is independently selected from the Fab fragment, Fv fragment, scFv fragment, and single-chain Fv fragment (scFv). In certain embodiments, the binding domain of the antibody of the present invention is independently selected from the Fab fragment and the single-chain Fv fragment (scFv). In other specific embodiments, the VL and VH domains of the scFv fragment are stabilized by interdomain disulfide bonds, in particular, the VH domain containing a single cysteine ​​residue at position 51 (AHo numbering), and the VL domain containing a single cysteine ​​residue at position 141 (AHo numbering).

[0055] Appropriately, the antibody variable domain of the present invention is an isolated variable domain. Similarly, the multispecific antibody of the present invention is an isolated antibody. As used herein, the terms “isolated variable domain” or “isolated antibody” refer to a variable domain or antibody that substantially does not contain other variable domains or other antibodies having different antigen specificities (for example, an isolated antibody variable domain that specifically binds to IL-4R substantially does not contain an antibody variable domain that specifically binds to antigens other than IL-4R). Furthermore, an isolated antibody variable domain or isolated antibody may substantially not contain other cellular material and / or chemical substances.

[0056] More appropriately, the antibody variable domain and multispecific antibody of the present invention are monoclonal antibody variable domains and antibodies. As used herein, the terms “monoclonal antibody variable domain” or “monoclonal antibody” refer to variable domains or antibodies having substantially identical amino acid sequences or originating from the same gene source. Monoclonal variable domains or antibodies exhibit binding specificity and affinity to a particular epitope, or multiple binding specificity and multiple affinities to multiple specific epitopes.

[0057] The antibody variable domain and multispecific antibody of the present invention are not particularly limited to, but include, chimeric antibodies, human antibodies, and humanized antibody variable domains and antibodies.

[0058] As used herein, the term “chimeric antibody variable domain” refers to an antibody molecule or antibody variable domain in which (a) a constant region or part thereof has been modified, substituted, or exchanged so as to be linked to a constant region of a different or modified class, effector function, and / or species, or (b) a variable region or part thereof has been modified, substituted, or exchanged with a variable region having a different or modified antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with the constant region of a human immunoglobulin. Due to the substitution with the human constant region, the chimeric antibody can retain its specificity for recognizing the antigen, while its antigenicity in humans is reduced compared to the original mouse antibody.

[0059] As used herein, the terms “human antibody” or “human antibody variable domain” are intended to include antibodies or antibody variable domains having a variable region in which both the framework region and the CDR region are derived from human sequences. Furthermore, if the antibody or antibody variable domain includes a constant region, that constant region is also derived from such a human sequence, e.g., a human germline sequence or a mutated version of a human germline sequence. The human antibodies and antibody variable domains of the present invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by site-directed mutagenesis in vitro or somatic mutation in vivo). This definition of human antibodies or antibody variable domains specifically excludes humanized antibodies or antibody variable domains that include non-human antigen-binding residues. Human antibodies and antibody variable domains can be generated using various techniques known in the art, e.g., phage-represented libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1992); Marks et al, J. Mol. Biol, 222:581 (1991)). Furthermore, the methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al, J. Immunol, 147(l):86-95 (1991) can also be used for the preparation of human monoclonal antibodies and human monoclonal antibody variable domains. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodies and human antibody variable domains are modified to produce such antibodies and antibody variable domains in response to antigen attack, but their endogenous gene loci have been deactivated. They can be prepared by administering an antigen to transgenic animals, such as immunized xenomouses (see, for example, U.S. Patents 6,075,181 and 6,150,584 relating to XENOMOUSE® technology).For information on human antibodies produced via human B-cell hybridoma technology, see, for example, Li et al, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0060] As used herein, the terms “humanized” antibody or “humanized” antibody variable domain refer to an antibody or antibody variable domain that retains the reactivity of a non-human antibody or antibody variable domain but exhibits low immunogenicity in humans. This can be achieved, for example, by retaining a non-human CDR region and replacing the rest of the antibody or antibody variable domain with a human counterpart (i.e., the constant region and the framework portion of the variable region). Further modifications of the framework region can be made within the human framework sequence and within the CDR sequence derived from the germline of another mammalian species. The humanized antibodies and antibody variable domains of the present invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo, or by conservative substitutions to enhance stability or production). See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239: 1534-1536, 1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec. Immun., 31: 169-217, 1994. Other examples of ergonomic techniques include, but are not limited to, the Xoma techniques disclosed in U.S. Patent No. 5,766,886.

[0061] As used herein, the terms “recombinant humanized antibody” or “recombinant humanized antibody variable domain” include all human antibodies and human antibody variable domains prepared, expressed, produced or isolated by recombinant means, for example, antibodies and human antibody variable domains isolated from host cells transformed to express a humanized antibody or human antibody variable domain from, for example, a transfectoma, and antibodies and human antibody variable domains prepared, expressed, produced or isolated by other means, including splicing all or part of a human immunoglobulin gene or sequence with another DNA sequence.

[0062] Preferably, the antibody variable domain and multispecific antibody of the present invention are humanized. More preferably, the antibody variable domain and multispecific antibody of the present invention are humanized and contain rabbit-derived CDRs.

[0063] As used herein, the term “multispecific antibody” refers to an antibody that binds to two or more different epitopes on at least two or more different targets (e.g., IL-4R and IL-31). Preferably, the multispecific antibody of the present invention is bispecific. As used herein, the term “bispecific antibody” refers to an antibody that binds to at least two different epitopes on two different targets (e.g., IL-4R and IL-31).

[0064] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes are typically composed of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural and charge properties. "Stereoscopic" epitopes and "linear" epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not.

[0065] As used herein, the term “structural epitope” refers to the amino acid residues of an antigen that assemble on the surface when a polypeptide chain folds to form a native protein.

[0066] The term "linear epitope" refers to an epitope where all interaction points between a protein and interacting molecules (such as antibodies) are linearly (continuously) located along the primary amino acid sequence of the protein.

[0067] As used herein, the term “recognize” refers to an antibody or antibody variable domain that finds its three-dimensional epitope and interacts with it (e.g., binds to it).

[0068] Preferably, the multispecific antibody of the present invention comprises one or two antibody-variable domains that specifically bind to IL-4R, as defined herein. In particular, the multispecific antibody of the present invention comprises two antibody-variable domains that specifically bind to IL-4R, as defined in the specification.

[0069] The term "IL-4R" specifically refers to human IL-4R having UniProt ID number P24394. The antibody variable domain of the present invention targets human IL-4R. In particular, the antibody variable domain of the present invention targets human and cynomolgus monkey (Macaca fascicularis) IL-4R. More specifically, the antibody variable domain of the present invention targets human, cynomolgus monkey (Macaca fascicularis), and marmoset (Callithrix jacchus) IL-4R.

[0070] When the antibody variable domain of the present invention is in scFv format, it is characterized by the following parameters: a. When measured by surface plasmon resonance (SPR), human IL-4R has a monovalent dissociation constant (K) of 0.1 to 200 pM. D ), especially K at 0.1-100 pM, especially 0.1-50 pM D Combined, b. Cross-reactivity with cynomolgus monkey (Macaca fascicularis) IL-4R is observed, and in particular, when measured by SPR, cynomolgus monkey IL-4R contains 1 pM to 5 nM, especially 1 pM to 3 nM, and especially 1 pM to 2 nM of monovalent potassium. D Combined, c. When measured by differential scanning fluorescence (DSF), it has a melting temperature (Tm) of at least 63°C, particularly at least 65°C, particularly at least 67°C, particularly at least 69°C, particularly at least 70°C, particularly at least 71°C, and particularly at least 72°C, where the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl.

[0071] In a specific embodiment, the antibody variable domain of the present invention, when in scFv format, is characterized by the following parameters: a. When measured by surface plasmon resonance (SPR), human IL-4R has a monovalent dissociation constant (K) of 0.1 to 200 pM. D ), especially K at 0.1-100 pM, especially 0.1-50 pM D Combined, b. Cross-reactivity with cynomolgus monkey (Macaca fascicularis) IL-4R is observed, and in particular, when measured by SPR, cynomolgus monkey IL-4R shows 1 pM to 5 nM, especially 1 pM to 3 nM, and especially 1 pM to 2 nM of monovalent potassium. D Combined, c. When measured in HEK-Blue cells using a stat-6 reporter gene assay, human IL-4-induced signaling was measured in IC50 cells with an IC50 of 0.01-5 ng / ml. 50 And especially ICs of 0.01~3 ng / ml 50 And in particular, ICs with a concentration of 0.01 to 1.5 ng / ml 50 Neutralize with, d. When measured in HEK-Blue cells using a stat-6 reporter gene assay, human IL-13-induced signaling was measured in IC50 cells of 0.01-10 ng / ml. 50 And especially ICs of 0.01~5 ng / ml 50 And especially ICs with concentrations of 0.01 to 2.5 ng / ml 50 Neutralize with, e. When measured by differential scanning fluorescence (DSF), it has a melting temperature (Tm) of at least 63°C, particularly at least 65°C, particularly at least 67°C, particularly at least 69°C, particularly at least 70°C, particularly at least 71°C, and particularly at least 72°C, in particular, where the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl. f. When the initial concentration of the scFv is 10 mg / ml, the loss of monomer content after storage at 4°C for 4 weeks is less than 5%, and in particular, the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl.

[0072] In a more specific embodiment, the antibody variable domain of the present invention, when in scFv format, is characterized by the following parameters: a. When measured by surface plasmon resonance (SPR), human IL-4R has a monovalent dissociation constant (K) of 0.1 to 200 pM. D ), especially K at 0.1-100 pM, especially 0.1-50 pM D Combined, b. Cross-reactivity with cynomolgus monkey (Macaca fascicularis) IL-4R is observed, and in particular, when measured by SPR, cynomolgus monkey IL-4R shows 1 pM to 5 nM, especially 1 pM to 3 nM, and especially 1 pM to 2 nM of monovalent potassium. D Combined, c. When measured in HEK-Blue cells using a stat-6 reporter gene assay, human IL-4-induced signaling was measured in IC50 cells with an IC50 of 0.01-5 ng / ml. 50 And especially ICs of 0.01~3 ng / ml 50 And in particular, ICs with a concentration of 0.01 to 1.5 ng / ml 50 Neutralize with, d. When measured in HEK-Blue cells using a stat-6 reporter gene assay, human IL-13-induced signaling was measured in IC50 cells of 0.01-10 ng / ml. 50 And especially ICs of 0.01~5 ng / ml 50 And especially ICs with concentrations of 0.01 to 2.5 ng / ml 50 Neutralize with, e. When measured by competitive ELISA, the binding of human IL-4 to human IL-4R was measured in IC50 at concentrations of 0.01 to 10 ng / ml. 50 And especially ICs of 0.01~5 ng / ml 50 And especially ICs of 0.01~2 ng / ml 50 inhibited by, f. When measured by differential scanning fluorescence (DSF), it has a melting temperature (Tm) of at least 63°C, particularly at least 65°C, particularly at least 67°C, particularly at least 69°C, particularly at least 70°C, particularly at least 71°C, and particularly at least 72°C, in particular, where the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl. g. When the initial concentration of the scFv is 10 mg / ml, the loss of monomer content after storage at 4°C for 4 weeks is less than 5%, and in particular, the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl. h. When the initial concentration of scFv is 10 mg / ml, the loss of protein content after storage at 40°C for 4 weeks is less than 5%, particularly less than 4%, particularly less than 3%, particularly less than 2%, particularly less than 1%, and in particular, here the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl.

[0073] In a more specific embodiment, the antibody variable domain of the present invention, when in scFv format, is characterized by the following parameters: a. When measured by surface plasmon resonance (SPR), human IL-4R has a monovalent dissociation constant (K) of 0.1 to 200 pM. D ), especially K at 0.1-100 pM, especially 0.1-50 pM D Combined, b. Cross-reactivity with cynomolgus monkey (Macaca fascicularis) IL-4R is observed, and in particular, when measured by SPR, cynomolgus monkey IL-4R shows 1 pM to 5 nM, especially 1 pM to 3 nM, and especially 1 pM to 2 nM of monovalent potassium. D Combined, c. Marmoset (Callithrix jacchus) shows cross-reactivity with IL-4R, and in particular, when measured by SPR, marmoset IL-4R contains 1 pM to 5 nM, especially 1 pM to 3 nM, and especially 1 pM to 2 nM of monovalent potassium. D Combined, d. In HEK-Blue cells, measurement using the stat-6 reporter gene assay revealed that human IL-4-induced signaling was reduced to 0.01-5 ng / ml IC50. 50 And especially ICs of 0.01~3 ng / ml 50 And in particular, ICs with a concentration of 0.01 to 1.5 ng / ml 50 Neutralize with, In e.HEK-Blue cells, the stat-6 reporter gene assay measured human IL-13-induced signaling, with IC50 ranging from 0.01 to 10 ng / ml. 50 And especially ICs of 0.01~5 ng / ml 50 And especially ICs with concentrations of 0.01 to 2.5 ng / ml 50 Neutralize with, f. When measured by competitive ELISA, the binding of human IL-4 to human IL-4R was measured in IC50 at concentrations of 0.01 to 10 ng / ml. 50 And especially ICs of 0.01~5 ng / ml 50 And especially ICs of 0.01~2 ng / ml 50 inhibited by, g. When measured by differential scanning fluorescence (DSF), it has a melting temperature (Tm) of at least 63°C, particularly at least 65°C, particularly at least 67°C, particularly at least 69°C, particularly at least 70°C, particularly at least 71°C, and particularly at least 72°C, in particular, where the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl. h. When the initial concentration of the scFv is 10 mg / ml, the loss of monomer content after storage at 4°C for 4 weeks is less than 5%, and in particular, the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl. i. When the initial concentration of scFv is 10 mg / ml, the loss of protein content after storage at 40°C for 4 weeks is less than 5%, particularly less than 4%, particularly less than 3%, particularly less than 2%, particularly less than 1%, and in particular, here the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl. j. After 5 freeze-thaw cycles, if the initial concentration of the scFv is 10 mg / ml, the loss of monomer content is less than 2%, particularly less than 1%, and in particular, the scFv is prepared in a 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl.

[0074] As used herein, the terms “HEK-Blue cells” or “HEK-Blue” refer to commercially available human embryonic kidney cells transfected with and stably expressed with an optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter induced by an NF-κB transcription factor. The level of SEAP protein released into the culture medium is typically used as a measure of NF-κB activation.

[0075] As used herein, the term "affinity" refers to the strength of the interaction between an antibody or antibody variable domain and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody or antibody variable domain "arm" interact with the antigen at many sites via weak non-covalent bonds. The more interactions there are, the stronger the affinity.

[0076] "Binding affinity" generally refers to the sum of the strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antibody variable domain) and its binding partner (e.g., an antigen, or more specifically, an epitope or antigen on the antigen). Unless otherwise specified, as used herein, "binding affinity," "bind to," "binds to," or "binding to" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of the binding pair (e.g., the antibody variable domain and the antigen). The affinity of molecule X for partner Y is generally expressed by the dissociation constant (K).D ) can be expressed as. Affinity can be measured by common methods known in the art, including the methods described herein. Low-affinity antibodies and antibody variable domains generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and maintain a longer binding state. Various methods for measuring binding affinity are known in the art and any of them can be used for the purposes of the present invention. Specific exemplary embodiments for measuring binding affinity, i.e., binding strength, are described below.

[0077] The term "K" as used in this specification assoc "K" a " or "K on The term "K" is intended to refer to the association rate of a particular antibody-antigen interaction, whereas "K" as used herein refers to the association rate of a particular antibody-antigen interaction. dis "K" d " or "K off The term "K" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. In one embodiment, "K" as used herein is used to mean "K D The term "K" is d vs K a The ratio of (i.e., K d / K a The term "K" according to the present invention is intended to refer to the dissociation constant obtained from and expressed in terms of molar concentration (M). D " or "K D The value, or KD, or KD value, is measured in one embodiment using a surface plasmon resonance assay.

[0078] The affinity for recombinant human IL-4R, recombinant cynomolgus monkey IL-4R, and recombinant marmoset IL-4R was measured by surface plasmon resonance (SPR) measurements, as described in paragraphs

[0197] to

[0199] (scFv) and

[0238] (multispecific molecules).

[0079] The antibody-variable domain of the present invention acts as an antagonist of IL-4R. In other words, the antibody-variable domain of the present invention is an inhibitor of IL-4R signaling. As used herein, the terms “blocker,” “inhibitor,” or “antagonist” refer to an antibody or antibody-variable domain that inhibits or reduces the biological activity of the antigen to which it binds. The antibody-variable domain of the present invention binds to IL-4R, thereby blocking the binding of IL-4 to IL-4R, particularly the binding of both IL-4 and IL-13 to IL-4R, thereby reducing IL-4R function.

[0080] DSF has already been described (Egan, et al., MAbs, 9(1) (2017), 68-84; Niesen, et al., Nature Protocols, 2(9) (2007) 2212-2221). The midpoint of the thermal unfolding transition of the scFv construct is determined by differential scanning fluorescence (SIMRI) using the fluorescent dye SYPRO® orange (see Wong & Raleigh, Protein Science 25 (2016) 1834-1840). The sample in phosphate-citrate buffer (pH 6.4) is prepared to a final protein concentration of 50 μg / ml, with a total volume of 100 μl containing 5x SYPRO® orange. 25 μl of the prepared sample is added in triples to a white-walled AB gene PCR plate. This assay is performed on a qPCR machine used as a thermal cycler, and fluorescence emission is detected using a custom dye calibration routine in the software. PCR plates containing the test sample are heated from 25°C to 96°C in 1°C increments, with a 30-second pause after each temperature increase. The total assay time is approximately 2 hours. Tm is calculated using the GraphPadPrism software, employing the mathematical second derivative method to determine the inflection point of the curve. The reported Tm is the average of three measurements.

[0081] The loss of monomer content is determined by calculating the area under the curve of the SE-HPLC chromatogram. SE-HPLC is a separation technique based on a solid stationary phase and a liquid mobile phase, outlined in Chapter 621 of the United States Pharmacopeia (USP). This method utilizes a hydrophobic stationary phase and an aqueous mobile phase to separate molecules based on their size and shape. Molecular separation is determined by the void volume (V0) and total osmotic volume (V0) of a particular column. T This occurs between ). SE-HPLC measurements are performed using a Chromaster HPLC system (Hitachi High-Technologies Corporation) equipped with automated sample injection and a UV detector set to a detection wavelength of 280 nm. This instrument is controlled by EZChrom Elite software (Agilent Technologies, version 3.3.2SP2), which also supports the analysis of the resulting chromatograms. Protein samples are clarified by centrifugation, kept at a temperature of 4-6°C in the autosampler, and then injected. For the analysis of scFv samples, a Shodex KW403-4F column (Showa Denko Inc., #F6989202) is used with a standardized buffered saline mobile phase (50 mM sodium phosphate pH 6.5, 300 mM sodium chloride) at a recommended flow rate of 0.35 ml / min. The amount of target sample per injection was 5 μg. The sample is detected by the UV detector at a wavelength of 280 nm, and the data is recorded by an appropriate software suite. The resulting chromatograms are V0-V T The analysis is performed within this range, thereby excluding matrix-related peaks with elution times exceeding 10 minutes.

[0082] More appropriately, the antibody variable domains of the present invention are the binding domains provided in this disclosure. These include, but are not limited to, humanized antibody variable domains derived from rabbit antibody clone 44-34-C10, the sequences of which are listed in Table 1.

[0083] The term "multivalent antibody" refers to a single binding molecule with multiple valencies, where "valency" is described as the number of antigen-binding sites that bind to an epitope on the target molecule. Therefore, a single binding molecule can bind to multiple binding sites on a target molecule and / or to multiple target molecules due to the presence of multiple copies of the corresponding antigen-binding site. Examples of multivalent antibodies, though not limited to them, include bivalent, trivalent, tetravalent, pentavalent, and hexavalent antibodies.

[0084] As used herein, the term "monovalent antibody" refers to an antibody that binds to a single target molecule, or more specifically, to a single epitope on a target molecule. Similarly, as used herein, the term "binding domain" or "monovalent binding domain" refers to a binding domain that binds to a single epitope on a target molecule.

[0085] In certain embodiments, the multispecific antibody of the present invention comprises one antibody variable domain that specifically binds to IL-4R, as defined herein, and one binding domain that binds to a target different from IL-4R, i.e., the multispecific antibody of the present invention is monovalent with respect to both IL-4R and a target different from IL-4R.

[0086] In a more specific embodiment, the multispecific antibody of the present invention comprises one antibody variable domain that specifically binds to IL-4R as defined herein, and one binding domain that has the same binding specificity but binds to a target different from IL-4R, i.e., the multispecific antibody of the present invention is monovalent with respect to IL-4R specificity and bivalent with respect to targets different from IL-4R.

[0087] In a more specific embodiment, the multispecific antibody of the present invention comprises one antibody variable domain that specifically binds to IL-4R, as defined herein, and one binding domain that binds to a target other than IL-4R, i.e., the multispecific antibody of the present invention is divalent with respect to IL-4R specificity and monovalent with respect to a target other than IL-4R.

[0088] In a preferred embodiment, the multispecific antibody of the present invention comprises two antibody-variable domains that specifically bind to IL-4R as defined herein, and two binding domains that have the same binding specificity but specifically bind to targets other than IL-4R, i.e., the multispecific antibody of the present invention is divalent with respect to IL-4R specificity and divalent with respect to targets other than IL-4R.

[0089] When the multispecific antibody of the present invention comprises two binding domains, which have the same binding specificity and specifically bind to a target different from IL-4R, the two binding domains bind to the same or different epitopes on the target molecule. Preferably, the two binding domains bind to the same epitope on the target molecule.

[0090] As used herein, the term “same epitope” refers to individual protein determinants on a protein that can specifically bind to multiple antibodies, where the individual protein determinants are identical, i.e., they consist of the same chemically active surface group of molecules such as amino acids or sugar side chains having the same three-dimensional structural properties and the same charge properties for each of the antibodies.

[0091] As used herein in relation to specific protein targets, the term “different epitopes” refers to individual protein determinants on a protein, each capable of specifically binding to a different antibody, where these individual protein determinants consist of non-identical chemically active surface groups of molecules, such as amino acids or sugar side chains, that are not identical to different antibodies, i.e., have different three-dimensional structural properties and different charge properties. These different epitopes may or may not overlap.

[0092] In certain embodiments, the multispecific antibody of the present invention is bispecific and bivalent.

[0093] Furthermore, in certain embodiments, the multispecific antibody of the present invention is bispecific and trivalent.

[0094] Preferably, the multispecific antibody of the present invention is bispecific and quadrivalent, that is, bivalent for IL-4R and bivalent for targets other than IL-4R.

[0095] In a particular embodiment, the present invention relates to a multispecific antibody comprising: a) Two antibody variable domains as described herein; b) Two binding domains that have the same binding specificity to a target different from IL-4R and specifically bind to it, in particular, where the binding domain is hSA-BD or IL4R-BD, Here, the multispecific antibody includes an IgG region.

[0096] Other variable domains used in the present invention include mutated amino acid sequences in which, compared to the CDR region shown in the sequences in Table 1, 1, 2, 3, 4, or 5 or fewer amino acids are mutated within the CDR region, however, such other variable domains exhibit the functional features of paragraph

[0070] and optionally paragraphs

[0071] to

[0073] .

[0097] Appropriately, the VH domain of the binding domain of the present invention belongs to the VH3 or VH4 family. In one embodiment, the binding domain used in the present invention includes a VH domain belonging to the VH3 family. In the context of the present invention, the term “belonging to the VHx family (or VLx family)” means that the framework sequences FR1 to FR3 show the highest homology to the VHx family (or VLx, respectively). Examples of the VH and VL families are described in Knappik et al., J. Mol. Biol. 296 (2000) 57-86, or International Publication No. 2019 / 057787. A specific example of a VH domain belonging to the VH3 family is shown in Sequence ID No. 19, and a specific example of a VH domain belonging to the VH4 family is shown in Sequence ID No. 20. In particular, the framework regions FR1 to FR3 from Sequence ID No. 19 belong to the VH3 family (Table 2, regions marked in non-bold). Appropriately, the VHs belonging to the VH3 family as used herein are VHs that include FR1-FR3 of Sequence ID No. 19 and FR1-FR3 having at least 90%, more specifically at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity. Alternative examples of VH3 and VH4 sequences, and examples of other VHx sequences, can be found in Knappik et al., J. Mol. Biol. 296 (2000) 57-86, or in International Publication No. 2019 / 057787.

[0098] Preferably, the VL domain of the binding domain used in the present invention includes a framework FR4 selected from Vκ frameworks FR1, FR2, and FR3, particularly Vκ1 or Vκ3 frameworks, particularly Vκ1 frameworks FR1 to FR3, and Vκ FR4. If the binding domain is in scFv format, the binding domain includes a framework FR4 selected from Vκ frameworks FR1, FR2, and FR3, particularly Vκ1 or Vκ3 frameworks, particularly Vκ1 frameworks FR1 to FR3, and Vκ FR4 and Vλ FR4, particularly Vλ FR4.

[0099] A suitable Vκ1 framework FR1-FR3 and an exemplary Vλ FR4 are shown in SEQ ID NO: 21 (Table 2, FR regions are marked in non-bold). Alternative examples of the Vκ1 sequence, and examples of the Vκ2, Vκ3, or Vκ4 sequences, can be found in Knappik et al., J. Mol. Biol. 296 (2000) 57-86. A suitable Vκ1 framework FR1-FR3 includes amino acid sequences corresponding to FR1-FR3 that have at least 80, 90, and 95 percent identity with the amino acid sequence obtained from SEQ ID NO: 21 (Table 2, FR regions are marked in non-bold). A suitable Vλ FR4 is described in SEQ ID NOs. 22-28 and SEQ ID NO: 29, and in particular includes a single cysteine ​​residue where a second single cysteine ​​is present, especially at position 51 (AHo numbering) of the corresponding VH chain, for the formation of an interdomain disulfide bond. In one embodiment, the VL domain of the binding domain of the present invention, when in scFv format, includes a Vλ FR4 having at least 80, 90, or 95 percent identity with an amino acid sequence selected from any of SEQ ID NOs. 22 to 29, particularly SEQ ID NOs. 22 or 29.

[0100] The antibody variable domain of the present invention comprises a VH domain listed in Table 1. Preferably, the antibody variable domain of the present invention comprises a VH amino acid sequence listed in Table 1, wherein five or fewer amino acids, particularly four or fewer amino acids, particularly three or fewer amino acids, particularly two or fewer amino acids, and particularly one or fewer amino acids in the framework sequence (i.e., a sequence that is not a CDR sequence) are mutated (wherein mutation is, in various non-limiting examples, addition, substitution, or deletion). Other binding domains used in the present invention include mutated amino acids that have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the VH region shown in the corresponding sequences listed in Table 1, and include a VL domain that includes at least positions 5-140 (AHo numbering), particularly at least positions 3-145, of one of the sequences shown in Table 1, provided that such other variable domains exhibit the functional features of paragraph

[0070] and optionally, additionally, paragraphs

[0071] -

[0073] .

[0101] In particular, the antibody variable domain of the present invention comprises a VL domain listed in one of Table 1. Preferably, the antibody variable domain of the present invention comprises a VL amino acid sequence listed in one of Table 1, wherein five or fewer amino acids, particularly four or fewer amino acids, particularly three or fewer amino acids, particularly two or fewer amino acids, and particularly one or fewer amino acids are mutated in the framework sequence (i.e., a sequence that is not a CDR sequence) (where mutation is, in various non-limiting examples, addition, substitution, or alteration). Other binding domains used in the present invention include mutated amino acids that have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the VL region shown in the sequences listed in Table 1, and include a VL domain that includes at least positions 5-140 (AHo numbering), particularly at least positions 3-145, of one of the sequences shown in Table 1, provided that such other variable domains exhibit the functional features of paragraphs

[0071] -

[0073] , and optionally, additionally, the functional features of paragraphs

[0071] -

[0073] .

[0102] Specific but non-limiting examples of the antibody variable domains of the present invention are scFv PRO1517, PRO1535, PRO1747, PRO1896, PRO1897, PRO1898, and PRO1899, the sequences of which are listed in Table 3.

[0103] Preferably, at least one binding domain of the multispecific antibody of the present invention is selected from the group consisting of Fab, Fv, dsFv, and scFv.

[0104] Appropriately, the antibody-variable domains and binding domains contained in the multispecific antibodies of the present invention can bind simultaneously to their respective antigens or receptors. As used in this context, the term “simultaneously” refers to the simultaneous binding of at least one antibody-variable domain that specifically binds to IL-4R and at least one binding domain that has specificity for a target different from IL-4R.

[0105] Ideally, the antibody-variable domain and binding domain contained in the multispecific antibody of the present invention are operably linked.

[0106] As used herein, the term “operably linked” indicates that two molecules (e.g., polypeptides, domains, binding domains) are linked in a manner that allows each molecule to retain its functional activity. Two molecules can be “functionally linked” directly or indirectly (e.g., via a linker, via a molar, via a linker to a molar). The term “linker” refers to a peptide or other molar optionally located between binding domains or antibody-variable domains as used in this invention. Many strategies can be used to covalently link molecules. These include, but are not limited to, polypeptide linking between the N-terminus and C-terminus of a protein or protein domain, linking via disulfide bonds, and linking via chemical crosslinking reagents. In one embodiment of this invention, the linker is a peptide bond produced by recombinant technology or peptide synthesis. The choice of a linker suitable for a particular case in which two polypeptide chains are linked depends on a variety of parameters, but is not limited to, the properties of the two polypeptide chains (e.g., whether they are naturally oligomerized), the distance between the N-terminus and C-terminus to be linked (if known), and / or the stability of the linker against proteolysis and oxidation. Furthermore, the linker may contain amino acid residues that provide flexibility.

[0107] In the context of this invention, the term “polypeptide linker” refers to a linker consisting of a chain of amino acid residues linked by peptide bonds connecting two domains, each attached to one end of the linker. The polypeptide linker must be long enough to link two molecules in such a way that the two molecules assume the correct three-dimensional structures relative to each other and maintain the desired activity. In certain embodiments, the polypeptide linker has a continuous chain of 2 to 30 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues). Furthermore, the amino acid residues selected to be included in the polypeptide linker must exhibit properties that do not significantly impede the activity of the polypeptide. Therefore, the linker peptide as a whole should not exhibit a charge inconsistent with the polypeptide's activity, interfere with internal folding, or form bindings or other interactions with one or more monomer amino acid residues that severely hinder the binding of the receptor monomer domain. In certain embodiments, the polypeptide linker is an unstructured polypeptide. Useful linkers include glycine-serine, or GS linkers. A "Gly-Ser" or "GS" linker is a polymer of sequential glycine and serine (e.g., (Gly-Ser) n (GSGGS) n (GGGGS) n , and (GGGS) nThis includes (where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers understood by those skilled in the art (e.g., tethers for shaker potassium channels, and a wide variety of other flexible linkers). Glycine-serine polymers are preferred because the oligopeptides containing these amino acids are relatively unstructured and therefore can function as neutral tethers between components. Secondly, serine is hydrophilic and can solubilize what may be spherical glycine chains. Thirdly, similar chains have been shown to be effective in binding subunits of recombinant proteins, such as single-chain antibodies.

[0108] In one group of embodiments, the multispecific antibody of the present invention comprises an immunoglobulin Fc region and is in a format selected from (scFv)2-Fc-(scFv)2 fusion (ADAPTIR), DVD-Ig, DART®, and TRIDENT®. The term "DART®" refers to an antibody format developed by MacroGenics, which comprises an immunoglobulin Fc region polypeptide and one or two bispecific Fv-binding domains fused to the N-terminus of one heavy chain of the Fc region or to the N-terminuses of both Fc regions. The term "TRIDENT®" refers to an antibody format developed by MacroGenics, which comprises an immunoglobulin Fc region polypeptide, one bispecific Fv-binding domain, and one Fab fragment. Both the bispecific Fv-binding domain and the Fab fragment are fused to the N-terminuses of the two heavy chains of the Fc region polypeptide.

[0109] In a group of other embodiments, the format of the multispecific antibody of the present invention is selected from a bivalent bispecific IgG format, a trivalent bispecific IgG format, and a tetravalent bispecific IgG format. In particular, the format of the multispecific antibody is KiH-based IgG, e.g., DuoBodies (bispecific IgG prepared by Duobody technology) (MAbs. 2017 Feb / Mar;9(2):182-212. doi: 10.1080 / 19420862.2016.1268307); DVD-Ig; IgG-scFv fusions, e.g., CODV-IgG, Morrison (IgG CH3-scFv fusion (Morrison-H), or IgG) The following are selected: CL-scFv fusion (Morrison-L), bsAb (scFv bound to the C-terminus of the light chain), Bs1Ab (scFv bound to the N-terminus of the light chain), Bs2Ab (scFv bound to the N-terminus of the heavy chain), Bs3Ab (scFv bound to the C-terminus of the heavy chain), Ts1Ab (scFv bound to the N-terminus of both the heavy and light chains), and Ts2Ab (dsscFv bound to the C-terminus of the heavy chain). More specifically, the format of the multispecific antibody is selected from KiH-based IgG, such as DuoBodies;DVD-Ig;CODV-IgG, and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)), and more specifically from DVD-Ig and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)).

[0110] In certain embodiments of the present invention, the format of the multispecific antibody is selected from Morrison formats, namely Morrison-L format and Morrison-H format. The Morrison-L and Morrison-H formats used in the present invention are tetravalent bispecific molecular formats having an IgGFc region, particularly an IgG4 Fc region. Two highly stable scFv binding domains (the light chain contains Vκ FR1~FR3 in combination with Vλ FR4 (λcap), also referred to here as λcap scFv) are fused to the C-terminus of the heavy chain (Morrison-H) or the light chain (Morrison-L) via linker L1 (see Figure 2).

[0111] Linker L1 is a peptide consisting of 2 to 30 amino acids, more specifically 5 to 25 amino acids, and most specifically 10 to 20 amino acids. In a particular embodiment, linker L1 is one or more units of four glycine amino acid residues and one serine amino acid residue (GGGGS). n This includes n=1, 2, 3, 4, or 5, in particular n=2.

[0112] In a special embodiment of the present invention, the multispecific antibody has the Morrison-L format defined above. In another special embodiment of the present invention, the multispecific antibody has the Morrison-H format defined above.

[0113] When the antibody variable domain contained in the multispecific antibody of the present invention is in the form of an scFv fragment, these scFv fragments include a variable heavy chain domain (VH) and a variable light chain domain (VL) linked by a linker L2.

[0114] Linker L2 is a peptide consisting of 10-40 amino acids, more specifically 15-30 amino acids, and most specifically 20-25 amino acids. In a particular embodiment, linker L2 consists of four glycine amino acid residues and one serine amino acid residue (GGGGS). n (where n=1, 2, 3, 4, 5, 6, 7, or 8, in particular n=4) and one or more units.

[0115] Specific but non-limiting examples of the multispecific antibodies of the present invention, in which the antibody variable domain contained therein is a Fab fragment, are Morrison-L antibodies PRO2198 and PRO2199, whose sequences are listed in Table 4.

[0116] The antibody variable domains and multispecific antibodies of the present invention can be produced using any convenient antibody production method known in the art (for the production of bispecific constructs, see, for example, Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14; for bispecific diabodies and tandem scFv, see Hornig, N. & Farber-Schwarz, A., Methods Mol. Biol. 907 (2012) 713-727 and International Publication No. WO99 / 57150). Specific examples of appropriate methods for preparing bispecific constructs include, in particular, the Genmab technique (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150) and the Merus technique (see de Kruif et al., Biotechnol. Bioeng. 106 (2010) 741-750). Methods for producing bispecific antibodies containing a functional antibody Fc moiety are also known in the art (see, for example, Zhu et al., Cancer Lett. 86 (1994) 127-134, and Suresh et al., Methods Enzymol. 121 (1986) 210-228).

[0117] These methods typically involve generating monoclonal antibodies or monoclonal antibody variable domains by, for example, hybridoma technology to fuse myeloma cells with spleen cells from mice immunized with a desired antigen (see, e.g., Yokoyama et al., Curr. Protoc. Immunol. Chapter 2, Unit 2.5, 2006), or by recombinant antibody engineering (repertory cloning or phage display / yeast display) (see, e.g., Chames & Baty, FEMS Microbiol. Letters 189 (2000) 1-8), and combining antigen-binding domains or fragments or parts thereof of two or more different monoclonal antibodies to obtain bispecific or multispecific constructs using known molecular cloning techniques.

[0118] The multispecific antibodies of the present invention can be prepared by linking the binding specificities of their constituent elements using methods known in the art. For example, each binding specificity of a bispecific molecule can be generated separately and then linked together. When the binding specificity is that of a protein or peptide, various coupling agents or crosslinking agents can be used for covalent bonding. Examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-5-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al., 1984 J. Exp. Med. 160: 1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science 229:81-83; and Glennie et al., 1987 J. Immunol. 139: 2367-2375. The binders are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, Ill, USA).

[0119] Alternatively, two or more binding specificities can be encoded within the same vector and expressed and constructed in the same host cell. This method is particularly useful when the bispecific molecule is an mAb×Fab, mAb×scFv, mAb×dsFv, or mAb×Fv fusion protein. Methods for preparing multispecific antibodies and molecules are described, for example, in U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858.

[0120] The binding of antibody variable domains and multispecific antibodies to these specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex by using a labeling reagent (e.g., antibody) specific to the complex of interest.

[0121] In a further embodiment, the present invention provides one or two nucleic acids encoding the antibody variable domain or a multispecific antibody of the present invention. Such nucleic acids can be optimized for expression in mammalian cells.

[0122] As used herein, the term “nucleic acid” is used interchangeably with the term “polynucleotide” and refers to one or more single-stranded or double-stranded deoxyribonucleotides or ribonucleotides and their polymers. This term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bindings, which are synthetic, natural, and unnatural, possess similar binding properties to a reference nucleic acid, and are metabolized in a similar manner to the reference nucleic acid. Examples of such analogs, but not limited to, include phosphorothioates, phosphoramides, methylphosphonates, chiral methyl phosphates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise explicitly stated, a particular nucleic acid also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. Specifically, as detailed below, degenerate codon substitution can be achieved by generating sequences in which the 3rd position of one or more selected (or all) codons is replaced with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0123] The present invention provides substantially purified nucleic acid molecules encoding polypeptides containing the above-described antibody variable domain or segments or domains of multispecific antibodies. When expressed from a suitable expression vector, the polypeptides encoded by these nucleic acid molecules can exhibit the antigen-binding ability of the multispecific antibodies of the present invention.

[0124] Polynucleotide sequences can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences encoding the multispecific antibodies of the present invention or their variable domains or binding domains (e.g., sequences described in the following examples). Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method of U.S. Patent No. 4,458,066. Methods for introducing mutations into polynucleotide sequences by PCR can be carried out as described in, for example, PCR Technology: Principles and Applications for DNA Amplification, HA Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0125] Furthermore, the present invention also provides an expression vector and host cells for producing the antibody variable domain or multispecific antibody of the present invention.

[0126] The term "vector" is intended to refer to a polynucleotide molecule that can transport another polynucleotide to which it is ligated. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment is ligated. Another type of vector is a viral vector, where the additional DNA segment may be ligated to a viral genome. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating alongside the host genome.

[0127] Furthermore, certain vectors can direct the expression of genes to which they are operably ligated. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Since plasmids are the most commonly used form of vectors, “plasmid” and “vector” may be used interchangeably herein. However, the present invention intends to include other forms of expression vectors, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions. In this particular context, the term “operably ligated” refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to a functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably ligated to a coding sequence if it stimulates or modulates the transcription of the coding sequence in a suitable host cell or other expression system. Generally, promoters and transcriptional regulatory sequences operably ligated to a transcription sequence are physically adjacent to the transcription sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically adjacent to or in close proximity to the coding sequences that enhance transcription.

[0128] Various expression vectors can be used to express polynucleotides encoding antibody-variable domains or multispecific antibody chains. Both virus-based and non-viral expression vectors can be used to produce antibodies or antibody-variable domains in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors typically having expression cassettes for expressing proteins or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat Genet. 15:345, 1997). For example, non-viral vectors useful for expressing IL-4R-binding polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHisA, B, and C, pcDNA3.1 / His, pEBVHisA, B, and C (Invitrogen, San Diego, CA, USA), MPSV vectors, and many other vectors known in the art for expressing other proteins. Useful viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, SV40-based vectors, papillomaviruses, HBP Epstein-Barr virus, vaccinia virus vectors, and Semryki Forest virus (SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68: 143, 1992.

[0129] The choice of expression vector depends on the host cells in which the vector is intended to be expressed. Typically, an expression vector includes a promoter and other regulatory sequences (e.g., enhancers) operably ligated to a polynucleotide encoding a multispecific antibody chain or variable domain. In one embodiment, an inducible promoter is used to prevent the expression of the insert sequence except under inducible conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducible conditions without biasing the population regarding coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may be necessary or desirable for the efficient expression of multispecific antibody chains or variable domains. These elements typically include the ATG start codon and adjacent ribosome-binding sites or other sequences. Furthermore, expression efficiency can be enhanced by including an appropriate enhancer in the cell line used (see, for example, Scharf et al., Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, expression in mammalian host cells can be increased using SV40 enhancers or CMV enhancers.

[0130] The vectors used typically encode an antibody-variable domain or the light and heavy chains of a multispecific antibody, including, if present, a constant region or a portion thereof. Such vectors allow the variable region to be expressed as a fusion protein with the constant region, thereby resulting in the production of an intact antibody and its antibody-variable domain. Typically, such a constant region is human.

[0131] The term “recombinant host cell” (or simply “host cell”) refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terminology is intended to refer not only to a specific target cell but also to the offspring of such cells. Such offspring may not be identical to the parent cell in practice, as mutations or environmental influences may cause certain modifications in subsequent generations, but they are still included within the scope of the term “host cell” as used herein.

[0132] The host cells for harboring and expressing the antibody-variable domain or multispecific antibodies of the present invention may be either prokaryotes or eukaryotes. Escherichia coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present invention. Other suitable microbial hosts include rod-shaped bacteria such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be constructed that typically contain expression regulatory sequences (e.g., replication origins) that are compatible with the host cell. Furthermore, there may be any number of different well-known promoters, such as lactose promoter systems, tryptophan (TRP) promoter systems, beta-lactamase promoter systems, or lambda phage-derived promoter systems. Promoters typically have, optionally selectively, operator sequences along with ribosome-binding site sequences for controlling expression and initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express the antibody-variable domain or multispecific antibodies of the present invention. Insect cells can also be used in combination with baculovirus vectors.

[0133] In one embodiment, mammalian host cells are used to express and produce the antibody variable domain or multispecific antibody of the present invention. These may be, for example, hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines having exogenous expression vectors. These include any normal mortal cells, or normal or abnormal immortal animal or human cells. For example, many suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures for polypeptide expression is outlined, for example, in Winnacker, FROM GENES TO CLONES, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may contain expression regulatory sequences, e.g., replication origins, promoters, and enhancers (see, e.g., Queen, et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites, e.g., ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and / or regulatory or controllable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRPpolIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (e.g., the human pre-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0134] The method for introducing an expression vector containing the target polynucleotide sequence varies depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cell hosts (see Green, MR, and Sambrook, J., Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012)). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation-nucleic acid complexes, naked DNA, artificial virions, fusion to the herpesvirus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced DNA incorporation, and ex vivo transduction. Stable expression is often desired for high yield production of recombinant proteins over long periods. For example, a cell line stably expressing the antibody variable domain or multispecific antibody of the present invention can be prepared using an expression vector of the present invention comprising a viral replication origin or endogenous expression element and a selection marker gene. After introducing the vector, the cells are grown in enriched medium for 1-2 days, and then switched to a selection medium. The purpose of the selection marker is to confer resistance to selection, and its presence enables the growth of cells that successfully express the introduced sequence in the selection medium. The resistant and stably transfected cells can be grown using tissue culture techniques appropriate to the cell type. Accordingly, the present invention provides a method for producing the variable domain or multispecific antibody of the present invention, the method comprising the step of culturing a host cell containing a nucleic acid or vector encoding the antibody variable domain or multispecific antibody of the present invention, thereby expressing the antibody variable domain or multispecific antibody or a fragment thereof of the present disclosure.

[0135] In one embodiment, the present invention relates to a method for producing the antibody variable domain or multispecific antibody of the present invention, the method comprising the step of culturing host cells expressing nucleic acids encoding the antibody variable domain or multispecific antibody of the present invention. In particular, the present invention relates to a method for producing the antibody variable domain or multispecific antibody of the present invention, the method comprising the steps of (i) providing one or two nucleic acids encoding the antibody variable domain or multispecific antibody of the present invention, or one or two vectors encoding the antibody variable domain or multispecific antibody of the present invention, expressing the one or more nucleic acids or the one or more vectors, and collecting the antibody variable domain or multispecific antibody from the expression system, or (ii) providing one or more host cells expressing one or two nucleic acids encoding the antibody variable domain or multispecific antibody of the present invention, culturing the one or more host cells, and collecting the antibody variable domain or multispecific antibody from the cell culture.

[0136] In a further embodiment, the present invention relates to a pharmaceutical composition comprising the multispecific antibody of the present invention and a pharmaceutically acceptable carrier. "pharmaceutically acceptable carrier" means a medium or diluent that does not interfere with the structure of the antibody. The pharmaceutically acceptable carrier enhances or stabilizes the composition or facilitates the preparation of the composition. pharmaceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, and the like.

[0137] Some of these carriers enable the formulation of pharmaceutical compositions as, for example, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral administration by subjects. Some of these carriers enable the formulation of pharmaceutical compositions for injection, infusion, or topical administration. For example, a pharmaceutically acceptable carrier may be a sterile aqueous solution.

[0138] The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route and / or method of administration varies depending on the desired outcome. Administration may be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered near the target site. In certain embodiments, administration is intramuscular or subcutaneous, particularly subcutaneous. A pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion), particularly intramuscular or subcutaneous administration. Depending on the route of administration, the active compound, i.e., the multispecific antibody of the present invention, may be coated with a material that protects the compound from the action of acids that can inactivate the compound and from other natural conditions.

[0139] The pharmaceutical compositions of the present invention can be prepared according to methods well known and routinely practiced in the art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or effective dose of the multispecific antibody of the present invention is used in the pharmaceutical composition of the present invention. The multispecific antibody of the present invention is prepared into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosing plan is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, or it may be administered in several divided doses over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and dose uniformity, it is particularly advantageous to prepare the parenteral composition in unit dosage forms. As used herein, a dosage unit refers to a physically distinct unit suitable as a unit dose for a subject being treated. Each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect when combined with the necessary pharmaceutical carrier.

[0140] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be modified to obtain an amount of the active ingredient effective in achieving a desired therapeutic response for a particular patient, composition, and method of administration without causing toxicity to the patient. The selected dose level depends on various pharmacokinetic factors, such as the activity of the particular composition of the present invention used, or its ester, salt, or amide; the route of administration; the time of administration; the excretion rate of the particular compound used; the duration of treatment; other drugs, compounds, and / or materials used in combination with the particular composition used; the age, sex, weight, condition, general health status, and medical history of the patient receiving treatment; and similar factors.

[0141] The multispecific antibodies of the present invention are typically administered multiple times. The interval between single doses can be weekly, monthly, or yearly. The interval may be irregular, as indicated by the measurement of blood levels of the multispecific antibodies of the present invention in the patient. Alternatively, the multispecific antibodies of the present invention can be administered as a sustained-release formulation, in which case the required frequency of administration will be reduced. The dosage and frequency of administration will vary depending on the half-life of the antibody in the patient. Humanized antibodies generally exhibit a longer half-life than chimeric antibodies or non-human antibodies. The dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic use, relatively low doses are administered over a long period at relatively infrequent intervals. Some patients may continue treatment for life. For therapeutic use, relatively high doses may be administered at relatively short intervals until the progression of the disease is reduced or stopped, preferably until the patient shows partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be given prophylactic therapy.

[0142] In one embodiment, the present invention relates to a multispecific antibody or pharmaceutical composition of the present invention for use as a pharmaceutical agent. In a suitable embodiment, the present invention provides a multispecific antibody or pharmaceutical composition for use in the treatment of allergic diseases, inflammatory diseases, and autoimmune diseases, particularly diseases selected from inflammatory diseases and autoimmune diseases.

[0143] In another aspect, the present invention provides pharmaceutical compositions for use in the manufacture of agents for the treatment of allergic diseases, inflammatory diseases, or autoimmune diseases, particularly inflammatory diseases or autoimmune diseases.

[0144] In another embodiment, the present invention relates to the use of multispecific antibodies or pharmaceutical compositions for treating allergic diseases, inflammatory diseases, or autoimmune diseases, particularly for treating inflammatory diseases or autoimmune diseases in subjects requiring treatment.

[0145] In another embodiment, the present invention relates to a method for treating a subject, comprising administering a therapeutically effective amount of the multispecific antibody of the present invention to the subject. In a suitable embodiment, the present invention relates to a method for treating an allergic disease, an inflammatory disease, or an autoimmune disease in a subject, particularly a method for treating an inflammatory disease or an autoimmune disease, comprising administering a therapeutically effective amount of the multispecific antibody of the present invention to the subject.

[0146] The term "subject" includes both humans and non-human animals.

[0147] The term “animal” includes all vertebrates, such as non-human mammals, and non-human mammals such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” and “subject” are used interchangeably herein.

[0148] As used herein, terms such as “treatment,” “treating,” “treat,” and “treated” refer to obtaining a desired pharmacological and / or physiological effect. This effect may be therapeutic in the sense of partial or complete cure of the disease and / or side effects caused by the disease, or delay in the progression of the disease. As used herein, “treatment” encompasses all treatments of diseases in mammals, such as humans, and includes: (a) inhibiting the disease, i.e., preventing its onset; and (b) reducing the disease, i.e., regressing the disease.

[0149] The terms "therapeutic dose" or "effective dose" refer to the amount of a drug that, when administered to a mammal or other subject to treat a disease, is sufficient to have an effect on treating such disease. The therapeutic dose varies depending on the drug, the disease and its severity, and the age and weight of the subject receiving treatment.

[0150] In one embodiment, allergic diseases, inflammatory diseases, and autoimmune diseases are selected from allergic diseases that cause itching, inflammatory diseases that cause itching, and autoimmune diseases that cause itching, particularly from inflammatory diseases that cause itching and autoimmune diseases that cause itching. The terms “pruritus” and “itching” are used synonymously herein and refer to the sensation that causes the urge or reflex to scratch. In the case of chronic skin diseases that cause itching, such as atopic dermatitis, cutaneous mycoses, psoriasis and urticaria, scratching can be particularly problematic because the persistent itchy irritation causes the patient to constantly and / or excessively scratch the affected area of ​​skin, leading to skin damage and further deterioration of the skin surface.

[0151] As used herein, the terms “allergic disease” or “allergy” refer to a number of conditions caused by hypersensitivity of the immune system to substances in the environment that are normally harmless.

[0152] As used herein, the term “inflammatory disease” often refers to a vast number of inflammatory disorders, namely inflammatory abnormalities characterized by prolonged inflammation, known as chronic inflammation. The term “inflammation” refers to the complex biological response of body tissues to harmful stimuli such as pathogens, damaged cells, or irritants. This is a defense response involving immune cells, blood vessels, and molecular mediators. While regular inflammatory responses are essential for the body to eliminate the initial cause of cell injury, remove necrotic cells and tissues damaged during the initial injury or inflammatory process, and initiate tissue repair, inflammatory diseases are generally characterized by persistent inflammation even without harmful stimuli.

[0153] As used herein, the term “autoimmune disease” refers to a condition resulting from an abnormal immune response against a functioning part of the body. It is a consequence of autoimmunity, or an autoreactive immune response (e.g., autoantibodies, autoreactive T cells), and is usually limited to a specific organ or involves specific tissues in various locations, with or without the presence of resulting damage or pathology.

[0154] In certain embodiments, the inflammatory or autoimmune disease causing pruritus is selected from atopic dermatitis, acute allergic contact dermatitis, chronic spontaneous urticaria, bullous pemphigoid, alopecia areata, dermatomyositis, nodular prurigo, psoriasis, and atopic asthma, particularly from atopic dermatitis.

[0155] In another embodiment, allergic, inflammatory, and autoimmune diseases are selected from atopic dermatitis, acute allergic contact dermatitis, chronic spontaneous urticaria, bullous pemphigoid, alopecia areata, dermatomyositis, nodular prurigo, psoriasis, and atopic asthma, particularly from atopic dermatitis.

[0156] In another embodiment, allergic diseases, inflammatory diseases, and autoimmune diseases are selected from allergic asthma, allergic rhinitis, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, Crohn's disease, chronic nonhistamine-associated urticaria, antihistamine-unresponsive mastocytosis, chronic lichen simplex, seborrheic dermatitis, xerosis, herpetiform dermatitis, lichen planus, and ulcerative colitis.

[0157] In another embodiment, the present invention provides a multispecific antibody or pharmaceutical composition as defined herein for use in the treatment of a disease which is a neuropathic pruritus selected from postherpetic neuralgia, postherpetic itching, paresthesia, multiple sclerosis, and brachioradialis pruritus.

[0158] In another embodiment, the present invention provides multispecific antibodies or pharmaceutical compositions for use as antipruritic agents for the treatment of systemic diseases accompanied by itching, said diseases including cholestasis, chronic kidney disease, Hodgkin's disease, cutaneous T-cell lymphoma and other lymphomas or leukemias accompanied by chronic itching, polycythemia vera, hyperthyroidism, chronic arthropod posterior pruritus (Id reaction), pregnancy-induced chronic pruritus (e.g., PUPPP), eosinophilic pustular folliculitis, drug hypersensitivity reactions, and chronic conditions in the elderly. Selected from pruritus or dry skin itching (localized, systemic), itching in burn scars (post-burn itching), hereditary or nevus-related chronic itching (e.g., Netherton syndrome, Darier disease (Morbus Darier), Hailey-Hailey disease, inflammatory linear verruca nevus (ILVEN), familial primary cutaneous amyloidosis, Olmsted syndrome), pruritus erythematous, fiberglass dermatitis, myxoid chronic itching, chemotherapy-induced itching, and HIV. Sequence listing (mutations specified according to the AHo numbering scheme; CDRs defined according to the NumabCDR definition unless otherwise specified) [Table 1] [Table 2] [Table 3] [Table 4]

[0159] In the event of any inconsistency between the text of the specification (e.g., Tables 1-4) and the sequence listings throughout this application, the text of the specification shall prevail.

[0160] For clarity, it is understood that some features of the present invention described in relation to separate embodiments may be provided in combination in a single embodiment. Conversely, various features of the present invention described in relation to a single embodiment for brevity may also be provided individually or in any suitable partial combination. All combinations of embodiments according to the present invention are specifically encompassed in the present invention and are disclosed herein as if every combination were individually and expressly disclosed. Furthermore, all partial combinations of various embodiments and their elements are also specifically encompassed in the present invention and are disclosed herein as if each of such partial combinations were individually and expressly disclosed herein.

[0161] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention beyond those described herein will be apparent to those skilled in the art from the above description. Such modifications are intended to be included in the appended claims.

[0162] To the extent permitted by the respective patent laws, all patents, applications, publications, test methods, documents, and other materials cited herein are incorporated herein by reference.

[0163] The following examples illustrate the present invention as described above, but are not intended to limit the scope of the invention in any way. Other test models known to those skilled in the art can also be used to determine the beneficial effects of the claimed invention. [Examples]

[0164] Examples Example 1: Production and testing of anti-IL-4R molecules : Project objectives The goal of this project was to generate a humanized monoclonal antibody variable domain that specifically binds to human IL-4R and neutralizes its biological effects. A further goal was to identify an anti-IL-4R antibody variable domain that is potent and stable enough to be incorporated into a multispecific antibody format.

[0165] 1.1.Immunity To obtain an optimal immune response to human IL-4R, two different immunization protocols were applied to a total of six rabbits. The rabbits were immunized with recombinant, purified IL-4R (Sino Biological, catalog number 10402-H08H). Prior to immunization, the quality of recombinant human IL-4R was analyzed by the manufacturer for its bioactivity by 1) purity by SDS-page and 2) binding ability to IL-4 by functional ELISA. Group 1 (Protocol 1), consisting of three rabbits, received four injections of 200 μg each over 70 days. Group 2 (Protocol 2), consisting of three rabbits, received five injections of 200 μg each over 112 days. During the immunization process, the intensity of the humoral immune response to the antigen was qualitatively assessed by determining the maximum dilution (titer) of the serum in each rabbit that still yielded detectable binding of polyclonal serum antibodies to the antigen. Serum antibody titers against immobilized antigen (recombinant human IL-4R) were evaluated using enzyme-linked immunosorbent assay (ELISA). All six rabbits immunized with purified human IL-4R showed titers up to 3 × 10⁶. 7 It showed a high titer of EC5.

[0166] 1.2. Selection Prior to the hit identification procedure, a flow cytometry-based screening procedure using protein G beads was performed in the presence of R-phycoerythrin (RPE)-labeled IL-4R. This enabled the specific detection and isolation of high-affinity IL-4R-binding B cells. This screening campaign involved a total of 1.05 × 10⁶ cells derived from four rabbits. 7Numerous lymphocytes were analyzed. Of these, a total of 3,520 B cells expressing IL-4R-specific antibody (IgG) were isolated and cultured individually as single clones for 3–4 weeks.

[0167] 1.3. Identifying Hits General Overview To identify hits, direct ELISA screening was performed to evaluate binding to human and mouse IL-4R, while binding to cynomolgus monkey IL-4R was evaluated solely by SPR. Direct ELISA revealed that 483 clones bound to human IL-4R, and 164 clones showed cross-reactivity with mouse IL-4R.

[0168] ELISA-based binding to human IL-4R To identify hits, i.e., B cell clones that produce antibodies that bind to IL-4R, the cell culture supernatants of the 3,520 B cell clones mentioned above were screened for the presence of antibodies that bind to human IL-4R using ELISA. Because individual rabbit antibodies cannot be purified at the scale of high-capacity culture, this primary hit identification procedure was performed using unpurified antibodies from the cell culture supernatant. Using the cell supernatant allows for the ranking of a large number of antibodies. The ELISA method used evaluates the "amount" of rabbit IgG bound to recombinant human IL-4R, but does not provide information on antibody affinity or concentration. In this assay, the supernatants of 483 B cell clones generated signals above the background.

[0169] Measurement of binding affinity to human IL-4R In the secondary hit identification procedure, information regarding the binding affinity of 483 monoclonal rabbit antibodies identified as positive during the primary screening to human IL-4R was determined by surface plasmon resonance (SPR).

[0170] For these affinity screenings by SPR, an antibody specific for the Fc region of rabbit IgG was immobilized onto a sensor chip (SPR-2 Affinity Sensor, High Capacity Amine, Sierra Sensors) using standard amine coupling procedures. Rabbit monoclonal antibodies in the B cell supernatants were captured by the immobilized anti-rabbit IgG antibody. To enable sufficient capture, it was necessary to minimize the IgG concentration in the B cell supernatants. After capturing the monoclonal antibodies, human IL-4R ECD (extracellular domain) was injected into the flow cell at a concentration of 90 nM for 3 minutes, and dissociation of the protein from the IgG captured on the sensor chip was allowed to proceed for 5 minutes. The apparent dissociation (k d ) rate constant and association (k a ) rate constant, as well as the apparent dissociation equilibrium constant (K D ), were calculated with MASS-2 analysis software (Analyzer, Sierra Sensors) using a 1:1 Langmuir binding model.

[0171] For 475 anti-IL-4R antibodies, the binding affinity for human IL-4R could be measured. These showed dissociation constants (K -12 ) in the range of less than 2.07×10 -8 M to 2.95×10 D M. 32% of the antibodies analyzed had a K D of less than 0.5 nM, and 17% showed a higher affinity than dupilumab.

[0172] Neutralization of human IL-4R in stat-6 reporter gene cell assays and competitive ELISA All 483 human IL-4R-binding supernatants were further analyzed for their potential to neutralize the biological activity of IL-4R in a cell-based stat-6 reporter gene assay and for their ability to block the interaction between human IL-4 and human IL-4R in a competitive ELISA.

[0173] For this purpose, cell-based stat-6-dependent reporter gene assays and receptor-ligand competitive ELISAs using HEK-Blue cells were developed and adapted for use with B cell supernatant. The cell-based assays allow for the evaluation of blockade of IL-4 and IL-13-induced signaling, while the competitive ELISA evaluates only the interaction between IL-4 and IL-4R. Both assays were used for screening because they can be performed using B cell supernatant as a matrix and are highly sensitive and accurate. Mouse IL-13 present in the B cell supernatant was blocked with a commercially available anti-mouse IL-13 antibody to ensure that signaling is induced only by human IL-13 in the cell-based assays. The inhibitory activity of each B cell supernatant was tested using single-well analysis (no dose-response analysis was performed). Therefore, the degree of inhibition observed depends not only on the properties of the IgG but also on the concentration of rabbit IgG in the B cell supernatant.

[0174] Of the 483 clones, 122 blocked the interaction between human IL-4R and human IL-4 by more than 90%, and 178 clones inhibited it by more than 80%. Based on the >30% inhibition threshold, in the HEK-Blue assay, 119 and 132 clones inhibited IL-4 and IL-13-induced signaling, respectively, and 86 hits inhibited both IL-4-induced and IL-13-induced signaling.

[0175] Species cross-reactivity (binding of IL-4R to cynomolgus monkeys and mice via SPR) All 483 hits identified in the primary screening were analyzed by SPR for species cross-reactivity to cynomolgus monkey and mouse IL-4R. Binding affinity was determined by surface plasmon resonance (SPR) using a MASS-2 SPR device (Sierra Sensors), similar to the method described above for human IL-4R, except that 90 nM cynomolgus monkey or mouse IL-4R was used instead of human IL-4R.

[0176] 423 clones (87.5%) and 266 clones (55%) showed binding to cynomolgus and mouse IL-4R, respectively. The higher number of mouse IL-4R cross-reactive clones found by SPR compared to direct ELISA (266 vs 164) is due to the more stringent cut-off used in ELISA. The anti-cynomolgus IL-4R antibodies showed equilibrium dissociation constants (K -12 in the range of 1.14×10 -6 M to 9.36×10 D M. 11% of all the antibodies analyzed had a K D of less than 500 pM. A generally rather low affinity was determined for the anti-mouse IL-4R antibodies, and the K D values were in the range of 1.63×10 -11 M to 4.57×10 -5 [[ID=I4]]M. Overall, there was no good correlation between the K D values of the species, and there were only about 20 hits showing high affinity for both human and cynomolgus IL-4R.

[0177] 1.4. Hit Selection and Hit Confirmation Hit Selection and RT-PCR As a prerequisite for hit confirmation, gene sequence analysis, and subsequent humanization of rabbit antibodies, it is necessary to retrieve the genetic information encoding the rabbit antibody variable domains. This was achieved by reverse transcription (RT) of each messenger RNA into complementary DNA (cDNA), followed by amplification of the double-stranded DNA by polymerase chain reaction (PCR). The selection of B cell clones subjected to RT-PCR was mainly based on the affinity for human IL-4R of less than 500 pM and the neutralizing activity in the stat-6 reporter gene assay. As an additional criterion, cross-reactivity with cynomolgus IL-4R was considered.

[0178] 94 sets of rabbit CDRs corresponding to 94 independent clones were selected and identified. All rabbit CDR sequences from the 94 sequenced clones were clustered in a phylogenetic tree. 75 sequences were non-overlapping sequences based on the CDR region, and 26 sequences contained free cysteine ​​(considered to be liability for development). All clones with identical sequences or sequences differing by less than 4 amino acids were excluded. The affinity for human IL-4R was 400-500 pM, and the affinity for cynomolgus monkey IL-4R was low (K D Two clones (>3nM) were also excluded from selection, but eight additional clones with free cysteine ​​were included. Although clones with free cysteine ​​tend to aggregate after reformatting, these clones were not discarded at this stage due to their high affinity for cynomolgus monkey IL-4R. As a result, a total of 60 clones were selected for expression as recombinant IgG.

[0179] Cloning and production of monoclonal antibodies Following the selection of clones for hit confirmation, rabbit antibodies were cloned, expressed, and purified for further characterization. Cloning of the corresponding light and heavy chain variable domains required in vitro ligation of DNA fragments into appropriate mammalian expression vectors. Expression vectors for the rabbit antibody heavy and light chains were transfected into mammalian suspension cell lines for transient heterologous expression. Subsequently, secreted rabbit IgG was purified by affinity, and the final product was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), UV absorbance at 280 nm, and size exclusion high-performance liquid chromatography (SE-HPLC) to confirm identity, content, and purity. Of the 60 clones, 58 were successfully cloned and produced with high monomer content (95.9-99.6%) and moderate to very good titers (2-36 μg protein / ml).

[0180] 1.5. Pharmacological Characterization of Monoclonal Antibodies Affinity for human, cynomolgus monkey, marmoset, and mouse IL-4R The binding kinetics of 58 purified monoclonal rabbit antibodies to human IL-4R were determined by SPR(MASS-2) analysis. Each IgG was captured via anti-rabbit IgG bound to the surface of carboxylmethylated dextran, and the dose-response of the analyte was measured. High affinity binding to human IL-4R was observed for K2 antibodies with affinities ranging from picomoles to subpicomoles, with the exception of two IgGs with nanomolar affinities. D Confirmed in all antibodies with a value. Of the 57 target-binding antibodies, 4 had a K content of less than picomoles. D The value was shown.

[0181] The binding kinetics to cynomolgus monkey and marmoset monkey IL-4R were determined in the same manner as described above. Since marmoset IL-4R ECD was not commercially available, it was manufactured on demand by Sino Biological. Of the 58 IgG molecules, 44 and 48 bound to cynomolgus monkey and marmoset IL-4R, respectively. 35 IgG molecules showed cross-reactivity to both monkey species. Marmoset IL-4R showed higher overall affinity compared to human and cynomolgus monkey IL-4R.

[0182] Binding to mouse IL-4R was evaluated using direct ELISA instead of SPR. None of the IgGs showed strong binding, and most curves did not reach a maximum signal (the peak of the curve is undefined), so EC 50 The value could not be determined.

[0183] Stat-6 reporter gene assay in HEK-Blue cells (blockade of human IL-4 and IL-13 induced signaling) Furthermore, the effects of 58 rabbit monoclonal antibodies on IL-4 and IL-13-induced signaling via IL-4R were evaluated in a stat-6 reporter gene assay using HEK-Blue cells. The efficacy of neutralizing signaling induced by 0.02 ng / ml IL-4 or 0.3 ng / ml IL-13 (IC) was assessed.50 The titer of all antibodies was analyzed for serial dilutions and compared to the potency of dupilumab. Relative IC50 was used to compare IgG from different assay plates. 50 The values ​​used were the IC50 of IgG against the reference molecule dupilumab contained in each assay plate. 50 Determined by calibrating the values ​​(relative IC) 50 :I C 50 Dupilumab / IC 50 (Test antibody).

[0184] IgG inhibited IL-4 and IL-13-induced signaling with higher or less than five times the potency of dupilumab. Regarding the blockade of IL-4-induced signaling, seven IgGs showed superior efficacy, and four IgGs blocked both IL-4 and IL-13-induced signaling more potently than dupilumab.

[0185] Competitive ELISA (inhibition of hIL-4 binding to hIL-4R) The inhibition of human IL-4 binding to human IL-4R was evaluated by competitive ELISA. For this purpose, IL-4 was coated onto ELISA plates. Biotinylated IL-4R was pre-incubated with rabbit monoclonal antibody, and the mixture was added to the ELISA plate to bind to immobilized IL-4. The bound biotinylated IL-4R was then detected using streptavidin-HRP.

[0186] Considering the low concentrations of biotinylated IL-4R used in the assay, the competitive ELISA had sufficient sensitivity to distinguish the rabbit antibodies in terms of potency. Compared to dupilumab, 12 rabbit antibodies more potently inhibited the interaction between human IL-4 and human IL-4R, while 25 antibodies showed similar potency or up to 2.5 times lower IC50. 50 The values ​​were shown. For the six antibodies, relative IC was shown due to lack of inhibition or incomplete blockade of interaction. 50 It was not measured.

[0187] Selection of rabbit IgG for humanized scFv generation Based on the pharmacodynamic characteristics of 58 characterized rabbit monoclonal antibodies, the clone exhibiting the best performance was selected for humanization and lead candidate generation. The selection criteria for clones were: i) complete blockade of IL-4 and IL-13-induced signaling in the HEK-Blue assay, ii) high affinity for human IL-4R, iii) neutralization of the interaction of human IL-4R with human IL-4 in competitive ELISA, iv) cross-reactivity to cynomolgus monkey and / or marmoset IL-4R by SPR, and v) sequence diversity. In addition to these criteria, the primary sequences of all 58 clones were screened for the presence of unpaired cysteine ​​in the complementarity-determining region (CDR). In identifying unpaired cysteine, pairs of cysteine ​​commonly present in CDR-H1 and CDR-H2 were excluded. These cysteine ​​pairs are encoded in the rabbit germline repertoire and are thought to form disulfide crosslinks.

[0188] Because the affinity of most rabbit IgG was close to or above the detection limit of the instrument, the data obtained by SPR were carefully processed. As a result, the stat-6 reporter gene assay was considered in the selection of rabbit antibodies for humanization. In the stat-6 reporter gene assay, antibodies that neutralized IL-4 and IL-13-induced signaling more potently, or antibodies with up to 3.3-fold lower potency, were selected for humanization. A total of 18 promising clones were selected for reformatting and humanization.

[0189] Humanization of 1.6.scFv For the lead candidate generation, 18 rabbit monoclonal antibody clones were selected. Humanization of these clones involved transferring rabbit CDRs to one of Numab's proprietary human variable domain acceptor scaffolds. In this process, the amino acid sequences of six CDR regions were identified using the Numab CDR definition (Table 5), and transplanted into Numab's proprietary, highly stable, fully human Vk1 / VH3 lambda cap acceptor framework to obtain constructs called "CDR grafts." [Table 5]

[0190] Exclusive engraftment of rabbit CDRs into a human acceptor framework is the most basic transplantation strategy. However, in some cases, a particular set of CDRs requires mutations in specific rabbit framework residues to maintain its full functionality. Therefore, in addition to the "CDR graft," additional grafting variants containing defined patterns of rabbit framework residues were designed.

[0191] We designed a humanized scFv construct and ordered it from GeneUniversal (formerly General Biosystems) in 1 mg scale as a mammalian (CHO-S, pcDNA3.1) expression vector. We used the plasmid for transient transfection of CHO-S cells, as described below.

[0192] 1.7. Manufacturing of humanized scFv Expression in mammalian constructs was performed in CHO-S cells using the CHOgro transient transfection kit (Mirus). Five to seven days after expression at 37°C (when the cell viability reached less than 70%), the culture was harvested by centrifugation followed by filtration. Proteins were purified from the clarified culture supernatant by protein L affinity chromatography. Since all molecules showed at least one affinity chromatography fraction with a monomer content >95% after capture in the SE-HPLC analysis, there were no molecules that required finishing by size exclusion chromatography. Samples were re-buffered by dialysis into the final buffer (50 mM phosphate-citrate buffer containing 150 mM NaCl, pH 6.4). For quality control of the produced substances, standard analytical methods such as SE-HPLC, UV 280 , SDS-PAGE were applied. The production characteristics of 10 scFv molecules derived from 5 clones considered suitable for incorporation into the multispecific antibody format are summarized in Table 6. The production characteristics of CDR grafting (sc01) and full grafting (sc04) of clone 44-18-C11 (PRO1510) and its variants (PRO1549 - PRO1554) are summarized in Table 7.

Table 6

Table 7

[0193] 1.8. Pharmacodynamic characterization of appropriate anti-IL-4R binding domains (scFv format) The humanized scFv antibodies evaluated as suitable were analyzed for primary pharmacodynamic properties.

[0194] Affinity for human IL-4R The affinity of 16 humanized scFvs (obtained from five rabbit monoclonal antibodies) for human IL-4R was measured by SPR analysis using a T200 instrument (Biacore, GEHealthcare). Human IL-4R-Fc was captured via anti-human IgG-Fc bound to a carboxylmethylated dextran surface, and scFv was injected as the analyte. After each analyte injection cycle, the sensor tip was regenerated and new antigen was captured. scFv was measured using a dose-response multicycle kinetic assay with seven analyte concentrations ranging from 0.12 to 90 nM, diluted in running buffer. The resulting sensorgrams were fitted using a 1:1 binding model. The results are summarized in Table 8. [Table 8]

[0195] As can be seen from Table 8, the scFv antibody variable domain based on clone 4434-C10 showed the best overall binding affinity to human IL-4R, followed closely by the scFv antibody variable domain based on clone 44-32-F04.

[0196] Affinity for IL-4R in cynomolgus monkeys, marmosets, and mice Cross-reactivity to cynomolgus monkey IL-4R was measured using the same setup as for human IL-4R affinity analysis, but with the difference being that cynomolgus monkey IL-4R-Fc was captured instead of human IL-4R-Fc. Since only the extracellular domain (ECD) of marmoset IL-4R was available, a direct setup was used to measure affinity to marmoset IL-4R. The marmoset ECD was immobilized directly onto the sensor tip, and the scFv of the titration series was injected as the analyte. K values ​​were obtained for cynomolgus monkey and marmoset IL-4R. D To enable comparison between values, affinity for cynomolgus monkey IL-4R ECD was also determined using direct settings.

[0197] Only a small amount of scFv bound to the cynomolgus monkey IL-4R ECD. Compared to two scFv in the direct setting, four scFv showed cross-reactivity in the capture setting, meaning that the binding of two scFv was no longer measurable in the direct setting. This is likely because the K measured in the direct setting was not D This is thought to be a result of the lower values. The difference is due to the on-rate, which is probably because the on-rate slowed down due to reduced accessibility. The off-rate was similar for all scFv. Furthermore, cross-reactivity to marmoset IL-4R ECD was measured. The results are summarized in Table 9. As can be seen from Table 9, the scFv antibody variable domains based on clones 44-34-C10 and 44-32-F04 show excellent cross-reactivity to cynomolgus monkey IL-4R ECD and marmoset IL-4R ECD.

[0198] Binding to mouse IL-4R was also evaluated for scFv PRO1517, PRO1535, and PRO1538, but none showed cross-reactivity.

[0199] Stat-6 reporter gene assay in HEK-Blue cells (blockade of human IL-4 and IL-13 induced signaling) The HEK-Blue assay was used to test the ability of humanized scFv to inhibit IL-4 and IL-13-induced signaling via IL-4R. 50,000 cells per well were inoculated into a 96-well plate. Serial dilutions of scFv and the control antibody dupilumab were added to the plate in the presence of either 0.02 ng / ml IL-4 or 0.3 ng / ml IL-13. After incubation at 37°C and 5% CO2 for 24 hours, the supernatant was transferred to a new plate, and secreted fetal alkaline phosphatase was quantified using Quanti-Blue substrate.

[0200] scFv was analyzed in this HEK-Blue assay for its ability to block IL-4R-mediated signaling. The potency of the analyzed molecules was compared to dupilumab. All potency data are summarized in Table 10. Representative dose-response curves for PRO1515, PRO1517, PRO1524, PRO1535, and PRO1552 for blocking IL-4-induced and IL-13-induced signaling are shown in Figures 1 and 2, respectively. Relative IC 50 Values ​​were calculated in units of mass (ng / ml) of dupilumab and scFv. Ten scFv strains blocked IL-4 and IL-13-inducible signaling with high potency. ScrFv strains based on clones 44-34-C10 and 44-32-F04, as well as PRO1552, showed the highest overall blocking potency. PRO1515, PRO1517, PRO1524, PRO1533, PRO1535, PRO1538, and PRO1552 may block IL-4 and IL-13-inducible signaling with similar potency to dupilumab.

[0201] Competitive ELISA (inhibition of hIL-4 binding to hIL-4R) The potency of humanized scFvs was further determined using competitive ELISA. The potency of each scFv in inhibiting the interaction between human IL-4 and human IL-4R was evaluated by ELISA using the same procedure as described above. Similarly, for the stat-6 reporter gene assay, the individual ICs on each plate were evaluated. 50 The IC value of the reference molecule dupilumab 50 It was calibrated against this.

[0202] Blockade data are summarized in Table 11. Representative dose-response curves for PRO1515, PRO1517, PRO1533, PRO1535, and PRO1538 are shown in Figure 4. The six scFv inhibited the interaction between human IL-4 and human IL-4R with good to high potency. The scFv based on clones 44-34-C10 and 44-32-F04, and PRO1552, showed the best blocking ability overall. The blocking ability of PRO1515, PRO1517, and PRO1552 was similar to that of dupilumab. PRO1538, on the other hand, showed only moderate inhibition. Five scFv molecules showed no inhibition.

[0203] Overview of molecular selection for pharmacological characterization and feasibility assessment of scFv Pharmacological characterization of 16 scFvs derived from five different B cell clones provided a basis for molecular selection for extended biophysical characterization. Based on the overall superior performance in neutralizing IL-4 and IL-13-induced signaling in stat-6 reporter gene assays and IL-4 / IL-4R blockade ELISAs, seven molecules, namely PRO1515, PRO1517, PRO1533, PRO1535, PRO1524, PRO1506, and PRO1538, were selected for further extended characterization. All seven molecules showed affinity greater than 500 pM. With the exception of three molecules that showed very low affinity or no binding to cynomolgus monkey IL-4R, affinity for cynomolgus monkey IL-4R was 4 to 20 times lower compared to human IL-4R. In particular, scFvs based on clone 44-18-C11, namely PRO1510, PRO1528, and PRO1549-PRO1554, did not show binding to cynomolgus monkey IL-4R. In general, the affinity of these scFvs for marmoset monkey IL-4R was also lower compared to human IL-4R.

[0204] As a result of the affinity test, the scFvs based on clone 44-18-C11, namely PRO1510, PRO1528, and PRO1549 to PRO1554, were excluded from further evaluation because they showed no cross-reactivity with cynomolgus IL-4R, but PRO1552 showed very excellent inhibitory efficacy. For the same reason, PRO1520 was also excluded. However, PRO1538 based on the same clone 44-39-B07 was not excluded despite its weak binding affinity to cynomolgus IL-4R.

Table 9

Table 10

Table 11

[0205] 1.9. Biophysical Characterization of the Appropriate Anti-IL-4R Binding Domain (scFv Format) Production of Materials for Stability Measurement PRO1515, PRO1517, and PRO1538 were regenerated at a slightly larger scale (expression volume 0.25 l) using the same production process as above to generate sufficient materials for stability evaluation. For the other proteins selected for stability evaluation, the amount of previously generated materials was sufficient for performing the stability evaluation. After purification, the samples were prepared in 50 mM phosphate-citrate buffer (50 mM NaCiP, pH 6.4) containing 150 mM NaCl at pH 6.4. After purification and dialysis, the protein samples were concentrated to 10 mg / ml or more using a 5 MWCO centrifugal concentrator tube because this is a desirable concentration for performing the storage stability evaluation.

[0206] As shown in Table 12, monomer loss upon concentration to 10 mg / ml ranged from 0 to 12% depending on the molecule. The monomer content of PRO1506, PRO1515, PRO1524, and PRO1533 fell below the critical value of 95% for molecules to be included in the storage stability evaluation, and therefore these were excluded from the test. However, since the materials had already been prepared and everything was ready for the test, the stability of these molecules was evaluated along with the remaining molecules that showed monomer content exceeding 95% after concentration. The scFv based on clone 44-34-C10, namely PRO1517, PRO1535, and PRO1538, showed virtually no loss of monomer content upon concentration. [Table 12]

[0207] Storage stability test Humanized scFv was subjected to stability tests, including a 4-week stability test, where scFv was prepared at 10 mg / ml in aqueous buffer (50 mM NaCiP, 150 mM NaCl, pH 6.4) and stored for 4 weeks at <-80°C, 4°C, and 40°C. At a minimum, the proportion of monomers and oligomers in the preparation was assessed by integrating the SE-HPLC peak area at 1 week, 2 weeks, and the end of each test. Additional time points were recorded for most molecules. Table 13 compares the endpoint measurements obtained at d14 (day 14) and d28 (day 28) of the test.

[0208] As can be seen from Table 13, the scFv based on clone 44-34-C10, namely PRO1517 and PRO1535, showed good storage stability at 4°C. The loss of monomer content after 28 days of storage at 4°C was less than 5%. PRO1538 also showed good storage stability for at least 14 days. On the other hand, PRO1515 showed a loss of monomer content of >10% at 4°C, which is a clear reason for its exclusion. However, none of the stable molecules showed a significant loss of protein content at any temperature.

[0209] Freeze-thaw stability In addition to the storage stability tests described above, the compatibility (colloidal stability) of the scFv molecule with the best performance in freeze-thaw (F / T) cycles was evaluated. In the early stages of development, the active pharmaceutical ingredient is usually stored frozen, so some freezing and thawing operations are necessary for preparation and filling / finishing processes.

[0210] In the F / T stability evaluation, the same analytical methods (SE-HPLC, SDS-PAGE) and parameters (monomer content %) and monomer loss %) as in the storage stability test were applied to track the molecular quality over five F / T cycles. Table 14 shows the progress of monomer content loss %) over the five F / T cycles. Since no dedicated freeze-thaw tests were performed, the freeze-thaw data obtained from the -80°C samples of the storage stability test, acquired over 28 days, is shown. For some proteins, only four time points could be recorded, so only four F / T cycles were performed for these proteins. As can be seen from Table 14, apart from PRO1515 and PRO1533, none of the other scFvs tested showed significant loss of monomer content.

[0211] Thermal unfolding Thermal unfolding measurements were performed on five selected scFv constructs using differential scanning fluorescence (DSF). The midpoint (T) of the obtained thermal unfolding was measured. m ) and the unfolding start temperature (T) calculated at 10% of the maximum signal. onset The 10% value was determined by fitting the data to the Boltzmann equation. Table 15 summarizes the calculated melting temperatures measured by DSF. As can be inferred from Table 15, scFv based on clone 44-34-C10 shows a high melting temperature of over 63°C. PRO1538 also showed a high melting temperature. [Table 13] [Table 14] [Table 15]

[0212] Example 2: Selection and optimization of anti-IL-4R molecules for multispecific formats: 2.1. General explanation regarding the selection of anti-IL-4R domains for optimization The anti-IL-4R domains PRO1517(44-34-C10-sc01) and PRO1535(44-34-C10-sc04) were initially selected due to their very good pharmacodynamic properties and acceptable to good stability. PRO1538(44-39-B07-sc04) was similarly selected because it exhibited the best stability, although it had pharmacological properties that could be improved. As shown in 2.2 below, PRO1517(44-34-C10-sc01) underwent stability optimization, and PRO1538(44-39-B07-sc04) underwent pI balance optimization.

[0213] The anti-IL-4R binding domain applied to the final multispecific antibody showed exclusive cross-reactivity with cynomolgus monkeys and marmosets.

[0214] 2.2. Optimization of the anti-IL-4R domain The anti-IL-4R domains PRO1538 (44-39-B07-sc03) and PRO1517 (44-34-C10-sc01) were further optimized for assembly into a multispecific format.

[0215] Optimization of the anti-IL-4R domain 44-39-B07-sc04 (PRO1538) PRO1538 (44-39-B07-sc03) has a pI imbalance between VL and VH (pIV L =8.2 and pIV H(=5.0). pI imbalance correlates with high viscosity and can negatively affect stability. Therefore, two new variants of this molecule were designed. These variants are described below. Briefly, all residues of VH were analyzed in detail with the aim of designing a molecule in which pI imbalance is eliminated, without impairing binding affinity, and without introducing significant sequence defects (chemical and post-translational modifications), T cell epitopes, etc.

[0216] Two variants were designed, which are summarized in Table 16. [Table 16]

[0217] However, these variants of PRO1538 (44-39-B07-sc03) did not show the desired improvement in stability. PRO1538 had significantly lower efficacy in inhibiting IL-4 and IL-13-induced signaling, and its ability to block the interaction between IL-4 and IL-4R was more than 10 times lower than that of PRO1517. Therefore, PRO1538 and its variants were excluded from further investigation, and efforts were focused on improving RPO1517 (44-34-C10-sc01).

[0218] Optimization of the anti-IL-4R domain 44-34-C10-sc01 (PRO1517) PRO1517 (44-34-C10-sc01) is an anti-IL-4R scFv with good binding properties. During biophysical characterization, scFv PRO1517 showed good but still room-for-improvement solubility characteristics. Attempts were made to further improve PRO1517 in terms of solubility, long-term stability, and concentration behavior. Therefore, four new variants of this molecule were designed. These variants are described below. Briefly, the hydrophobic patch in the CDR or previous VH-CH interface was analyzed in detail, and the pI imbalance was reduced with the aim of designing molecules in which the hydrophobic patch was removed without impairing binding affinity and without introducing significant sequence defects (chemical and post-translational modifications), T cell epitopes, etc.

[0219] Four variants were designed, which are summarized in Table 17. [Table 17]

[0220] 2.3. Biophysical characterization of the optimized anti-IL-4R binding domain (scFv format) Storage stability test PRO1897, PRO1898, and PRO1899 were subjected to the storage stability tests described in Section 1.9. The results are summarized in Table 18.

[0221] As shown in Table 19, scFv PRO1898 and PRO1899 exhibit excellent storage stability. The loss of monomer content after 28 days of storage at 4°C was less than 5%. Furthermore, these molecules did not show significant loss of protein content at any temperature.

[0222] Thermal unfolding The thermal stability of PRO1897, PRO1898, and PRO1899 was analyzed by nanodynamic scanning fluorescence (nDSF) using NanoTemper, and the unfolding onset temperature (T onset ), midpoint of unfolding (T m1 ), and the scattering onset temperature could be determined. T of double / triple measurements m1 and T onset The results are summarized in Table 19. As can be inferred from Table 19, PRO1897, PRO1898, and PRO1899 exhibit high melting temperatures.

[0223] 2.4. Pharmacodynamic characterization of the optimized anti-IL-4R binding domain (scFv format) Affinity for human IL-4R As described above in Section 1.5 or 1.8, the affinity of the optimized anti-IL-4R scFv PRO1898 and PRO1899 for human IL-4R was measured by SPR analysis using a T200 instrument (Biacore, GEHealthcare). scFv was measured using a dose-response multicycle kinetic assay with seven analyte concentrations ranging from 0.12 to 90 nM, diluted in running buffer. The resulting sensorgrams were fitted using a 1:1 binding model.

[0224] As shown in Table 20, binding to human IL-4R and cynomolgus monkey IL-4R was confirmed for all optimized scFv. [Table 18] [Table 19] [Table 20]

[0225] Affinity for IL-4R in cynomolgus monkeys The affinity of optimized anti-IL-4R scFv PRO1898 and PRO1899 for cynomolgus monkey IL-4R was measured using the same setup as the human IL-4R affinity analysis, except that cynomolgus monkey IL-4R-Fc was captured instead of human IL-4R-Fc, as described above in Section 1.5 or 1.8. The results of this analysis are summarized in Table 21. [Table 21]

[0226] Stat-6 reporter gene assay in HEK-Blue cells (blockade of human IL-4 and IL-13 induced signaling) The optimized anti-IL-4R scFv PRO1898 and PRO1899 were analyzed in this HEK-Blue assay for their ability to block IL-4R-mediated signaling, as described above in Section 1.5 or 1.8. The potency of the analyzed molecules was compared to dupilumab. All potency data are summarized in Table 22. Relative IC50 50 The values ​​were calculated in units of mass (ng / ml) for dupilumab and scFv. The optimized scFv blocked IL-4 and IL-13-induced signaling with similar high potency to dupilumab (see Figure 3). [Table 22]

[0227] Competitive ELISA (inhibition of hIL-4 binding to hIL-4R) The potency of the optimized anti-IL-4R scFv PRO1898 and PRO1899scFv was measured using competitive ELISA. The potency of each scFv in inhibiting the interaction between human IL-4 and human IL-4R was evaluated by ELISA as described in Section 1.5 or 1.8. Similarly, for the stat-6 reporter gene assay, the individual ICs on each plate were evaluated. 50 The IC value of the reference molecule dupilumab 50 It was calibrated against this.

[0228] The results of the competitive ELISA analysis are summarized in Table 23. The blocking efficacy of PRO1898 and PRO1899 was similar to that of dupilumab (see Figure 5). [Table 23]

[0229] Example 3: Morrison-H IgG4-based anti-IL-4R × IL-31 bispecific antibody Next, we further tested whether the anti-IL-4R antibody variable domain of the present invention also provides advantageous biological and biophysical properties when incorporated into a multispecific antibody format. Accordingly, a multispecific antibody was designed based on a Morrison-H IgG4 format containing two anti-IL-4R antibody variable domains, as defined herein. Two IL-31 binding domains (IL31-BD) were selected as binding domains that specifically bind to targets different from IL-4R.

[0230] 3.1 Design of the Morrison Format A series of anti-IL-4R × IL-31 bispecific antibodies with the Morrison-H format were designed, in which the Fc region was derived from the IgG subclass IgG4.

[0231] Due to their cross-reactivity with cynomolgus monkey IL-4R, their excellent efficacy in blocking IL-4R-mediated signaling, and their superior biophysical properties, the anti-IL-4R scFv variable domains 44-34-C10-sc08 and 44-34-C10-sc09 were selected as the Fab arm binding domains for the Morrison-H construct. Similarly, due to their superior biological and biophysical properties, the anti-IL31 scFv 50-09-D07-sc04 was selected as the scFv domain fused to the C-terminus of the heavy chain of the Morrison-H antibody.

[0232] The identification, humanization, production, and final selection of the humanized anti-IL-31 binding domain 50-09-D07-sc04 were carried out using the same method as described above for the anti-IL-4R antibody variable domain.

[0233] Two IgG4-(scFv)2 Morrison-H molecules, namely PRO2198 and PRO2199 shown in Table 24, were designed (Morrison-H). [Table 24]

[0234] Expression of multispecific antibodies PRO2198 and PRO2199 was performed in FreeStyle CHO-S cells using a transient CHOgro expression system (Mirus). The target genes were optimized for mammalian expression, synthesized, and cloned into standard pcDNA3.1 vectors. Expression cultures were cultured in batches at 37°C for 6–7 days using shaking flasks (cell viability <70%). The culture supernatant was separated from the cells by centrifugation and subsequent 0.22 μm sterile filtration. Target proteins were captured from the clarified culture supernatant by protein L or A affinity chromatography followed by size exclusion chromatography (if a fraction containing the appropriate monomer content, as assessed by SE-HPLC, was not available after capture). SE-HPLC, SDS-PAGE, and UV were used for quality control of the manufactured materials. 280 Standard analytical methods such as those listed above were used.

[0235] 3.2. Affinity for IL-4R in humans and cynomolgus monkeys The binding kinetics (including affinity) of bispecific Morrison-H antibodies PRO2198 and PRO2199 to recombinant human IL-4R protein (His-tagged ECD, Sino Biological) were measured by SPR analysis using a T200 instrument (Biacore, Cytiva). In this SPR experiment, Morrison molecules were captured via anti-human IgG-Fc antibodies (Human Antibody Capture Kit; Biacore, Cytiva) covalently immobilized on a carboxymethylated dextran surface (CM5 sensor chip; Biacore, Cytiva), and human IL-4R ECD was injected as the analyte in a titer series. After each analyte injection cycle, the sensor chip was regenerated (MgCl2), and then a new Morrison antibody was recaptured. The binding kinetics to human IL-4R were measured using a multicycle kinetic assay with nine analyte concentrations ranging from 0.175 to 45 nM, diluted in running buffer (HEPS-buffered saline, 0.05% Tween-20, pH 7.5). The apparent dissociation constant (k) was also measured. d ) and meetings (k a) Rate constant, and apparent dissociation equilibrium constant (K D The IL-4R cross-reactivity was calculated using Biacore analysis software (Biacore Evaluation software version 3.2, Cytiva) with a one-to-one Langmuir binding model, and the quality of fit was tracked based on Chi2 and U values, measures of curve fitting quality. Binding levels were calculated as the maximum stable binding achieved, normalized against theoretical Rmax. Cross-reactivity to cynomolgus monkey IL-4R was measured using the same setup as the analysis of human IL-4R kinetics, except that recombinant cynomolgus monkey IL-4R (His-tagged ECD, Acro Biosystems) protein was used as the analyte instead of human IL-4R.

[0236] As shown in Table 25, high affinity binding of Morrison-H antibodies PRO2198 and PRO2199 to human IL-4R and cynomolgus monkey IL-4R was demonstrated. [Table 25] [Table 26] [Table 27]

[0237] 3.3. Evaluation of efficacy in inhibiting human IL-4 and IL-13 signaling (HEK-Blue® IL-4 / IL-13 cell stat-6 reporter gene assay) The HEK-Blue assay was used to test the ability of Morrison-H antibodies PRO2198 and PRO2199 to inhibit IL-4 and IL-13-induced signaling via IL-4R. 50,000 cells per well were inoculated into a 96-well plate. Three-fold serial dilutions of Morrison molecules and the reference antibody dupilumab, in the concentration range of 100–0.002 ng / ml, were added to the plate in the presence of either 0.05 ng / ml of IL-4 or 0.3 ng / ml of IL-13. After incubation at 37°C and 5% CO2 for 24 hours, the supernatant was transferred to a new plate, and secreted fetal alkaline phosphatase was quantified using Quanti-Blue substrate. Morrison-H PRO2198 and PRO2199 were also tested for inhibition of IL-4-induced signaling by excess IL-31 (10 nM) in the assay medium.

[0238] All efficacy data are summarized in Table 26 (IL-4 / IL-4R inhibition) and Table 27 (IL-13 / IL-4R inhibition). Mean relative IC from repeated analyses. 50 The values ​​(pM) are also shown. Morrison-H antibodies PRO2198 and PRO2199 efficiently blocked the interaction between IL-4 or IL-13 and IL4R with the same potency as dupilumab.

[0239] 3.4. Biophysical characterization of PRO2198 and PRO2199: 3.4.1.Thermal stability PRO2198 and PRO2199 were analyzed for their thermal stability between pH 5 and pH 8.5. Both thermal unfolding and the onset of thermal aggregation were measured. During thermal unfolding, all molecules exhibited multiple transitions due to their multi-domain structure. For simplicity, only the initial melting intermediate point is shown. The results are summarized in Table 28. [Table 28]

[0240] Start of unfolding (T onset ) and the first melting point (T m1The thermal stability was highest for all molecules at pH 7. Changing to pH 8.5 only slightly reduced thermal stability. Thermal stability decreased as the pH became more acidic. However, at pH 5, unfolding began at temperatures above 55°C for all molecules.

[0241] Thermal agglutination was observed only at pH 7 and pH 8.5. At pH 5.0, neither Morrison antibody formed larger aggregates, even in an unfolded state. At neutral and basic pH levels, aggregation initiation occurred above the initial melting point, suggesting a non-natural aggregation mechanism.

[0242] 3.4.2. High-Concentration Stability Test In the high-concentration stability test, PRO2198 was prepared using two different buffer solutions. Prepared buffer F1: 20 mM acetate, pH 5.5; Prepared buffer F2: 20 mM citrate, 50 mM NaCl, pH 5.5.

[0243] Solubility - Protein Concentration PRO2198 could be concentrated to over 100 mg / ml without any signs of precipitation or reaching its solubility limit. Furthermore, PRO2198 did not show any detectable decrease in protein concentration, meaning that PRO2198 was sufficiently soluble and reached and maintained its target concentration of over 100 mg / ml for at least 4 weeks at 4°C and 25°C.

[0244] Monomer stability at various temperatures PRO2198 was prepared using F1 and F2 and concentrated to over 100 mg / ml. The concentrated samples were stored at 4°C, 25°C, and 40°C for up to 4 weeks. Monomer content was analyzed at different time points by SE-HPLC. The results are summarized in Table 29. [Table 29]

[0245] 3.5. General methods used for biophysical characterization of PRO2198 and PRO2199 buffer exchange Buffer exchange was performed by dialysis. Antibodies were dialyzed in a Spectra Pro3 dialysis membrane (Spectrum Laboratories) using at least 200 times excess dialysis buffer.

[0246] Antibody enrichment Antibodies were concentrated using a centrifugal concentrator with a molecular weight cutoff (MWCO) of 10 kDa or 30 kDa. The samples were centrifuged in 5-minute increments at 22°C until the target concentration was reached. The samples were resuspended between increments.

[0247] Determination of protein concentration The concentration of protein samples was determined using a Tecan plate reader and NanoQuant plates. Buffers were used as a blank, subtracted from the absorbance measured at 280 nm. Each measurement was corrected for scattering caused by visible particles, measured at 310 nm. The corrected values ​​were standardized for a 1 cm path length, and the protein concentration was calculated using the theoretical extinction coefficient of the corresponding protein. For protein samples with concentrations exceeding 10 mg / ml, the sample was diluted at least 10-fold with the corresponding buffer, or to a nominal concentration of 1 mg / ml.

[0248] keep To evaluate the stability of proteins at various temperatures, samples were incubated at 4°C, 25°C, and 40°C. Storage at 4°C was performed in a refrigerator at a nominal temperature of 4°C. For storage at 25°C and 40°C, samples were placed in cabinets with controlled humidity levels of 65%rH and 75%rH, respectively.

[0249] Dynamic light scattering Dynamic light scattering is used to determine the diffusion coefficients of molecules and particles in solution. This allows for the calculation of the hydrodynamic radius (Rh) of molecules in solution and provides a highly sensitive method for detecting the formation of higher-order oligomers.

[0250] In high-concentration stability studies, Rh and polydispersity were determined at target concentrations. This provided information regarding potential increases in self-association, oligomerization, and viscosity. The measurements were not corrected for the viscosity of the buffer or sample; instead, the viscosity of water was used to calculate the Rh of the sample.

[0251] Thermal unfolding by nanoDSF Thermal unfolding using TSA was determined by the change in the fluorescence intensity of Sypro Orange. The fluorescence of the dye is sensitive to hydrophobic interactions. When the protein is unfolded, hydrophobic amino acids are exposed to the solvent, and the fluorescence of Sypro Orange increases. The onset temperature (Tm) was calculated at the midpoint (Tm) of thermal unfolding and at 10% of the maximum signal. onset This is the temperature at the minimum signal + 0.1 * (Maximum signal - Minimum signal) was determined by fitting the data to the Boltzmann equation.

[0252] Monomer content measured by SE-HPLC Monomer content was determined by analytical size exclusion chromatography using a Shodex KW403-4F column with 50 mM sodium phosphate, 300 mM NaCl, pH 6.5. For analysis, 5 μg of sample was injected and absorbance was recorded at 280 nm. Sample quality is expressed as relative percentage of monomer, HMWS, and LMWS.

Claims

1. The following are examples of substances that specifically bind to IL-4R: a) A VH chain having a sequence selected from sequence numbers 4, 5, 6, 7, 8, and 9, and b) A VL chain having a VL sequence present in a sequence selected from sequence numbers 33, 34, 35, 37, and 38, Antibody variable domains, including those mentioned above.

2. The antibody variable domain is as follows: a) A VH chain having the sequence of SEQ ID NO: 4 and a VL chain having the VL sequence present in the sequence of SEQ ID NO: 33, or b) A VH chain having the sequence of SEQ ID NO: 4 and a VL chain having the VL sequence present in the sequence of SEQ ID NO: 37, or c) A VH chain having the sequence of SEQ ID NO: 4 and a VL chain having the VL sequence present in the sequence of SEQ ID NO: 38, or d) A VH chain having the sequence of SEQ ID NO: 5 and a VL chain having the VL sequence present in the sequence of SEQ ID NO: 34, or e) A VH chain having the sequence of SEQ ID NO: 6 and a VL chain having the VL sequence present in the sequence of SEQ ID NO: 35, or f) A VH chain having the sequence of SEQ ID NO: 7 and a VL chain having the VL sequence present in the sequence of SEQ ID NO: 33, or g) A VH chain having the sequence of SEQ ID NO: 8 and a VL chain having the VL sequence present in the sequence of SEQ ID NO: 33, or h) A VH chain having the sequence of sequence number 9 and a VL chain having the VL sequence present in the sequence of sequence number 33, The antibody variable domain according to claim 1, comprising:

3. An antibody variable domain according to claim 1 or 2, selected from FAB, Fv, scFv, and dsFv.

4. The antibody variable domain according to claim 3, which is an scFv having a sequence selected from sequence numbers 33 to 39.

5. below: a) One or two antibody variable domains according to any one of claims 1 to 4; b) At least one binding domain that specifically binds to a target different from IL-4R, A multispecific antibody containing [specific antibody].

6. A multispecific antibody according to claim 5, which does not contain an immunoglobulin Fc region.

7. Tandem scDb (Tandab), linear dimer scDb (LD-scDb), cyclic dimer scDb (CD-scDb), tandem tri-scFv, tribody (Fab-(scFv)) ２ ), Fab-Fv ２ The multispecific antibody according to claim 6, which is in a format selected from the group consisting of triabody, scDb-scFv, tetrabody, diabody, tandem-di-scFv, and MATCH.

8. The multispecific antibody according to claim 5, comprising an immunoglobulin Fc region selected from IgG1 and IgG4, which are IgG subclasses, particularly from IgG4.

9. The format of the multispecific antibody is KiH-based IgG, DVD-IgG, CODV-IgG, and Morrison (IgG CH4). ３ A fused compound (Morrison-H) or an IgG CL-scFv fused compound (Morrison-L) is selected, particularly from Morrison-H and Morrison-L. The multispecific antibody according to claim 8.

10. A multispecific antibody according to any one of claims 5 to 9, comprising two antibody-variable domains according to any one of claims 1 to 4 and two binding domains, which specifically bind to a second target different from IL-4R.

11. One nucleic acid or two nucleic acids encoding an antibody variable domain according to any one of claims 1 to 4, or a multispecific antibody according to any one of claims 5 to 10.

12. A vector comprising one nucleic acid or two nucleic acids as described in claim 11, or a vector comprising two nucleic acids.

13. A host cell or a plurality of host cells comprising the one vector or the two vectors according to claim 12.

14. A pharmaceutical composition comprising an antibody variable domain according to any one of claims 1 to 4 or a multispecific antibody according to any one of claims 5 to 10, and a pharmaceutically acceptable carrier.

15. An antibody variable domain according to any one of claims 1 to 4, or a multispecific antibody according to any one of claims 5 to 10, for use as a pharmaceutical.

16. An antibody variable domain according to any one of claims 1 to 4 or a multispecific antibody according to any one of claims 5 to 10 for use in the treatment of a disease, particularly a human disease, more particularly a human disease selected from allergic diseases, inflammatory diseases, and autoimmune diseases, particularly from inflammatory diseases and autoimmune diseases.

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