High-affinity human antibodies against the human IL-4 receptor

Abstract

High affinity for human interleukin-4 receptor alpha (hIL-4Rα) (K D It binds to and can block hIL-4 and hIL-13 activity, isolated Providing human antibodies or antibody fragments thereof. [Solution] An antibody or antigen-binding fragment thereof that specifically binds to a human interleukin-4 receptor (hIL-4R) containing the amino acid sequence shown in SEQ ID NO: 274, comprising a heavy chain variable region (HCVR) containing the amino acid sequence shown in SEQ ID NO: 162 and a light chain variable region (LCVR) containing the amino acid sequence shown in SEQ ID NO: 164.

Description

[Background technology]

[0001] Interleukin-4 (also known as IL-4, B-cell stimulator, or BSF-1) was originally characterized by its ability to stimulate B cell proliferation in response to low concentrations of surface immunoglobulin-specific antibodies. IL-4 has been shown to have a wide range of biological activities, including stimulating the proliferation of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes. IL-4 induces the expression of class II major histocompatibility complex molecules in resting B cells and enhances the secretion of IgE and IgG1 isotypes by stimulated B cells.

[0002] The biological activity of IL-4 is mediated by a specific cell surface receptor for IL-4. Human IL-4 receptor alpha (hIL-4R) (SEQ ID NO: 274) is described, for example, in Patent Documents 1, 2, and 3. Antibodies against hIL-4R are described in Patent Documents 4 and 5.

[0003] Methods for producing antibodies useful as human therapeutic agents include generating chimeric antibodies and humanized antibodies (see, for example, Patent Document 6). For example, see Patent Documents 7 and 8, which describe methods for generating non-human transgenic mice capable of producing human antibodies.

[0004] Methods using antibodies against hIL-4R are described in Patent Document 9; Patent Document 10; and Patent Document 11. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] U.S. Patent No. 5,599,905 [Patent Document 2] U.S. Patent No. 5,767,065 [Patent Document 3] U.S. Patent No. 5,840,869 [Patent Document 4] U.S. Patent No. 5,717,072 [Patent Document 5] U.S. Patent No. 7,186,809 [Patent Document 6] U.S. Patent No. 6,949,245 [Patent Document 7] WO94 / 02602 [Patent Document 8] U.S. Patent No. 6,596,541 [Patent Document 9] U.S. Patent No. 5,714,146 [Patent Document 10] U.S. Patent No. 5,985,280 [Patent Document 11] U.S. Patent No. 6,716,587 [Overview of the project] [Means for solving the problem]

[0006] In the first aspect, the present invention provides a human antibody, preferably a recombinant human antibody, that specifically binds to the human interleukin-4 receptor (hIL-4R). This human antibody is characterized by its ability to bind to hIL-4R with high affinity and to neutralize hIL-4 activity. In certain embodiments, this human antibody can block the binding of the hIL-13 / hIL-13R1 complex to hIL-4R, thereby inhibiting hIL-13-mediated signaling. This antibody may be full-length (e.g., IgG1 or IgG4 antibody) or contain only an antigen-binding moiety (e.g., Fab, F(ab')2, or scFv fragment). To achieve the desired function, the device can be modified, for example, by eliminating any remaining effects functions (Reddy et al. (2000) J.Immunol. 164: 1925-1933).

[0007] In a typical embodiment, when measured by surface plasmon resonance in a monomer or dimer assay, the K is approximately 300 pM or less. D hIL-4R (Sequence No. 274) The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to ). In a more specific embodiment, the antibody or antigen-binding portion thereof has a K content of about 200 pM or less, about 150 or less, about 100 pM or less, or about 50 pM. D This shows various In this embodiment, the antibody or antigen-binding fragment has an IC50 of approximately 100 pM or less when measured by luciferase bioassay. 50 This blocks hIL-4 activity. In a more specific embodiment, the antibody or antigen-binding fragment has an IC of approximately 50 pM or less, approximately 30 pM or less, or approximately 25 pM or less, as measured by a STAT6 luciferase bioassay. 50 This shows that, in various embodiments, the antibody or antigen-binding fragment, when measured by STAT6 luciferase bioassay, has an IC of approximately 100 pM or less, approximately 90 pM or less, approximately 50 pM or less, or approximately 20 pM or less. 50 This blocks hIL-13 activity.

[0008] In the second aspect, the antibody of the present invention comprises a heavy chain variable region (HCVR) sequence selected from the group consisting of SEQ ID NOs: 2, 18, 22, 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114, 118, 122, 138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218, 234, 238, 242, 258, and 262, or a sequence substantially similar thereto.

[0009] In the third aspect, the antibody of the present invention comprises a light chain variable region (LCVR) sequence selected from the group consisting of SEQ ID NOs: 10, 20, 24, 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144, 154, 164, 168, 178, 188, 192, 202, 212, 216, 226, 236, 240, 250, 260, and 264, or a sequence substantially similar thereto.

[0010] In one embodiment, the antibody or antibody fragment of the present invention is sequence numbers 2 / 10, 18 / 20, 22 / 24, 26 / 34, 42 / 44, 46 / 48, 50 / 58, 66 / 68, 70 / 72, 74 / 82, 90 / 92, 94 / 96, 98 / 106, 114 / 116, 118 / 120, 122 / 130, 138 / 140, 142 / 144, 146 / 1 Includes HCVR and LCVR sequence pairs (HCVR / LCVR) selected from the group consisting of 54, 162 / 164, 166 / 168, 170 / 178, 186 / 188, 190 / 192, 194 / 202, 210 / 212, 214 / 216, 218 / 226, 234 / 236, 238 / 240, 242 / 250, 258 / 260, and 262 / 264. In a preferred embodiment, the antibody or antibody fragment comprises an HCVR / LCVR sequence pair SEQ ID NOs: 162 / 164, 210 / 212, or 18 / 20; typical antibodies having these HCVR / LCVR sequence pairs include antibodies called H4H098P (SEQ ID NOs: 162 / 164), H4H083P (SEQ ID NOs: 210 / 212), and H4H095P (SEQ ID NOs: 18 / 20).

[0011] In a fourth aspect, the present invention provides a nucleic acid molecule encoding HCVR, which is a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 17, 21, 25, 41, 45, 49, 65, 69, 73, 89, 93, 97, 113, 117, 121, 137, 141, 145, 161, 165, 169, 185, 189, 193, 209, 213, 217, 233, 237, 241, 257, and 261, or a substantially identical sequence having at least 95% homology thereto.

[0012] In a fifth embodiment, the present invention provides a nucleic acid molecule encoding LCVR, and this nucleic acid The molecule is a sequence selected from the group consisting of SEQ ID NOs: 9, 19, 23, 33, 43, 47, 57, 67, 71, 81, 91, 95, 105, 115, 119, 129, 139, 143, 153, 163, 167, 177, 187, 191, 201, 211, 215, 225, 235, 239, 249, 259, and 263, or a substantially identical sequence having at least 95% homology thereto.

[0013] In one embodiment, the antibodies of the present invention are SEQ ID NOs: 1 / 9, 17 / 19, 21 / 22, 25 / 33, 41 / 43, 45 / 47, 49 / 57, 65 / 67, 69 / 71, 73 / 81, 89 / 91, 93 / 95, 97 / 105, 113 / 115, 117 / 119, 121 / 129, 137 / 139, 141 / 143, 145 / 153, 161 / The antibody or antibody fragment comprises HCVR and LCVR encoded by a nucleotide sequence pair selected from the group consisting of 163, 165 / 167, 169 / 177, 185 / 187, 189 / 191, 193 / 201, 209 / 211, 213 / 215, 217 / 225, 233 / 235, 237 / 239, 241 / 249, 257 / 259, and 261 / 263. In a preferred embodiment, the antibody or antibody fragment comprises an HCVR / LCVR sequence encoded by a nucleic acid sequence selected from SEQ ID NOs: 161 / 163, 209 / 211, and 17 / 19. In a further preferred embodiment, the antibody or antibody fragment comprises an HCVR / LCVR encoded by the nucleic acid sequence SEQ ID NOs: 161 / 163.

[0014] In a sixth aspect, the present invention provides an antibody or antigen-binding fragment comprising HCDR3 and LCDR3, wherein the HCDR3 domain is selected from the group consisting of SEQ ID NOs: 8, 32, 56, 80, 104, 128, 152, 176, 200, 224, and 248; and the LCDR3 domain is selected from the group consisting of SEQ ID NOs: 16, 40, 64, 88, 112, 136, 160, 184, 208, 232, and 256. In a preferred embodiment, the HCDR3 / LCDR3 sequence is SEQ ID NOs: 152 / 160, 8 / 16, or 200 / 208. In a more preferred embodiment, the HCDR3 and LCDR3 sequences are SEQ ID NOs: 152 and 160.

[0015] In a further embodiment, the antibody or antibody fragment is an HCDR1 sequence selected from the group consisting of SEQ ID NOs: 4, 28, 52, 76, 100, 124, 148, 172, 196, 220, and 244, or a substantially similar sequence thereto; an HCDR2 sequence selected from the group consisting of SEQ ID NOs: 6, 30, 54, 78, 102, 126, 150, 174, 198, 222, and 246, or a substantially similar sequence thereto; an HCDR3 sequence selected from the group consisting of SEQ ID NOs: 8, 32, 56, 80, 104, 128, 152, 176, 200, 224, and 248, or a substantially similar sequence thereto The following are further included: substantially similar sequences; LCDR1 sequences selected from the group consisting of SEQ ID NOs: 12, 36, 60, 84, 108, 132, 156, 180, 204, 228 and 252, or substantially similar sequences; LCDR2 sequences selected from the group consisting of SEQ ID NOs: 14, 38, 62, 86, 110, 134, 158, 182, 206, 230 and 252, or substantially similar sequences; and LCDR3 sequences selected from the group consisting of SEQ ID NOs: 16, 40, 64, 88, 112, 136, 160, 184, 208, 232 and 256, or substantially similar sequences. In a preferred embodiment, the antibody or antigen-binding fragment includes HCDR sequences SEQ ID NOs: 148, 150, and 152 and LCDR sequences SEQ ID NOs: 156, 158, and 160; HCDR sequences SEQ ID NOs: 4, 6, and 8 and LCDR sequences SEQ ID NOs: 12, 14, and 16; and HCDR sequences SEQ ID NOs: 196, 198, and 200 and LCDR sequences SEQ ID NOs: 204, 206, and 208.

[0016] According to a particular embodiment, the present invention relates to an anti-hIL-4R antibody or its antigen-binding filament having an HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence selected from the group consisting of SEQ ID NOs: 148 / 150 / 152 / 156 / 158 / 160; 4 / 6 / 8 / 12 / 14 / 16; and 196 / 198 / 200 / 204 / 206 / 208. This provides a gen. Typical antibodies having these HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequences include antibodies called H4H098P (SEQ ID NOs: 148 / 150 / 152 / 156 / 158 / 160), H4H083P (SEQ ID NOs: 196 / 198 / 200 / 204 / 206 / 208), and H4H095P (SEQ ID NOs: 4 / 6 / 8 / 12 / 14 / 16).

[0017] In the seventh aspect, the present invention is characterized by a human antibody or antibody fragment comprising HCDR3 and LCDR3, wherein HCDR3 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 31, 55, 79, 103, 127, 151, 175, 199, 223 and 247, or a substantially identical sequence having at least 95% homology; and LCDR3 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 39, 63, 87, 111, 135, 159, 183, 207, 231 and 255, or a substantially identical sequence having at least 95% homology.

[0018] In further embodiments, the present invention relates to an HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 27, 51, 75, 99, 123, 147, 171, 195, 219, and 243, or a substantially identical sequence having at least 95% homology; an HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 29, 53, 77, 101, 125, 149, 173, 197, 221, and 245, or a substantially identical sequence having at least 95% homology; an HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 31, 55, 79, 103, 127, 151, 175, 199, 223, and 247, or a substantially similar sequence having at least 95% homology; SEQ ID NO: 11 The present invention features a human antibody or antibody fragment comprising: an LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of 35, 59, 83, 107, 131, 155, 179, 203, 227, and 251, or a substantially similar sequence having at least 95% homology; an LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 37, 61, 85, 109, 133, 157, 181, 205, 229, and 253, or a substantially similar sequence having at least 95% homology; and an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 39, 63, 87, 111, 135, 159, 183, 207, 231, and 255, or a substantially similar sequence having at least 95% homology. In a preferred embodiment, the antibody or antigen-binding fragment comprises HCDR and LCDR sequences encoded by nucleotide sequences SEQ ID NOs: 147, 149, 151, 155, 157 and 159; 195, 197, 199, 203, 205 and 207; and 3, 5, 7, 11, 13 and 15.

[0019] In certain embodiments, the anti-hIL-4R antibody or antigen-binding fragment of the invention comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 162 and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 164, and has a K of about 100 pM or less at 25° C and 37° C, respectively, D (monomeric substrate) or a K of 70 pM or less D (dimeric substrate); about 160 pM or less (monomeric substrate) or a K of 40 pM or less D (dimeric substrate); and an IC of about 10 pM or less (25 pM dimeric substrate) or about 100 pM or less (200 pM monomeric substrate) 50 characterized in that it can block both hIL-4 and hIL-13 activities with an IC of about 30 pM or less 50 (when measured in a bioassay), and cross-reacts with simian IL-4R.

[0020] In certain embodiments, the anti-hIL-4R antibody or antigen-binding fragment of the invention comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 18 and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 20, and has a K of about 450 pM or less D (monomer or dimeric substrate); and an IC of about 40 pM or less (25 pM dimeric substrate) or about 100 pM or less (200 pM monomeric substrate) 50 characterized in that it can block both hIL-4 and hIL-13 activities with an IC of about 100 pM or less 50 (when measured in a bioassay).

[0021] In certain embodiments, the anti-hIL-4R antibody or antigen-binding fragment of the invention comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 210 and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 212, and has a K of about 50 pM or less at 25° C and 37° C, respectively, D (monomeric substrate) or a K of 30 pM or less D(Dimer substrate); K at approximately 200 pM or less (monomer substrate) D or 40 pM or less K D (Dimer substrate); and ICs of approximately 10 pM or less. 50 (25 pM dimer) Substrate) or IC of approximately 90 pM or less 50 (Characterized by a 200 pM monomer substrate), which allows both hIL-4 and hIL-13 activity to be reduced to approximately 25 pM or less IC50. 50 It can be blocked (when measured by bioassay) and does not cross-react with monkey IL-4R.

[0022] In the eighth aspect, the present invention is characterized by an antibody or antigen-binding fragment of an antibody that specifically binds to hIL-4R, comprising three heavy chain complementarity-determining regions and three light chain complementarity-determining regions (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3), where HCDR1 is of formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (Sequence ID 265) contains the amino acid sequence, where X 1 =Gly;X 2 =Phe;X 3 =Thr; X 4 =Phe;X 5 =Asp or Arg;X 6 =Asp or Ser;X 7 =Tyr; and X 8 =Ala or G ly is; HCDR2 is equation X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (Sequence ID 266) contains the amino acid sequence, where X 1 =Ile or Leu, X 2=Ser, X 3 =Gly, T yr or Arg, X 4 =Ser, Asp or Thr, X 5 =Gly or Ser, X 6 =Gl y, Ser or Val, X 7 =Ser or Asn, and X 8 =Thr, Lys, or Ile; HCDR3 is formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 - X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 (Sequence ID 267) contains the amino acid sequence, where X 1 =Ala, X 2 =Lys, X 3 =Asp, Glu or Trp, X 4 =Gly or Arg, X 5 =Leu, Thr or Arg, X 6 =Gly, Arg, or Ser, X 7 =Ile also Gly, X 8 =Thr, Phe or Tyr, X 9 =Ile, Asp or Phe, X 10 =Arg, Tyr, or Asp, X 11 =Pro, Tyr is either present or not present, X 12 =Arg or does not exist, X 13 =Tyr is either true or does not exist, X 14 =Tyr is either true or does not exist, X 15 =Gly or not present, X 16 = Leu is either true or does not exist, X 17 =Asp or does not exist, and X 18=Val or does not exist; LCDR1 has the formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 (SEQ ID NO: 268) and contains the amino acid sequence, where X = Gln, X 1 = Gln, X 2 = Asp, Ser or Val, X 3 = Ile or Leu, X 4 = Ser, Leu or Asn, X 5 = Asn, Tyr or Ile, X 6 = Trp, Ser or Tyr; X 7 = Ile or does not exist; X 8 = Gly or does not exist; X 9 = Tyr or does not exist; X 10 = Asn or does not exist; and X 11 = Tyr or does not exist; LCDR2 has the formula X 1 -X 2 -X 3 (SEQ ID NO: 2 69) and contains the amino acid sequence, where X 1 = Leu, Ala or Val, X 2 = Ala or Gly, and X 3 = Ser; and LCDR3 has the formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 (SEQ ID NO: 270) and contains the amino acid sequence, where X 1 = Gln or Met, X 2 = Gln, X 3 = Ala or Tyr, X 4 = Leu or Asn, X5 =Gln or Ser, X 6 =Thr, Phe or His, X 7 =Pro, X 8 =Tyr, Ile, or Trp, And X 9 =Thr

[0023] In a more specific embodiment, HCDR1 is given by formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 - X 8 (Sequence ID 265) contains the amino acid sequence, where X 1 =Gly;X 2 =Phe;X 3 =T hr;X 4 =Phe;X 5 =Arg;X 6 =Asp or Ser;X 7 =Tyr; and X 8 = Ala or Gly; HCDR2 is formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (Sequence ID 266) contains the amino acid sequence, where X 1 =Ile, X 2 =Ser, X 3 =Glymata Tyr, X 4 =Ser or Thr, X 5 =Gly, X 6 =Gly or Ser, X 7 =Asn, and X 8 =Thr or Lys; HCDR3 is formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 (Sequence The amino acid sequence is 267), and X in the formula 1 =Ala, X 2 =Lys, X 3 =Asp or Gl u, X 4 =Gly or Arg, X 5 =Leu or Arg, X 6 =Gly or Ser, X 7 =Ile or Gly, X 8 =Thr or Phe, X 9 =Ile or Asp, X 10 =Arg or Tyr, X 11 =Pro or not present, X 12 =Arg or does not exist, X 13 =Tyr is either true or does not exist, X 14 =Tyr is either true or does not exist, X 15 =Gly or not present, X 16 = Leu is either true or does not exist, X 17 =Asp or does not exist, and X 18 =Val or does not exist; LCDR1 is expression X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 (Sequence ID 268) contains the amino acid sequence, where X 1 =Gln, X 2 =Ser or Val, X 3 =Ile or Leu, X 4 =Leu or Asn, X 5=Asn or Tyr, X 6 =Ser or Tyr;X 7 =Ile or not; X 8 =Gly or not present;X 9 =Tyr is either Tyr or does not exist; X 10 =Asn or does not exist; and X 11 =Tyr is either true or false; LCDR2 is expression X 1 - X 2 -X 3 (Sequence ID 269) contains the amino acid sequence, where X 1 =Leu or Ala, X 2 =Ala or Gly, and X 3 =Ser; and LCDR3 is equation X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 (SEQ ID NO: 270) contains the amino acid sequence, where X 1 =Gl n or Met, X 2 =Gln, X 3 =Ala or Tyr, X 4 =Leu or Asn, X 5 =Gln or Ser, X 6 =Thr or His, X 7 =Pro, X 8 =Tyr or Trp, and teX 9 =Thr

[0024] In another, more specific embodiment, HCDR1 is formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (Sequence ID 265) contains the amino acid sequence, where X 1 =Gly;X 2 =Phe;X3 =Thr; X 4 =Phe;X 5 =Asp or Arg;X 6 =Asp;X 7 =Tyr; and X 8 =Ala; HCDR2 is equation X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (Sequence The amino acid sequence is 266), and X in the formula 1 =Ile or Leu, X 2 =Ser, X 3 =Gl y or Arg, X 4 =Ser or Thr, X 5 =Gly or Ser, X 6 =Gly or Va l, X 7 =Ser or Asn, and X 8 =Thr or Ile; HCDR3 is formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 (Sequence ID 267) contains the amino acid sequence, where X 1 =Ala, X 2 =Lys, X 3 =Asp or Trp, X 4 =Gly or Arg, X 5 =Leu or Thr, X 6 =Arg or Ser, X 7 =Ile or Gly, X 8=Thr or Tyr, X 9 =Ile or P he, X 10 =Arg or Asp, X 11 =Pro, Tyr is either present or not present, X 12 =Arg or does not exist, X 13 =Tyr is either true or does not exist, X 14 =Tyr is either true or does not exist, X 15 =Gly or not present, X 16 = Leu is either true or does not exist, X 17 =Asp or does not exist, and X 18 =Val or does not exist; LCDR1 is expression X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 (Sequence No. 26) 8) Containing the amino acid sequence, where X 1 =Gln, X 2 =Asp or Ser, X 3 =Ile also is Leu, X 4 =Ser or Leu, X 5 =Tyr or Ile, X 6 =Trp or Ser; X 7 =Ile or not;X 8 =Gly or not present;X 9 =Tyr either exists or does not exist; X 10 =Asn or does not exist; and X 11 =Tyr is either true or false; LCDR2 is expression X 1 -X 2 -X 3 (SEQ ID NO: 269) contains the amino acid sequence , where X 1 =Leu or Val, X 2 =Ala or Gly, and X 3=Ser; so And LCDR3 is equation X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 (Sequence ID 270) The amino acid sequence is included in the formula, where X 1 =Gln or Met, X 2 =Gln, X 3 =Ala, X 4 =Leu or Asn, X 5 =Gln or Ser, X 6 =Thr or Phe, X 7 =Pro, X 8 =Tyr or Ile, and X 9 =Thr

[0025] In the ninth aspect, the present invention provides an antibody or antigen-binding fragment comprising HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequences from an HCVR and LCVR pair, where the HCVR / LCVR sequence is selected from the group consisting of SEQ ID NOs: 162 / 164, 210 / 212, and 18 / 20. In a more specific embodiment, the heavy and light chain CDR sequences are those contained in HCVR SEQ ID NO: 162 and LCVR SEQ ID NO: 164. In another more specific embodiment, the heavy and light chain CDR sequences are those contained in HCVR SEQ ID NO: 18 and LCVR SEQ ID NO: 20. In yet another specific embodiment, the heavy and light chain CDR sequences are those contained in HCVR SEQ ID NO: 210 and LCVR SEQ ID NO: 212.

[0026] The present invention encompasses anti-hIL-4R antibodies having modified glycosylation patterns. In some applications, modifications to remove undesirable glycosylation sites, or antibodies deleting the fucose portion present on the oligosaccharide chain, may be useful to enhance, for example, antibody-dependent cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications may be made to modify complement-dependent cytotoxicity (CDC).

[0027] In the tenth aspect, the present invention provides recombinant vectors having nucleic acid molecules of the present invention, and host cells containing such vectors, and also provides a method for producing antibodies or antigen-binding fragments of the present invention obtained by culturing the host cells of the present invention. The host cells may be prokaryotic or eukaryotic cells, and preferably the host cells are E. coli cells or mammalian cells, such as CHO cells.

[0028] In the eleventh aspect, the present invention is characterized by a composition comprising a recombinant human antibody that specifically binds to hIL-4R and an acceptable carrier.

[0029] In the twelfth aspect, the present invention is characterized by a method of inhibiting hIL-4 activity using the antibody of the present invention or its antigen-binding moiety. In certain embodiments, the antibody of the present invention also blocks the binding of the hIL-13 / hIL-13R1 complex to hIL-4R. In one embodiment, the method comprises contacting hIL-4R with the antibody of the present invention or its antigen-binding moiety so as to inhibit hIL-4 or hIL-4 / hIL-13 activity. In another embodiment, the method comprises administering the antibody of the present invention or its antigen-binding moiety to a human subject suffering from a disorder that is mitigated by inhibition of hIL-4 or hIL-4 / hIL-13 activity. The disorder treated is any disease or condition that is improved, mitigated, suppressed or prevented by the removal, inhibition or reduction of hIL-4 or hIL-4 / hIL-13 activity.

[0030] Examples of IL-4-related disorders treated with the antibody or antibody fragment of the present invention include arthritis (including septic arthritis), herpetiformis, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, lung disorders such as mild, moderate, or severe asthma, inflammatory disorders such as inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephrotic syndrome.

[0031] Other purposes and benefits will become clear from the following detailed explanation.

[0032] Detailed explanation Before describing the method of the present invention, it is obvious that the present invention is a specific method and The methods and conditions may vary, not limited to experimental conditions. Naturally, the scope of the present invention is limited only by the appended claims, and the terms used herein are for the purpose of describing specific embodiments and are not intended to limit them.

[0033] 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 which the present invention pertains. Any method and material similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein.

[0034] definition As used herein, the term “human IL4R” (hIL-4R) is intended to refer to the human cytokine receptor that specifically binds to interleukin-4 (IL-4), IL-4Rα (SEQ ID NO: 274). The term “human interleukin-13” (hIL-13) refers to the cytokine that specifically binds to the IL-13 receptor, and the “hIL-13 / hIL-13R1 complex” refers to the complex formed when hIL-13 binds to the hIL-13R1 complex, which then binds to the hIL-4 receptor and initiates biological activity.

[0035] As used herein, the term “antibody” is intended to refer to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are incorporated along with more conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0036] As used herein, the term “antigen-binding moiety” (or simply “antibody moiety” or “antibody fragment”) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hIL-4R). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed in the “antigen-binding moiety” of an antibody include: (i) the Fab fragment, a monovalent fragment consisting of VL, VH, CL1, and CH1 domains; (ii) the F(ab')2 fragment, a bivalent fragment containing two F(ab)' fragments linked by disulfide crosslinks in the hinge region; (iii) the Fd fragment consisting of VH and CH1 domains; (iv) the Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) the dAb fragment (Ward et al. (1989) Nature 241:544-546), which consists of a VH domain; and (vi) the CDR. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked together by a synthetic linker using recombination methods, which allows them to form a single continuous chain so that the VL and VH region pair forms a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term antibody's "antigen-binding portion." Other forms of single-chain antibodies (e.g., bispecific antibodies) are also included. For example, see Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90:6444-6448.

[0037] As used herein, “neutralizing” or “blocking” antibodies are intended to refer to antibodies whose binding to hIL-4R results in inhibition of the biological activity of hIL-4 and / or hIL-13. This inhibition of the biological activity of hIL-4 and / or IL-13 can be assessed by measuring one or more indicators of hIL-4 and / or hIL-13 biological activity known in the art, such as hIL-4 and / or IL-13-induced cell activation and the binding of hIL-4 to hIL-4R (see Examples below).

[0038] The "CDR" or complementarity-determining region is a highly variable region interspersed within a more conserved region called the "framework region" (FR). In various embodiments of the anti-hIL-4R antibody or fragment of the present invention, the FR may be identical to a human germline sequence, or it may be naturally or artificially modified.

[0039] The term "surface plasmon resonance" as used herein refers to, for example, BIACORE TM This refers to an optical phenomenon that enables real-time interaction analysis by detecting changes in protein concentration within a biosensor matrix using a system (Pharmacia Biosensor AB).

[0040] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, also known as a paratope. A single antigen may have more than one epitope. Epitopes can be steric or linear. Steric epitopes are generated by spatially juxtaposed amino acids from different parts of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may contain sugar, phosphoryl, or sulfonyl portions on the antigen.

[0041] When referring to nucleic acids or fragments thereof, the term “substantially identical” or “substantially identical” means that, when optimally aligned with another nucleic acid (or its complementary strand) including appropriate nucleotide insertions or deletions, nucleotide sequence identity exists at least about 95%, and more preferably at least about 96%, 97%, 98%, or 99% of nucleotide bases, as measured by any of the well-known sequence identity algorithms, e.g., FASTA, BLAST, or Gap, as considered below.

[0042] When applied to polypeptides, the term “substantial similarity” or “substantially identical” means that two peptide sequences share at least 95% sequence identity, and more preferably at least 98% or 99%, when optimally aligned using default gap weights, for example, by programmed GAP or BESTFIT. Preferably, the non-identical residue positions differ by conserved amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the sequence identity percentage or similarity may be adjusted upward to compensate for the conservation suitability of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids with side chains having similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains are cysteine ​​and methionine. Preferred conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conserved substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A “moderately conserved” substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0043] Sequence similarity (also called sequence identity) of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications (including conserved amino acid substitutions). For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides (e.g., homologous polypeptides from different species or between wild-type proteins and their variants). See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, and the programs in GCG version 6.1 (e.g., FASTA2 and FASTA3) provide alignment of the best overlap region between the query sequence and the search sequence and a sequence identity percentage (Pearson (2000), cited above). Another preferred algorithm when comparing the sequences of the present invention against a database containing numerous sequences from various biological origins is the computer program BLAST, particularly BLASTP or TBLASTN, which uses default parameters. See, for example, Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402.

[0044] Manufacturing of human antibodies Methods for generating human antibodies include, for example, those described in US 6,596,541, Green et al. (1994) Nature Genetics 7:13-21), US 5,545,807, and US 6,787,637.

[0045] Rodents can be immunized by any method known in the field (e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual 1988 Cold Spring Harbor Laboratory; Malik and Lillehoj (1994) Antibody (See Techniques, Academic Press, CA). The antibodies of the present invention are preferably VELOCIMMUNE TM The process is carried out using the technology (US6,596,541). Transgenic mice in which the endogenous immunoglobulin heavy and light chain variable regions are replaced with corresponding human variable regions are attacked with the target antigen, and lymphocytes (e.g., B cells) are recovered from the mice that express antibodies. The lymphocytes can be fused with myeloma cell lines to produce immortal hybridoma cell lines, and such hybridoma cell lines can be screened and selected to identify hybridoma cell lines that produce antibodies specific to the target antigen. The DNA encoding the heavy and light chain variable regions can be isolated and ligated to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, antigen-specific chimeric antibodies or DNA encoding the light and heavy chain variable regions can be isolated directly from antigen-specific lymphocytes.

[0046] DNA encoding the variable regions of the antibody's heavy and light chains was isolated to obtain the human heavy and light chain constant regions. The DNA encoding the region is operably ligated to it. This DNA is then expressed in cells capable of expressing a fully human antibody. In certain embodiments, the cells are CHO cells.

[0047] Antibodies may be therapeutically useful in blocking ligand-receptor interactions or inhibiting receptor component interactions, rather than in their involvement in complement-mediated cell killing (complement-dependent cell injury) (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The constant region of an antibody is crucial in its ability to bind complement and mediate cell-dependent cell injury. Therefore, antibody isotypes may be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0048] Human immunoglobulins can exist in two forms related to hinge heterogeneity. In one form, they contain a stable four-chain construct of approximately 150–160 kDa, where dimers are linked by interchain heavy-chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds, and a molecule of approximately 75–80 kDa is formed, consisting of covalently linked light and heavy chains (half-antibody). These forms are extremely difficult to separate, even after affinity purification. The frequency of occurrence of the second form in various intact IgG isotypes is attributed, to varying degrees, to structural differences related to the hinge region isotype of the antibody. Indeed, a single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using the human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention encompasses antibodies having one or more mutations in the hinge, CH2, or CH3 region, which may be desirable in production, for example, to improve the yield of a desired antibody morphology.

[0049] First, high-affinity chimeric antibodies having a human variable region and a mouse constant region are isolated. As described below, these antibodies are characterized and selected with respect to desirable features including binding affinity to hIL-4R, the ability to block the binding of hIL-4 to hIL-4R, and / or selectivity for human proteins. The mouse constant region is replaced with the desired human constant region to produce the fully human antibody of the present invention, e.g., wild-type or modified IgG4 or IgG1 (e.g., SEQ ID NOs. 271, 272, 273). The selected constant region may vary depending on the specific application, but the high-affinity antigen-binding and target specificity features reside in the variable region.

[0050] Epitope mapping and related technologies To screen for antibodies that bind to specific epitopes, Harlow and Cross-blocking assays, such as the one described by Lane (mentioned above), can be performed. Other methods include alanine scanning mutants, peptide blotting (Reineke (2004) Methods Mol Biol 248:443-63), or peptide cleavage analysis. Furthermore, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Protein Science:9:487-496).

[0051] Modification-Assisted Profiling (MAP), also known as Antigen Structure-Based Antibody Profiling (ASAP), is a method for classifying a number of monoclonal antibodies (mAbs) specific to the same antigen according to the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces (U.S. Patent Application Publication 2004 / 0101920). Each category is distinct from or partially overlaps with epitopes represented by other categories. - It can reflect specific epitopes that wrap around the antibody. This technique allows for rapid selection of genetically identical antibodies, enabling focus on characterization of genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones with desired features. Using MAP, the hIL-4R antibody of the present invention can be sorted into groups of various epitopes that bind to the antibody.

[0052] Useful agents for altering the structure of immobilized antigens include enzymes, such as proteolytic enzymes, and chemicals. The antigen protein may be immobilized on either the surface of a biosensor chip or polystyrene beads. The latter is, for example, multiple LUMINEX. TM It can be processed using assays such as detection assays (Luminex Corp., TX). LUMINEX handles multiplex analysis using up to 100 different types of beads. TM Due to its capabilities, LUMINEX TM This provides a nearly non-limiting antigen surface with various modifications, resulting in improved resolution in antibody epitope profiling compared to biosensor assays on antibody epitopes.

[0053] Bispecifics The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific to different epitopes of one target polypeptide, or they may contain antigen-binding domains specific to one or more target polypeptides. See, for example, Tutt et al. (1991) J. Immunol. 147:60-69. Human anti-IL-4R antibodies may be ligated to another functional molecule, such as another peptide or protein, or co-expressed with another functional molecule. For example, an antibody or its fragment may be ligated (e.g., by chemical coupling, gene fusion, non-covalent bond, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity.

[0054] Therapeutic administration and formulations This invention provides a therapeutic composition comprising the anti-IL-4R antibody or its antigen-binding fragment. Administration of the therapeutic composition according to this invention is performed together with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Numerous suitable formulations are available in the prescription collection known to all pharmacists: Remington's Pha These can be found in rmaceutical Sciences, Mack Publishing Company, Easton, PA. Examples of these formulations include powders, pastes, ointments, jellies, waxes, oils, lipids, and lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN). TM Examples include DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax (polyethylene glycol of various molecular weights) emulsions, semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA(1998)J Pharm Sci Technol 52:238-311.

[0055] The dosage may vary depending on the age and size of the subject being administered the antibody, the target disease, condition, and route of administration. When the antibody of the present invention is used to treat various IL-4R-related conditions and diseases in adult patients, it is advantageous to administer the antibody of the present invention intravenously as a single dose of approximately 0.01 to approximately 20 mg / kg body weight, more preferably approximately 0.02 to approximately 7, approximately 0.03 to approximately 5, or approximately 0.05 to approximately 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted.

[0056] Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention. For example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (e.g., Wu et al.) See al. (1987) J. Biol. Chem. 262:4429-4432). Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through the epithelium or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other bioactive agents. Administration may be systemic or topical.

[0057] This pharmaceutical composition can also be delivered by vesicles, particularly liposomes (Langer). (1990) Science 249:1527-1533; Treat et al. (1989) Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp.353-365; See also Lopez-Berestein, ibid., pp.317-327; See ibid. in general.

[0058] In certain circumstances, the pharmaceutical composition can be delivered by a controlled-release system. In one embodiment, a pump may be used (see Langer, previously cited; Sefton (1987) CRC Crit.Ref.Biomed.Eng.14:201). In another embodiment, a polymer material may be used (Medical Applications of Controlled Release, Langer and See Wise (ed.), CRC Pres., Boca Raton, Florida (1974). In yet another embodiment, the controlled-release system can be positioned close to the target of the composition and therefore requires only a small fraction of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release, ibid., vol.2, pp.115-138, 1984). Other controlled-release systems are discussed in the review by Langer (1990) in Science 249:1527-1533.

[0059] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, as well as intravenous infusion. These injectable preparations may be manufactured by generally known methods. For example, an injectable preparation may be manufactured by dissolving, suspending, or emulsifying, for example, the antibody or a salt thereof in a sterile aqueous medium or an oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, which may be used in combination with appropriate solubilizers, such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers, such as benzyl benzoate and benzyl alcohol. The injectable preparations thus manufactured are preferably filled into appropriate ampoules.

[0060] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are manufactured in appropriate unit dose formulations to match the dosage of the active ingredient. Examples of such unit dose formulations include tablets, pills, capsules, injections (ampoules), and suppositories. The amount of the aforementioned antibody contained is generally about 5 to 500 mg per unit dose formulation; in particular, it is preferable that the antibody is contained in an amount of about 5 to 100 mg in the form of an injection and about 10 to 250 mg for other formulations.

[0061] Monotherapy and combination therapy The antibodies and antibody fragments of the present invention are useful for treating diseases and disorders that are improved, suppressed, or mitigated by reducing IL-4 activity. These disorders include those characterized by abnormal or overexpression of IL-4, or abnormal host responses to IL-4 production. IL-4-related disorders treated with this antibody or antibody fragment include, for example, arthritis (including septic arthritis), herpetiform disease, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, lung disorders such as asthma (mild, moderate, or severe), inflammatory disorders such as inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, atopic dermatitis (dermatitis), ulcerative colitis, fibrosis, and nephrotic syndrome (see US7,186,809).

[0062] The present invention encompasses combination therapy, in which an anti-IL-4R antibody or antibody fragment is administered in combination with a second therapeutic agent. Co-administration and combination therapy include treatment regimens in which an anti-IL-4R antibody or antibody fragment is administered at least once during a course of treatment, which includes administering at least one other therapeutic agent to the patient, but is not limited to simultaneous administration. The second therapeutic agent may be another IL-4 antagonist, e.g., another antibody / antibody fragment, or a soluble cytokine receptor, IgE antagonist, or an anti-asthmatic agent that can be delivered by inhalation or other suitable means (corticosteroids, nonsteroidal antisteroids, beta-agonists, leukotriene antagonists, xanthines, fluticasone, salmeterol, albuterol). In certain embodiments, the anti-IL-4R antibody or antibody fragment of the present invention may be administered together with an IL-1 antagonist, e.g., lilonacept, or an IL-13 antagonist. The second category of drugs may include one or more leukotriene receptor antagonists for treating allergic inflammatory diseases, such as asthma and allergies. Examples of leukotriene receptor antagonists include, but are not limited to, montelukast, pranlukast, and zafirlukast. The second category of drugs may also include cytokine inhibitors, such as TNF(etanercept, ENBREL). TM ), may include one or more of the following: IL-9, IL-5, or IL-17 antagonists.

[0063] The present invention also includes the use of any anti-IL-4R antibody or antigen-binding fragment described herein in the manufacture of a pharmaceutical for the treatment of a disease or disorder, wherein the disease or disorder is improved, mitigated, or suppressed by the removal, inhibition, or reduction of human interleukin-4 (hIL-4) activity. Examples of such diseases or disorders include, for example, arthritis, herpetiform disease, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, lung disorders, asthma, inflammatory disorders, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, nephrotic syndrome, atopic dermatitis, and asthma (athsma). [Examples]

[0064] The following examples are provided to the art to provide a complete disclosure and description of the methods and compositions of the present invention and how they are prepared and used, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), but some degree of experimental error and deviation is to be expected. Unless otherwise indicated, parts are parts by mass, molecular weight is the average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0065] Example 1. Preparation of human antibodies against the human IL-4 receptor. VELOCIMMUNE TM Mice (Regeneron Pharmaceuticals, Inc.; US6,596,541) were immunized with human IL-4R (hIL-4R, SEQ ID NO: 274) or a combination of hIL-4R and monkey (Macaca fascicularis) IL-4R (mfIL-4R, SEQ ID NO: 275) protein or DNA. To obtain an optimal immune response, the animals were subsequently boosted every 3-4 weeks, and blood was collected 10 days after each boost to assess the progression of the anti-antigen response.

[0066] Once the mice reached their maximum immune response, antibody-expressing B cells were collected and fused with mouse myeloma cells to form hybridomas. Alternatively, antigen-specific antibodies were isolated directly from B cells without fusion to myeloma cells, as described in U.S. Patent Application Publication 2007 / 0280945A1 (which is incorporated herein by reference in its entirety). Stable recombinant antibody-expressing CHO cell lines were established from the isolated appropriate recombinants. Functionally desirable monoclonal antibodies were selected by screening the conditioned medium of hybridomas or transfected cells for specificity, antigen-binding affinity, and efficacy in blocking the binding of hIL-4 to hIL-4R (as described below).

[0067] Several anti-hIL-4R antibodies were obtained by the method described above, including typical antibodies named H4H083P, H4H094P, H4H095P, H4H098P, and H4H099P. These typical anti-hIL-4R antibodies and their biological properties are described in more detail in the following examples.

[0068] Example 2. Determination of antigen binding affinity At either 25°C or 37°C, the binding affinity (K) of the selected antibody for hIL-4R is measured. D ) is used in real-time biosensor surface plasmon resonance assays (BIACORE TM 200 Determined using 0). Briefly, the antibody was BIACORE TM A capture antibody surface was formed by capturing the goat anti-hFc polyclonal antibody, which was produced by direct coupling to a chip. Various concentrations (ranging from 50 nM to 12.5 nM) of monomer hIL-4R (R&D Systems) or dimer hIL-4R-mFc were injected onto the capture antibody surface at a rate of 10 μl / min for 2.5 minutes at either 25°C or 37°C. Antigen binding to the antibody and dissociation of the binding complex were monitored in real time. The parallel dissociation constant (K) was also measured. D ) and dissociation rate The number was confirmed by performing kinetic analysis using BIA evaluation software. The BIA evaluation software was used to determine the half-life (T) of the antigen / antibody complex dissociation. 1 / 2 It is also used to calculate ) The results are shown in Table 1. NB: Antibody-antigen binding was not observed under experimental conditions. Control: Fully human anti-IL-4R antibody (US Patent Nos. 7,186, 809; Sequence IDs: 10 and 12).

[0069] [Table 1]

[0070] Regarding cynomolgus monkey (Macaca fascicularis) IL-4R (mfIL-4R), the binding affinity (K) of selected antibodies at either 25°C or 37°C is D ) mo ma Furthermore, the results were determined using the real-time biosensor surface plasmon resonance assay described above, with monomer mfIL-4R-myc-myc-his (mfIL-4R-mmh) or dimer mfIL-4R-mFc at various concentrations (ranging from 100 nM to 25 nM). Only antibody H4H098P was used to test on both the monomer and dimer mfIL-4R at 25°C with K2 at concentrations of 552 nM and 9.08 nM, respectively. D It was possible to bind with it. Furthermore, antibody H4H098P Furthermore, a 24.3 nM K2440 D Combined with H4H0. 83P had a very weak binding to the dimer mfIL-4R.

[0071] Antibody-antigen binding affinity was also evaluated using an ELISA-based solution competition assay. Briefly, 96-well MAXISORP TMThe plates were first coated overnight with 5 μg / ml avidin, followed by BSA blocking for 1 hour. The avidin-coated plates were then incubated with 250 ng / ml biotin-hIL4 for 2 hours. Using these plates, either free hIL-4R-mFc (dimer hIL-4R) or free hIL-4R-myc-myc-his (hIL4R-mmh, monomer hIL4R) was measured in the antibody titration sample solution. To prepare the antibody titration sample, a fixed amount of either 25 pM hIL-4R-mFc or 200 pM hIL-4R-mmh was pre-mixed with various amounts of antibody in serial dilutions ranging from 0 to approximately 10 nM, and then incubated at room temperature for 1 hour to reach antibody-antigen equilibrium. The equilibrated sample solution was then transferred to a plate coated with hIL-4 for measurement of either free hIL-4R-mFc or free hIL-4R-mmh. After 1 hour of binding, the plates were washed, and the bound hIL-4R-mFc was detected using either an HRP-conjugated mouse anti-mFc polyclonal antibody or an HRP-conjugated goat anti-myc polyclonal antibody. 50 The values ​​were determined (Table 2).

[0072] [Table 2]

[0073] Cross-reactivity of the antibody against monkey IL-4R was also determined using an ELISA-based solution competition assay. Antibody H4H098P showed a 300 pM IC50 for mfIL-4R-mFc. 50 And for mfIL-4R-mmh, an IC with a 20nM pulse rate. 50 This was shown.

[0074] Example 3. Neutralization of the biological effects of hIL-4 and hIL-13 in vitro. To determine in vitro the ability of purified anti-hIL-4R antibodies to neutralize hIL-4R-mediated cellular function, we developed a bioassay using an HK293 cell line modified to contain human STAT6 and a STAT6 luciferase reporter. Inhibition of hIL-4R-induced luciferase activity was determined as follows: cells were placed in 96-well plates in medium at a rate of 1 × 10⁶ 4 Cells were seeded in wells and incubated overnight at 37°C and 5% CO2. Antibody proteins ranging from 0 to 20 nM were added to the cells in serial dilutions, along with either 10 pM hIL-4 or 40 pM hIL-13. Cells were then incubated at 37°C and 5% CO2 for 6 hours. The cells were incubated for an extended period. The degree of cellular response was measured by a luciferase assay (Promega Biotech). The results are shown in Table 3. NB: Luciferase activity was not blocked under the above experimental conditions. Furthermore, H4H098P inhibited mfIL-4R-mediated cellular function with 150 nM IC in the presence of 360 fM mfIL-4. 50 It was possible to block it.

[0075] [Table 3]

Claims

1. An antibody or its antigen-binding fragment that specifically binds to the human interleukin-4 receptor (hIL-4R) (SEQ ID NO: 274), which includes a heavy chain variable region (HCVR) and a light chain variable region (LCVR): (a) Affinity for hIL-4R at approximately 100 pM or less (K D ) features HCVR having the amino acid sequence shown in SEQ ID NO: 162 and LCVR having the amino acid sequence shown in SEQ ID NO: 164; (b) Affinity for hIL-4R at approximately 300 pM or less (K D ) features HCVR having the amino acid sequence shown in SEQ ID NO: 18 and LCVR having the amino acid sequence shown in SEQ ID NO: 20; or (c) Affinity for hIL-4R at approximately 50 pM or less (K D ) is a characteristic of HCVR having the amino acid sequence shown in SEQ ID NO: 210 and LCVR having the amino acid sequence shown in SEQ ID NO: 212 The above antibody or antigen-binding fragment, including the above.

2. An antibody or antigen-binding fragment thereof that specifically binds to the human interleukin-4 receptor (hIL-4R) (SEQ ID NO: 274), comprising a heavy chain complementarity-determining region 3 (HCDR3) and a light chain complementarity-determining region 3 (LCDR3): (a) HCDR3 having the amino acid sequence shown in SEQ ID NO: 152 and LCDR3 having the amino acid sequence shown in SEQ ID NO: 160; (b) HCDR3 having the amino acid sequence shown in SEQ ID NO: 8 and LCDR3 having the amino acid sequence shown in SEQ ID NO: 16; or (c) HCDR3 having the amino acid sequence shown in SEQ ID NO: 200 and LCDR3 having the amino acid sequence shown in SEQ ID NO: 208; The above antibody or antigen-binding fragment, including the above.

3. An antibody or antigen-binding fragment of an antibody that specifically binds to human IL-4R, comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), 3 (HCDR3) and light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), 3 (LCDR3), HCDR1 contains the amino acid sequence of formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (SEQ ID NO: 265), where X 1 = Gly; X 2 = Phe; X 3 = Thr; X 4 = Phe; X 5 = A sp or Arg; X 6 = Asp or Ser; X 7 = Tyr; and X 8 = Ala or Gly And; HCDR2 is given by equation X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 The formula includes the amino acid sequence of (SEQ ID NO: 266), where X 1 =Ile or Leu, X 2 = Ser, X 3 = Gly, Tyr or Arg, X 4 = Ser, Asp or Thr, X 5 = Gly or Ser, X 6 = Gly, S er or Val, X 7 = Ser or Asn, and X 8 = Thr, Lys, or Ile; HCDR3 is given by formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6- X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 The formula includes the amino acid sequence of (SEQ ID NO: 267), where X 1 = Ala, X 2 = Lys, X 3 = Asp, Glu or Trp, X 4 = Gly or Arg, X 5 = Leu, Thr, or Arg, X 6 = Gly, Arg, or Ser, X 7 = Ile or G ly, X 8 = Thr, Phe or Tyr, X 9 = Ile, Asp or Phe, X 10 = Arg, Tyr or Asp, X 11 = Pro, Tyr is either present or not present, X 12 = Arg or does not exist, X 13 = Tyr is either true or does not exist, X 14 = Tyr is either true or does not exist, X 15 = Gly or not exist, X 16 = Leu is either true or does not exist, X 17 = Asp or not exist, and X 18 = Val is either true or does not exist; LCDR1 is, Equation X 1 -X 2 -X 3 -X 4 -X 5 -X 6- X 7 -X 8 -X 9 -X 10 -X 11 (array The formula includes the amino acid sequence of number 268, where X 1 = GLn, X 2 = Asp, Ser, or Va l, X 3 =Ile or Leu, X 4 = Ser, Leu or Asn, X 5 = Asn, Tyr is Ile, X 6 = Trp, Ser or Tyr; X 7 = Ile or does not exist; X 8 = Gly or not; X 9 = Tyr is either true or does not exist; X 10 = Asn or not exist; and X 11 = Tyr is either true or does not exist; The LCDR2 contains the amino acid sequence of formula X 1 -X 2 -X 3 (SEQ ID NO: 269), wherein X 1 = Leu, Ala or Val, X 2 = Ala or Gly, and X 3 = Ser; and LCDR3 is, Equation X 1 -X 2 -X 3 -X 4 -X 5 -X 6- X 7 -X 8 -X 9 The formula includes the amino acid sequence of (SEQ ID NO: 270), where X 1 = GLN or Met, X 2 = GLn, X 3 = Ala or T yr, X 4 = Leu or Asn, X 5 = Gln or Ser, X 6 = Thr, Phe or Hi s, X 7 = Pro, X 8 = Tyr, Ile or Trp, and X 9 = Thr, An antibody or an antigen-binding fragment of an antibody.

4. The heavy and light chain CDR amino acid sequences (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) are as follows: (a) Sequence IDs 148, 150, 152, 156, 158, 160; (b) Sequence IDs 4, 6, 8, 12, 14, 16; and (c) Sequence IDs 196, 198, 200, 204, 206, 208 An antibody or antigen-binding fragment according to claim 3, selected from the group consisting of the following.

5. A nucleic acid sequence encoding the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody or antigen-binding fragment according to any one of claims 1 to 4.

6. A vector comprising the nucleic acid sequence described in claim 5.

7. A host vector system for the production of an antibody or an antigen-binding fragment of an antibody, comprising the vector described in claim 6.

8. A method for producing an antibody or its antigen-binding fragment that specifically binds to human interleukin-4 receptor alpha (hIL-4R), The method comprising growing cells of the host vector system described in claim 7 under conditions in which an antibody or fragment is expressed, and recovering the expressed anti-hIL-4 antibody.

9. The method according to claim 8, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

10. The method according to claim 9, wherein the host cell is an E. coli cell or a CHO cell.

11. Use of an antibody or antigen-binding fragment as defined in claim 1, 2, 3, or 4 in the manufacture of a pharmaceutical product for the treatment of a disease or disorder, wherein the disease or disorder is improved, mitigated, or suppressed by the removal, inhibition, or reduction of human interleukin-4 (hIL-4) activity.

12. Use as defined in claim 11, wherein the disease or disorder is selected from the group consisting of arthritis, herpetiform disease, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, lung disorder, asthma, inflammatory disorders, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephrotic syndrome.

13. The use as defined in claim 11, wherein the disease or disorder is asthma or atopic dermatitis.

14. A method for treating a disease or disorder which is improved, reduced or suppressed by the removal, inhibition or reduction of human interleukin-4 (hIL-4) activity, comprising administering an antibody or antigen-binding fragment according to any one of claims 1 to 4 to a patient in need thereof.

15. The method according to claim 14, wherein the disease or disorder is selected from the group consisting of arthritis, herpetiform disease, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, lung disorder, asthma, inflammatory disorders, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephrotic syndrome.

16. The method according to claim 14, wherein the disease or disorder is asthma or atopic dermatitis.

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