High-affinity human antibody against human IL-4 receptor

Abstract

To provide an isolated human antibody or antibody fragment thereof which binds to human interleukin-4 receptor alpha (hIL-4Rα) with high affinity (KD), capable of blocking hIL-4 and hIL-13 activity.SOLUTION: An antibody or antigen-binding fragment thereof that specifically binds to human interleukin-4 receptor (hIL-4R) including an amino acid sequence set forth in SEQ ID NO:274 comprises a heavy chain variable region (HCVR) including an amino acid sequence set forth in SEQ ID NO:162 and a light chain variable region (LCVR) including an amino acid sequence set forth in SEQ ID NO:164.SELECTED DRAWING: None

Description

[Background technology]

[0001] Interleukin-4 (IL-4, also known as B cell stimulating factor or BSF-1) was originally characterized for its ability to stimulate the proliferation of B cells in response to low concentrations of antibodies specific for surface immunoglobulin. 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 its specific cell surface receptor. Human IL-4 receptor alpha (hIL-4R) (SEQ ID NO: 274) is described, for example, in U.S. Patent Nos. 5,629,999, 5,729,963, 5,739,974, and 5,823,262. Antibodies against hIL-4R are described in U.S. Patent Nos. 5,629,999 and 5,823,262.

[0003] Methods for producing antibodies useful as human therapeutics include generating chimeric and humanized antibodies (see, e.g., U.S. Patent No. 6,223,999). See, e.g., U.S. Patent No. 6,223,999 and U.S. Patent No. 6,223,999, which describe methods for generating non-human transgenic mice capable of producing human antibodies.

[0004] Methods using antibodies against hIL-4R are described in US Pat. Nos. 5,629,999; 5,729,999; and 5,729,999. [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 Summary of the Invention [Means for solving the problem]

[0006] In a 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). The human antibody is characterized by binding to hIL-4R with high affinity and the ability to neutralize hIL-4 activity. In a specific embodiment, the human antibody is capable of blocking the binding of the hIL-13 / hIL-13R1 complex to hIL-4R, thereby inhibiting hIL-13-mediated signal transduction. The antibody may be full-length (e.g., an IgG1 or IgG4 antibody) or may contain only the antigen-binding portion (e.g., a Fab, F(ab')2, or scFv fragment), and may be a recombinant human antibody. The antibody can be modified to achieve its function by eliminating residual effector function, for example, by eliminating residual effector function (Reddy et al. (2000) J. Immunol. 164:1925-1933).

[0007] In a general embodiment, the K is about 300 pM or less as measured by surface plasmon resonance in a monomer or dimer assay. D hIL-4R (SEQ ID NO: 274 In more specific embodiments, the antibody or antigen-binding portion thereof has a K of about 200 pM or less, about 150 or less, about 100 pM or less, or about 50 pM. D Shows various In one embodiment, the antibody or antigen-binding fragment has an IC of about 100 pM or less as measured by luciferase bioassay. 50 In more specific embodiments, the antibody or antigen-binding fragment has an IC50 of about 50 pM or less, about 30 pM or less, or about 25 pM or less as measured by a STAT6 luciferase bioassay. 50 In various embodiments, the antibody or antigen-binding fragment has an IC of about 100 pM or less, about 90 pM or less, about 50 pM or less, or about 20 pM or less as measured by a STAT6 luciferase bioassay. 50 to block hIL-13 activity.

[0008] In a second aspect, the antibody of the 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 a third aspect, an antibody of the 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 invention is selected from the group consisting of SEQ ID NOs: 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 / 150, 152 / 154, 156 / 158, 158 / 160, 162 / 164, 164 / 166, 166 / 168, 168 / 170, 168 / 172, 168 / 174, 168 / 176, 168 / 178, 170 / 172, 170 / 176, 170 / 178 ... 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 preferred embodiments, the antibody or antibody fragment comprises the HCVR / LCVR sequence pair SEQ ID NO: 162 / 164, 210 / 212, or 18 / 20; exemplary antibodies having these HCVR / LCVR sequence pairs include the antibodies designated H4H098P (SEQ ID NO: 162 / 164), H4H083P (SEQ ID NO: 210 / 212), and H4H095P (SEQ ID NO: 18 / 20).

[0011] In a fourth aspect, the present invention provides a nucleic acid molecule encoding an HCVR, the nucleic acid molecule being 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 thereof having at least 95% homology.

[0012] In a fifth embodiment, the present invention provides a nucleic acid molecule encoding an LCVR, the 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 thereof having at least 95% homology.

[0013] In one embodiment, the antibodies of the invention are selected from the group consisting of 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 / 162, 163 / 164, 165 / 166, 167 / 168, 168 / 169, 170 / 171, 172 / 173, 174 / 175, 176 / 177, 178 / 179, 180 / 181, 182 / 183, 184 / 185, 186 / 187, 188 / 189, 190 / 191, 192 / 193, 200 / 201, 203 / 204, 205 / 206, 207 / 208, 209 / 210, 211 / 212, 213 / 214, 215 / 216, 217 / 218, 218 / 219, 220 / 221, 222 / 223, 224 / 225, 226 / 227, 228 / 229, 230 / 231, 232 / 233, 234 / 235, 236 / 237, 238 / 239, 240 / 241, 24 In a preferred embodiment, the antibody or antibody fragment comprises an HCVR / LCVR encoded by a nucleic acid sequence pair selected from the group consisting of SEQ ID NOs: 161 / 163, 209 / 211, and 17 / 19. In an even more preferred embodiment, the antibody or antibody fragment comprises an HCVR / LCVR encoded by a nucleic acid sequence pair selected from the group consisting of SEQ ID NOs: 161 / 163, 209 / 211, and 17 / 19.

[0014] In a sixth aspect, the present invention provides an antibody or antigen-binding fragment comprising an HCDR3 and an 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 NO: 152 / 160, 8 / 16 or 200 / 208. In an even 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 has 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 sequence substantially similar 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 sequence substantially similar 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 sequence substantially similar thereto. an LCDR1 sequence selected from the group consisting of SEQ ID NOs: 12, 36, 60, 84, 108, 132, 156, 180, 204, 228 and 252, or a sequence substantially similar thereto; an LCDR2 sequence selected from the group consisting of SEQ ID NOs: 14, 38, 62, 86, 110, 134, 158, 182, 206, 230 and 252, or a sequence substantially similar thereto; and an LCDR3 sequence selected from the group consisting of SEQ ID NOs: 16, 40, 64, 88, 112, 136, 160, 184, 208, 232 and 256, or a sequence substantially similar thereto. In preferred embodiments, the antibody or antigen-binding fragment comprises 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 specific embodiment, the present invention provides an anti-hIL-4R antibody or antigen-binding fragment thereof 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. Exemplary antibodies having these HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequences include antibodies designated 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 a seventh aspect, the invention features a human antibody or antibody fragment comprising an HCDR3 and an LCDR3, wherein the 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 thereof with at least 95% homology; and the 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 thereof with at least 95% homology.

[0018] In a further embodiment, the present invention provides 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 thereof with 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 thereof with 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 thereof with at least 95% homology; , 35, 59, 83, 107, 131, 155, 179, 203, 227, and 251, or a substantially similar sequence thereof with 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 thereof with 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 thereof with at least 95% homology. In a preferred embodiment, the antibody or antigen-binding fragment comprises the HCDR and LCDR sequences encoded by the 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 present invention comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 162 and an 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 (monomer substrate) or K of 70 pM or less D (dimeric substrate); K of about 160 pM or less (monomeric substrate) or 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 which exhibits both hIL-4 and hIL-13 activity with an IC of about 30 pM or less 50 (as measured in bioassays) and cross-reacts with monkey IL-4R.

[0020] In certain embodiments, the anti-hIL-4R antibody or antigen-binding fragment of the present invention comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 18 and an 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 which exhibits both hIL-4 and hIL-13 activity with an IC of about 100 pM or less 50 (as measured by bioassay) can be blocked.

[0021] In certain embodiments, the anti-hIL-4R antibody or antigen-binding fragment of the present invention comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 210 and an 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 (monomer substrate) or a K of 30 pM or less D(dimeric substrate); K of about 200 pM or less (monomeric substrate) D or 40 pM or less K D (dimeric substrate); and an IC of about 10 pM or less 50 (25pM dimer substrate) or an IC of about 90 pM or less 50 (200 pM monomeric substrate), which exhibited both hIL-4 and hIL-13 activity with IC values ​​of approximately 25 pM or less. 50 (as measured by bioassay) and does not cross-react with monkey IL-4R.

[0022] In an eighth aspect, the invention features 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 represented by the formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (SEQ ID NO: 265), wherein 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; HCDR2 is of formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (SEQ ID NO: 266), wherein 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 of the 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 (SEQ ID NO: 267), wherein 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 or absent, X 12 = Arg or absent, X 13 = Tyr or absent, X 14 = Tyr or absent, X 15 = Gly or absent, X 16 =Leu or absent, X 17 =Asp or absent and X 18= Val or absent; LCDR1 is of 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) comprising an amino acid sequence, wherein 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 absent; X 8 = Gly or absent; X 9 = Tyr or absent; X 10 =Asn or absent; and X 11 = Tyr or absent; LCDR2 has the formula X 1 -X 2 -X 3 (SEQ ID NO:2 69), wherein X 1 = Leu, Ala or Val, X 2 = Ala or Gly and X 3 = Ser; and LCDR3 is of formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 (SEQ ID NO: 270), wherein 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 particular embodiment, HCDR1 is of formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 - X 8 (SEQ ID NO: 265), wherein 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 of formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (SEQ ID NO: 266), wherein 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 of the 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 number 267), wherein X 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 absent, X 13 = Tyr or absent, X 14 = Tyr or absent, X 15 = Gly or absent, X 16 =Leu or absent, X 17 =Asp or absent and X 18 = Val or absent; LCDR1 is of 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), wherein 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 absent; X 8 = Gly or absent; X 9 = Tyr or absent; X 10 = Asn or absent; and X 11 = Tyr or absent; LCDR2 has the formula X 1 - X 2 -X 3 (SEQ ID NO: 269), wherein X 1 =Leu or Ala, X 2 = Ala or Gly and X 3 = Ser; and LCDR3 is of formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 (SEQ ID NO: 270), wherein 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 particular embodiment, HCDR1 is of formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (SEQ ID NO: 265), wherein 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 has the formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (Sequence number 266), wherein X 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 of the 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 (SEQ ID NO: 267), wherein 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 or absent, X 12 = Arg or absent, X 13 = Tyr or absent, X 14 = Tyr or absent, X 15 = Gly or absent, X 16 =Leu or absent, X 17 =Asp or absent and X 18 = Val or absent; LCDR1 is of 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:26 8), wherein X 1 = Gln, X 2 = Asp or Ser, X 3 =Ile also Leu, X 4 = Ser or Leu, X 5 = Tyr or Ile, X 6 =Trp or Ser; X 7 = Ile or absent; X 8 = Gly or absent; X 9 = In Tyr Present or absent; X 10 = Asn or absent; and X 11 = Tyr or absent; LCDR2 has the formula X 1 -X 2 -X 3 (SEQ ID NO: 269) , where X 1 =Leu or Val, X 2 = Ala or Gly and X 3= Ser; And LCDR3 is formula X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 (SEQ ID NO: 270) wherein 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 a 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, wherein the HCVR / LCVR sequences are 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 the sequences 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 the sequences 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 the sequences contained in HCVR SEQ ID NO: 210 and LCVR SEQ ID NO: 212.

[0026] The present invention encompasses anti-hIL-4R antibodies with altered glycosylation patterns. In some applications, modifications to remove undesired glycosylation sites or antibodies lacking fucose moieties present on the oligosaccharide chains may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modifications of galactosylation may be made to alter complement-dependent cytotoxicity (CDC).

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

[0028] In an eleventh aspect, the invention features a composition including a recombinant human antibody that specifically binds to hIL-4R and an acceptable carrier.

[0029] In a twelfth aspect, the invention features a method of inhibiting hIL-4 activity using an antibody of the invention, or an antigen-binding portion thereof. In certain embodiments, the antibody of the invention also blocks binding of the hIL-13 / hIL-13R1 complex to hIL-4R. In one embodiment, the method comprises contacting hIL-4R with an antibody of the invention, or an antigen-binding portion thereof, such that hIL-4 or hIL-4 / hIL-13 activity is inhibited. In another embodiment, the method comprises administering an antibody of the invention, or an antigen-binding portion thereof, to a human subject suffering from a disorder that is alleviated by inhibition of hIL-4 or hIL-4 / hIL-13 activity. The disorder to be treated is any disease or condition that is ameliorated, alleviated, suppressed, or prevented by removal, inhibition, or reduction of hIL-4 or hIL-4 / hIL-13 activity.

[0030] IL-4 associated disorders that may be treated with the antibodies or antibody fragments of the invention include, for example, arthritis (including septic arthritis), herpetiformis, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, pulmonary 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, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephrosis.

[0031] Other objects and advantages will become apparent from a consideration of the following detailed description.

[0032] Detailed Description Before describing the methods of the present invention, it should be understood that the present invention is not limited to the specific methods and methods described. It is not limited to experimental conditions, and as such methods and conditions may vary. It is also to be understood that the scope of the present invention will be limited only by the appended claims, and the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0034] definition As used herein, the term "human IL4R" (hIL-4R) is intended to refer to a human cytokine receptor that specifically binds interleukin-4 (IL-4), IL-4Rα (SEQ ID NO: 274). The term "human interleukin-13" (hIL-13) refers to a cytokine that specifically binds to the IL-13 receptor, and "hIL-13 / hIL-13R1 complex" refers to the complex formed by the binding of hIL-13 to the hIL-13R1 complex, which 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, inter-connected 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 regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). VH and VL each comprise three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0036] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion" or "antibody fragment") 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 by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL1, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two F(ab)' fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 241:544-546), which consists of the VH domain; and (vi) a CDR. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows the pair of VL and VH domains to be combined into a single continuous chain to form a monovalent molecule (known as a 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 encompassed by the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies (e.g., bispecific antibodies) are also encompassed ( See, e.g., Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90:6444-6448).

[0037] As used herein, a "neutralizing" or "blocking" antibody is intended to refer to an antibody 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 cellular activation and hIL-4 binding to hIL-4R (see Examples below).

[0038] "CDRs," or complementarity-determining regions, are regions of hypervariability interspersed within more conserved regions called "framework regions" (FRs). In various embodiments of the anti-hIL-4R antibodies or fragments of the present invention, the FRs may be identical to human germline sequences or may be naturally or artificially modified.

[0039] The term "surface plasmon resonance" as used herein refers to, for example, BIACORE TM It refers to an optical phenomenon that allows 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, known as the paratope. A single antigen may have more than one epitope. Epitopes can be conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different parts of a linear polypeptide chain. Linear epitopes are those generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include saccharide, phosphoryl, or sulfonyl moieties on the antigen.

[0041] The terms "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand), including appropriate nucleotide insertions or deletions, there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases as measured by any well-known sequence identity algorithm, e.g., FASTA, BLAST, or Gap, as discussed below.

[0042] As applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned using default gap weights, for example, with the programs GAP or BESTFIT, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with 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 a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids with side chains that have similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative 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 conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0043] Sequence similarity (also called sequence identity) for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications (including conservative 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 a wild-type protein and its variants). See, e.g., GCG version 6.1. Polypeptide sequences can also be compared using FASTA, using default or recommended parameters; programs in GCG version 6.1 (e.g., FASTA2 and FASTA3) provide alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention against databases containing a large number of sequences from various organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0044] Human antibody production Methods for producing human antibodies include, for example, those described in US Pat. No. 6,596,541, Green et al. (1994) Nature Genetics 7:13-21), US Pat. No. 5,545,807, and US Pat. No. 6,787,637.

[0045] Rodents can be immunized by any method known in the art (e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual 1988 Cold Spring Harbor Laboratory; Malik and Lillehoj (1994) Antibody Immunizations. (See Techniques, Academic Press, CA). The antibodies of the present invention preferably contain VELOCIMMUNE TM Antibody-specific chimeric antibodies are produced using techniques such as those described in U.S. Pat. No. 6,596,541. Transgenic mice in which endogenous immunoglobulin heavy and light chain variable regions have been replaced with corresponding human variable regions are challenged with an antigen of interest, and lymphocytes (e.g., B cells) expressing the antibody are collected from the mice. The lymphocytes can be fused with a myeloma cell line to produce immortal hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines producing antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions can be isolated and linked to constant regions of the desired heavy and light chain isotypes. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the light and heavy chain variable regions can be isolated directly from antigen-specific lymphocytes.

[0046] DNA encoding the heavy and light chain variable regions of the antibody was isolated to generate human heavy and light chain constant regions. The antibody is operably linked to DNA encoding the target region. This DNA is then expressed in a cell capable of expressing a fully human antibody. In a specific embodiment, the cell is a CHO cell.

[0047] Antibodies may be therapeutically useful in blocking ligand-receptor interactions or inhibiting receptor component interactions, rather than participating in cell killing by complement fixation (complement-dependent cytotoxicity) (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype can 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. One form contains a stable four-chain construct of approximately 150-160 kDa, in which the dimer is held together by interchain heavy chain disulfide bonds. In the second form, the dimer is not linked via interchain disulfide bonds, forming a molecule of approximately 75-80 kDa composed of covalently linked light and heavy chains (half-antibody). These forms are very difficult to separate, even after affinity purification. The frequency of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the antibody hinge region isotype. Indeed, a single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to levels typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention encompasses antibodies with one or more mutations in the hinge, CH2, or CH3 regions, which may be desirable in manufacturing, for example, to improve the yield of the desired antibody form.

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

[0050] Epitope mapping and related techniques To screen for antibodies that bind to specific epitopes, Harlow and Cross-blocking assays such as those described in Lane (supra) can be performed. Other methods include alanine scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63), or peptide truncation analysis. Additionally, 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 multiple monoclonal antibodies (mAbs) specific for the same antigen according to the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces (U.S. Patent Application Publication No. 2004 / 0101920). Each category contains epitopes that are clearly distinct or partially overlapping with those represented in other categories. MAP may reflect the specific epitopes that bind to the antibodies. This technique allows for the rapid selection of genetically identical antibodies, allowing characterization to be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones with desired characteristics. MAP can be used to separate the hIL-4R antibodies of the invention into groups of different epitopes that bind to the antibodies.

[0052] Useful agents for altering the structure of immobilized antigens include enzymes, such as proteases, and chemical agents. The antigen protein may be immobilized either on a biosensor chip surface or on polystyrene beads. The latter can be used, for example, in multiplexed LUMINEX. TM The assays can be processed using assays such as the LUMINEX detection assay (Luminex Corp., TX). LUMINEX can handle multiplexed assays using up to 100 different types of beads. TM Due to its capabilities, LUMINEX TM provides an almost unlimited antigen surface with various modifications, leading to improved resolution in antibody epitope profiling compared to biosensor assays.

[0053] Bispecifics The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al. (1991) J. Immunol. 147:60-69. A human anti-IL-4R antibody may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be linked (e.g., by chemical coupling, genetic fusion, noncovalent bonding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to generate a bispecific or multispecific antibody having a second binding specificity.

[0054] Therapeutic Administration and Formulations The present invention provides therapeutic compositions comprising the anti-IL-4R antibodies or antigen-binding fragments thereof of the present invention. Therapeutic compositions according to the present invention are administered together with suitable carriers, additives, and other agents that are incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Many suitable formulations are listed in the formulary known to all pharmacists: Remington's Pharmaceuticals, Inc. These formulations can be found in Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN TM Excipients for parenteral formulations include acetaminophen, 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 to be administered, the target disease, condition, route of administration, etc. When the antibodies of the present invention are used to treat various conditions and diseases associated with IL-4R in adult patients, it is advantageous to administer the antibodies of the present invention intravenously, typically at a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. The frequency and duration of treatment may be adjusted depending on the severity of the condition.

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

[0057] The pharmaceutical compositions can also be delivered in vesicles, in particular 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 Lopez-Berestein, ibid., pp. 317-327; see generally ibid.

[0058] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used (Medical Applications of Controlled Release, Langer and Wise (ed.), CRC Pres., Boca Raton, Florida (1974). In yet another embodiment, a controlled-release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984). Other controlled-release systems are discussed in the review by Langer (1990), Science 249:1527-1533.

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

[0060] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in a unit dosage form suitable for the dosage of the active ingredient. Examples of such unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained therein is generally about 5 to 500 mg per unit dosage form; particularly, in the form of injections, the amount of the above-mentioned antibody is preferably about 5 to 100 mg, and for other dosage forms, about 10 to 250 mg.

[0061] Monotherapy and Combination Therapy The antibodies and antibody fragments of the invention are useful for treating diseases and disorders that are ameliorated, inhibited, or alleviated by reducing IL-4 activity, including disorders characterized by aberrant or excessive expression of IL-4 or an aberrant host response to IL-4 production. IL-4 associated disorders that may be treated with the antibody or antibody fragment of the present invention include, for example, arthritis (including septic arthritis), herpetiform disease, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, pulmonary 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, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, atopic dermatitis, ulcerative colitis, fibrosis, and nephrosis (see U.S. Pat. No. 7,186,809).

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

[0063] The present invention also includes the use of any of the anti-IL-4R antibodies or antigen-binding fragments described herein in the manufacture of a medicament for the treatment of a disease or disorder that is ameliorated, alleviated, or suppressed by removing, inhibiting, or reducing human interleukin-4 (hIL-4) activity, including, for example, arthritis, herpetiform disease, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, pulmonary disorders, asthma, inflammatory disorders, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, preeclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, nephrosis, atopic dermatitis, and asthma. [Example]

[0064] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of this invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some degree of experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0065] Example 1. Preparation of human antibodies against human IL-4 receptor Velocimmune TM Mice (Regeneron Pharmaceuticals, Inc.; US Pat. No. 6,596,541) 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 optimal immune responses, animals were subsequently boosted every 3-4 weeks and bled 10 days after each boost to assess the progression of the anti-antigen response.

[0066] When the mice reached a maximal immune response, antibody-expressing B cells were harvested 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 No. 2007 / 0280945A1 (specifically 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 conditioned medium from hybridomas or transfected cells for specificity, antigen-binding affinity, and potency in blocking hIL-4 binding to hIL-4R (described below).

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

[0068] Example 2. Antigen binding affinity determination The binding affinity (K) of selected antibodies for hIL-4R was measured at either 25°C or 37°C. D ) was analyzed by real-time biosensor surface plasmon resonance assay (BIACORE TM 200 Briefly, antibodies were analyzed using BIACORE TM The capture antibody surface was formed by capturing the antibody on a goat anti-hFc polyclonal antibody surface generated by direct coupling to the chip. Various concentrations (ranging from 50 nM to 12.5 nM) of monomeric hIL-4R (R&D Systems) or dimeric hIL-4R-mFc were injected over the capture antibody surface at 10 μl / min for 2.5 min at either 25°C or 37°C. Binding of the antigen to the antibody and dissociation of the bound complex were monitored in real time. The equilibrium dissociation constant (K D ) and dissociation rate constant The number of antibodies was confirmed by performing kinetic analysis using BIA evaluation software. The BIA evaluation software was used to calculate the half-life of antigen / antibody complex dissociation (T 1 / 2 ) is also used to calculate The results are shown in Table 1. NB: No antibody-antigen binding was observed under the experimental conditions. Control: Fully human anti-IL-4R antibody (U.S. Patent No. 7,186,809; SEQ ID NOs: 10 and 12).

[0069] [Table 1]

[0070] The binding affinity (K ) of selected antibodies for the cynomolgus monkey (Macaca fascicularis) IL-4R (mfIL-4R) at either 25°C or 37°C D )Moma Also, various concentrations (ranging from 100 nM to 25 nM) of monomeric mfIL-4R-myc-myc-his (mfIL-4R-mmh) or dimeric mfIL-4R-mFc were determined using the real-time biosensor surface plasmon resonance assay described above. Only antibody H4H098P bound to both monomeric and dimeric mfIL-4R with K values ​​of 552 nM and 9.08 nM at 25°C, respectively. D Furthermore, antibody H4H098P The dimeric mfIL-4R also exhibits a K of 24.3 nM at 37 °C. D Bonded with H4H0 83P had very weak binding to dimeric mfIL-4R.

[0071] Antibody-antigen binding affinity was also assessed using an ELISA-based solution competition assay. Briefly, 96-well MAXISORP TMPlates were first coated with 5 μg / ml avidin overnight, followed by 1 hour of BSA blocking. The avidin-coated plates were then incubated with 250 ng / ml biotin-hIL4 for 2 hours. This plate was used to measure either free hIL-4R-mFc (dimeric hIL-4R) or free hIL-4R-myc-myc-his (hIL-4R-mmh, monomeric hIL-4R) in antibody titration sample solutions. To prepare antibody titration samples, a fixed amount of either 25 pM hIL-4R-mFc or 200 pM hIL-4R-mmh was premixed with various amounts of antibody in serial dilutions ranging from 0 to approximately 10 nM, followed by 1 hour of incubation at room temperature to allow antibody-antigen binding to reach equilibrium. The equilibrated sample solution was then transferred to a hIL-4-coated plate for measurement of either free hIL-4R-mFc or free hIL-4R-mmh. After 1 hour of binding, the plates were washed and 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] The cross-reactivity of the antibodies to monkey IL-4R was also determined using an ELISA-based solution competition assay. Antibody H4H098P had an IC of 300 pM for mfIL-4R-mFc. 50 , and an IC of 20 nM for mfIL-4R-mmh 50 showed.

[0074] Example 3. Neutralization of the biological effects of hIL-4 and hIL-13 in vitro To determine the ability of purified anti-hIL-4R antibodies to neutralize hIL-4R-mediated cellular functions in vitro, a bioassay was developed using an HK293 cell line engineered to contain human STAT6 and a STAT6 luciferase reporter. Inhibition of hIL-4R-induced luciferase activity was determined as follows: cells were plated in 96-well plates at 1 × 10 in medium. 4 Cells / well were seeded and incubated overnight at 37°C, 5% CO2. Serial dilutions of antibody protein ranging from 0 to 20 nM were added to the cells along with either 10 pM hIL-4 or 40 pM hIL-13. The cells were then incubated at 37°C, 5% CO2 for 6 hours. The cells were incubated for 1 h. The extent of the cellular response was measured by 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 an IC of 150 nM in the presence of 360 fM mfIL-4. 50 I was able to block it.

[0075] [Table 3]

Claims

1. 1. Use of an antibody or antigen-binding fragment thereof that specifically binds to human interleukin-4 receptor (hIL-4R) in the manufacture of a medicament for the treatment of a disease or disorder that is ameliorated, alleviated, or suppressed by removal, inhibition, or reduction of human interleukin-4 (hIL-4) activity, comprising: wherein the disease or disorder is ulcerative colitis or inflammatory bowel disease; The above use, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 162 and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

164.

2. 1. Use of an antibody or antigen-binding fragment thereof that specifically binds to human interleukin-4 receptor (hIL-4R) in the manufacture of a medicament for the treatment of a disease or disorder that is ameliorated, alleviated, or suppressed by removal, inhibition, or reduction of human interleukin-4 (hIL-4) activity, comprising: wherein the disease or disorder is ulcerative colitis or inflammatory bowel disease; The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (LCVR) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3); The amino acid sequences of HCDR1, HCDR2, and HCDR3 are those shown in SEQ ID NOs: 148, 150, and 152, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are those shown in SEQ ID NOs: 156, 158, and 160, respectively.

3. The use of claim 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:

162.

4. The use of claim 2, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR) comprising an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:

164.

5. The use according to any one of claims 1 to 4, wherein the antibody is a full-length IgG4 antibody.

6. The use according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously.

7. The use according to any one of claims 1 to 6, wherein the disease or disorder is ulcerative colitis.

8. The use according to any one of claims 1 to 6, wherein the disease or disorder is inflammatory bowel disease.

9. 1. A pharmaceutical comprising an antibody or antigen-binding fragment thereof that specifically binds to human interleukin-4 receptor (hIL-4R) for use in a method for treating a disease or disorder that is ameliorated, alleviated, or suppressed by removal, inhibition, or reduction of human interleukin-4 (hIL-4) activity, wherein the disease or disorder is ulcerative colitis or inflammatory bowel disease; The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 162 and a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

164.

10. 1. A pharmaceutical comprising an antibody or antigen-binding fragment thereof that specifically binds to human interleukin-4 receptor (hIL-4R) for use in a method for treating a disease or disorder that is ameliorated, alleviated, or suppressed by removal, inhibition, or reduction of human interleukin-4 (hIL-4) activity, wherein the disease or disorder is ulcerative colitis or inflammatory bowel disease; The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (LCVR) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3); The pharmaceutical composition as described above, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 are those shown in SEQ ID NOs: 148, 150, and 152, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are those shown in SEQ ID NOs: 156, 158, and 160, respectively.

11. The pharmaceutical of claim 10, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:

162.

12. The pharmaceutical of claim 10, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR) comprising an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:

164.

13. The pharmaceutical according to any one of claims 9 to 12, wherein the antibody is a full-length IgG4 antibody.

14. The pharmaceutical according to any one of claims 9 to 13, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously.

15. The pharmaceutical composition according to any one of claims 9 to 14, wherein the disease or disorder is ulcerative colitis.

16. The pharmaceutical composition according to any one of claims 9 to 14, wherein the disease or disorder is inflammatory bowel disease.

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