RU2825304C2 - Antibody capable of binding to thymic stromal lymphopoietin, and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an antibody to TSLP (thymic stromal lymphopoietin), as well as to a composition containing same. Also disclosed is a nucleic acid molecule coding said antibody, as well as a cell containing it.

EFFECT: invention is effective for treating diseases associated with TSLP.

19 cl, 5 dwg, 40 tbl, 9 ex

Description

AREA OF TECHNOLOGY

The present invention relates to the field of antibody-based agents. In particular, the present invention relates to agents based on antibodies to TSLP (thymic stromal lymphopoietin) and their use.

LEVEL OF TECHNOLOGY

The statements in this section relate only to general information related to the present invention and do not necessarily constitute prior art.

Asthma is a serious chronic inflammatory disease of the airways. There are about 334 million asthma patients worldwide and about 30 million asthma patients in China, where the mortality rate is much higher than in developed countries. As the environment deteriorates and air pollution increases, more and more people may suffer from this disease, which poses a serious threat to human life and health.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell cytokine produced in response to proinflammatory stimuli. It mainly promotes allergic inflammation through its activity on dendritic cells and mast cells. TSLP is a type of interleukin 7 (IL-7)-like cytokine that was first discovered in the conditioned medium of mouse thymic stromal cells. TSLP is mainly expressed in lung, skin and intestinal epithelial cells. TSLP consists of 4 α-helices and two AB and CD loops. Its molecule contains three pairs of disulfide bonds consisting of six cysteines, two N-glycosylation sites, and the molecular weight is about 15-20 kDa. The TSLP receptor is a complex consisting of two parts, one is TSLPR and the other is IL7Rα. TSLP first binds to TSLPR with relatively low affinity, then recruits IL7Rα binding with high affinity, and finally activates stat5 and other signaling pathways, leading to DC maturation and T cell differentiation.

Myeloid dendritic cells (mDCs) are the primary effector cells for TSLP. TSLP acts on immature mDCs that secrete the cytokines IL-8, eotaxin-2, TARC, and MDC while expressing high levels of OX40L. In the absence of IL-12, OX40L binds to naïve CD4+ T cells, leading to their differentiation into Th2 cells. Th2 cells then secrete Th2 cytokines such as IL-5, IL-4, IL-9, IL-9, and TNF, inducing a Th2-type inflammatory response in the body. In addition, TSLP can also induce DCs to produce the cytokine IL-8, which in turn recruits neutrophils, leading to a neutrophilic inflammatory response of innate immunity. TSLP can also induce eotaxin-2 production by DCs, which recruits eosinophils and acts in concert with IL5 to prompt the body to rapidly initiate an inflammatory process of eosinophilic infiltration. TSLP also acts on mast cells and natural killer cells and mediates the innate inflammatory response by inducing the production of IL-4, IL-6, IgE, etc. Thus, TSLP can induce the innate inflammatory response and Th2 inflammation simultaneously, which in turn increases mucus production in tissues, remodels the airways resulting in tracheal stenosis, and leads to severe cellular fibrosis. The inflammation gradually develops into three major allergic diseases: asthma, allergic dermatitis, and allergic rhinitis. Therefore, blocking TSLP is a potentially effective strategy for the treatment of diseases such as asthma, allergic dermatitis, etc.

Currently known antibodies to TSLP are disclosed in WO 2008155365, WO 2009035577, WO 2011056772, WO 2016142426 and WO 2017004149. However, the corresponding antibodies are not available on the market. Therefore, it is necessary to continue developing effective drugs for the treatment of diseases associated with TSLP.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to an antibody to TSLP.

In some embodiments, an anti-TSLP antibody as described above comprises a heavy chain variable region and a light chain variable region of an antibody, wherein:

i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 47, respectively, and the light chain variable region comprises LCDR1, LCDR2 as set forth in SEQ ID NO: 17, SEQ ID NO: 18, respectively, and LCDR3 as set forth in SEQ ID NO: 48 or 55;

wherein the sequence of SEQ ID NO: 47 is EDYDYDGYAMDX ₁ , the sequence of SEQ ID NO: 48 is QQWSSX ₂ RT, the sequence of SEQ ID NO: 55 is QQSDX ₃ X ₄ RX ₅ , wherein X ₁ is H or Y, X ₂ is N or D, X ₃ is N or S, X ₄ is V or G, X ₅ is G or E; or

ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 76, SEQ ID NO: 24 and SEQ ID NO: 25, respectively;

wherein the sequence SEQ ID NO: 76 is RASESVDX ₆ SGLSFMH, where X ₆ is selected from N, S or Q; or

iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 96 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 118 and SEQ ID NO: 31, respectively;

wherein the sequence of SEQ ID NO: 96 is VIDPGX ₇ X ₈ DTNYNE, the sequence of SEQ ID NO: 118 is X ₉ VX ₁₀ X ₁₁ X ₁₂ X ₁₃ T, wherein X ₇ is selected from N, Q and V, X ₈ is G or V; X ₉ is Y or E, X ₁₀ is selected from S, D and E, X ₁₁ is selected from N, Q, D and E, X ₁₂ is selected from H, Y, D and E, X ₁₃ is E or Y; or

iv) the variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively, and the variable region of the light chain comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, respectively.

In some embodiments, an anti-TSLP antibody as described above comprises a heavy chain variable region and a light chain variable region, wherein:

i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively; or

ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 45, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 46, respectively; or

iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 45, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 53, respectively; or

iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 45, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 54, respectively; or

v) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively; or

vi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 70, SEQ ID NO: 24 and SEQ ID NO: 25, respectively; or

vii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 71, SEQ ID NO: 24 and SEQ ID NO: 25, respectively; or

viii) the heavy chain variable region comprises HCDR1 and HCDR3 as shown in SEQ ID NO: 26, SEQ ID NO: 28, respectively, and HCDR2 as shown in SEQ ID NO: 27, 93, 94 or 95, and the light chain variable region comprises LCDR1 and LCDR3 as shown in SEQ ID NO: 29, SEQ ID NO: 31, respectively, and LCDR2 as shown in SEQ ID NO: 30, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117.

In some embodiments, an anti-TSLP antibody as described above comprises a heavy chain variable region and a light chain variable region, wherein:

a) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; or

b) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 93 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; or

c) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 94 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; or

d) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 95 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; or

e) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 108 and SEQ ID NO: 31, respectively; or

f) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 109 and SEQ ID NO: 31, respectively; or

g) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 110 and SEQ ID NO: 31, respectively; or

h) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 111 and SEQ ID NO: 31, respectively; or

i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 112 and SEQ ID NO: 31, respectively; or

j) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 113 and SEQ ID NO: 31, respectively; or

k) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 114 and SEQ ID NO: 31, respectively; or

l) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 115 and SEQ ID NO: 31, respectively; or

m) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 116 and SEQ ID NO: 31, respectively; or

n) the variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the variable region of the light chain comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 29, SEQ ID NO: 117 and SEQ ID NO: 31, respectively.

In some embodiments of the TSLP antibody as described above, the TSLP antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

In some embodiments of the TSLP antibody as described above, the TSLP antibody comprises a framework region(s) derived from a human antibody, or the TSLP antibody comprises a light chain variable region and/or a heavy chain variable region selected from those described in (a), (b), (c), or (d) below:

a) the heavy chain variable region comprises HCDR1 and HCDR2 as shown in SEQ ID NO: 14, SEQ ID NO: 15, respectively, and HCDR3 as shown in SEQ ID NO: 16 or 45, and its framework region(s) comprises no more than 10 backmutations, preferably the backmutation is selected from one or more of 38K, 48I, 67A, 69L, 71V and 73K; and/or the light chain variable region comprises LCDR1 and LCDR2 as shown in SEQ ID NO: 17, SEQ ID NO: 18, respectively, and LCDR3 as shown in SEQ ID NO: 19, 46, 53 or 54, and its framework region(s) comprises no more than 10 amino acid back mutations, preferably the back mutation is selected from one or more of 46P, 47W, 58V, 70S and 71Y;

b) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and its framework region(s) comprises no more than 10 backmutations, preferably the backmutation is selected from one or more of 2A, 27F, 38K, 39H, 48I, 67A, 69L, 71V and 76R; and/or the light chain variable region comprises LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25, respectively, and LCDR1 as shown in SEQ ID NO: 23, 70 or 71, and its framework region(s) comprises no more than 10 amino acid back mutations, preferably the back mutation is one or more of 1D, 4L, 43P, 48L and 58I;

c) the heavy chain variable region comprises HCDR1 and HCDR3 as shown in SEQ ID NO: 26, SEQ ID NO: 28, respectively, and HCDR2 as shown in SEQ ID NO: 27, 93, 94 or 95, and its framework region(s) comprises no more than 10 backmutations, preferably the backmutation is selected from one or more of 27Y, 28A, 38K, 48I, 66K, 67A, 69L, 80I and 82bR; and/or the light chain variable region comprises LCDR1 and LCDR3 as shown in SEQ ID NO: 29, SEQ ID NO: 31, respectively, and LCDR2 as shown in SEQ ID NO: 30, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117, and its framework region(s) comprises no more than 10 backmutations, preferably the backmutation is selected from one or more of 1S, 43S, 67Y and 73F; or

d) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively, and its framework region(s) comprises no more than 10 backmutations, preferably the backmutation is selected from one or more of 38K, 48I, 66K, 67A, 69L, 71V, 73K and 78A; and/or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, respectively, and its framework region(s) comprises no more than 10 backmutations, preferably the backmutation is selected from one or more of 43S, 45Q, 48V, 66V and 70Q.

In some embodiments, an anti-TSLP antibody as described above comprises a heavy chain variable region and a light chain variable region, wherein:

i) the heavy chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 6, 42, 43, 44, or 50, and the light chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the light chain variable region shown in the amino acid sequence of SEQ ID NO: 7, 38, 39, 40, 41, 49, 51, or 52; or

ii) the heavy chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 8, 62, 63, 64, 65, 66, 67, 68, or 69, and the light chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the light chain variable region shown in the amino acid sequence of SEQ ID NO: 9, 56, 57, 58, 59, 60, 61, 72, 73, 74, or 75; or

iii) the heavy chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 10, 85, 86, 87, 88, 89, 90, 91, 92, or 97, and the light chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the light chain variable region shown in the amino acid sequence of SEQ ID NO: 11, 77, 78, 79, 80, 81, 82, 83, 84, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 119; or

iv) the variable region of the heavy chain has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the variable region of the heavy chain presented in the amino acid sequence of SEQ ID NO: 12, 126, 127, 128, 129, 130, 131 or 132, and the variable region of the light chain has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the variable region of the light chain presented in the amino acid sequence of SEQ ID NO: 13, 120, 121, 122, 123, 124 or 125.

In some embodiments of the anti-TSLP antibody as described above, the anti-TSLP antibody is a humanized antibody comprising a framework region(s) derived from a human antibody, or a framework region variant thereof, wherein said framework region variant has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid back mutations in the light chain framework region(s) and/or the heavy chain framework region(s) of the human antibody, respectively.

In some embodiments of the TSLP antibody as described above, the framework variant comprises backmutations selected from those described in (a), (b), (c) or (d) below:

a) one or more amino acid back mutations selected from the group consisting of 46P, 47W, 58V, 70S and 71Y contained in the framework region(s) of the light chain variable region presented in SEQ ID NO: 38, 49, 51 or 52, and/or one or more amino acid back mutations selected from the group consisting of 38K, 48I, 67A, 69L, 71V and 73K contained in the framework region(s) of the heavy chain variable region presented in SEQ ID NO: 42 or 50;

b) one or more amino acid back mutations selected from the group consisting of 1D, 4L, 43P, 48L and 58I contained in the framework region(s) of the light chain variable region presented in SEQ ID NO: 56, 59, 72, 73, 74 or 75, and/or one or more amino acid back mutations selected from the group consisting of 2A, 27F, 38K, 39H, 48I, 67A, 69L, 71V and 76R contained in the framework region(s) of the heavy chain variable region presented in SEQ ID NO: 62;

c) one or more amino acid back mutations selected from the group consisting of 1S, 43S, 67Y and 73F contained in the framework region(s) of the variable light chain presented in SEQ ID NO: 77, 81, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 119 and/or one or more amino acid back mutations selected from the group consisting of 27Y, 28A, 38K, 48I, 66K, 67A, 69L, 80I and 82bR contained in the framework region(s) of the variable heavy chain presented in SEQ ID NO: 85, 90, 91, 92 or 97;

d) one or more amino acid back mutations selected from the group consisting of 43S, 45Q, 48V, 66V and 70Q contained in the framework region(s) of the light chain variable region presented in SEQ ID NO: 120, and/or one or more amino acid back mutations selected from the group consisting of 38K, 48I, 66K, 67A, 69L, 71V, 73K and 78A contained in the framework region of the heavy chain variable region presented in SEQ ID NO: 126.

In some embodiments, an anti-TSLP antibody as described above comprises a heavy chain variable region and a light chain variable region, wherein:

i) the amino acid sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 6, 42, 43, 44 or 50, and the amino acid sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 7, 38, 39, 40, 41, 49, 51 or 52; or

ii) the amino acid sequence of the variable region of the heavy chain is as set forth in SEQ ID NO: 8, 62, 63, 64, 65, 66, 67, 68 or 69, and the amino acid sequence of the variable region of the light chain is as set forth in SEQ ID NO: 9, 56, 57, 58, 59, 60, 61, 72, 73, 74 or 75; or

iii) the amino acid sequence of the variable region of the heavy chain is as set forth in SEQ ID NO: 10, 85, 86, 87, 88, 89, 90, 91, 92 or 97, and the amino acid sequence of the variable region of the light chain is as set forth in SEQ ID NO: 11, 77, 78, 79, 80, 81, 82, 83, 84, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 119; or

iv) the amino acid sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 12, 126, 127, 128, 129, 130, 131 or 132, and the amino acid sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 13, 120, 121, 122, 123, 124 or 125.

In some embodiments, the anti-TSLP antibody as described above comprises a heavy chain variable region and a light chain variable region as shown below:

(a) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 6, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 7;

(b) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 42, 43 or 44, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 39, 40 or 41;

(c) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 43, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 38;

(d) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 50, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 49, 51 or 52;

(e) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 8, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 9;

(f) the heavy chain variable region sequence is as set forth in SEQ ID NO: 62, 63, 64 or 65, and the light chain variable region sequence is as set forth in SEQ ID NO: 56, 57 or 58;

(g) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 64, 66, 67, 68 or 69, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 59, 60 or 61;

(h) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 64, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 72 or 73;

(i) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 69, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 74;

(j) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 10, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 11;

(k) the heavy chain variable region sequence is as set forth in SEQ ID NO: 85, and the light chain variable region sequence is as set forth in SEQ ID NO: 77, 78, 102, or 104;

(l) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 86 or 88, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 77 or 78;

(m) the heavy chain variable region sequence is as set forth in SEQ ID NO: 87, and the light chain variable region sequence is as set forth in SEQ ID NO: 77, 78, 79, 81, 82, 83, 84, 98, 99, 100, 101, 103, 105, 106, or 107;

(n) the heavy chain variable region sequence is as set forth in SEQ ID NO: 89, and the light chain variable region sequence is as set forth in SEQ ID NO: 79, 81, 82, 83, or 84;

(o) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 90, 91 or 92, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 78;

(p) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 97, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 119;

(q) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 12, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 13;

(r) the heavy chain variable region sequence is as set forth in SEQ ID NO: 127, 128, 129, 130, 131 or 132 and the light chain variable region sequence is as set forth in SEQ ID NO: 120, 121, 123, 124 or 125; or

(s) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 132, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 125.

In some embodiments of the TSLP antibody as described above, the combinations of the variable light chain and variable heavy chain regions of the antibodies are as follows:

Table 1. Combinations of light and heavy chain variable regions of humanized mAb3 antibodies

Antibody VH (SEQ ID NO) VL(SEQ ID NO) hu3-01 42 39 hu3-02 42 40 hu3-03 42 41 hu3-04 43 38 hu3-05 43 39 hu3-06 43 40 hu3-07 43 41 hu3-08 44 39 hu3-09 44 40 hu3-10 44 41 hu3-11 50 49 hu3-12 50 51 hu3-13 50 52

Table 2. Combinations of light and heavy chain variable regions of humanized antibodies mAb119

Antibody VH (SEQ ID NO) VL(SEQ ID NO) hu119-01 62 56 hu119-02 63 56 hu119-03 64 56 hu119-04 65 56 hu119-05 62 57 hu119-06 63 57 hu119-07 64 57 hu119-08 65 57 hu119-09 62 58 hu119-10 63 58 hu119-11 64 58 hu119-12 65 58 hu119-13 64 59 hu119-14 66 59 hu119-15 67 59 hu119-16 68 59 hu119-17 69 59 hu119-18 64 60 hu119-19 66 60 hu119-20 67 60 hu119-21 68 60 hu119-22 69 60 hu119-23 64 61 hu119-24 66 61 hu119-25 67 61 hu119-26 68 61 hu119-27 69 61 hu119-28 64 72 hu119-29 64 73 hu119-30 69 74

Table 3. Combinations of light and heavy chain variable regions of humanized antibodies mAb179

Antibody VH (SEQ ID NO) VL(SEQ ID NO) hu179-01 85 77 hu179-02 85 78 hu179-03 86 77 hu179-04 86 78 hu179-05 87 77 hu179-06 87 78 hu179-07 87 79 hu179-08 87 81 hu179-09 87 82 hu179-10 87 83 hu179-11 87 84 hu179-12 88 77 hu179-13 88 78 hu179-14 89 79 hu179-15 89 80 hu179-16 89 81 hu179-17 89 82 hu179-18 89 83 hu179-19 89 84 hu179-20 90 78 hu179-21 91 78 hu179-22 92 78 hu179-23 85 102 hu179-24 85 104 hu179-25 87 98 hu179-26 87 99 hu179-27 87 100 hu179-28 87 101 hu179-29 87 103 hu179-30 87 105 hu179-31 87 106 hu179-32 87 107 hu179-33 97 119

Table 4. Combinations of light and heavy chain variable regions of humanized antibodies mAb199

Antibody VH (SEQ ID NO) VL(SEQ ID NO) hu199-01 127 120 hu199-02 127 121 hu199-03 127 122 hu199-04 127 123 hu199-05 127 124 hu199-06 127 125 hu199-07 128 120 hu199-08 128 121 hu199-09 128 122 hu199-10 128 123 hu199-11 128 124 hu199-12 128 125 hu199-13 129 120 hu199-14 129 121 hu199-15 129 122 hu199-16 129 123 hu199-17 129 124 hu199-18 129 125 hu199-19 130 120 hu199-20 130 121 hu199-21 130 122 hu199-22 130 123 hu199-23 130 124 hu199-24 130 125 hu199-25 131 120 hu199-26 131 121 hu199-27 131 122 hu199-28 131 123 hu199-29 131 124 hu199-30 131 125 hu199-31 132 120 hu199-32 132 121 hu199-33 132 122 hu199-34 132 123 hu199-35 132 124 hu199-36 132 125

In some embodiments of the TSLP antibody as described above, said antibody further comprises an antibody constant region(s); preferably, the heavy chain constant region of the antibody constant regions is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, the light chain constant region of the antibody constant regions is selected from the group consisting of human κ and λ chain constant regions and conventional variants thereof; more preferably, the antibody comprises the heavy chain constant region shown in SEQ ID NO: 133 and the light chain constant region shown in SEQ ID NO: 134.

In some embodiments, the anti-TSLP antibody as described above comprises a heavy chain and a light chain as shown below:

(a) the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 135 or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to said sequence, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 136 or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to said sequence;

(b) the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 137 or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to said sequence, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 138 or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to said sequence;

(c) the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 139 or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to said sequence, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 140 or has at least 90% identity to said sequence; or

(d) the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 141 or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to said sequence, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 142 or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to said sequence.

In some embodiments, the anti-TSLP antibody as described above comprises a heavy chain and a light chain as shown below:

(a) the amino acid sequence of the heavy chain is as set forth in SEQ ID NO: 135, and the amino acid sequence of the light chain is as set forth in SEQ ID NO: 136;

(b) the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 137, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 138;

(c) the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 139, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 140; or

(d) the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 141, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 142.

In some embodiments, said antibody competes with an anti-TSLP antibody as described above, or an antigen-binding fragment thereof, for binding to human TSLP.

In another aspect, the present invention also relates to a nucleic acid molecule encoding an antibody to TSLP as described above.

In another aspect, the present invention also relates to an expression vector comprising a nucleic acid molecule as described above.

In another aspect, the present invention also relates to a host cell comprising a nucleic acid molecule as described above or an expression vector as described above, preferably said cell is a bacterial cell, a fungal cell, an insect cell or a mammalian cell.

In some embodiments, the present invention provides a method for producing an antibody to TSLP as described above.

In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-TSLP antibody as described above, or a nucleic acid molecule as described above, or a host cell as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. Preferably, the therapeutically effective amount is 0.1-3000 mg or 1-1000 mg of an anti-TSLP antibody as described above contained in a single dose of the composition.

In some embodiments, the present invention provides a method for immunodetection or determination of TSLP in vitro or ex vivo, comprising the step of using an antibody to TSLP as described above.

In some embodiments, the present invention provides the use of an antibody to TSLP as described above to prepare reagents for the immunodetection of human TSLP.

In some embodiments, the present invention provides an anti-TSLP antibody as described above for use in immunodetection or determination of TSLP.

In some embodiments, the present invention provides a kit comprising an anti-TSLP antibody as described above.

In some embodiments, the present invention provides the use of an anti-TSLP antibody as described above, or a nucleic acid molecule as described above, or a host cell as described above, or a pharmaceutical composition as described above, for the preparation of a medicament for the treatment of TSLP-associated diseases, wherein the TSLP-associated disease includes, but is not limited to: asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic sinusitis, urticaria, Netherton syndrome, eosinophilic esophagitis, food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungal sinusitis, chronic pruritus, cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, rheumatoid arthritis, chronic obstructive pulmonary disease, systemic sclerosis, multiple sclerosis, keloidosis, ulcerative colitis, nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, Churg-Strauss syndrome, eosinophilia-myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, scleroderma, interstitial lung disease, fibrosis due to chronic hepatitis B or C, fibrosis due to radiation, and fibrosis due to wound healing.

In some embodiments, the present invention provides a method of treating TSLP-associated diseases comprising administering to a subject a therapeutically effective amount of an anti-TSLP antibody as described above, or a nucleic acid molecule as described above, or a host cell as described above, or a pharmaceutical composition as described above, asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic sinusitis, urticaria, Netherton syndrome, eosinophilic esophagitis, food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungal sinusitis, chronic pruritus, cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, rheumatoid arthritis, chronic obstructive pulmonary disease, systemic sclerosis, multiple sclerosis, keloidosis, ulcerative colitis, nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, Churg-Strauss syndrome, eosinophilia-myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, scleroderma, interstitial lung disease, fibrosis due to chronic hepatitis B or C, fibrosis due to radiation, and fibrosis due to wound healing.

In some embodiments, the present invention provides an anti-TSLP antibody for use as a medicament, wherein the anti-TSLP antibody is for use in the treatment of a TSLP-associated disease, wherein the TSLP-associated disease includes, but is not limited to, asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic sinusitis, urticaria, Netherton syndrome, eosinophilic esophagitis, food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungal sinusitis, chronic pruritus, cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, rheumatoid arthritis, chronic obstructive pulmonary disease, systemic sclerosis, multiple sclerosis, keloidosis, ulcerative colitis, nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, Churg-Strauss syndrome, eosinophilia-myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, scleroderma, interstitial lung disease, fibrosis due to chronic hepatitis B or C, fibrosis due to radiation, and fibrosis due to wound healing.

DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1: Result regarding blocking of TSLP binding activity to TSLP receptor by antibody.

Fig. 2: Result regarding the blocking activity of TSLP binding to the TSLP receptor on the cell surface by the antibody.

Fig. 3: Antibody inhibits TSLP-induced proliferative activity of BaF3 cells.

Fig. 4A shows the activity of the antibody in inhibiting TSLP-induced TARC chemokine production; Fig. 4B shows the activity of the antibody in inhibiting TSLP-induced OPG chemokine production.

Fig. 5A shows the activity of the antibody in inhibiting the production of the Th2 cytokine IL-13; Fig. 5B shows the activity of the antibody in inhibiting the production of the Th2 cytokine IL-4; Fig. 5C shows the activity of the antibody in inhibiting the production of the Th2 cytokine TNF-α; Fig. 5D shows the activity of the antibody in inhibiting the production of the Th2 cytokine IL-5.

DETAILED DESCRIPTION OF DISCLOSURE

Detailed description of the invention

Terminology

To facilitate understanding of the present invention, definitions of some technical and scientific terms are specifically provided below. Unless otherwise explicitly defined herein, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present invention belongs.

The three-letter codes and single-letter codes for amino acids used in this description correspond to those described in J. biol. chem, 243, p 3558 (1968).

The term "thymic stromal lymphopoietin (TSLP)" is a type I cytokine containing a bundle of four α-helices, also known as an epithelial cell cytokine, produced in response to proinflammatory stimuli. It is closely related to interleukin-7 (IL-7), triggers allergic reactions by stimulating dendritic cells (DCs), and is an important factor in regulating the immune response in humans. The term "TSLP" includes variants, isoforms, homologs, orthologs, and paralogs of TSLP.

"Antibody" as described herein refers to an immunoglobulin, typically an intact antibody has a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of the immunoglobulin heavy chain have different amino acid compositions and rankings, and therefore have different antigenicity. Accordingly, immunoglobulins can be divided into five types, otherwise called immunoglobulin isotypes, namely, IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same Ig type can be further subdivided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of the disulfide bonds of the heavy chain. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. The light chain is classified into κ chain or λ chain according to the difference in the constant region. Each of the five Ig types can have either κ chain or λ chain.

The sequence of approximately 110 amino acids near the N-terminus of the heavy and light chains of an antibody varies greatly and is known as the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and constitutes the constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 framework regions (FRs) with relatively conserved sequences. The 3 hypervariable regions determine the specificity of the antibody and are also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDRs and 4 FRs. The order from amino terminus to carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; The 3 CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3.

The antibodies of the present invention include murine antibodies, chimeric antibodies and humanized antibodies.

The term "mouse antibody" in the present invention refers to a monoclonal antibody to human TSLP obtained in accordance with the knowledge and skills in the art. When obtained, the subject is administered with the TSLP antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated. In a preferred embodiment of the present invention, the mouse antibody to TSLP or an antigen-binding fragment thereof may further comprise a constant region of a light chain of a mouse κ, λ chain or variants thereof, or further comprise a constant region of a heavy chain of a mouse IgG1, IgG2, IgG3 or variants thereof.

The term "chimeric antibody" means an antibody formed by fusing a variable region of a mouse antibody with a constant region of a human antibody, which can weaken the immune response induced by the mouse antibody. The creation of a chimeric antibody requires first creating a hybridoma secreting mouse-specific monoclonal antibodies, then cloning a variable region gene from mouse hybridoma cells, then cloning a constant region gene of a human antibody, if necessary, linking the mouse variable region gene to the human constant region gene to form a chimeric gene, inserting it into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present invention, the antibody light chain of the chimeric anti-TSLP antibody further comprises a constant region of a light chain of a human κ, λ chain, or a variant thereof. The heavy chain of the antibody of the chimeric antibody to TSLP further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably comprises a heavy chain constant region of human IgG1, IgG2 or IgG4, or IgG1, IgG2 or IgG4 variants with amino acid mutations (e.g. L234A and/or L235A mutations and/or S228P mutations).

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody prepared by grafting murine CDR sequences onto the variable region framework of a human antibody, i.e., an antibody obtained from different types of human germline antibody framework sequences. It can overcome the heterogeneous response induced by the chimeric antibody because it carries a large number of murine protein components. Such framework sequences can be obtained from publicly available DNA databases or published sources that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the human germline sequence database "VBase" (available at www.mrccpe.com.ac.uk/vbase) and in Kabat, E.A., et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity simultaneously caused by the decrease in immunogenicity, the framework sequence of the variable region of the human antibody can be subjected to minimal reversible mutations or back mutations to maintain activity. The humanized antibody of the present invention also includes humanized antibodies in which the affinity maturation of the CDR is carried out by yeast display.

Grafting of CDRs may result in a decrease in the affinity of the resulting antibody or antigen-binding fragment thereof for that antigen due to alteration of the framework residues in contact with the antigen. Such interactions may result from hypermutation in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids onto the framework of a humanized antibody. Amino acid residues involved in antigen binding that are derived from non-human antibodies or antigen-binding fragments thereof can be identified by examining the sequence and structure of the variable region of the animal monoclonal antibody. Residues in the donor CDR framework that differ from the germline can be considered related. If the closest germline cannot be determined, the sequence can be compared to a subclass consensus sequence or an animal antibody sequence with a high percentage of similarity. Rare framework residues are thought to result from hypermutation in somatic cells and therefore play an important role in binding.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof may further comprise a constant region of a light chain of a human or mouse κ, λ chain or a variant thereof, or further comprise a constant region of a heavy chain of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof; preferably comprises a constant region of a heavy chain of a human IgG1, IgG2 or IgG4, or variants of IgG1, IgG2 or IgG4 with amino acid mutations (e.g., a L234A/L235A mutation, a S228P mutation, a YTE mutation).

A "traditional variant" of a human antibody heavy chain constant region and a human antibody light chain constant region described herein refers to a variant of a heavy chain constant region or a light chain constant region disclosed in the art that does not alter the structure and function of the antibody variable region. Examples of variants include variants of an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region with site-directed modifications and amino acid substitutions in the heavy chain constant region. Particular substitutions are such as YTE mutations, L234A and/or L235A mutations, S228P mutations and/or knob-into-hole mutations (which converts the antibody heavy chain into a knob-Fc and hole-Fc combination) known in the art. These mutations have been shown to confer new properties to the antibody without altering the function of the antibody variable region.

The terms "human HuMAb antibody", "antibody of human origin", "fully human antibody" and "fully human antibody" may be used interchangeably and may refer to antibodies obtained from a human or antibodies obtained from a genetically modified organism that has been "engineered" to produce specific human antibodies in response to stimulation with an antigen, and may be produced by any method known in the art. In some technologies, elements of the human heavy chain and light chain gene loci are introduced into cell lines of organisms derived from embryonic stem cell lines in which the endogenous heavy chain and light chain genetic loci have been purposefully disrupted. Transgenic organisms can synthesize human antibodies specific for human antigens, and these organisms can be used to produce hybridomas that secrete human antibodies. A human antibody may also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more sources of human DNA. A fully human antibody can also be constructed by gene or chromosome transfection techniques and phage display technology, or constructed by in vitro activated B cells, all of which are known in the art.

The terms "full-length antibody," "intact antibody," "whole antibody," and "complete antibody" are used interchangeably herein and refer to an antibody in substantially intact form, as opposed to antigen-binding fragments, as defined below. These terms refer exclusively to an antibody in which the light chain and heavy chain comprise a constant region. An "antibody" according to the present invention includes a "full-length antibody" and antigen-binding fragments thereof.

In some embodiments, the full-length antibody of the present invention includes antibodies formed by linking a light chain variable region to a light chain constant region and linking a heavy chain variable region to a heavy chain constant region, as shown in the light and heavy chain combinations in Tables 1 through 4 below. Those skilled in the art can select different light chain constant regions and heavy chain constant regions derived from the antibody according to actual needs, such as light chain constant regions and heavy chain constant regions derived from a human antibody.

The term "antigen-binding fragment" or "functional 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., TSLP). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies. Examples of a binding fragment encompassed by the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab') ₂ fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment, consisting of the VH and CH1 domains; (iv) an Fv fragment, consisting of the VH and VL domains of one arm of an antibody; (v) dsFv - a stable antigen-binding fragment formed by interchain disulfide bonds between VH and VL; (vi) diabody - a bispecific antibody and multispecific antibody containing fragments such as scFv, dsFv, Fab, etc. Furthermore, although the two VL and VH domains of the Fv fragment are encoded by separate genes, recombinant techniques can be used to join them using synthetic linkers so that it can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called 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 encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are produced using conventional techniques known to those skilled in the art and are screened in the same manner as intact antibodies. The antigen-binding fragment may be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulin. The antibodies may be of different isotypes, such as IgG antibodies (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.

Fab is an antibody fragment having a molecular weight of approximately 50,000 and possessing antigen-binding activity, and refers to fragments obtained by treating IgG antibody molecules with papain (which cleaves the amino acid residue at position 224 of the H chain), in which approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

F(ab')2 is an antibody fragment that has a molecular weight of about 100,000, has antigen-binding activity, and contains two Fab regions linked at the hinge position, and refers to fragments obtained by cleaving the lower portion of two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, obtained by cleaving a disulfide bond in the hinge region of F(ab')2. Fab' of the present invention can be obtained by using reducing agents, such as dithiothreitol, to treat F(ab')2 of the present invention, which specifically recognizes TSLP and binds to the amino acid sequence of the extracellular domain or its three-dimensional structure.

In addition, Fab' can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic organism or a eukaryotic organism to express Fab'.

The term "single chain antibody", "single chain Fv" or "scFv" refers to molecules comprising an antibody heavy chain variable domain (or region, VH) and an antibody light chain variable domain (or region, VL) linked by a linker. Such scFv molecules may have the general structure: _NH2 -VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. Suitable linkers known in the art consist of repeating amino acid sequences GGGGS or variants thereof, for example, variants with 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

A diabody is an antibody fragment in which an scFv or Fab is dimerized and is an antibody fragment with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens may be the same or different.

Bispecific antibody and multispecific antibody refer to an antibody that can simultaneously bind to two or more antigens or antigenic determinants, including scFv or Fab fragments that can bind to TSLP.

The diabody according to the present invention can be obtained by the following steps: obtaining a cDNA encoding VH and VL of the monoclonal antibody according to the present invention that specifically recognizes human TSLP and binds to the amino acid sequence of the extracellular domain or its three-dimensional structure, constructing a DNA encoding scFv so that the length of the amino acid sequence of the peptide linker is 8 residues or less, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or a eukaryotic organism to express the diabody.

dsFv is obtained by linking VH and VL polypeptides in which one amino acid residue in each of them is replaced by a cysteine residue, via disulfide bonds between said cysteine residues. The amino acid residues replaced by cysteine residues can be selected according to known methods (Protein Engineering, 7, 697 (1994)), based on the prediction of the three-dimensional structure of the antibody.

A full-length antibody or an antigen-binding fragment thereof according to the present invention can be obtained by the following steps: obtaining a VH and VL encoding cDNA of a monoclonal antibody according to the present invention that specifically recognizes human TSLP and binds to the amino acid sequence of the extracellular domain or its three-dimensional structure, constructing a DNA encoding the full-length antibody or an antigen-binding fragment thereof, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or a eukaryotic organism for expression.

The term "amino acid difference" or "amino acid mutation" refers to the presence of amino acid changes or mutations in a variant protein or polypeptide compared to a reference protein or polypeptide, including the presence of 1, 2, 3 or more amino acid insertions, deletions or substitutions relative to the reference protein or polypeptide.

The term "antibody framework" or "FR region" refers to the portion of the VL or VH variable domain that serves as a framework for the CDR of the variable domain. It is essentially a variable domain without the CDR.

The term "complementarity determining region", "CDR" or "hypervariable region" refers to one of six hypervariable regions in the variable domain of an antibody that primarily contribute to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each variable region of the heavy chain and three CDRs (LCDR1, LCDR2, LCDR3) in each variable region of the light chain. Any of the well-known schemes can be used to define the amino acid sequence boundaries of the CDRs, including the "Kabat" numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5 ^th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering system (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImmunoGenTics (IMGT) numbering system (Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP, et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, for the classical format according to the Kabat system, the amino acid residue numbers of the CDRs in the variable domain of the heavy chain (VH) are 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the amino acid residue numbers of the CDRs in the variable domain of the light chain (VL) are 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to the Chothia system, the amino acid residue numbers of the CDRs in VH are 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residue numbers of the VL are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). When combining the CDR definitions according to Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. According to the IMGT system, the amino acid residue numbers of the CDRs in VH are approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the amino acid residue numbers of the CDRs in VL are approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). According to the IMGT system, the CDR regions of an antibody can be determined using the IMGT/DomainGap Align program.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on TSLP molecules). Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or nonconsecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E.Morris, Ed. (1996).

The terms "specifically binds,""selectivelybinds,""bindsselectively," and "binds specifically" refer to the binding of an antibody to an epitope on a selected antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 ^-8 M, such as less than about 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M, or less.

The term "KD" refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. Typically, an antibody of the present invention binds to TSLP with an affinity (KD) of less than about ^10-7 M, such as less than about ^10-8 M or ^10-9 M, for example, in the present invention, the affinity of an antibody for a cell surface antigen is determined by FACS or Biacore to determine the KD value.

When the term "competition" is used in the context of antigen-binding proteins (e.g., a neutralizing antigen-binding protein or a neutralizing antibody) that compete for the same epitope, it refers to competition between antigen-binding proteins that is determined by the following assay: the antigen-binding proteins to be tested (e.g., antibodies or immunologically functional fragments thereof) prevent or inhibit (e.g., reduce) the specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., a TSLP antigen or fragment thereof). Many types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme-linked immunosorbent assay (EIA), competitive sandwich assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9: 242–253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137: 3614–3619), solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct-labeling RIA with I-125 labeling (see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176: 546-552); and direct-labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, the assays involve the use of either unlabeled test antigen-binding proteins and labeled reference antigen-binding proteins to bind purified antigens bound to a solid surface or cells. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or to cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antigen-binding proteins identified by a competitive assay (competing antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as a reference antigen-binding protein; and antigen-binding proteins that bind to adjacent epitopes located sufficiently close to the epitope to which the reference antigen-binding protein binds that the two epitopes spatially hinder each other's binding. Typically, when a competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antigen-binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70%-75%, or 75%, or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97%, or more.

The term "nucleic acid molecule" as used herein refers to a DNA molecule and an RNA molecule. A nucleic acid molecule may be single-stranded or double-stranded, and is preferably double-stranded DNA or single-stranded mRNA or modified mRNA. When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is "functionally linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the coding sequence.

"Identity" of an amino acid sequence refers to the percentage of amino acid residues that are identical between a first and second sequence when the amino acid sequences are aligned (with gaps introduced if necessary) to achieve the maximum percent sequence identity, and no conservative substitutions are considered part of the sequence identity. To determine the percent amino acid sequence identity, the alignment can be achieved in a variety of ways within the technical capabilities of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art are able to determine parameters suitable for measuring alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded loop of DNA to which additional DNA segments can be linked. In another embodiment, the vector is a viral vector in which additional DNA segments can be linked to a viral genome. The vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (e.g., bacterial vectors with a bacterial origin of replication and mammalian episomal vectors), or can be integrated into the host cell genome after introduction into the host cell to replicate along with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, see, for example, Antibody Experiment Technical Guide, Cold Spring Harbor, Chapters 5-8 and 15. For example, mice can be immunized with human TSLP or a fragment thereof, and the resulting antibodies can be annealed and purified, and amino acid sequencing can be performed using conventional methods. Antigen-binding fragments can also be produced using conventional methods. The antibody or antigen-binding fragment of the present invention is genetically engineered to add one or more human FR regions to non-human CDR regions. Human germline FR sequences can be obtained from the ImmunoGeneTics (IMGT) website https://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and the MOE software, or obtained from The Immunoglobulin FactsBook, 2001ISBN012441351.

The term "host cell" refers to a cell into which the expression vector has been introduced. Host cells may include bacteria, microorganisms, plant cells, or animal cells. Bacteria that can be readily transformed include members of the Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells (a Chinese hamster ovary cell line), 293 cells, and NS0 cells.

The genetically modified antibodies or antigen-binding fragments of the present invention can be produced and purified by conventional methods. For example, cDNA sequences encoding the heavy chain and light chain can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems can result in glycosylation of antibodies, particularly at the highly conserved N-terminal sites of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human TSLP. Positive clones are expanded in serum-free antibody bioreactors. The medium into which the antibodies are secreted can be purified by conventional methods. For example, using a Sepharose FF A or G column with an adjusted buffer for purification. Non-specifically bound components are washed away. The bound antibodies are then eluted by a pH gradient method and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

"Administering," "giving," "treating," and "treating," when used with animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with animals, humans, subjects, cells, tissues, organs, or biological fluids. "Administering," "giving," "treating," and "treating" may refer to, for example, treatment, pharmacokinetics, diagnostics, research, and experimental methods. Treating cells includes contacting reagents with cells and contacting reagents with fluids, where the fluids contact the cells. "Administering," "giving," "treating," and "treating" also refer to treating, for example, cells with reagents, diagnostics, binding compositions, or another cell in vitro and ex vivo. "Treatment", when applied to humans, veterinary subjects, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, investigational and diagnostic applications.

"Treatment" refers to the administration of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present invention, to a patient with one or more symptoms of a disease in which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, to cause a regression of such symptoms or to suppress the progression of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any particular symptom of a disease (also referred to as a "therapeutically effective amount") may vary depending on various factors, such as the pathological condition, the age and body weight of the patient, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed using any of the clinical testing methods commonly used by physicians or other healthcare professionals to assess the severity or progression of symptoms. Although embodiments of the present invention (e.g., treatment methods or products) may not be effective in alleviating the symptom(s) of the target disease in every patient, they should alleviate the symptom(s) of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, the chi-square test, the Mann-Whitney U-test, the Kruskal-Wallis (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

"Conservative modification" or "conservative substitution or replacement" refers to the replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein. Those skilled in the art will recognize that, in general, substitution of a single amino acid in non-essential regions of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, ( ^4th edition)). In addition, substitution with amino acids of similar structure or function is unlikely to affect biological activity. Illustrative conservative amino acid substitutions are provided in the table "Illustrative Conservative Amino Acid Substitutions" below.

Table 5. Illustrative conservative amino acid substitutions

Initial balance Conservative replacement Ala(A) Gly;Ser Arg(R) Lys;His Asn(N) Gln; His; Asp Asp(D) Glu;Asn Cys(C) Ser; Ala; Val Gln(Q) Asn;Glu Glu(E) Asp; Gln Gly(G) Ala His(H) Asn;Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg;His Met(M) Leu; Ile; Tyr Phe(F) Tyr; Met; Leu Pro(P) Ala Ser(S) Th Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

"An effective amount" or "an effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For prophylactic purposes, beneficial or desired results include the elimination or reduction in the risk of, the reduction in the severity of, or the delay in the onset of a disease, including the biochemical, histological, and/or behavioral manifestations of the disease, its complications, and the intermediate pathological phenotypes that occur during the development of the disease. For therapeutic purposes, beneficial or desired results include clinical results such as a decrease in the incidence of various disorders associated with a target antigen according to the present invention or an improvement in one or more symptoms of a disorder, a decrease in the dose of other agents required to treat the disorder, an increase in the therapeutic efficacy of another agent, and/or a delay in the progression of disorders in a patient associated with a target antigen according to the present invention.

"Exogenous" refers to substances produced outside of organisms, cells, or human bodies, as the case may be. "Endogenous" refers to substances produced inside cells, organisms, or human bodies, as the case may be.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in the two sequences being compared are occupied by the same base or subunit of an amino acid monomer, for example, if a position in each of two DNA molecules is occupied by an adenine, then the molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions common to the two sequences divided by the number of positions being compared, then multiplied by 100. For example, in an optimal sequence alignment, if 6 out of 10 positions in the two sequences are matching or homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences are matching or homologous, then the two sequences are 95% homologous. Typically, when aligning two sequences, comparisons are made to obtain the maximum percentage homology. For example, the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are chosen to ensure the maximum match for each sequence over the entire length of each reference sequence. The following references refer to the BLAST algorithm, which is often used for sequence analysis: BLAST ALGORITHMS: Altschul, S. F. et al., (1990) J. Mol. Biol. 215:403–410; Gish, W. et al., (1993) Nature Genet. 3:266–272; Madden, T. L. et al., (1996) Meth. Enzymol. 266:131–141; Altschul, S. F. et al., (1997) Nucleic Acids Res. 25:3389–3402; Zhang, J. et al., (1997) Genome Res. 7:649–656. Other traditional BLAST algorithms, such as those available from NCBI BLAST, are also well known to those skilled in the art.

The terms "cell", "cell line" and "cell culture" as used herein may be used interchangeably and all such names include progeny. Accordingly, the words "transformant" and "transformed cell" include the primary test cells and the cultures derived therefrom, regardless of the number of passages. It should also be understood that, due to deliberate or accidental mutations, all progeny may not be exactly alike in terms of DNA content. Mutant progeny with the same function or biological activity as that found in screening the original transformed cells are included. Where another term is intended, it will be clear from the context.

"Polymerase chain reaction" or "PCR" as used herein refers to a procedure or technique that amplifies a trace amount of a specific fragment of nucleic acid, RNA and/or DNA, as described, for example, in U.S. Patent No. 4,683,195. In general, it is necessary to obtain sequence information at the end or outside of the target region in order to be able to design oligonucleotide primers; these primers are the same or similar in sequence to the corresponding strand of the template to be amplified. The 5'-terminal nucleotides of two primers may be identical to the ends of the substance to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, as well as cDNA sequences transcribed from total cellular RNA, phage or plasmid sequences, etc. See generally Mullis et al. (1987) Cold Spring Harbor, Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR as used herein is considered an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a test nucleic acid sample, and this method involves the use of known nucleic acids as primers and nucleic acid polymerases to amplify or produce a specified fragment of nucleic acid.

"Isolated" refers to a purified state and in this case means that the designated molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other substances, such as cellular debris and growth medium. In general, the term "isolated" does not imply the complete absence of these substances or the absence of water, buffer, or salt, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compound as described herein.

"Optional" or "optional" means that the event or circumstance described below may, but does not necessarily, occur, and the description includes instances in which the event or circumstance does or does not occur.

"Pharmaceutical composition" means a mixture containing one or more compounds described herein, or physiologically/pharmaceutically acceptable salts or prodrugs thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compound to organisms, thereby promoting absorption of the active ingredient, resulting in its biological activity.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in the formulation for delivering antibodies or antigen-binding fragments. The carrier may be an anti-adhesive agent, a binder, a coating, a disintegrating agent, a filler or diluent, a preservative (such as an antioxidant, antibacterial or antifungal agent), a sweetener, an absorption delaying agent, a wetting agent, an emulsifier, a buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, a polyhydric alcohol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oil (e.g., olive oil), saline, buffer, buffered saline and an isotonic agent such as sugar, a polyhydric alcohol, sorbitol and sodium chloride.

In addition, the present invention includes agents for treating TSLP-related diseases comprising the TSLP antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

The TSLP-related disease is not limited in the present invention as long as it is a TSLP-related disease. For example, the therapeutic response induced by the molecule of the present invention can be achieved by binding to human TSLP and then blocking the binding of TSLP to its receptors, or killing cells overexpressing TSLP.

In addition, the present invention relates to methods for immunodetection or detection of a target antigen (e.g. TSLP), reagents for immunodetection or detection of a target antigen (e.g. TSLP), methods for immunodetection or detection of cells expressing a target antigen (e.g. TSLP), and diagnostic agents for diagnosing diseases associated with cells positive for a target antigen (e.g. TSLP), which include an antibody or antibody fragment according to the present invention as an active ingredient that specifically recognizes a target antigen (e.g. human TSLP) and binds to the amino acid sequence of the extracellular domain or its three-dimensional structure.

In the present invention, the method used to detect or measure the amount of the target antigen (e.g., TSLP) may be any known method. For example, it includes immunodetection or measurement methods.

Immunodetection or measurement methods are methods for detecting or measuring the amount of an antibody or antigen using labeled antigens or antibodies. Examples of immunodetection or measurement methods include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay, Western blotting, physicochemical methods, etc.

The above-mentioned TSLP-related diseases can be diagnosed by detecting or measuring TSLP-expressing cells using the antibody or antibody fragment of the present invention.

To detect cells expressing the said polypeptide, known immunodetection methods can be used, preferably using immunoprecipitation, fluorescent cell staining, immunohistochemical staining, etc. In addition, a fluorescent staining method with antibodies using the FMAT8100HTS system (Applied Biosystem) can be used.

In the present invention, there is no particular limitation on the in vivo sample used for detecting or measuring the target antigen (e.g. TSLP), provided that it may contain cells expressing the target antigen (e.g. TSLP), such as histocytes, blood, plasma, serum, pancreatic juice, urine, feces, interstitial fluid or culture fluid.

Depending on the required diagnostic method, the diagnostic agent containing the monoclonal antibody or fragment thereof according to the present invention may also contain reagents for carrying out the antigen-antibody reaction or reagents for detecting such a reaction. Reagents used for carrying out the antigen-antibody reaction include buffers, salts, etc. Reagents used for detection include agents commonly used in immunodetection or measurement methods, such as labeled secondary antibodies recognizing the monoclonal antibody, antibody fragment, or conjugate thereof, and substrates corresponding to the label, etc.

The foregoing description provides details of one or more embodiments of the present invention. Although any methods and materials similar or identical to those described herein can be used to make or test the present invention, the preferred methods and materials are described below. Other features, objects, and advantages of the present invention will be apparent from the description and claims. In the description and claims, unless the context clearly dictates otherwise, the singular form includes the plural of the term referred to. Unless clearly defined otherwise, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present invention pertains. All patents and publications cited in the description are incorporated by reference. The following examples are presented to more fully illustrate the preferred embodiments of the present invention. These examples should not be construed as limiting the scope of the present invention in any way, and the scope of the present invention is defined by the claims.

Examples

The following examples are included to further describe the present invention, but these examples do not limit the scope of the present invention.

Experimental methods for which conditions are not specifically indicated in the Examples or Test Examples of the present invention generally correspond to conventional conditions or correspond to conditions recommended by the manufacturer of the raw material or product. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Current Protocols Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents not specifically indicated by sources are common reagents purchased commercially.

Example 1. Expression of TSLP and TSLP receptor

Sequences encoding His-tagged human and cynomolgus TSLP, human IgG1-Fc-tagged human and cynomolgus TSLP, and TSLP receptor extracellular domain sequences were loaded onto the phr vector to construct expression plasmids, which were then transfected into HEK293. The specific transfection steps were as follows: the day before, HEK293E cells were seeded in Freestyle expression medium (containing 1% FBS) at a density of 0.8 x 10 ⁶ /ml, placed on a constant temperature shaker at 37°C (120 rpm), and cultured for 24 h. After 24 h, the transfection plasmid and PEI transfection reagent were sterilized through 0.22 μm filters. Then, the transfection plasmid was adjusted to a concentration of 100 μg/100 ml cells, and the weight ratio of PEI (1 mg/ml) and plasmid was 3:1. Taking the transfection of 200 ml HEK293E cells as an example, 10 ml of Opti-MEM and 200 μg of plasmid were taken, mixed well and left for 5 min; another 10 ml of Opti-MEM and 600 μg of PEI were taken, mixed well and left for 5 min. The plasmid and PEI were mixed well and left for 15 min, preferably no more than 20 min. The mixture of plasmid and PEI was slowly added to 200 ml of HEK293E cells and placed on a shaker at 8% _CO2 , 120 rpm and 37°C for culture. On day 3 of transfection, 10% of the volume of additional medium was added to the culture. Until day 6 of transfection, samples were taken and centrifuged at 4500 rpm for 10 min to collect the cell supernatant. The supernatant was filtered and purified to obtain recombinant TSLP and TSLP receptor proteins according to Example 2. The purified proteins can be used in the experiments of each of the examples below. The corresponding sequences are as follows.

1. Amino acid sequence of his-tagged human TSLP antigen (huTSLP-his)

Note: Underlining represents the signal peptide sequence; italicized portion represents the Flag-His6 tag.

SEQ ID NO:1

2. Amino acid sequence of Fc-tagged human TSLP antigen (huTSLP-Fc)

Note: Underlining represents the signal peptide sequence; italicized portion represents the linker-human Fc tag.

SEQ ID NO:2

3. Amino acid sequence of his-tagged TSLP antigen of cynomolgus macaque (cynoTSLP-his)

Note: Underlining represents the signal peptide sequence; italicized portion represents the flag-His6 tag.

SEQ ID NO:3

4. Amino acid sequence of Fc-tagged TSLP antigen from cynomolgus monkey (cynoTSLP-Fc)

Note: Underlining represents the signal peptide sequence; italicized portion represents the linker-human Fc tag.

SEQ ID NO:4

5. Amino acid sequence of the extracellular domain of the Fc-tagged human TSLP receptor (human-TSLPR-Fc-ECD):

Note: The underlined portion represents the extracellular domain of human TSLPR, and the italicized portion represents the linker-human Fc tag.

SEQ ID NO:5

Example 2. Purification of recombinant TSLP and TSLP receptor (TSLPR) proteins

2.1 Purification of His-tagged recombinant TSLP proteins of each species

Supernatant samples containing cell expression products were centrifuged at high speed to remove contaminants and filtered. Ni columns were equilibrated with PBS and washed with 10× column volume. Filtered supernatant samples were injected onto the columns. Columns were washed with PBS containing 30 mM imidazole until the _A280 reading dropped to baseline. Target proteins were then eluted with PBS containing 300 mM imidazole and the elution peaks were collected. Proteins were concentrated, medium was exchanged with PBS and aliquoted for use after they were identified as correct by LC-MS. His-labeled human and cynomolgus monkey TSLP were obtained.

2.1 Purification of recombinant proteins of human Fc-tagged TSLP of each species and the extracellular domain of the human TSLP receptor

Supernatant samples containing cell expression products were centrifuged at high speed to remove contaminants. The supernatant containing the recombinant antibody expression product was purified on protein A columns. The columns were washed with PBS until the absorbance at 280 nm dropped to baseline. The target proteins were eluted with 100 M acetate buffer, pH 3.5, and neutralized with 1 M Tris-HCl, pH 8.0. The resulting proteins were concentrated, the medium was replaced with fresh solution, and aliquots were taken for use after they were identified as correct by electrophoresis and LC-MS.

Example 3. Construction and identification of cell lines expressing recombinant TSLP receptor and IL7Rα receptor

To screen for antibodies capable of blocking TSLP binding to the TSLP receptor, CHO-K1 and BaF3 cell lines co-expressing both human TSLP receptor and human IL7Rα (TSLPR/IL7Rα) were constructed. The target TSLPR/IL7 Rα gene was packaged by lentivirus and cloned into the target cell lines to generate stable, high-expressing cell lines. First, the human TSLPR and human IL7Rα genes were cloned into pCDH-CMV-MCS-EF1-puro and pCDH-CMV-MCS-EF1-Neo (SBI, CD500B-1) plasmids, respectively. Then, lentivirus infection method was used to insert human TSLPR into CHO-K1 and BaF3 cell lines, which were cultured under selection pressure of 10 μg/ml puromycin (Gibco, USA) for three weeks. Then, a second round of infection was performed. Human IL7Rα gene was inserted into the cell lines and screened using 1 mg/ml G418 (Gibco, USA) and 10 μg/ml puromycin for two to three weeks. Finally, monoclonal CHO-K1 and BaF3 cell lines with simultaneous high expression of TSLPR and IL7Rα were screened by flow cytometric sorting method.

Example 4. Production and screening of antibodies to human TSLP

Monoclonal antibodies to human TSLP were obtained by immunization of laboratory white mice SJL, females aged 6-8 weeks (Beijing Charles River Laboratory Animal Technology Co., Ltd., Animal Husbandry License Number: SCXK (Beijing) 2012-0001). Maintenance conditions: free from pathogenic microflora. After purchase, the mice were kept in laboratory conditions for 1 week, with a light/dark cycle of 12/12 hours at a temperature of 20-25°C; humidity 40-60%. Mice that adapted to the environment were immunized with recombinant proteins huTSLP-Fc (25 μg), huTSLP-his (12.5 μg) and cyno TSLP-his (12.5 μg) and TiterMax, Alum or CpG adjuvant. After 4-5 immunizations, mice with high serum antibody titers and titers tending to reach a plateau were selected and sacrificed. Spleen cells were collected and fused with myeloma cells. Spleen lymphocytes were fused with Sp2/0 myeloma cells (ATCC ^® CRL-8287™) to generate hybridoma cells using an optimized PEG-mediated fusion procedure.

For initial screening, ELISA binding assays were performed against human and cynomolgus monkey TSLP, blockade assays of binding of human TSLP to its receptor TSLPR, and TSLP-induced BaF3 cell proliferation inhibition experiments were performed. After transfer of hybridoma cells to 24-well plates, the supernatant was rescreened. Hybridoma clones were obtained after two rounds of subcloning of selected positive clones and used for antibody production and purification was performed by affinity-based methods.

After screening, monoclonal hybridoma cell lines #3, #119, #179 and #199 with good activity were obtained, and hybridoma cells were collected in the logarithmic growth phase. RNA was isolated using NucleoZol (MN) and reverse transcription was performed (PrimeScript™ Reverse Transcriptase, Takara, Cat. #2680A). The cDNA obtained as a result of reverse transcription was amplified by PCR using a mouse Ig primer kit (Novagen, TB326 Rev.B 0503) and sent to a sequencing service company for sequencing. After sequencing, mouse anti-TSLP antibodies were obtained: mab3, mab119, mab179 and mab199 sequences, the amino acid sequences of their variable regions are as follows:

>mouse mab3 heavy chain variable region sequence:

SEQ ID NO:6

>mouse mab3 light chain variable region sequence:

SEQ ID NO:7

>mouse mab119 heavy chain variable region sequence:

SEQ ID NO:8

>mouse mab119 light chain variable region sequence:

SEQ ID NO:9

>mouse mab179 heavy chain variable region sequence:

SEQ ID NO:10

>mouse mab179 light chain variable region sequence:

SEQ ID NO: 11

>mouse mab199 heavy chain variable region sequence:

SEQ ID NO:12

>mouse mab199 light chain variable region sequence:

SEQ ID NO:13

The amino acid sequences of the CDR regions, obtained according to the Kabat numbering system, are presented in the following table:

Table 6. Sequences of the CDR regions of the heavy chain and light chain of antibodies from hybridoma clones

Antibody Heavy chain Light chain mab3 HCDR1 DDYMN
SEQ ID NO:14 LCDR1 RASSSVSYMH
SEQ ID NO:17 HCDR2 IISPYNGGTSYNQKFKG
SEQ ID NO:15 LCDR2 ATSNLAS
SEQ ID NO:18 HCDR3 EDYDYDGYAMDH
SEQ ID NO:16 LCDR3 QQWSSNRT
SEQ ID NO:19 mab119 HCDR1 TYNMH
SEQ ID NO:20 LCDR1 RASESVDNSGLSFMH
SEQ ID NO:23 HCDR2 AIYPGNGETSYNQKFKD
SEQ ID NO:21 LCDR2 RASNLGS
SEQ ID NO:24 HCDR3 EDDYGEGYFDV
SEQ ID NO:22 LCDR3 QQINTDPLT
SEQ ID NO:25 mab179 HCDR1 NYLIE
SEQ ID NO:26 LCDR1 KASQSVSSDVT
SEQ ID NO:29 HCDR2 VIDPGNGDTNYNENFKG
SEQ ID NO:27 LCDR2 YVSNHYT
SEQ ID NO:30 HCDR3 EDNTGTAFDY
SEQ ID NO:28 LCDR3 QQHHRFPLT
SEQ ID NO:31 mab199 HCDR1 TYWMH
SEQ ID NO:32 LCDR1 RASENIYSYLA
SEQ ID NO:35 HCDR2 MIDPSDSETTLIKKFKD
SEQ ID NO:33 LCDR2 FACTS
SEQ ID NO:36 HCDR3 TLDGYYDY
SEQ ID NO:34 LCDR3 QHHYGTPWT
SEQ ID NO:37

Chimeric antibodies were formed by linking the variable regions of the light and heavy chains of the above-mentioned mouse antibody with the constant regions of the light and heavy chains of a human antibody (such as the kappa constant region shown in SEQ ID NO: 134 and the IgG1-YTE constant region shown in SEQ ID NO: 133). The chimeric antibody corresponding to the mab3 clone was named Ch3, and so on for other antibodies.

Example 5. Design of humanization of monoclonal antibodies to human TSLP

In order to reduce the immunogenicity of mouse antibodies, the selected antibodies mab3, mab119, mab179 and mab199 with excellent in vivo and in vitro activity were humanized. The humanization of mouse monoclonal antibodies was carried out according to the methods published in many documents in the art. Briefly, the constant domains of human antibody were used to replace the parental constant domains (mouse antibody), the germline sequences of human antibodies were selected according to the homology between mouse and human antibodies, and CDR grafting was performed. Then, based on the three-dimensional structure of mouse antibody, the amino acid residues of VL and VH were backmutated, and the constant regions of mouse antibody were replaced with human constant regions to obtain the final humanized molecule.

5.1 Selection and backmutation of human FR regions for mab3

(1) Selection and back mutations of human FR regions

For mab3, the humanized VH template was IGHV1-3*01+IGHJ6*01, and the humanized VL template was IGKV3-20+IGKJ4*01. The CDRs of mab3 were grafted onto the human template, and the variable region sequences obtained after grafting were as follows:

VL hu3 with grafted CDRs:

SEQ ID NO:38

VH hu3 with grafted CDRs:

SEQ ID NO:42

The design of the humanized mab3 antibody back mutations is presented in the following table:

Table 7. Reverse mutations of the humanized antibody mab3

VL hu3 VH hu3 hu3VL1 Grafted hu3VH1 Grafted hu3VL2 L46P, F71Y hu3VH2 I69L, R71V, T73K hu3VL3 L46P, L47W, I58V, F71Y hu3VH3 R38K, M48I, V67A, I69L, R71V, T73K hu3VL4 L46P, L47W, I58V, D70S, F71Y

Note: "grafted" means grafting of a mouse antibody CDR into a human germline FR region sequence. L46P means that according to the Kabat numbering system, L at position 46 is back-mutated to P.

The sequences of the variable regions of the humanized mab3 antibody are as follows:

> hu3VL1 (VL hu3 with grafted CDRs)

SEQ ID NO:38

Note: Single underlines denote CDR regions and double underlines denote back mutation sites.

The above-mentioned variable regions of the light and heavy chain were combined with the sequences of the constant regions of the light and heavy chain of the human germline to form the final complete sequences of the light and heavy chains, thereby obtaining an antibody with a complete sequence. For example, for the humanized mab3 antibody in the present invention, the constant region of the heavy chain is the constant region of IgG1-YTE shown in SEQ ID NO: 133, and the constant region of the light chain is the constant region of the kappa chain shown in SEQ ID NO: 134, but they can also be replaced by other constant regions known in the art.

The sequences of the variable regions of the heavy and light chains of the obtained humanized mab3 antibodies are presented in the following table:

Table 8. Sequences of the variable region of the heavy and light chains of the humanized antibody mab3

Antibody VH (SEQ ID NO:) VL (SEQ ID NO:) hu3-01 42 39 hu3-02 42 40 hu3-03 42 41 hu3-04 43 38 hu3-05 43 39 hu3-06 43 40 hu3-07 43 41 hu3-08 44 39 hu3-09 44 40 hu3-10 44 41

The binding activity of humanized mab3 antibody to human TSLP was determined by ELISA, and the results showed that humanized mab3 antibody had very good binding ability to human TSLP.

(2) Point mutation of the hu3 antibody

By determination, the presence of hot spots was found on the MDH HCDR3 sequence and the NTR LCDR3 sequence of humanized mab3 antibody. Therefore, the corresponding hot spots were mutated. The sequences of the CDR regions of humanized mab3 antibodies obtained after introducing mutations are as follows:

Table 9. Sequences of HCDR3 and LCDR3 after introduction of mutations

hu3HCDR3-H110Y
SEQ ID NO:45 hu3LCDR3-N93D
SEQ ID NO:46

Note: The positions of the mutation sites in Table 9 are numbered according to the natural order of the variable region sequences.

It can be concluded that the CDR sequences of the humanized mab3 antibody are as follows:

Table 10. CDR after introduction of mutations into the humanized antibody mab3

Heavy chain Light chain HCDR1 DDYMN
SEQ ID NO:14 LCDR1 RASSSVSYMH
SEQ ID NO:17 HCDR2 IISPYNGGTSYNQKFKG
SEQ ID NO:15 LCDR2 ATSNLAS
SEQ ID NO:18 HCDR3 (general formula) EDYDYDGYAMDX1
SEQ ID NO:47 LCDR3 (general formula 1) QQWSSX2RT
SEQ ID NO:48

Where X ₁ is selected from H or Y, X ₂ is selected from N or D.

As an example, the CDRs and variable regions of the heavy and light chains of the humanized hu3-11 antibody obtained after introducing mutations are as follows:

Table 11. CDR regions of hu3-11

Heavy chain Light chain HCDR1 DDYMN
SEQ ID NO:14 LCDR1 RASSSVSYMH
SEQ ID NO:17 HCDR2 IISPYNGGTSYNQKFKG
SEQ ID NO:15 LCDR2 ATSNLAS
SEQ ID NO:18 HCDR3-H110Y EDYDYDGYAMDY
SEQ ID NO:45 LCDR3-N93D QQWSSDRT
SEQ ID NO:46

>Variable region light chain hu3-11 (hu3VL4-N93D)

SEQ ID NO:49

>Variable region heavy chain hu3-11 (hu3VH2-H110Y)

SEQ ID NO:50

The variable regions of the light and heavy chains, after introducing hotspot mutations, were recombined with human germline light and heavy chain constant region sequences to form the final complete light and heavy chain sequences, thus producing a complete sequence antibody.

The binding activity of the antibody obtained after introducing mutations to human TSLP was determined by ELISA. The results showed that the affinity activity of hu3-11 to human TSLP remained high, indicating that the mutations in the HCDR3 and LCDR3 hot spots of the humanized mab3 antibody do not affect the activity of the antibody.

(3) Affinity maturation of hu3-11 antibody

The hu3-11 molecule was subjected to affinity maturation. The affinity maturation process was as follows:

Yeast library construction: Degenerate primers were designed and the designed mutant amino acids were introduced into the hu3-11 scFv mutant antibody libraries by PCR, where the size of each library was about 10 ⁹ . The constructed yeast libraries were checked for their diversity by sequencing.

In the first round of screening, approximately 5 × 10 ¹⁰ cells from the hu3-11-scFv mutant libraries and biotinylated TSLP-Fc protein (1-10 μg/ml) were incubated in 50 ml of sodium phosphate buffer with 0.1% bovine serum albumin (BSA) (PBSA) for 1 h at room temperature. The mixture was then washed with 0.1% PBSA to remove unbound antibody fragments. Then, 100 μl of streptomycin beads (Milenyi Biotec, Auburn, CA) were added to the hu3-11-scFv mutant antibody libraries coupled to biotinylated TSLP-Fc and loaded onto the AutoMACS system for sorting. Cells with high affinity for TSLP-Fc were collected from the antibody library and induced at 250 rpm and 20°C for 18 h. The resulting enriched library was subjected to a second round of screening against biotinylated recombinant TSLP-Fc protein.

For the third and fourth rounds of screening, library cells from the previous round were incubated with biotinylated recombinant TSLP-Fc protein (0.1-1 μg/ml) and 10 μg/ml mouse anti-cMyc antibody (9E10, sigma) in 0.1% PBSA at room temperature for 1 h. The mixture was washed with 0.1% PBSA to remove unbound antibody fragments. Goat anti-mouse antibody conjugated with Alexa488 (A-11001, life technologies) and streptavidin-PE (S-866, Life technologies) were added and incubated at 4°C for 1 h. The mixture was washed with 0.1% PBSA to remove unbound antibody fragments. Finally, high affinity antibodies were screened using FACS screening (BD FACSAriaTM FUSION).

The hu3-11-scFv mutant libraries were screened in 2 rounds by MACS and 2 rounds by FACS using biotinylated TSLP-Fc antigen. Approximately 400 individual yeast clones were then selected for culture and expression induction. Binding of individual yeast clones to TSLP-Fc antigen was determined by FACS, and high-affinity individual yeast clones were selected and verified by sequencing. Sequenced clones were compared and analyzed. After removal of duplicate sequences, non-redundant sequences were converted to full-length antibodies for expression in mammalian cells.

The light chain variable region sequences obtained by affinity maturation are as follows:

The resulting light chain variable regions were recombined with the heavy chain variable regions of the humanized mab3 antibody to produce a new humanized mab3 antibody. For example, huVL5 and huVL6 were combined with hu3VH2-H110Y, respectively, to produce new antibody molecules hu3-12 and hu3-13, as detailed below:

Table 12. Antibodies obtained by affinity maturation

Antibody VH hu3 hu3VL hu3-12 hu3VH2-H110Y hu3VL5 hu3-13 hu3VH2-H110Y hu3VL6

The CDR sequences of the humanized mab antibody obtained by affinity maturation are as follows:

Table 13. Antibody CDRs of the humanized mab3 antibody obtained by affinity maturation

Antibody Heavy chain Light chain hu3-12 HCDR1 DDYMN
SEQ ID NO:14 LCDR1 RASSSVSYMH
SEQ ID NO:17 HCDR2 IISPYNGGTSYNQKFKG
SEQ ID NO:15 LCDR2 ATSNLAS
SEQ ID NO:18 HCDR3-H110Y EDYDYDGYAMDY
SEQ ID NO:45 LCDR3-V1 QQSDNVRG
SEQ ID NO:53 hu3-13 HCDR1 DDYMN
SEQ ID NO:14 LCDR1 RASSSVSYMH
SEQ ID NO:17 HCDR2 IISPYNGGTSYNQKFKG
SEQ ID NO:15 LCDR2 ATSNLAS
SEQ ID NO:18 HCDR3-H110Y EDYDYDGYAMDY
SEQ ID NO:45 LCDR3-V2 QQSDSGRE
SEQ ID NO:54

The resulting novel humanized mab3 antibody was subjected to ELISA to determine its binding activity to human TSLP. The results showed that hu3-12 and hu3-13 retained high binding affinity to human TSLP. They showed that LCDR3 changes did not affect the activity of hu3 series antibodies.

Thus, the CDRs of the humanized mab3 antibody have the following sequences:

Table 14. General formula of the CDR sequences of the humanized mab3 antibody

Heavy chain Light chain HCDR1 DDYMN
SEQ ID NO:14 LCDR1 RASSSVSYMH
SEQ ID NO:17 HCDR2 IISPYNGGTSYNQKFKG
SEQ ID NO:15 LCDR2 ATSNLAS
SEQ ID NO:18 HCDR3 (general formula) EDYDYDGYAMDX ₁
SEQ ID NO:47 LCDR3 (general formula 2) QQSDX ₃ X ₄ RX ₅
SEQ ID NO:55

Where X ₁ is H or Y, X ₃ is N or S, X ₄ is V or G, X ₅ is G or E.

The combinations of heavy and light chain variable regions of the humanized mab3 antibody after introduction of mutations into mutagenesis hot spots and affinity maturation are presented in the following table:

Table 15. Antibody sequences after affinity maturation

Antibody VH (SEQ ID NO) VL(SEQ ID NO) hu3-11 50 49 hu3-12 50 51 hu3-13 50 52

5.2 Selection and backmutation of human FR regions for mab119

For mab119, IGHV1-69*02 and HJ6*01 were selected as templates for VH, and IGKV4-1*01 and IGKJ2*01, as well as IGKV3-11*01 and IGKJ2*01 were selected as templates for VL. The CDR regions of the mouse antibody were grafted onto the selected humanized templates, and the FR regions were backmutated to generate different light and heavy chain variable regions. The variable region sequences resulting from the CDR grafting are as follows:

The back mutations of the humanized antibody mab119 are presented in the following table:

Table 16. Reverse mutations of mab119

hu119VL hu119VH hu119VL1 Grafted (IGKV4-1*01) hu119VH1 Vaccinated (IGHV1-69*02) hu119VL2 Grafted (IGKV4-1*01)+M4L hu119VH2 G27F, I69L, A71V hu119VL3 Grafted (IGKV4-1*01)+I48L, V58I hu119VH3 G27F, M48I, V67A, I69L, A71V hu119VL4 Grafted (IGKV3-11*01) hu119VH4 G27F, R38K, Q39H, M48I, V67A, I69L, A71V hu119VL5 Grafted (IGKV3-11*01)
+A43P, I48L hu119VH5 M48I, V67A, I69L, A71V hu119VL6 Grafted (IGKV3-11*01)+E1D, A43P, I48L hu119VH6 V2A, G27F, M48I, V67A, I69L, A71V hu119VH7 M48I, V67A, I69L, A71V, S76R hu119VH8 V2A, G27F, M48I, V67A, I69L, A71V, S76R

Note: For example, M4L indicates that according to the Kabat numbering system, the M at position 4 is back-mutated to L. "Grafted" means that the CDR of the mouse antibody is implanted into the human germline FR region sequence.

The specific sequences of the variable regions of the humanized antibody mab119 are as follows:

>hu119VL1 (grafted (IGKV4-1*01))

>hu119VL2

SEQ ID NO:57

>hu119VL3

SEQ ID NO:58

>hu119VL4 (grafted, IGKV3-11*01)

SEQ ID NO:59

>hu119VL5

SEQ ID NO:60

>hu119VL6

SEQ ID NO:61

Note: Single underlines denote variable regions, and double underlines denote back mutations.

The above-mentioned variable regions of the light and heavy chain were combined with the sequences of the constant regions of the light and heavy chain of the human germline to form the final complete sequences of the light and heavy chains, thereby obtaining an antibody with a complete sequence. For example, for the humanized antibody mab119 in the present invention, the constant region of the heavy chain is the constant region of IgG1-YTE shown in SEQ ID NO: 133, and the constant region of the light chain is the constant region of the kappa chain shown in SEQ ID NO: 134, but they can also be replaced by other constant regions known in the art.

The variable regions of the heavy and light chains of the humanized antibody mab119 are presented in Table 17.

Table 17. Variable regions of the heavy and light chains of the humanized antibody mab119

Antibody VH (SEQ ID NO) VL(SEQ ID NO) hu119-01 62 56 hu119-02 63 56 hu119-03 64 56 hu119-04 65 56 hu119-05 62 57 hu119-06 63 57 hu119-07 64 57 hu119-08 65 57 hu119-09 62 58 hu119-10 63 58 hu119-11 64 58 hu119-12 65 58 hu119-13 64 59 hu119-14 66 59 hu119-15 67 59 hu119-16 68 59 hu119-17 69 59 hu119-18 64 60 hu119-19 66 60 hu119-20 67 60 hu119-21 68 60 hu119-22 69 60 hu119-23 64 61 hu119-24 66 61 hu119-25 67 61 hu119-26 68 61 hu119-27 69 61

The binding activity of the humanized antibody to human TSLP was determined by ELISA, and the results showed that the humanized mab119 antibody was able to specifically bind to human TSLP.

(2) hu119 mutations

By detection, a hot spot was found in the LCDR1 DNS sequence of the humanized antibody mab119, so the corresponding site was mutated to N31S or N31Q. The LCDR1 sequences obtained after introducing the mutations are as follows:

Table 18. LCDR1 after introduction of mutations into the site of the humanized antibody mab119

hu119 LCDR1-N31S RASESVDSSGLSFMH
SEQ ID NO:70 hu119 LCDR1-N31Q RASESVDQSGLSFMH
SEQ ID NO:71

Note: The positions of mutation sites in Table 19 are numbered according to natural order.

As an example, the mutant sequences of hu119VL2, hu119VL6 obtained after introducing mutations are as follows:

Note: Single underlines denote variable regions, and double underlines denote back mutations.

The obtained mutants hu119VL2, hu119VL6 were combined with hu119VH to obtain new humanized hu119 antibodies. For example, hu119VL2-N31S, hu119VL2-N31Q were respectively combined with hu119VH3 to obtain antibodies hu119-28 and hu119-29; hu119VL3-N31S was combined with hu119VH8 to obtain antibody hu119-30. Examples of combinations of variable regions of mutated antibodies are as follows:

Table 19. Combinations of variable regions of humanized antibodies after introduction of mutations into mutagenesis hot spots

hu119VH hu119VL hu119-28 hu119VH3 hu119VL2-N31S hu119-29 hu119VH3 hu119VL2-N31Q hu119-30 hu119VH8 hu119VL6-N31S

The affinity of the antibody obtained after introducing the mutations to human TSLP was determined by ELISA. The results showed that the hu119-28 and hu119-29 antibodies retained relatively high affinity to human TSLP, indicating that the N31S and N31Q mutations in LCDR2 will not affect the activity of the antibody to TSLP.

Thus, the CDRs of the humanized antibody mab119 have the following sequences:

Table 20. CDR of the humanized antibody mab119

HCDR1 TYNMH
SEQ ID NO:20 LCDR1-
general formula RASESVDX ₆ SGLSFMH
SEQ ID NO:76 HCDR2 AIYPGNGETSYNQKFKD
SEQ ID NO:21 LCDR2 RASNLGS
SEQ ID NO:24 HCDR3 EDDYGEGYFDV
SEQ ID NO:22 LCDR3 QQINTDPLT
SEQ ID NO:25

Where X ₆ is chosen from N, S and Q.

5.3. Humanization mab179

(1) Selection of template and back mutations for humanization of mouse antibody mab179

For mab179, IGHV1-69*02 and IGHJ6*01 were chosen as templates for VH, and IGKV4-1*01 and IGKJ2*01 or IGKV2-29*02 and IGKJ2*01 were chosen as templates for VL. The CDR regions of the mouse antibody were grafted onto the selected humanized templates, and the FR regions were backmutated to generate light and heavy chain variable regions with different sequences. The humanized variable region sequences and backmutations are as follows:

Table 21. Templates and backmutations for humanization of mab179

hu179VL1VL hu179VH hu179VL1 Grafted (IGKV4-1*01) hu179VH1 Vaccinated (IGHV1-69*02) hu179VL2 Grafted (IGKV4-1*01)
P43S hu179VH2 G27Y, I69L hu179VL3 Grafted (IGKV4-1*01) P43S,L73F hu179VH3 G27Y, M48I, V67A, I69L, M80I hu179VL4 Grafted (IGKV4-1*01) D1S,P43S hu179VH4 G27Y,R38K, M48I, R66K, V67A, I69L, M80I, S82bR hu179VL5 Grafted (IGKV2-29*02) hu179VH5 G27Y,T28A,M48I,V67A,I69L hu179VL6 Grafted (IGKV2-29*02), D1S hu179VL7 Grafted (IGKV2-29*02) D1S,L73F hu179VL8 Grafted (IGKV2-29*02) D1S,S67Y

Note: For example, P43S indicates that according to the Kabat numbering system, the P at position 43 is back-mutated to S. "Grafted" means that the CDR of the mouse antibody is implanted into the human germline FR region sequence.

The variable regions of the humanized antibody mab179 are as follows:

>hu179VL1 (Grafted (IGKV4-1*01))

Note: Single underlines denote CDR regions and double underlines denote back mutation sites.

The above-mentioned variable regions of the light and heavy chain were combined with the sequences of the constant regions of the light and heavy chain of the human germline to form the final complete sequences of the light and heavy chains, thereby obtaining an antibody with a complete sequence. For example, for the humanized antibody mab199 in the present invention, the constant region of the heavy chain is the constant region of IgG1-YTE shown in SEQ ID NO: 133, and the constant region of the light chain is the constant region of the kappa chain shown in SEQ ID NO: 134, but they can also be replaced by other constant regions known in the art.

Table 22. Combinations of variable regions of the heavy and light chains of the humanized antibody mab179

Antibody VH (SEQ ID NO) VL(SEQ ID NO) hu179-01 85 77 hu179-02 85 78 hu179-03 86 77 hu179-04 86 78 hu179-05 87 77 hu179-06 87 78 hu179-07 87 79 hu179-08 87 81 hu179-09 87 82 hu179-10 87 83 hu179-11 87 84 hu179-12 88 77 hu179-13 88 78 hu179-14 89 79 hu179-15 89 80 hu179-16 89 81 hu179-17 89 82 hu179-18 89 83 hu179-19 89 84

The affinity of humanized mab179 antibody to human TSLP was determined by ELISA, and the results showed that humanized mab179 antibody has very good affinity for human TSLP.

(2) Mutations of the hu179 antibody

By determination, the presence of hot spots on the HCDR2 and LCDR2 sequences of the humanized antibody mab179 was found. Therefore, the corresponding hot spots were mutated to eliminate the risk of modifying the molecule.

In one embodiment, the GNG HCDR2 of hu179VH1 has been amino acid mutated and the sequences of hu179VH1 after the introduction of the mutations are as follows:

hu179VH1- N55Q

SEQ ID NO:90

Note: Single underlines denote CDR regions and double underlines denote back mutation sites.

The sequences of the HCDR2 regions of the humanized antibody mab179 obtained after the introduction of mutations are as follows:

Table 23. HCDR2 mutants of the humanized antibody mab179

hu179HCDR2-N55Q VIDPGQGDTNYNENFKG
SEQ ID NO:93 hu179HCDR2-N55V VIDPGVGDTNYNENFKG
SEQ ID NO:94 hu179HCDR2-G56V VIDPGNVDTNYNENFKG
SEQ ID NO:95

Note: The positions of mutation sites in Table 24 are numbered according to natural order.

The CDR regions of the humanized mab179 antibody can be derived from the above and are as follows:

Table 24. CDR of humanized antibody mab179 after introduction of mutations

HCDR1 NYLIE
SEQ ID NO:26 LCDR1 KASQSVSSDVT
SEQ ID NO:29 HCDR2 (general formula) VIDPGX ₇ X ₈ DTNYNENFKG
SEQ ID NO:96 LCDR2 YVSNHYT
SEQ ID NO:30 HCDR3 EDNTGTAFDY
SEQ ID NO:28 LCDR3 QQHHRFPLT
SEQ ID NO:31

Where X ₇ is selected from N, Q or V, X ₈ is selected from G or V.

The hu179VH1 mutants obtained after introducing the mutations were combined with humanized hu179VL to produce new humanized mab179 antibodies. Examples of antibodies obtained from the combination of the hu179VH1 mutant and hu179VL2 are as follows:

Table 25. Combinations of antibody variable regions after the introduction of mutations

Variable region hu179VH1-N55Q hu179VH1-N55V hu179VH1-G56V hu179VL2 hu179-20 hu179-21 hu179-22

The affinity of the antibody obtained after introducing mutations to human TSLP was determined by ELISA. The results showed that the antibodies after introducing mutations in HCDR2 retained a relatively high affinity to human TSLP. They showed that the point mutations N55Q, N55V and G56V in HCDR2 of the humanized antibody mab179 will basically not affect the affinity of the antibody to TSLP.

In the same manner, point mutations N55Q, N55V and G56V (numbered in natural order) were made in hu179VH2, hu179VH3, hu179VH4 and hu179VH5, respectively, and the heavy and light chain variable regions obtained by introducing these mutations were recombined to produce new humanized antibodies mab179. As an example, the mutated sequence of hu179VH3 is as follows:

Note: Single underlines denote CDR regions and double underlines denote back mutation sites.

In some other examples, the LCDR2 humanized antibody mab179 was subjected to the introduction of amino acid mutations. As an example, the sequences of hu179VL2 obtained after the introduction of mutations are as follows:

Note: Single underlines denote CDR regions and double underlines denote back mutation sites.

The LCDR2 sequences of the humanized antibody mab179 obtained after introducing mutations are as follows:

Table 26. LCDR2 mutants of the humanized antibody mab179

Mutant Subsequence hu179 LCDR2-Y50E EVSNHIT
SEQ ID NO: 108 hu179 LCDR2-S52D YVDNHYT
SEQ ID NO: 109 hu179 LCDR2-S52E YVENHYT
SEQ ID NO: 110 hu179LCDR2-N53Q YVSQHYT
SEQ ID NO: 111 hu179 LCDR2-N53D YVSDHYT
SEQ ID NO: 112 hu179 LCDR2-N53E YVSEHYT
SEQ ID NO: 113 hu179 LCDR2-H54Y YVSNYYT
SEQ ID NO: 114 hu179 LCDR2-H54D YVSNDYT
SEQ ID NO: 115 hu179 LCDR2-H54E YVSNEYT
SEQ ID NO: 116 hu179 LCDR2-Y55E YVSNHET
SEQ ID NO: 117

From the above, it is seen that the general formula of LCDR2 of the humanized antibody mab179 is: X ₉ VX ₁₀ X ₁₁ X ₁₂ X ₁₃ T (SEQ ID NO: 118), where X ₉ is selected from Y or E, X ₁₀ is selected from S, D or E, X ₁₁ is selected from N, Q, D or E; X ₁₂ is selected from H, Y, D or E; X ₁₃ is selected from E or Y. The CDR regions of the humanized antibody mab179 are shown in the following table:

Table 27. CDR of the humanized antibody mab179

HCDR1 NYLIE
SEQ ID NO:26 LCDR1 KASQSVSSDVT
SEQ ID NO:29 HCDR2 VIDPGX ₇ X ₈ DTNYNENFKG
SEQ ID NO:96 LCDR2 X ₉ VX ₁₀ X ₁₁ X ₁₂ X ₁₃ T
SEQ ID NO: 118 HCDR3 EDNTGTAFDY
SEQ ID NO:28 LCDR3 QQHHRFPLT
SEQ ID NO:31

Where X ₇ is selected from N, Q or V, X ₈ is selected from G or V; X ₉ is selected from Y or E; X ₁₀ is selected from S, D or E; X ₁₁ is selected from N, Q, D or E; X ₁₂ is selected from H, Y, D or E; X ₁₃ is selected from E or Y.

The hu179VL2 mutants obtained after introducing the mutations were combined with the heavy chain variable regions of humanized hu179 to obtain new humanized mab179 antibodies. As an example, the hu179VL2 mutants were combined with hu179VH1, hu179VH3 and CDR, and the combinations of the heavy and light chain variable regions of the resulting humanized mab179 antibodies are as follows:

Table 28. Sequences of the CDR regions of the humanized antibody mab179 after the introduction of mutations in LCDR2

HCDR1 NYLIE
SEQ ID NO:26 LCDR1 KASQSVSSDVT
SEQ ID NO:29 HCDR2 VIDPGNGDTNYNENFKG
SEQ ID NO:27 LCDR2 X ₅ VX ₆ X ₇ X ₈ X ₉ T
SEQ ID NO: 118 HCDR3 EDNTGTAFDY
SEQ ID NO:28 LCDR3 QQHHRFPLT
SEQ ID NO:31

Where X ₅ is selected from Y or E; X ₆ is selected from S, D or E; X ₇ is selected from N, Q, D or E; X ₈ is selected from H, Y, D or E; X ₉ is selected from E or Y.

Table 29. Combinations of variable regions of the heavy and light chains of the humanized antibody mab179 obtained after introducing mutations into LCDR2

Antibody VH (SEQ ID NO) VL(SEQ ID NO) hu179-23 85 102 hu179-24 85 104 hu179-25 87 98 hu179-26 87 99 hu179-27 87 100 hu179-28 87 101 hu179-29 87 103 hu179-30 87 105 hu179-31 87 106 hu179-32 87 107

The affinity of humanized mab179 antibodies obtained by introducing mutations in LCDR2 to human TSLP was determined by ELISA. The results showed that antibodies obtained by introducing mutations in the mutagenesis hot spots of LCDR2 retained relatively good affinity to human TSLP. They indicated that mutations in the hot spots of LCDR2 would not affect the binding activity of humanized mab179 antibodies.

In the same manner, mutations N53Q, N53D, N53S, H54Y, Y50E, S52D, S52E, N53E, H54D, H54E, Y55E were made in LCDR2 hu179VL3, hu179VL4, hu179VL5, hu179VL6, hu179VL7 and hu179VL8. The variable regions of the light chain and the variable regions of the heavy chain after introducing the mutations were combined to form new humanized antibodies mab. In one embodiment, the sequence of hu179VL8 after introducing the mutations is as follows:

hu179VL8-N53E and hu179VH3-N55V, obtained by introducing mutations, were combined to obtain a new antibody molecule hu179-33, the CDR sequences of which are as follows:

Table 30. CDR regions of the hu179-33 antibody

HCDR1 NYLIE
SEQ ID NO:26 LCDR1 KASQSVSSDVT
SEQ ID NO:29 HCDR2-N55V LCDR2-N53E HCDR3 EDNTGTAFDY
SEQ ID NO:28 LCDR3 QQHHRFPLT
SEQ ID NO:31

The binding activity of the antibodies obtained after introducing the mutations towards human TSLP was determined using Biacore. Illustrative binding activity of the antibodies is as follows:

Table 31. Affinity of hu179-33 for human TSLP

Antibody KD affinity for huTSLP (M) AMG157 8,12E-12 hu179-33 9.03E-13

The results showed that hu179-33 antibody has relatively high specific binding activity to human TSLP. This indicates that the hot spot point mutations in both HCDR2 and LCDR2 will not affect the affinity of humanized mab179 antibody to human TSLP. It can be seen that in the humanized mab179 antibody molecule, the mutations N55Q, N55V, G56V made in HCDR2 and the mutations N53Q, N53D, N53S, H54Y, Y50E, S52D, S52E, N53E, H54D, H54E, Y55E made in LCDR2 will not affect the binding of the antibody to human TSLP, i.e., will not affect the activity of antibodies to TSLP.

5.4 Selection and backmutation of human FR regions for the mab199 antibody

For mab199, IGHV1-46*01 and HJ6*01 were selected as templates for VH, and IGKV1-39*01 and IGKJ4*01 were selected as templates for VL. The CDR regions of the mouse antibody were grafted onto the selected humanized templates, and the FR regions were backmutated to produce light and heavy chain variable regions with different sequences. The backmutations are presented in Table 32.

Table 32. Back mutation design for mab199

hu199VL hu199VH hu199VL1 Grafted hu199VH1 Grafted hu199VL2 I48V hu199VH2 R71V, T73K, V78A hu199VL3 A43S, K45Q, I48V, D70Q hu199VH3 M69L, R71V, T73K, V78A hu199VL4 G66V hu199VH4 M48I, V67A, M69L, R71V, T73K, V78A hu199VL5 I48V, G66V hu199VH5 R38K, M48I, R66K, V67A, M69L, R71V, T73K, V78A hu199VL6 A43S, K45Q, I48V, G66V, D70Q hu199VH6 R38K, R66K, R71V, T73K, V78A hu199VH7 R38K, R67K, M69L, R71V, T73K, V78A

Note: For example, I48V indicates that according to the Kabat numbering system, I at position 48 is backmutated to V. "Grafted" means that the CDR of the mouse antibody is implanted into the human germline FR region sequence.

The variable regions of the humanized antibody mab199 are as follows:

Note: Single underlines denote CDR regions and double underlines denote back mutation sites.

The above-mentioned variable regions of the light and heavy chain were combined with the sequences of the constant regions of the light and heavy chain of the human germline to form the final complete sequences of the light and heavy chains, thereby obtaining an antibody with a complete sequence. For the humanized mab199 antibodies, unless clearly described herein, the constant region of the light chain is the constant region shown in SEQ ID NO: 134, and the constant region of the heavy chain is the constant region shown in SEQ ID NO: 133.

The resulting humanized mab199 antibodies are as follows:

Table 33. Sequences of the variable regions of the heavy and light chains of humanized antibodies mab199

Antibody VH (SEQ ID NO) VL(SEQ ID NO) hu199-01 127 120 hu199-02 127 121 hu199-03 127 122 hu199-04 127 123 hu199-05 127 124 hu199-06 127 125 hu199-07 128 120 hu199-08 128 121 hu199-09 128 122 hu199-10 128 123 hu199-11 128 124 hu199-12 128 125 hu199-13 129 120 hu199-14 129 121 hu199-15 129 122 hu199-16 129 123 hu199-17 129 124 hu199-18 129 125 hu199-19 130 120 hu199-20 130 121 hu199-21 130 122 hu199-22 130 123 hu199-23 130 124 hu199-24 130 125 hu199-25 131 120 hu199-26 131 121 hu199-27 131 122 hu199-28 131 123 hu199-29 131 124 hu199-30 131 125 hu199-31 132 120 hu199-32 132 121 hu199-33 132 122 hu199-34 132 123 hu199-35 132 124 hu199-36 132 125

The activity of humanized mab199 antibodies blocking TSLP binding to TSLP receptor was determined by ELSA, and the results are as follows:

Table 34. Activity of humanized antibody mab199 in blocking TSLP binding to TSLP receptor

Antibody IC ₅₀ (nM) Antibody IC ₅₀ (nM) Antibody IC ₅₀ (nM) Antibody IC ₅₀ (nM) hu199-01 0,1912 hu199-10 0.3910 hu199-191 0.6584 hu199-28 0.4619 hu199-02 0.2193 hu199-11 0.3648 hu199-20 0.4001 hu199-29 0.5543 hu199-03 0.2077 hu199-12 0,3700 hu199-21 0.5353 hu199-30 0.3493 hu199-04 0.4242 hu199-13 0.2395 hu199-22 0.3449 hu199-31 0.3044 hu199-05 0.4726 hu199-14 0.3112 hu199-23 0.3370 hu199-32 0.2870 hu199-06 0.3806 hu199-15 0.2866 hu199-24 0.4960 hu199-33 0.2055 hu199-07 0.2834 hu199-16 0.7367 hu199-25 0.2460 hu199-34 0.7107 hu199-08 0.2828 hu199-17 0.6111 hu199-26 0.3651 hu199-35 0.4849 hu199-09 0.2732 hu199-18 0.4806 hu199-27 0.3544 hu199-36 0.7273 Ch199 0.4266

The results showed that humanized mab199 antibodies retained relatively high activity in blocking TSLP binding to the TSLP receptor.

5.5 Constant regions of antibodies

The constant region of the heavy chain of the humanized antibody and the chimeric antibody can be selected from the group consisting of constant regions of IgG1, IgG2, IgG4 and their variants. For example, the constant region of IgG1-YTE was used in the present invention, and its sequence is presented in SEQ ID NO: 133. The constant region of the light chain can be selected from the constant regions of the human light κ-, λ-chain or their variants. For example, the constant region of the human κ-chain was used in the present invention, and its sequence is presented in SEQ ID NO: 134.

>IgG1 heavy chain constant region-YTE:

Note: Underlining refers to the developed mutations M252Y, S254T, T256E

> constant region of the κ light chain:

The humanized heavy and light chain variable regions of the present invention were recombined with the above-mentioned constant regions to produce full-length heavy and light chain sequences. Examples of antibody sequences are as follows:

heavy chain of antibody hu3-13:

light chain of antibody hu3-13:

heavy chain antibody hu119-30

light chain of antibody hu119-30

heavy chain antibody hu179-33

light chain of antibody hu179-33

heavy chain antibody hu199-36

light chain of antibody hu199-36

Note: The underlined part is the CDR and the italicized part is the constant region.

AMG157 was used as a positive control for the present invention, and its sequence corresponds to that shown in SEQ ID NO: 143 and SEQ ID NO: 144.

AMG157 heavy chain sequence

AMG157 light chain sequence

In addition, when testing the activity of antibodies in the present invention, human TSLP receptor and human IL7Rα were also used to construct cell lines, and their sequences are as follows:

Full-length amino acid sequence of the human TSLP receptor:

Note: The underlined part refers to the signal peptide.

SEQ ID NO: 145

Full-length amino acid sequence of human IL7Rα (Uniprot ID: P16871)

Note: The underlined part refers to the signal peptide.

SEQ ID NO: 146

The antibodies of the present invention can be cloned, expressed and purified using conventional gene cloning and recombinant expression techniques.

Test examples:

In vitro biological activity assessment

Test Case 1: Determination of binding of TSLP antibodies to human TSLP using ELSA

Human TSLP-his (SEQ ID NO: 1) was diluted to 1 μg/mL with PBS buffer pH 7.4 (Shanghai BasalMedia, B320), added at 100 μg/well to 96-well microtiter plates (Corning, CLS3590-100EA), and incubated overnight at 4°C. After removing the liquid, 200 μL/well of blocking solution with 5% skim milk (Bright Dairy skim milk powder) diluted in PBS was added and incubated in an incubator at 37°C for 2 hours for blocking. After blocking was completed, the blocking solution was removed and the plates were washed 3 times with PBST buffer (PBS containing 0.1% Tween 20, pH 7.4). Test antibodies and positive control antibody AMG157 at various concentrations diluted with sample diluent were added at 100 µl/well and incubated in an incubator at 37°C for 1 hour. After incubation, the plates were washed 3 times with PBST. HRP (horseradish peroxidase)-labeled secondary goat anti-mouse antibody (Jackson Immuno Research, 115-035-003) diluted with sample diluent was added at 100 µl/well and incubated at 37°C for 1 hour. After washing the plate 6 times with PBST, 50 μl/well of TMB chromogenic substrate (KPL, 52-00-03) was added and incubated at room temperature for 10-15 min, followed by the addition of 50 μl/well of _{1 MH2SO4} to stop the reaction. The absorbance was read using a NOVOStar microplate reader at 450 nm. The EC50 value of binding of anti-TSLP antibodies to TSLP was calculated and the results are shown in the following table.

Table 35. Results on the binding activity of antibodies to human TSLP

Antibody EC ₅₀ (nM) Antibody EC ₅₀ (nM) Antibody EC ₅₀ (nM) Ch3 0.4929 hu119-14 0.345 hu179-09 0,1573 hu3-01 0.8494 hu119-15 0.3497 hu179-10 0.19 hu3-02 0.6285 hu119-16 0.366 hu179-11 0.1369 hu3-03 0.5545 hu119-17 0.3515 hu179-12 0.1437 hu3-04 0.4353 hu119-18 0.3455 hu179-13 0,2011 hu3-05 0.5168 hu119-19 0.3533 hu179-14 0.2053 hu3-06 0.594 hu119-20 0.3412 hu179-15 0.2035 hu3-07 0.3853 hu119-21 0.3987 hu179-16 0.2287 hu3-08 0.4687 hu119-22 0.351 hu179-17 0.218 hu3-09 0.4941 hu119-23 0.3404 hu179-18 0.2458 hu3-10 0.3879 hu119-24 0.3446 hu179-19 0.1616 hu3-12 0.1519 hu119-25 0.3575 hu179-20 0.7077 hu3-13 0.1477 hu119-26 0.3782 hu179-21 0.9784 Ch119 0.851 hu119-27 0.3347 hu179-22 0.7519 hu119-01 0.107 hu119-28 0.2648 hu179-23 0.997 hu119-02 0,1938 hu119-29 0.2729 hu179-24 0.6358 hu119-03 0.1593 hu119-28 0.2648 hu179-25 0,1313 hu119-04 0,1881 hu119-29 0.2729 hu179-26 0,2006 hu119-05 0.1445 Ch179 0,2023 hu179-27 0.1799 hu119-06 0.2206 hu179-01 0.1248 hu179-28 0,0906 hu119-07 0.2132 hu179-02 0.1697 hu179-29 0.2041 hu119-08 0,2015 hu179-03 0.138 hu179-30 0.246 hu119-09 0.1492 hu179-04 0,1886 hu179-31 0,2012 hu119-10 0.2329 hu179-05 0.1416 hu179-32 0.145 hu119-11 0.174 hu179-06 0.2188 Ch199 0.5157 hu119-12 0.2034 hu179-07 0.4478 AMG157 0.7219 hu119-13 0.3438 hu179-08 0.1615

The results showed that the antibodies of the present invention have very good binding activity to human TSLP.

Test Case 2: Determination of the Affinity of Humanized TSLP Antibodies to TSLP from Different Species Using Biacore Technology

The affinity of humanized antibodies to TSLP to be tested with human and cynomolgus monkey TSLP was determined using a Biacore T200 (GE) instrument.

The test molecules were affinity captured onto protein A biosensor chips (Cat. No. 29127556, GE). Then, the antigens (huTSLP-his, cynoTSLP-his, prepared in Example 1) were allowed to flow over the chip surface and the reaction signal was determined in real time using a Biacore T200 instrument to obtain binding and dissociation curves. After the dissociation was completed in each experimental cycle, the biosensor chips were washed and regenerated with glycine-hydrochloric acid regeneration solution (pH 1.5, Cat. No. BR-1003-54, GE). The data were fitted with the Langmuir model (1:1) using BIAevaluation version 4.1 software, GE to obtain the affinity value presented in the following tables.

Table 36. Affinity of TSLP antibodies to TSLP from different species

Antibody KD affinities
to huTSLP
(M) KD affinities
to TSLP of the cynomolgus macaque
(M) AMG157 8,12E-12 9.22E-12 hu179-33 9.03E-13 3.04E-11 hu3-13 1,0E-12 3.40E-10 hu119-30 5.0E-12 1,95E-09 hu199-36 10,5E-12 1.72E-11

The results showed that the anti-TSLP antibodies of the present invention have a relatively high affinity for human TSLP and can also bind to cynomolgus macaque TSLP.

Test Case 3: ELISA-based experiment to investigate the blocking of TSLP binding to TSLP receptor by TSLP antibodies

The TSLP receptor has two subunits, TSLPR and IL7R, of which TSLPR is a specific receptor for TSLP and IL7R is a common receptor for TSLP and IL7. TSLP first binds to TSLPR and then to IL7R. This test case was used to determine whether TSLP antibodies could block TSLP binding to the extracellular domain of recombinantly expressed TSLPR receptor protein.

ELISA plates were coated with human TSLPR-Fc-ECD (2 μg/ml, SEQ ID NO: 5) and incubated overnight at 4°C. After removing the liquid, 200 μl/well of blocking solution with 5% skim milk diluted in PBS were added and incubated in an incubator at 37°C for 2 hours for blocking. After blocking was completed, the blocking solution was removed and the plates were washed 3 times with PBST buffer (PBS containing 0.05% Tween 20, pH 7.4). Biotin-labeled huTSLP-Fc antigen was prepared at a concentration of 3 nM and serial dilutions of the tested antibodies starting from 200 nM. Antigen and antibody were mixed 1:1 and then placed at 37°C for 15 min, 100 μl/well were added to microtiter plates and placed at 37°C for 1 h. The plates were washed 3 times with PBST. Streptavidin peroxidase polymer diluted to 1:4000 with sample diluent was added at 100 μl/well and incubated at 37°C for 1 h. After washing the plates 5 times with PBST, 100 μl/well of TMB chromogenic substrate (KPL, 52-00-03) were added and incubated at room temperature for 3-10 min, after which 100 μl/well of 1 _{MH2SO4 was} added to stop the reaction. The absorbance was read using a NOVOStar microplate reader at 450 nm. The IC ₅₀ value of blocking TSLP binding to TSLPR by anti-TSLP antibodies was calculated and the results are presented in Table 37 and Fig. 1.

Table 37. Results regarding the blocking activity of antibodies

Antibody hu179-33 hu119-30 hu3-13 hu199-36 IC ₅₀ (nM) 0.5038 0.5192 0.4975 0.5693

The results showed that all the antibodies of the present invention were capable of inhibiting the binding of TSLP to its receptor TSLPR at a high level.

Test Case 5: FACS-based experiment to investigate the blockade of TSLP binding to TSLP receptor by TSLP antibody

This test case was used to determine whether TSLP antibodies could adequately block TSLP binding to the TSLPR/IL7R receptors on the surface of the CHOK1 cell line.

Specifically, the method was as follows: CHOK1-TSLPR/IL7R were cultured with DME/F12 containing 10% FBS, 1 mg/ml G418, and 10 μg/ml puromycin. The CHOK1-TSLPR/IL7R cells in good condition were centrifuged (1000 rpm, 5 min), washed once with 2% FBS in PBS. The cells were counted and adjusted to a cell concentration of 1 × 10 ⁶ /ml. 50 μl of cells were added to 96-well round-bottom plates. The test antibodies were diluted with PBS containing 2% BSA at an initial concentration of 20 nM and 8 gradients at a ratio of 1:4. Biotin-labeled TSLP-Fc antigen was prepared at a concentration of 2 nM. Antigen and antibody were mixed at a ratio of 1:1 and placed at 37°C for 15 min. The mixture was added at 50 μl per well to 96-well plates in which the cells were seeded and incubated at 4°C for 1 hour. After incubation, the plates were centrifuged at 4°C (800 g, 5 min) and the supernatant was removed. The plates were washed twice with 200 μl of pre-chilled PBS by centrifugation. Secondary antibody PE-SA diluted 1:1000 was added and incubated at 4°C in the dark for 40 minutes. Then the plates were centrifuged at 4°C (800 g, 5 min) and the supernatant was removed. 200 μl of pre-chilled PBS were added to expand the cells and they were washed three times by centrifugation at 4°C. 100 μl of PBS was added and the plate was loaded into a plate reader. The _IC50 value of blocking TSLP binding to TSLPR/IL7R by anti-TSLP antibodies was calculated according to the fluorescence signals. The results are shown in Table 38.

Table 38. Results of TSLPR blocking with antibodies on the cell surface

Antibody AMG157 hu179-33 hu119-30 hu3-13 hu199-36 IC ₅₀ (nM) 0.2068 0,1867 0,1368 0.1325 0.2270

The results showed that all the antibodies of the present invention were able to inhibit the binding of TSLP to TSLPR/IL7R on the cell surface at a relatively high level.

Test Case 6: Anti-TSLP antibodies inhibited TSLP-induced chemokine production

TSLP can induce the maturation of naïve myeloid dendritic cells (mDCs) and the secretion of thymus-associated regulatory chemokine (TARC) and osteoprotegerin (OPG), thereby further mediating the innate and adaptive immune inflammatory response. This test case was used to verify that the resulting antibodies are able to block TSLP-induced mDC chemokine production, thereby blocking the occurrence of the innate and adaptive inflammatory response.

Naïve myeloid mDCs were isolated and purified from human peripheral blood mononuclear cells (PBMCs) using magnetic bead sorting method (CD1c (BDCA-1) + Dendritic Cell Isolation Kit, Miltenyi Biotec). The obtained mDCs were seeded in 96-well cell culture plates. Serially diluted samples of antibodies and human TSLP (huTSLP-his, final concentration 50 ng/mL) were pre-incubated for approximately 45 min (37°C) and then respectively added to each cell culture well containing mDCs to stimulate mDCs in vitro. The plates were placed in a culture incubator for 48 h. The cell culture supernatant was collected and diluted appropriately, and then its chemokine content was determined by ELISA. TARC was determined using the human CCL17/TARC Quantikine ELISA kit from R&D Company; OPG content was determined using the human CCL22/MDC Quantikine ELISA kit (R&D), and the results are shown in Fig. 4A-Fig. 4B.

The results showed that all the antibodies of the present invention were able to significantly inhibit TSLP-induced production of TARC and OPG chemokines, indicating that the antibodies of the present invention were able to block the occurrence of innate and adaptive inflammatory response.

Test Case 7: Anti-TSLP antibodies blocked native TSLP-induced BaF3-TLSPR/IL7R cell proliferation

BaF3-hTSLPR/hIL7R cells can proliferate when stimulated with native TSLP. Binding of antibodies to native TSLP reduces the stimulatory effect of TSLP on BaF3-hTSLPR/hIL7R cells.

NHLF cells (BeNa Culture Collection, BNCC340764) and HLF1 cells (BeNa Culture Collection, BNCC337730) were cultured until the cells grew to 80% and the supernatant was removed. Human lung fibroblasts, NHLF (BeNa Culture Collection, BNCC340764) and HLF1 (BeNa Culture Collection, BNCC337730) were stimulated with 10 ng/ml human IL1-β (Sino Biological GMP-10139-HNAE), 20 ng/ml IL13 (R&D 213-ILB-005), 20 ng/ml TNF-α (PEPROTECH 300-01A) for 72 hours to induce native TSLP production. After stimulation, the cell supernatant was collected and centrifuged at 4500 rpm for 5 min to remove cellular debris. The supernatant was collected, concentrated approximately 10-fold on concentration columns, and filtered for further use.

BaF3-hTSLPR/hIL17R cells were cultured in RPMI1640 with 10% FBS (10 ng/ml mIL3, R&D 213-ILB-005), adjusted to a density of 1×10 ⁴ cells/ml and cultured in an incubator at 37°C, 5% CO ₂ until logarithmic growth phase. The cells were harvested, centrifuged at 800 rpm for 5 min and the supernatant was removed; the cells were washed three times with PBS to remove cytokines that stimulate their proliferation from the culture medium. The cells were resuspended in RPMI1640 with 4% FBS, seeded in 96-well plates at 4000 cells/50 μl/well and cultured in an incubator for 2 h. The antibodies to be tested were serially diluted 10-fold using native TSLP with an initial antibody concentration of 100 nM, resulting in 3 dilution gradients: 100 nM, 10 nM, and 1 nM. 50 μl/well of the diluted antibody/antigen mixture with a final antibody concentration of 50 nM, 5 nM, 0.5 nM were added to the cells. The plates were incubated in a 5% _CO2 incubator at 37°C for 72 h. Then 30 μl of CellTiter-Glo (Promega) were added to each well and incubated in the dark at room temperature for 10 min, and detection was performed using the luminescence program on the Cytation5 cell imager. The results are presented in the following table.

Table 39. Results of inhibition of BaF3-TLSPR/IL7R cell proliferation by TSLP antibodies

Antibody AMG157 hu179-33 hu3-13 hu119-30 IC ₅₀ (nM) 3,379 0.02279 0.2888 1,533

The results showed that all the antibodies obtained in the present invention were able to significantly inhibit the activity of native TSLP in stimulating BaF3 proliferation, especially hu179-33, whose activity was more than 100 times higher than that of AMG157.

Test Case 8: Experiment on inhibition of TSLP-induced proliferation of TSLPR/IL7R-overexpressing BaF3 cells by TSLP antibodies

TSLP can bind to TSLPR/IL7R on the surface of BaF3, thereby promoting the proliferation of BaF3. This test example was used to determine whether the antibodies of the present invention can block the activity of TSLP in inducing the proliferation of BaF3.

Specifically, BaF3 cells overexpressing TSLPR/IL7R were cultured in RPMI1640 with 10% FBS and 2 ng/mL rhIL3 (MultiSciences, Cat. No. 96-AF-300-03-20) and cultured in an incubator at 37°C, 5% _CO2 at a cell density of no more than 1× ¹⁰⁶ cells/mL. Upon antibody detection, the cells in logarithmic growth phase were washed three times with PBS and centrifuged at 800 rpm for 5 min. The cell density was adjusted to 8000 cells/well/90 μL with RPMI1640 (2% FBS, recombinant human TSLP-Fc: 40 ng/mL). 10 μl of serially diluted antibodies for testing were added to 96-well plates and cultured for 2 days. 30 μl of cell titer was added and mixed for detection. IC ₅₀ was calculated according to the readings. The results are shown in Table 40 and Fig. 3.

Table 40. Inhibition of proliferative activity of BaF3 cells by antibodies

Antibody AMG157 hu179-33 hu119-30 hu3-13 hu199-36 IC ₅₀ (nM) 0.5730 0.4092 0.4305 0.4436 0.4769

The results showed that all the antibodies of the present invention had a relatively strong ability to inhibit TSLP-mediated proliferation of BaF3 cells.

Test Case 9: Humanized TSLP antibodies blocked TSLP-induced differentiation of naive CD4 ⁺ T cells into Th2 cells

TSLP can induce maturation of primary myeloid mDC cells. Mature mDC cells highly express OX40 ligand, which can bind to OX40 on the surface of naive CD4 ⁺ T cells, thereby differentiating naive CD4 ⁺ T cells into Th2 cells that produce immune response-related factors such as IL4/IL5/IL13, etc., leading to a Th2-type inflammatory response in the body. This test example was used to determine whether the antibodies produced in the present invention could block TSLP-induced Th2 cell differentiation.

Naïve myeloid mDCs were isolated and purified from human peripheral blood mononuclear cells (PBMCs) using magnetic bead sorting method (CD1c (BDCA-1) + Dendritic Cell Isolation Kit, Miltenyi Biotec). The obtained mDCs were seeded in 96-well cell culture plates. Serially diluted samples of antibodies and recombinantly expressed human TSLP (huTSLP-his, final concentration 50 ng/ml) were pre-incubated (37°C) for approximately 45 min and then respectively added to each cell culture well containing mDCs and cultured at 37°C for 24 h. Mature mDCs after stimulation were collected and washed twice with PBS. CD4 ⁺ CD45RA ⁺ naive T cells were extracted from PBMCs by magnetic bead separation method (Myltenyi, Biotec). Naive T cells obtained by separation and mature mDCs were mixed and seeded in 96-well cell culture plates at a ratio of 5:1 and co-cultured for 6 days. The cells were harvested and seeded in 96-well plates pre-coated with anti-CD3 antibody (10 μg/ml) and anti-CD28 antibody (1 μg/ml) was added to restimulate differentiated T cells. The cells were cultured for 24 h and finally the cell culture supernatant was collected. Th2-related cytokines secreted by the cells in the supernatant were detected by ELISA. Cytokines IL-4 and IL-5 were detected by ELISA kits from R&D, and TNF-α and IL-13 were detected by ELISA kits from NeoBioscience. The results are shown in Fig. 5A-Fig. 5D.

The results showed that the antibodies of the present invention were able to significantly inhibit the production of Th2 cytokines IL4, IL5, IL13 and TNF-α, indicating that the antibodies of the present invention were able to block the differentiation of Th2 cells induced by TSLP.

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SEQUENCE LIST

<110> JIANGSU HANGRUI MEDICAL CO., LTD.

SHANGHAI HANGRUI PHARMACEUTICAL CO., LTD.

<120> ANTIBODY CAPABLE OF BINDING TO THYMIC STROMAL

LYMPHOPOIETIN AND ITS USE

<130> 702048CPCT

<160> 146

<170> SIPOSequenceListing 1.0

<210> 1

<211> 176

<212> PROTEIN

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> Amino acid sequence of his-tagged

human TSLP antigen (huTSLP-his)

<400> 1

Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys

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Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr Asp Phe Thr

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Asn Cys Asp Phe Glu Lys Ile Lys Ala Ala Tyr Leu Ser Thr Ile Ser

35 40 45

Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser Thr Glu Phe Asn

50 55 60

Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu Thr Glu Ile Gln

65 70 75 80

Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser Leu Ala Lys Glu

85 90 95

Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly

100 105 110

Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Ala Arg

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Lys Ser Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu

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Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln Gly

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Ser Ser Asp Tyr Lys Asp Asp Asp Asp Lys His His His His His

165 170 175

<210> 2

<211> 398

<212> PROTEIN

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> Amino acid sequence of Fc-tagged

human TSLP antigen (huTSLP-Fc)

<400> 2

Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys

1 5 10 15

Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr Asp Phe Thr

20 25 30

Asn Cys Asp Phe Glu Lys Ile Lys Ala Ala Tyr Leu Ser Thr Ile Ser

35 40 45

Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser Thr Glu Phe Asn

50 55 60

Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu Thr Glu Ile Gln

65 70 75 80

Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser Leu Ala Lys Glu

85 90 95

Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly

100 105 110

Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Ala Arg

115 120 125

Lys Ser Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu

130 135 140

Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln Asp

145 150 155 160

Ile Glu Gly Arg Met Asp Glu Pro Lys Ser Ser Asp Lys Thr His Thr

165 170 175

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

180 185 190

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

195 200 205

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

210 215 220

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

225 230 235 240

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

245 250 255

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

260 265 270

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

275 280 285

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

290 295 300

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

305 310 315 320

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

325 330 335

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

340 345 350

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

355 360 365

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

370 375 380

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

385 390 395

<210> 3

<211> 168

<212> PROTEIN

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> Amino acid sequence of his-tagged TSLP

cynomolgus macaque (cynoTSLP-his)

<400> 3

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

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Gly Ser Thr Gly Tyr Asp Phe Thr Asn Cys Asp Phe Gln Lys Ile Glu

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Ala Asp Tyr Leu Arg Thr Ile Ser Lys Asp Leu Ile Thr Tyr Met Ser

35 40 45

Gly Thr Lys Ser Thr Asp Phe Asn Asn Thr Val Ser Cys Ser Asn Arg

50 55 60

Pro His Cys Leu Thr Glu Ile Gln Ser Leu Thr Phe Asn Pro Thr Pro

65 70 75 80

Arg Cys Ala Ser Leu Ala Lys Glu Met Phe Ala Arg Lys Thr Lys Ala

85 90 95

Thr Leu Ala Leu Trp Cys Pro Gly Tyr Ser Glu Thr Gln Ile Asn Ala

100 105 110

Thr Gln Ala Met Lys Lys Ala Arg Lys Ser Lys Val Thr Thr Asn Lys

115 120 125

Cys Leu Glu Gln Val Ser Gln Leu Leu Gly Leu Trp Arg Arg Phe Ile

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Arg Thr Leu Leu Lys Gln Gln Gly Ser Ser Asp Tyr Lys Asp Asp Asp

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Asp Lys His His His His His

165

<210> 4

<211> 390

<212> PROTEIN

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> Amino acid sequence of Fc-tagged TSLP

cynomolgus macaque (cyno TSLP-Fc)

<400> 4

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Tyr Asp Phe Thr Asn Cys Asp Phe Gln Lys Ile Glu

20 25 30

Ala Asp Tyr Leu Arg Thr Ile Ser Lys Asp Leu Ile Thr Tyr Met Ser

35 40 45

Gly Thr Lys Ser Thr Asp Phe Asn Asn Thr Val Ser Cys Ser Asn Arg

50 55 60

Pro His Cys Leu Thr Glu Ile Gln Ser Leu Thr Phe Asn Pro Thr Pro

65 70 75 80

Arg Cys Ala Ser Leu Ala Lys Glu Met Phe Ala Arg Lys Thr Lys Ala

85 90 95

Thr Leu Ala Leu Trp Cys Pro Gly Tyr Ser Glu Thr Gln Ile Asn Ala

100 105 110

Thr Gln Ala Met Lys Lys Ala Arg Lys Ser Lys Val Thr Thr Asn Lys

115 120 125

Cys Leu Glu Gln Val Ser Gln Leu Leu Gly Leu Trp Arg Arg Phe Ile

130 135 140

Arg Thr Leu Leu Lys Gln Gln Asp Ile Glu Gly Arg Met Asp Glu Pro

145 150 155 160

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

165 170 175

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

180 185 190

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

195 200 205

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

210 215 220

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

225 230 235 240

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

245 250 255

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

260 265 270

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

275 280 285

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

290 295 300

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

305 310 315 320

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

325 330 335

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

340 345 350

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

355 360 365

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

370 375 380

Ser Leu Ser Pro Gly Lys

385 390

<210> 5

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> Amino acid sequence of the extracellular domain

Fc-tagged human TSLP receptor (human-TSLPR-Fc-ECD)

<400> 5

Gly Ala Ala Glu Gly Val Gln Ile Gln Ile Ile Tyr Phe Asn Leu Glu

1 5 10 15

Thr Val Gln Val Thr Trp Asn Ala Ser Lys Tyr Ser Arg Thr Asn Leu

20 25 30

Thr Phe His Tyr Arg Phe Asn Gly Asp Glu Ala Tyr Asp Gln Cys Thr

35 40 45

Asn Tyr Leu Leu Gln Glu Gly His Thr Ser Gly Cys Leu Leu Asp Ala

50 55 60

Glu Gln Arg Asp Asp Ile Leu Tyr Phe Ser Ile Arg Asn Gly Thr His

65 70 75 80

Pro Val Phe Thr Ala Ser Arg Trp Met Val Tyr Tyr Leu Lys Pro Ser

85 90 95

Ser Pro Lys His Val Arg Phe Ser Trp His Gln Asp Ala Val Thr Val

100 105 110

Thr Cys Ser Asp Leu Ser Tyr Gly Asp Leu Leu Tyr Glu Val Gln Tyr

115 120 125

Arg Ser Pro Phe Asp Thr Glu Trp Gln Ser Lys Gln Glu Asn Thr Cys

130 135 140

Asn Val Thr Ile Glu Gly Leu Asp Ala Glu Lys Cys Tyr Ser Phe Trp

145 150 155 160

Val Arg Val Lys Ala Met Glu Asp Val Tyr Gly Pro Asp Thr Tyr Pro

165 170 175

Ser Asp Trp Ser Glu Val Thr Cys Trp Gln Arg Gly Glu Ile Arg Asp

180 185 190

Ala Cys Ala Glu Thr Pro Thr Pro Pro Lys Pro Lys Leu Ser Lys Asp

195 200 205

Ile Glu Gly Arg Met Asp Glu Pro Lys Ser Ser Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 6

<211> 121

<212> PROTEIN

<213> Mus musculus

<220>

<221> PEPTIDE

<223> Mouse heavy variable region sequence

mab3 chains

<400> 6

Glu Val Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp

20 25 30

Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Ile Ile Ser Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Tyr Asp Tyr Asp Gly Tyr Ala Met Asp His Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 7

<211> 105

<212> PROTEIN

<213> Mus musculus

<220>

<221> PEPTIDE

<223> Mouse mab3 variable region sequence

light chain

<400> 7

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Arg Thr Phe

85 90 95

Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 8

<211> 120

<212> PROTEIN

<213> Mus musculus

<220>

<221> PEPTIDE

<223> Mouse mab119 heavy chain variable region sequence

<400> 8

Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Phe Ala Phe Thr Thr Tyr

20 25 30

Asn Met His Trp Val Lys His Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Arg Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val Trp Gly Ala

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 9

<211> 111

<212> PROTEIN

<213> Mus musculus

<220>

<221> PEPTIDE

<223> Mouse mab119 light chain variable region sequence

<400> 9

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Leu Tyr Arg Ala Ser Asn Leu Gly Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Leu Asn

65 70 75 80

Pro Val Glu Thr Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 10

<211> 119

<212> PROTEIN

<213> Mus musculus

<220>

<221> PEPTIDE

<223> Mouse mab179 heavy chain variable region sequence

<400> 10

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Gly Asn Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Ile Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 11

<211> 107

<212> PROTEIN

<213> Mus musculus

<220>

<221> PEPTIDE

<223> Mouse mab179 light chain variable region sequence

<400> 11

Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn His Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 12

<211> 117

<212> PROTEIN

<213> Mus musculus

<220>

<221> PEPTIDE

<223> Mouse mab199 heavy chain variable region sequence

<400> 12

Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Ile Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 13

<211> 107

<212> PROTEIN

<213> Mus musculus

<220>

<221> PEPTIDE

<223> Mouse mab199 light chain variable region sequence

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Val Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 14

<211> 5

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223>HCDR1 mab3

<400> 14

Asp Asp Tyr Met Asn

1 5

<210> 15

<211> 17

<212> PROTEIN

<213> Mus musculus

<220>

<221> PEPTIDE

<223>HCDR2 mab3

<400> 15

Ile Ile Ser Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 16

<211> 12

<212> PROTEIN

<213> Mus musculus

<220>

<221> PEPTIDE

<223>HCDR3 mab

<400> 16

Glu Asp Tyr Asp Tyr Asp Gly Tyr Ala Met Asp His

1 5 10

<210> 17

<211> 10

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR1 mab3

<400> 17

Arg Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210> 18

<211> 7

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR2 mab

<400> 18

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 19

<211> 8

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR3 mab3

<400> 19

Gln Gln Trp Ser Ser Asn Arg Thr

1 5

<210> 20

<211> 5

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223>HCDR1 mab119

<400> 20

Thr Tyr Asn Met His

1 5

<210> 21

<211> 17

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223>HCDR2 mab119

<400> 21

Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 22

<211> 11

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223>HCDR3 mab119

<400> 22

Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val

1 5 10

<210> 23

<211> 15

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR1 mab119

<400> 23

Arg Ala Ser Glu Ser Val Asp Asn Ser Gly Leu Ser Phe Met His

1 5 10 15

<210> 24

<211> 7

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR2 mab119

<400> 24

Arg Ala Ser Asn Leu Gly Ser

1 5

<210> 25

<211> 9

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR3 mab119

<400> 25

Gln Gln Ile Asn Thr Asp Pro Leu Thr

1 5

<210> 26

<211> 5

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223>HCDR1 mab179

<400> 26

Asn Tyr Leu Ile Glu

1 5

<210> 27

<211> 17

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223>HCDR2 mab179

<400> 27

Val Ile Asp Pro Gly Asn Gly Asp Thr Asn Tyr Asn Glu Asn Phe Lys

1 5 10 15

Gly

<210> 28

<211> 10

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223>HCDR3 mab179

<400> 28

Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr

1 5 10

<210> 29

<211> 11

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR1 mab179

<400> 29

Lys Ala Ser Gln Ser Val Ser Ser Asp Val Thr

1 5 10

<210> 30

<211> 7

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR2 mab179

<400> 30

Tyr Val Ser Asn His Tyr Thr

1 5

<210> 31

<211> 9

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR3 mab179

<400> 31

Gln Gln His His Arg Phe Pro Leu Thr

1 5

<210> 32

<211> 5

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223>HCDR1 mab199

<400> 32

Thr Tyr Trp Met His

1 5

<210> 33

<211> 17

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223>HCDR2 mab199

<400> 33

Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Ile Gln Lys Phe Lys

1 5 10 15

Asp

<210> 34

<211> 8

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223>HCDR3 mab199

<400> 34

Thr Leu Asp Gly Tyr Tyr Asp Tyr

1 5

<210> 35

<211> 11

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR1 mab199

<400> 35

Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala

1 5 10

<210> 36

<211> 7

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR2 mab199

<400> 36

Phe Ala Lys Thr Leu Ala Glu

1 5

<210> 37

<211> 9

<212> PROTEIN

<213> Mus musculus

<220>

<221> DOMAIN

<223> LCDR3 mab199

<400> 37

Gln His His Tyr Gly Thr Pro Trp Thr

1 5

<210> 38

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> VL hu3 with grafted CDRs

<400> 38

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Arg Thr Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 39

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3VL2

<400> 39

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Arg Thr Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 40

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3VL3

<400> 40

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Arg Thr Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 41

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3VL4

<400> 41

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Arg Thr Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 42

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3VH1

<400> 42

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Ile Ile Ser Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Tyr Asp Tyr Asp Gly Tyr Ala Met Asp His Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 43

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3VH2

<400> 43

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Ile Ile Ser Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Tyr Asp Tyr Asp Gly Tyr Ala Met Asp His Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 44

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3VH3

<400> 44

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp

20 25 30

Tyr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Ile Ile Ser Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Tyr Asp Tyr Asp Gly Tyr Ala Met Asp His Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 45

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3 HCDR3-H110Y

<400> 45

Glu Asp Tyr Asp Tyr Asp Gly Tyr Ala Met Asp Tyr

1 5 10

<210> 46

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3LCDR3-N93D

<400> 46

Gln Gln Trp Ser Ser Asp Arg Thr

1 5

<210> 47

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR3 hu3 (general formula)

<220>

<221> DOMAIN

<222> (12)..(12)

<223> Xaa is chosen from His or Tyr.

<400> 47

Glu Asp Tyr Asp Tyr Asp Gly Tyr Ala Met Asp Xaa

1 5 10

<210> 48

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR hu3 (general formula 1)

<220>

<221> DOMAIN

<222> (6)..(6)

<223> Xaa is selected from Asn or Asp.

<400> 48

Gln Gln Trp Ser Ser Xaa Arg Thr

1 5

<210> 49

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3-11 light chain variable region sequence

<400> 49

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asp Arg Thr Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 50

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence of the variable region of the heavy chain of hu3-11

<400> 50

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Ile Ile Ser Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Tyr Asp Tyr Asp Gly Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 51

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3VL5

<400> 51

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asp Asn Val Arg Gly Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 52

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu3VL6

<400> 52

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asp Ser Gly Arg Glu Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 53

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3-V1 hu3

<400> 53

Gln Gln Ser Asp Asn Val Arg Gly

1 5

<210> 54

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3-V2 hu3

<400> 54

Gln Gln Ser Asp Ser Gly Arg Glu

1 5

<210> 55

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3 hu3 (general formula 2)

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa is selected from Asn or Ser.

<220>

<221> DOMAIN

<222> (6)..(6)

<223> Xaa is selected from Val or Gly.

<220>

<221> DOMAIN

<222> (8)..(8)

<223> Xaa is selected from Gly or Glu.

<400> 55

Gln Gln Ser Asp Xaa Xaa Arg Xaa

1 5

<210> 56

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> VL hu119 with grafted CDR(IGKV4-1*01)

<400> 56

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Asn Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Gly Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 57

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VL2

<400> 57

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Asn Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Gly Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 58

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VL3

<400> 58

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Asn Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Leu Tyr Arg Ala Ser Asn Leu Gly Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 59

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VL4 (Grafted, IGKV3-11*01)

<400> 59

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Gly Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 60

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VL5

<400> 60

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Leu Tyr Arg Ala Ser Asn Leu Gly Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 61

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VL6

<400> 61

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Leu Tyr Arg Ala Ser Asn Leu Gly Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 62

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VH1 (Grafted)

<400> 62

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 63

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VH2

<400> 63

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 64

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VH3

<400> 64

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Asn Met His Trp Val Lys His Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 65

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VH4

<400> 65

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Asn Met His Trp Val Lys His Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 66

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VH5

<400> 66

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 67

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VH6

<400> 67

Glu Ala Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 68

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VH7

<400> 68

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Arg Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 69

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VH8

<400> 69

Glu Ala Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Arg Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 70

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119 LCDR1-N31S

<400> 70

Arg Ala Ser Glu Ser Val Asp Ser Ser Gly Leu Ser Phe Met His

1 5 10 15

<210> 71

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119 LCDR1-N31Q

<400> 71

Arg Ala Ser Glu Ser Val Asp Gln Ser Gly Leu Ser Phe Met His

1 5 10 15

<210> 72

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VL2-N31S

<400> 72

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Ser Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Gly Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 73

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VL2-N31Q

<400> 73

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Gln Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Gly Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 74

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VL6-N31S

<400> 74

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Leu Tyr Arg Ala Ser Asn Leu Gly Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 75

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119VL6-N31Q

<400> 75

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Gln Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Leu Tyr Arg Ala Ser Asn Leu Gly Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 76

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu119 LCDR1-general formula

<220>

<221> DOMAIN

<222> (9)..(9)

<223> Xaa is selected from Asn, Ser or Gln.

<400> 76

Arg Ala Ser Glu Ser Val Asp Xaa Ser Gly Leu Ser Phe Met His

1 5 10 15

<210> 77

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL1 (Grafted (IGKV4-1*01))

<400> 77

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 78

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2

<400> 78

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 79

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> VL3 hu179

<400> 79

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 80

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> VL4 hu179

<400> 80

Ser Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 81

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL5 (Grafted(IGKV2-29*02))

<400> 81

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 82

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL6

<400> 82

Ser Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 83

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL7

<400> 83

Ser Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 84

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL8

<400> 84

Ser Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 85

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VH1 (Grafted)

<400> 85

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asp Pro Gly Asn Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 86

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VH2

<400> 86

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asp Pro Gly Asn Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 87

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VH3

<400> 87

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Gly Asn Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Ile Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 88

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VH4

<400> 88

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Gly Asn Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Ile Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 89

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VH5

<400> 89

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Gly Asn Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 90

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VH1- N55Q

<400> 90

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asp Pro Gly Gln Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 91

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VH1- N55V

<400> 91

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asp Pro Gly Val Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 92

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VH1- G56V

<400> 92

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asp Pro Gly Asn Val Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 93

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223>HCDR2-N55Qhu179

<400> 93

Val Ile Asp Pro Gly Gln Gly Asp Thr Asn Tyr Asn Glu Asn Phe Lys

1 5 10 15

Gly

<210> 94

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223>HCDR2-N55Vhu179

<400> 94

Val Ile Asp Pro Gly Val Gly Asp Thr Asn Tyr Asn Glu Asn Phe Lys

1 5 10 15

Gly

<210> 95

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223>HCDR2-G56V hu179

<400> 95

Val Ile Asp Pro Gly Asn Val Asp Thr Asn Tyr Asn Glu Asn Phe Lys

1 5 10 15

Gly

<210> 96

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 hu179 (general formula)

<220>

<221> DOMAIN

<222> (6)..(6)

<223> Xaa is selected from Asn, Gln or Val.

<220>

<221> DOMAIN

<222> (7)..(7)

<223> Xaa is selected from Gly or Val.

<400> 96

Val Ile Asp Pro Gly Xaa Xaa Asp Thr Asn Tyr Asn Glu Asn Phe Lys

1 5 10 15

Gly

<210> 97

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VH3-N55V

<400> 97

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Gly Val Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Ile Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 98

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2-Y50E

<400> 98

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Glu Val Ser Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 99

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2-S52D

<400> 99

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Asp Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 100

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2-S52E

<400> 100

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Glu Asn His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 101

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2-N53Q

<400> 101

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Gln His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 102

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2-N53D

<400> 102

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asp His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 103

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2-N53E

<400> 103

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Glu His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 104

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2-H54Y

<400> 104

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn Tyr Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 105

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2-H54D

<400> 105

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn Asp Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 106

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2-H54E

<400> 106

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn Glu Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 107

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL2-Y55E

<400> 107

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Asn His Glu Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 108

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179 LCDR2-Y50E

<400> 108

Glu Val Ser Asn His Tyr Thr

1 5

<210> 109

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179 LCDR2-S52D

<400> 109

Tyr Val Asp Asn His Tyr Thr

1 5

<210> 110

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179 LCDR2-S52E

<400> 110

Tyr Val Glu Asn His Tyr Thr

1 5

<210> 111

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179 LCDR2-N53Q

<400> 111

Tyr Val Ser Gln His Tyr Thr

1 5

<210> 112

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179 LCDR2-N53D

<400> 112

Tyr Val Ser Asp His Tyr Thr

1 5

<210> 113

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179 LCDR2-N53E

<400> 113

Tyr Val Ser Glu His Tyr Thr

1 5

<210> 114

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179 LCDR2-H54Y

<400> 114

Tyr Val Ser Asn Tyr Tyr Thr

1 5

<210> 115

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179 LCDR2-H54D

<400> 115

Tyr Val Ser Asn Asp Tyr Thr

1 5

<210> 116

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179 LCDR2-H54E

<400> 116

Tyr Val Ser Asn Glu Tyr Thr

1 5

<210> 117

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179 LCDR2-Y55E

<400> 117

Tyr Val Ser Asn His Glu Thr

1 5

<210> 118

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 humanized antibody mab179 general formula

<220>

<221> DOMAIN

<222> (1)..(1)

<223> Xaa is selected from Try or Glu.

<220>

<221> DOMAIN

<222> (3)..(3)

<223> Xaa is selected from Ser, Asp or Glu.

<220>

<221> DOMAIN

<222> (4)..(4)

<223> Xaa is selected from Asn, Gln, Asp or Glu.

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa is selected from His, Tyr, Asp or Glu.

<220>

<221> DOMAIN

<222> (6)..(6)

<223> Xaa is selected from Glu or Tyr.

<220>

<221> DOMAIN

<223> hu179VL8-N53E

<400> 118

Xaa Val Xaa Xaa Xaa Xaa Thr

1 5

<210> 119

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu179VL8-N53E

<400> 119

Ser Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Glu His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 120

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VL1 (Grafted)

<400> 120

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 121

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VL2

<400> 121

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val

35 40 45

Tyr Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 122

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VL3

<400> 122

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 123

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VL4

<400> 123

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Val Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 124

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VL5

<400> 124

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val

35 40 45

Tyr Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Val Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 125

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VL6

<400> 125

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Val Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 126

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VH1 (Grafted)

<400> 126

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Ile Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 127

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VH2

<400> 127

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Ile Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 128

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VH3

<400> 128

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Ile Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 129

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VH4

<400> 129

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Ile Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 130

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VH5

<400> 130

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Ile Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 131

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VH6

<400> 131

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Ile Gln Lys Phe

50 55 60

Lys Asp Lys Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 132

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu199VH7

<400> 132

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Ile Gln Lys Phe

50 55 60

Lys Asp Lys Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 133

<211> 330

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> IgG1 heavy chain constant region-YTE

<400> 133

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 134

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain constant region kappa

<400> 134

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 135

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu3-13 antibody heavy chain

<400> 135

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Ile Ile Ser Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Tyr Asp Tyr Asp Gly Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr

245 250 255

Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 136

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain of the antibody hu3-13

<400> 136

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asp Ser Gly Arg Glu Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 137

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> Antibody heavy chain hu119-30

<400> 137

Glu Ala Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Glu Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Arg Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asp Tyr Gly Glu Gly Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile

245 250 255

Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 138

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> Light chain of the antibody hu119-30

<400> 138

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Ser

20 25 30

Gly Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Leu Tyr Arg Ala Ser Asn Leu Gly Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ile Asn

85 90 95

Thr Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 139

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Antibody heavy chain hu179-33

<400> 139

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Gly Val Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Ile Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr

245 250 255

Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 140

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain of the antibody hu179-33

<400> 140

Ser Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Glu His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 141

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> Antibody heavy chain hu199-36

<400> 141

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Ile Gln Lys Phe

50 55 60

Lys Asp Lys Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 142

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain of antibody hu199-36

<400> 142

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Val Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 143

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> AMG157 heavy chain sequence

<400> 143

Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val

290 295 300

Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 144

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> AMG157 light chain sequence

<400> 144

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 145

<211> 371

<212> PROTEIN

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> Amino acid sequence of the full-length

human TSLP receptor sequences

<400> 145

Met Gly Arg Leu Val Leu Leu Trp Gly Ala Ala Val Phe Leu Leu Gly

1 5 10 15

Gly Trp Met Ala Leu Gly Gln Gly Gly Ala Ala Glu Gly Val Gln Ile

20 25 30

Gln Ile Ile Tyr Phe Asn Leu Glu Thr Val Gln Val Thr Trp Asn Ala

35 40 45

Ser Lys Tyr Ser Arg Thr Asn Leu Thr Phe His Tyr Arg Phe Asn Gly

50 55 60

Asp Glu Ala Tyr Asp Gln Cys Thr Asn Tyr Leu Leu Gln Glu Gly His

65 70 75 80

Thr Ser Gly Cys Leu Leu Asp Ala Glu Gln Arg Asp Asp Ile Leu Tyr

85 90 95

Phe Ser Ile Arg Asn Gly Thr His Pro Val Phe Thr Ala Ser Arg Trp

100 105 110

Met Val Tyr Tyr Leu Lys Pro Ser Ser Pro Lys His Val Arg Phe Ser

115 120 125

Trp His Gln Asp Ala Val Thr Val Thr Cys Ser Asp Leu Ser Tyr Gly

130 135 140

Asp Leu Leu Tyr Glu Val Gln Tyr Arg Ser Pro Phe Asp Thr Glu Trp

145 150 155 160

Gln Ser Lys Gln Glu Asn Thr Cys Asn Val Thr Ile Glu Gly Leu Asp

165 170 175

Ala Glu Lys Cys Tyr Ser Phe Trp Val Arg Val Lys Ala Met Glu Asp

180 185 190

Val Tyr Gly Pro Asp Thr Tyr Pro Ser Asp Trp Ser Glu Val Thr Cys

195 200 205

Trp Gln Arg Gly Glu Ile Arg Asp Ala Cys Ala Glu Thr Pro Thr Pro

210 215 220

Pro Lys Pro Lys Leu Ser Lys Phe Ile Leu Ile Ser Ser Leu Ala Ile

225 230 235 240

Leu Leu Met Val Ser Leu Leu Leu Leu Ser Leu Trp Lys Leu Trp Arg

245 250 255

Val Lys Lys Phe Leu Ile Pro Ser Val Pro Asp Pro Lys Ser Ile Phe

260 265 270

Pro Gly Leu Phe Glu Ile His Gln Gly Asn Phe Gln Glu Trp Ile Thr

275 280 285

Asp Thr Gln Asn Val Ala His Leu His Lys Met Ala Gly Ala Glu Gln

290 295 300

Glu Ser Gly Pro Glu Glu Pro Leu Val Val Gln Leu Ala Lys Thr Glu

305 310 315 320

Ala Glu Ser Pro Arg Met Leu Asp Pro Gln Thr Glu Glu Lys Glu Ala

325 330 335

Ser Gly Gly Ser Leu Gln Leu Pro His Gln Pro Leu Gln Gly Gly Asp

340 345 350

Val Val Thr Ile Gly Gly Phe Thr Phe Val Met Asn Asp Arg Ser Tyr

355 360 365

Val Ala Leu

370

<210> 146

<211> 459

<212> PROTEIN

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> Amino acid sequence of the full-length

human IL7R alpha receptor sequences

<400> 146

Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln

1 5 10 15

Val Val Ser Gly Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp

20 25 30

Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val

35 40 45

Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val

50 55 60

Asn Thr Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val

65 70 75 80

Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr

85 90 95

Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly

100 105 110

Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys

115 120 125

Pro Glu Ala Pro Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn

130 135 140

Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val

145 150 155 160

Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn

165 170 175

Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln

180 185 190

Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile

195 200 205

Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr

210 215 220

Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro

225 230 235 240

Ile Leu Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu

245 250 255

Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys Pro Ile Val

260 265 270

Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His Leu Cys Lys

275 280 285

Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu

290 295 300

Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala Arg Asp Glu Val

305 310 315 320

Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu Ser Glu

325 330 335

Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn Cys Pro Ser Glu

340 345 350

Asp Val Val Ile Thr Pro Glu Ser Phe Gly Arg Asp Ser Ser Leu Thr

355 360 365

Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser

370 375 380

Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro His Val

385 390 395 400

Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro

405 410 415

Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala

420 425 430

Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala

435 440 445

Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln

450 455

<---

Claims (53)

1. An antibody to TSLP (thymic stromal lymphopoietin) containing a variable region of the heavy chain and a variable region of the light chain, where: i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 45, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 54, respectively; or ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 45, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 53, respectively; or iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 70, SEQ ID NO: 24 and SEQ ID NO: 25, respectively; or iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 71, SEQ ID NO: 24 and SEQ ID NO: 25, respectively; or v) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 94 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 113 and SEQ ID NO: 31, respectively; or vi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 94 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; or vii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively; or viii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 45, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 46, respectively; or ix) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively; or x) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; or xi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 93 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; or xii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 95 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; or xiii) the variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the variable region of the light chain comprises LCDR1, LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 31, respectively, and LCDR2 as set forth in SEQ ID NO: 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117. 2. The TSLP antibody of claim 1, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 94 and SEQ ID NO: 28, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 29, SEQ ID NO: 113 and SEQ ID NO: 31, respectively. 3. The TSLP antibody according to claim 1 or 2, which is a murine antibody, a chimeric antibody or a humanized antibody. 4. The anti-TSLP antibody of claim 3, wherein the anti-TSLP antibody comprises a framework region(s) derived from a human antibody, wherein the anti-TSLP antibody comprises a light chain variable region and/or a heavy chain variable region selected from those described in (a), (b), (c) or (d) below: a) the variable region of the heavy chain comprises HCDR1 and HCDR2 as set forth in SEQ ID NO: 14 and SEQ ID NO: 15, respectively, and HCDR3 as set forth in SEQ ID NO: 16 or 45, and its framework region(s) comprises(are) no more than 10 backmutations; and/or the variable region of the light chain comprises LCDR1 and LCDR2 as set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and LCDR3 as set forth in SEQ ID NO: 19, 46, 53 or 54, and its framework region(s) comprises(are) no more than 10 amino acid backmutations; b) the variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and its framework region(s) comprises no more than 10 backmutations; and/or the variable region of the light chain comprises LCDR2 and LCDR3 as set forth in SEQ ID NO: 24 and SEQ ID NO: 25, respectively, and LCDR1 as set forth in SEQ ID NO: 23, 70 or 71, and its framework region(s) comprises no more than 10 amino acid backmutations; c) the variable region of the heavy chain comprises HCDR1 and HCDR3 as shown in SEQ ID NO: 26 and SEQ ID NO: 28, respectively, and HCDR2 as shown in SEQ ID NO: 27, 93, 94 or 95, and its framework region(s) comprises no more than 10 backmutations; and/or the variable region of the light chain comprises LCDR1 and LCDR3 as shown in SEQ ID NO: 29 and SEQ ID NO: 31, respectively, and LCDR2 as shown in SEQ ID NO: 30, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117, and its framework region(s) comprises no more than 10 backmutations. 5. The anti-TSLP antibody of claim 3, wherein the anti-TSLP antibody comprises a framework region(s) derived from a human antibody, wherein the anti-TSLP antibody comprises a light chain variable region and/or a heavy chain variable region selected from those described in (a), (b), (c) or (d) below: a) the heavy chain variable region comprises HCDR1 and HCDR2 as set forth in SEQ ID NO: 14 and SEQ ID NO: 15, respectively, and HCDR3 as set forth in SEQ ID NO: 16 or 45, and its framework region(s) comprises no more than 10 backmutations, wherein the backmutation is selected from one or more of 38K, 48I, 67A, 69L, 71V and 73K; and/or the light chain variable region comprises LCDR1 and LCDR2 as set forth in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and LCDR3 as set forth in SEQ ID NO: 19, 46, 53 or 54, and its framework region(s) comprises no more than 10 amino acid back mutations, wherein the back mutation is selected from one or more of 46P, 47W, 58V, 70S and 71Y; b) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and its framework region(s) comprises no more than 10 backmutations, wherein the backmutation is selected from one or more of 2A, 27F, 38K, 39H, 48I, 67A, 69L, 71V and 76R; and/or the light chain variable region comprises LCDR2 and LCDR3 as set forth in SEQ ID NO: 24 and SEQ ID NO: 25, respectively, and LCDR1 as set forth in SEQ ID NO: 23, 70 or 71, and its framework region(s) comprises no more than 10 amino acid back mutations, wherein the back mutation is selected from one or more of 1D, 4L, 43P, 48L and 58I; c) the heavy chain variable region comprises HCDR1 and HCDR3 as shown in SEQ ID NO: 26 and SEQ ID NO: 28, respectively, and HCDR2 as shown in SEQ ID NO: 27, 93, 94 or 95, and its framework region(s) comprises no more than 10 backmutations, wherein the backmutation is selected from one or more of 27Y, 28A, 38K, 48I, 66K, 67A, 69L, 80I and 82bR; and/or the light chain variable region comprises LCDR1 and LCDR3 as shown in SEQ ID NO: 29 and SEQ ID NO: 31, respectively, and LCDR2 as shown in SEQ ID NO: 30, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117, and its framework region(s) comprises no more than 10 backmutations, wherein the backmutation is selected from one or more of 1S, 43S, 67Y and 73F. 6. The TSLP antibody according to claim 3, comprising a heavy chain variable region and a light chain variable region, wherein: i) the variable region of the heavy chain has at least 90% sequence identity to the variable region of the heavy chain shown in the amino acid sequence of SEQ ID NO: 6, 42, 43, 44 or 50, and the variable region of the light chain has at least 90% sequence identity to the variable region of the light chain shown in the amino acid sequence of SEQ ID NO: 7, 38, 39, 40, 41, 49, 51 or 52; or ii) the variable region of the heavy chain has at least 90% sequence identity to the variable region of the heavy chain shown in the amino acid sequence of SEQ ID NO: 8, 62, 63, 64, 65, 66, 67, 68 or 69, and the variable region of the light chain has at least 90% sequence identity to the variable region of the light chain shown in the amino acid sequence of SEQ ID NO: 9, 56, 57, 58, 59, 60, 61, 72, 73, 74 or 75; or iii) the variable region of the heavy chain has at least 90% sequence identity with the variable region of the heavy chain presented in the amino acid sequence of SEQ ID NO: 10, 85, 86, 87, 88, 89, 90, 91, 92 or 97, and the variable region of the light chain has at least 90% sequence identity with the variable region of the light chain presented in the amino acid sequence of SEQ ID NO: 11, 77, 78, 79, 80, 81, 82, 83, 84, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 119. 7. The TSLP antibody of claim 6, comprising a heavy chain variable region and a light chain variable region, wherein: i) the amino acid sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 6, 42, 43, 44 or 50, and the amino acid sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 7, 38, 39, 40, 41, 49, 51 or 52; or ii) the amino acid sequence of the variable region of the heavy chain is as set forth in SEQ ID NO: 8, 62, 63, 64, 65, 66, 67, 68 or 69, and the amino acid sequence of the variable region of the light chain is as set forth in SEQ ID NO: 9, 56, 57, 58, 59, 60, 61, 72, 73, 74 or 75; or iii) the amino acid sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 10, 85, 86, 87, 88, 89, 90, 91, 92 or 97, and the amino acid sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 11, 77, 78, 79, 80, 81, 82, 83, 84, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 119. 8. The TSLP antibody of claim 7, comprising a heavy chain variable region and a light chain variable region as follows: a) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 50, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 52; or b) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 50, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 51; or c) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 69, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 74; or d) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 64, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 73; or e) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 97, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 119; or f) the sequence of the variable region of the heavy chain corresponds to that presented in SEQ ID NO: 91, and the sequence of the variable region of the light chain corresponds to that presented in SEQ ID NO: 78. 9. The TSLP antibody of claim 8, comprising a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region corresponds to that presented in SEQ ID NO: 97, and the sequence of the light chain variable region corresponds to that presented in SEQ ID NO: 119. 10. An antibody to TSLP or an antigen-binding fragment thereof according to any one of claims 1-9, wherein the antibody comprises a heavy chain constant region and a light chain constant region of an antibody. 11. An antibody to TSLP or an antigen-binding fragment thereof according to any one of claims 1-10, wherein the constant region of the heavy chain is selected from the group consisting of the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conventional variants, the constant region of the light chain is selected from the group consisting of the constant regions of the κ- and λ-chain of a human antibody and their conventional variants. 12. An antibody to TSLP or an antigen-binding fragment thereof according to any one of claims 1-11, wherein the antibody comprises a heavy chain constant region presented in the sequence SEQ ID NO: 133 and a light chain constant region presented in the sequence SEQ ID NO: 134. 13. The TSLP antibody of claim 12, comprising a heavy chain and a light chain as follows: a) the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 135 or has at least 90% identity to said sequence, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 136 or has at least 90% identity to said sequence; or b) the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 137 or has at least 90% identity to said sequence, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 138 or has at least 90% identity to said sequence; or c) the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 139 or has at least 90% identity to said sequence, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 140 or has at least 90% identity to said sequence. 14. The TSLP antibody of claim 13, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain corresponds to that presented in SEQ ID NO: 139, and the amino acid sequence of the light chain corresponds to that presented in SEQ ID NO: 140. 15. A nucleic acid molecule encoding an antibody to TSLP according to any one of claims 1-14. 16. A host cell for replicating nucleic acid molecules, containing a nucleic acid molecule according to claim 15. 17. A pharmaceutical composition for treating diseases associated with TSLP, comprising a therapeutically effective amount of an antibody to TSLP according to any one of claims 1-14, or a nucleic acid molecule according to claim 15, or a host cell according to claim 16, as well as one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. 18. A method for treating diseases associated with TSLP, comprising administering to a subject a therapeutically effective amount of an antibody to TSLP according to any one of claims 1-14, or a nucleic acid molecule according to claim 15, or a host cell according to claim 16, or a pharmaceutical composition according to claim 17. 19. The method of claim 18, wherein the disease associated with TSLP is selected from the group consisting of: asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic sinusitis, urticaria, Netherton syndrome, eosinophilic esophagitis, food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungal sinusitis, chronic pruritus, cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, rheumatoid arthritis, chronic obstructive pulmonary disease, systemic sclerosis, multiple sclerosis, keloidosis, ulcerative colitis, nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, Churg-Strauss syndrome, eosinophilia-myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, scleroderma, interstitial lung disease, fibrosis due to chronic hepatitis B or C, fibrosis due to radiation, and fibrosis due to wound healing.