相關申請案之交互參考Cross-references to Related Applications
本申請案主張於2023年8月25日提申之美國臨時申請號63/578,947的權益及優先權，其內容通過引用整體併入本文。This application claims the benefit of and priority to U.S. Provisional Application No. 63/578,947, filed on August 25, 2023, the contents of which are incorporated herein by reference in their entirety.
電子序列表之參考References to Electronic Sequence Listings
電子序列表之內容(253272000240seqlist.xml；大小：121,903位元；及創建日期：2024年8月19日)以引用方式整體併入本文。The contents of the electronic sequence listing (253272000240seqlist.xml; size: 121,903 bytes; and creation date: August 19, 2024) are incorporated herein by reference in their entirety.
本發明係有關特異性地辨識胸腺基質淋巴球生成素(thymic stromal lymphopoietin，TSLP)的分離的抗體構築體(例如，抗TSLP抗體構築體)、含有該分離的抗TSLP抗體構築體的醫藥組成物、使用該分離的抗TSLP抗體構築體治療發炎性疾病的方法及含有該分離的抗TSLP抗體構築體的套組。亦提供製造其之方法。The present invention relates to an isolated antibody construct (e.g., an anti-TSLP antibody construct) that specifically recognizes thymic stromal lymphopoietin (TSLP), a pharmaceutical composition containing the isolated anti-TSLP antibody construct, a method of using the isolated anti-TSLP antibody construct to treat inflammatory diseases, and a kit containing the isolated anti-TSLP antibody construct. Methods for making the same are also provided.
許多異常細胞與組織以及許多疾病皆表現出獨特的抗原，該些抗原可用於進行免疫細胞媒介的清除，包括發炎性疾病，諸如自體免疫疾病或氣喘。此等發炎性疾病可各自表現出一或多種不同的標靶抗原或相同標靶抗原之一或多種不同的表位。舉例而言，一些抗原在發炎的組織中被過度表現、誘變或選擇性地誘變。因此，全身性或局部性發炎疾病中存在的抗體標靶特異性抗可用作治療劑。Many abnormal cells and tissues and many diseases express unique antigens that can be used to clear immune cell mediators, including inflammatory diseases such as autoimmune diseases or asthma. These inflammatory diseases can each express one or more different target antigens or one or more different epitopes of the same target antigen. For example, some antigens are overexpressed, induced, or selectively induced in inflamed tissues. Therefore, antibodies with target specificity present in systemic or localized inflammatory diseases can be used as therapeutic agents.
胸腺基質淋巴球生成素(TSLP) 為一種多效性細胞激素，其具有淋巴球生長因子的特徵。TSLP結合至胸腺基質淋巴球生成素受體(TSLPR)鏈與介白素7受體-α (IL-7Rα)組成的高親和力異質複合體。TSLP主要由腸道與肺上皮細胞、皮膚角質細胞及樹突細胞(DC)表現，但其亦可由(例如)呼吸道平滑肌細胞、肥大細胞、單核球、巨噬細胞、顆粒細胞、滑液膜纖維母細胞(synovial fibroblast)等產生。取決於TSLP靶向的免疫細胞，據報導TSLP不僅可促進T輔助2型細胞(Th2)反應，還與自體免疫病症相關。Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine with characteristics of a lymphocyte growth factor. TSLP binds to a high-affinity heterocomplex consisting of the thymic stromal lymphopoietin receptor (TSLPR) chain and interleukin-7 receptor-α (IL-7Rα). TSLP is primarily expressed by intestinal and lung epithelial cells, skin keratinocytes, and dendritic cells (DCs), but it can also be produced by, for example, airway smooth muscle cells, mast cells, monocytes, macrophages, granulocytes, synovial fibroblasts, etc. Depending on the immune cells targeted by TSLP, TSLP has been reported to not only promote T helper type 2 (Th2) responses but also be associated with autoimmune disorders.
特澤佩魯單抗(Tezepelumab，TezspireTM)為一種完整人類單株抗體，其結合至TSLP並防止其與TSLPR鏈相互作用。隨後，IL-7Rα鏈不會被招募，且不會形成功能性TSLP受體複合體。參見H.K. Lehman and C.M. Sabella，「Allergic and Immunologic Diseases, A Practical Guide to the Evaluation, Diagnosis and Management of Allergic and Immunologic Diseases」，2022，第1111-1145頁，「第38章 - New biologics in allergy」。該抗體被US-FDA核准作為嚴重氣喘的附加(add-on)維持治療。Tezepelumab (Tezspire™ ) is a fully human monoclonal antibody that binds to TSLP and prevents it from interacting with the TSLPR chain. Subsequently, the IL-7Rα chain is not recruited and a functional TSLP receptor complex is not formed. See HK Lehman and CM Sabella, "Allergic and Immunologic Diseases, A Practical Guide to the Evaluation, Diagnosis and Management of Allergic and Immunologic Diseases", 2022, pp. 1111-1145, "Chapter 38 - New biologics in allergy". The antibody is approved by the US-FDA as an add-on maintenance treatment for severe asthma.
在本發明之一態樣中，提供一種分離的抗體構築體(「抗TSLP抗體構築體」)，其包含特異性地辨識胸腺基質淋巴球生成素的抗體部分(TSLP；「抗TSLP抗體部分」)，其中該抗TSLP抗體部分包含重鏈可變區(VH)及輕鏈可變區(VL)，且其中：(a)該VH包含(i)含有SEQ ID NO:2之胺基酸序列的CDR-H1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:3之胺基酸序列的CDR-H2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:4之胺基酸序列的CDR-H3或其含有至多3個胺基酸變異的變體；且該VL包含(i)含有SEQ ID NO:6之胺基酸序列的CDR-L1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:7之胺基酸序列的CDR-L2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:8之胺基酸序列的CDR-L3或其含有至多3個胺基酸變異的變體；(b)該VH包含(i)含有SEQ ID NO:12之胺基酸序列的CDR-H1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:13之胺基酸序列的CDR-H2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:4之胺基酸序列的CDR-H3或其含有至多3個胺基酸變異的變體；且該VL包含(i)含有SEQ ID NO:6之胺基酸序列的CDR-L1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:7之胺基酸序列的CDR-L2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:8之胺基酸序列的CDR-L3或其含有至多3個胺基酸變異的變體；(c)該VH包含(i)含有SEQ ID NO:20之胺基酸序列的CDR-H1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:21之胺基酸序列的CDR-H2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:22之胺基酸序列的CDR-H3或其含有至多3個胺基酸變異的變體；且該VL包含(i)含有SEQ ID NO:24之胺基酸序列的CDR-L1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:25之胺基酸序列的CDR-L2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:26之胺基酸序列的CDR-L3或其含有至多3個胺基酸變異的變體；(d)該VH包含(i)含有SEQ ID NO:28之胺基酸序列的CDR-H1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:29之胺基酸序列的CDR-H2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:30之胺基酸序列的CDR-H3或其含有至多3個胺基酸變異的變體；且該VL包含(i)含有SEQ ID NO:32之胺基酸序列的CDR-L1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:33之胺基酸序列的CDR-L2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:34之胺基酸序列的CDR-L3或其含有至多3個胺基酸變異的變體；(e)該VH包含(i)含有SEQ ID NO:36之胺基酸序列的CDR-H1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:37之胺基酸序列的CDR-H2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:38之胺基酸序列的CDR-H3或其含有至多3個胺基酸變異的變體；且該VL包含(i)含有SEQ ID NO:40之胺基酸序列的CDR-L1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:41之胺基酸序列的CDR-L2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:42之胺基酸序列的CDR-L3或其含有至多3個胺基酸變異的變體；或(f)該VH包含(i)含有SEQ ID NO:44之胺基酸序列的CDR-H1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:45之胺基酸序列的CDR-H2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:46之胺基酸序列的CDR-H3或其含有至多3個胺基酸變異的變體；且該VL包含(i)含有SEQ ID NO:48之胺基酸序列的CDR-L1或其含有至多3個胺基酸變異的變體；(ii)含有SEQ ID NO:49之胺基酸序列的CDR-L2或其含有至多3個胺基酸變異的變體；及(iii)含有SEQ ID NO:50之胺基酸序列的CDR-L3或其含有至多3個胺基酸變異的變體。In one aspect of the present invention, an isolated antibody construct ("anti-TSLP antibody construct") is provided, which comprises an antibody portion that specifically recognizes thymic stromal lymphopoietin (TSLP; "anti-TSLP antibody portion"), wherein the anti-TSLP antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein: (a) the VH comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof containing up to 3 amino acid variations; and the VL comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof containing up to 3 amino acid variations; NO:6 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-L2 containing an amino acid sequence of SEQ ID NO:7 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-L3 containing an amino acid sequence of SEQ ID NO:8 or a variant thereof containing up to 3 amino acid variations; (b) the VH comprises (i) a CDR-H1 containing an amino acid sequence of SEQ ID NO:12 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 containing an amino acid sequence of SEQ ID NO:13 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 containing an amino acid sequence of SEQ ID NO:4 or a variant thereof containing up to 3 amino acid variations; and the VL comprises (i) a CDR-H1 containing an amino acid sequence of SEQ ID NO:12 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 containing an amino acid sequence of SEQ ID NO:13 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 containing an amino acid sequence of SEQ ID NO:4 or a variant thereof containing up to 3 amino acid variations; NO:6 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-L2 containing an amino acid sequence of SEQ ID NO:7 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-L3 containing an amino acid sequence of SEQ ID NO:8 or a variant thereof containing up to 3 amino acid variations; (c) the VH comprises (i) a CDR-H1 containing an amino acid sequence of SEQ ID NO:20 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 containing an amino acid sequence of SEQ ID NO:21 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 containing an amino acid sequence of SEQ ID NO:22 or a variant thereof containing up to 3 amino acid variations; and the VL comprises (i) a CDR-H1 containing an amino acid sequence of SEQ ID NO:20 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 containing an amino acid sequence of SEQ ID NO:21 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 containing an amino acid sequence of SEQ ID NO:22 or a variant thereof containing up to 3 amino acid variations; NO:24 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-L2 containing an amino acid sequence of SEQ ID NO:25 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-L3 containing an amino acid sequence of SEQ ID NO:26 or a variant thereof containing up to 3 amino acid variations; (d) the VH comprises (i) a CDR-H1 containing an amino acid sequence of SEQ ID NO:28 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 containing an amino acid sequence of SEQ ID NO:29 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 containing an amino acid sequence of SEQ ID NO:30 or a variant thereof containing up to 3 amino acid variations; and the VL comprises (i) a CDR-H1 containing an amino acid sequence of SEQ ID NO:28 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 containing an amino acid sequence of SEQ ID NO:29 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 containing an amino acid sequence of SEQ ID NO:30 or a variant thereof containing up to 3 amino acid variations; NO:32 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-L2 containing an amino acid sequence of SEQ ID NO:33 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-L3 containing an amino acid sequence of SEQ ID NO:34 or a variant thereof containing up to 3 amino acid variations; (e) the VH comprises (i) a CDR-H1 containing an amino acid sequence of SEQ ID NO:36 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 containing an amino acid sequence of SEQ ID NO:37 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 containing an amino acid sequence of SEQ ID NO:38 or a variant thereof containing up to 3 amino acid variations; and the VL comprises (i) a CDR-H1 containing an amino acid sequence of SEQ ID NO:36 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 containing an amino acid sequence of SEQ ID NO:37 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 containing an amino acid sequence of SEQ ID NO:38 or a variant thereof containing up to 3 amino acid variations; NO:40 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-L2 containing an amino acid sequence of SEQ ID NO:41 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-L3 containing an amino acid sequence of SEQ ID NO:42 or a variant thereof containing up to 3 amino acid variations; or (f) the VH comprises (i) a CDR-H1 containing an amino acid sequence of SEQ ID NO:44 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 containing an amino acid sequence of SEQ ID NO:45 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 containing an amino acid sequence of SEQ ID NO:46 or a variant thereof containing up to 3 amino acid variations; and the VL comprises (i) a CDR-H1 containing an amino acid sequence of SEQ ID NO:44 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-H2 containing an amino acid sequence of SEQ ID NO:45 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-H3 containing an amino acid sequence of SEQ ID NO:46 or a variant thereof containing up to 3 amino acid variations; (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:48 or a variant thereof containing up to 3 amino acid variations; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:49 or a variant thereof containing up to 3 amino acid variations; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:50 or a variant thereof containing up to 3 amino acid variations.
在根據上述分離的抗TSLP抗體構築體的一些實施例中，該抗TSLP抗體部分包含：(a) VH，其包含(i)含有SEQ ID NO:2之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:3之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:4之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:6之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:7之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:8之胺基酸序列的CDR-L3；(b) VH，其包含(i)含有SEQ ID NO:12之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:13之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:4之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:6之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:7之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:8之胺基酸序列的CDR-L3；(c) VH，其包含(i)含有SEQ ID NO:20之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:21之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:22之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:24之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:25之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:26之胺基酸序列的CDR-L3；(d) VH，其包含(i)含有SEQ ID NO:28之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:29之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:30之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:32之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:33之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:34之胺基酸序列的CDR-L3；(e) VH，其包含(i)含有SEQ ID NO:36之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:37之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:38之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:40之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:41之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:42之胺基酸序列的CDR-L3；或 (f) VH，其包含(i)含有SEQ ID NO:44之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:45之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:46之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:48之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:49之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:50之胺基酸序列的CDR-L3。In some embodiments of the anti-TSLP antibody construct isolated above, the anti-TSLP antibody portion comprises: (a) a VH comprising (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 2; (ii) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 3; and (iii) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 4; and a VL comprising (i) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 6; (ii) a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 7; and (iii) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 8; (b) a VH comprising (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 12; (ii) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 13; and (iii) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 14. NO:4; and VL, which comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:6; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:7; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8; (c) VH, which comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:21; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and VL, which comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:24; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:25; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:26; (d) VH, which comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:21; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22 (e) a VH comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38; and a VL comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34; NO:41; and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO:42; or (f) VH, which comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:44; (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:45; and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:46; and VL, which comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO:48; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO:49; and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO:50.
在根據上述分離的抗TSLP抗體構築體之任一者的一些實施例中，(a)該VH包含與SEQ ID NO:1具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:5具有至少約80%序列一致性的胺基酸序列；(b)該VH包含與SEQ ID NO:9具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:10具有至少約80%序列一致性的胺基酸序列；(c)該VH包含與SEQ ID NO:11具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:15具有至少約80%序列一致性的胺基酸序列；(d)該VH包含與SEQ ID NO:19具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:23具有至少約80%序列一致性的胺基酸序列；(e)該VH包含與SEQ ID NO:27具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:31具有至少約80%序列一致性的胺基酸序列；(f)該VH包含與SEQ ID NO:35具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:39具有至少約80%序列一致性的胺基酸序列；(g)該VH包含與SEQ ID NO:43具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:47具有至少約80%序列一致性的胺基酸序列；(h)該VH包含與SEQ ID NO:63具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:53具有至少約80%序列一致性的胺基酸序列；(i)該VH包含與SEQ ID NO:63具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:54具有至少約80%序列一致性的胺基酸序列；(j)該VH包含與SEQ ID NO:56具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:52具有至少約80%序列一致性的胺基酸序列；(k)該VH包含與SEQ ID NO:55具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:51具有至少約80%序列一致性的胺基酸序列；(l)該VH包含與SEQ ID NO:56具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:51具有至少約80%序列一致性的胺基酸序列；(m)該VH包含與SEQ ID NO:63具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:51具有至少約80%序列一致性的胺基酸序列；(n)該VH包含與SEQ ID NO:64具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:51具有至少約80%序列一致性的胺基酸序列；(o)該VH包含與SEQ ID NO:55具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:52具有至少約80%序列一致性的胺基酸序列；(p)該VH包含與SEQ ID NO:58具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:52具有至少約80%序列一致性的胺基酸序列；(q)該VH包含與SEQ ID NO:60具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:52具有至少約80%序列一致性的胺基酸序列；(r)該VH包含與SEQ ID NO:64具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:52具有至少約80%序列一致性的胺基酸序列；(s)該VH包含與SEQ ID NO:55具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:53具有至少約80%序列一致性的胺基酸序列；(t)該VH包含與SEQ ID NO:57具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:53具有至少約80%序列一致性的胺基酸序列；(u)該VH包含與SEQ ID NO:58具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:53具有至少約80%序列一致性的胺基酸序列；(v)該VH包含與SEQ ID NO:62具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:53具有至少約80%序列一致性的胺基酸序列；(w)該VH包含與SEQ ID NO:64具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:53具有至少約80%序列一致性的胺基酸序列；(x)該VH包含與SEQ ID NO:55具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:54具有至少約80%序列一致性的胺基酸序列；(y)該VH包含與SEQ ID NO:61具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:54具有至少約80%序列一致性的胺基酸序列；(z)該VH包含與SEQ ID NO:62具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:54具有至少約80%序列一致性的胺基酸序列；(aa)該VH包含與SEQ ID NO:64具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:54具有至少約80%序列一致性的胺基酸序列；(bb)該VH包含與SEQ ID NO:63具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:52具有至少約80%序列一致性的胺基酸序列；(cc)該VH包含與SEQ ID NO:188具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:189具有至少約80%序列一致性的胺基酸序列；或(dd)該VH包含與SEQ ID NO:188具有至少約80%序列一致性的胺基酸序列，且該VL包含與SEQ ID NO:190具有至少約80%序列一致性的胺基酸序列。In some embodiments according to any of the above isolated anti-TSLP antibody constructs, (a) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:1, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:5; (b) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:9, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:10; (c) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:11, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:15; (d) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:19, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:19; NO:23 has at least about 80% sequence identity; (e) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:27, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:31; (f) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:35, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:39; (g) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:43, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:47; (h) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:63, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:53; (i) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: NO:63 has an amino acid sequence that is at least about 80% identical to SEQ ID NO:54, and the VL comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:54; (j) the VH comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:56, and the VL comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:52; (k) the VH comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:55, and the VL comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:51; (l) the VH comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:56, and the VL comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:51; (m) the VH comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:63, and the VL comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: NO:51 has at least about 80% sequence identity; (n) the VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:64, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:51; (o) the VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:55, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:52; (p) the VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:58, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:52; (q) the VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:60, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:52; (r) the VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:54. NO:64 has an amino acid sequence that has at least about 80% sequence identity, and the VL comprises an amino acid sequence that has at least about 80% sequence identity with SEQ ID NO:52; (s) the VH comprises an amino acid sequence that has at least about 80% sequence identity with SEQ ID NO:55, and the VL comprises an amino acid sequence that has at least about 80% sequence identity with SEQ ID NO:53; (t) the VH comprises an amino acid sequence that has at least about 80% sequence identity with SEQ ID NO:57, and the VL comprises an amino acid sequence that has at least about 80% sequence identity with SEQ ID NO:53; (u) the VH comprises an amino acid sequence that has at least about 80% sequence identity with SEQ ID NO:58, and the VL comprises an amino acid sequence that has at least about 80% sequence identity with SEQ ID NO:53; (v) the VH comprises an amino acid sequence that has at least about 80% sequence identity with SEQ ID NO:62, and the VL comprises an amino acid sequence that has at least about 80% sequence identity with SEQ ID NO: NO:53 has an amino acid sequence having at least about 80% sequence identity; (w) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:64, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:53; (x) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:55, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:54; (y) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:61, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:54; (z) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:62, and the VL comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:54; (aa) the VH comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: NO:64 has an amino acid sequence that has at least about 80% sequence identity, and the VL comprises an amino acid sequence that has at least about 80% sequence identity to SEQ ID NO:54; (bb) the VH comprises an amino acid sequence that has at least about 80% sequence identity to SEQ ID NO:63, and the VL comprises an amino acid sequence that has at least about 80% sequence identity to SEQ ID NO:52; (cc) the VH comprises an amino acid sequence that has at least about 80% sequence identity to SEQ ID NO:188, and the VL comprises an amino acid sequence that has at least about 80% sequence identity to SEQ ID NO:189; or (dd) the VH comprises an amino acid sequence that has at least about 80% sequence identity to SEQ ID NO:188, and the VL comprises an amino acid sequence that has at least about 80% sequence identity to SEQ ID NO:190.
在根據上述分離的抗TSLP抗體構築體之任一者的一些實施例中，(a)該VH含有SEQ ID NO:1之胺基酸序列，且該VL含有SEQ ID NO:5之胺基酸序列；(b)該VH含有SEQ ID NO:9之胺基酸序列，且該VL含有SEQ ID NO:10之胺基酸序列；(c)該VH含有SEQ ID NO:11之胺基酸序列，且該VL含有SEQ ID NO:15之胺基酸序列；(d)該VH含有SEQ ID NO:19之胺基酸序列，且該VL含有SEQ ID NO:23之胺基酸序列；(e)該VH含有SEQ ID NO:27之胺基酸序列，且該VL含有SEQ ID NO:31之胺基酸序列；(f)該VH含有SEQ ID NO:35之胺基酸序列，且該VL含有SEQ ID NO:39之胺基酸序列；(g)該VH含有SEQ ID NO:43之胺基酸序列，且該VL含有SEQ ID NO:47之胺基酸序列；(h)該VH含有SEQ ID NO:63之胺基酸序列，且該VL含有SEQ ID NO:53之胺基酸序列；(i)該VH含有SEQ ID NO:63之胺基酸序列，且該VL含有SEQ ID NO:54之胺基酸序列；(j)該VH含有SEQ ID NO:56之胺基酸序列，且該VL含有SEQ ID NO:52之胺基酸序列；(k)該VH含有SEQ ID NO:55之胺基酸序列，且該VL含有SEQ ID NO:51之胺基酸序列；(l)該VH含有SEQ ID NO:56之胺基酸序列，且該VL含有SEQ ID NO:51之胺基酸序列；(m)該VH含有SEQ ID NO:63之胺基酸序列，且該VL含有SEQ ID NO:51之胺基酸序列；(n)該VH含有SEQ ID NO:64之胺基酸序列，且該VL含有SEQ ID NO:51之胺基酸序列；(o)該VH含有SEQ ID NO:55之胺基酸序列，且該VL含有SEQ ID NO:52之胺基酸序列；(p)該VH含有SEQ ID NO:58之胺基酸序列，且該VL含有SEQ ID NO:52之胺基酸序列；(q)該VH含有SEQ ID NO:60之胺基酸序列，且該VL含有SEQ ID NO:52之胺基酸序列；(r)該VH含有SEQ ID NO:64之胺基酸序列，且該VL含有SEQ ID NO:52之胺基酸序列；(s)該VH含有SEQ ID NO:55之胺基酸序列，且該VL含有SEQ ID NO:53之胺基酸序列；(t)該VH含有SEQ ID NO:57之胺基酸序列，且該VL含有SEQ ID NO:53之胺基酸序列；(u)該VH含有SEQ ID NO:58之胺基酸序列，且該VL含有SEQ ID NO:53之胺基酸序列；(v)該VH含有SEQ ID NO:62之胺基酸序列，且該VL含有SEQ ID NO:53之胺基酸序列；(w)該VH含有SEQ ID NO:64之胺基酸序列，且該VL含有SEQ ID NO:53之胺基酸序列；(x)該VH含有SEQ ID NO:55之胺基酸序列，且該VL含有SEQ ID NO:54之胺基酸序列；(y)該VH含有SEQ ID NO:61之胺基酸序列，且該VL含有SEQ ID NO:54之胺基酸序列；(z)該VH含有SEQ ID NO:62之胺基酸序列，且該VL含有SEQ ID NO:54之胺基酸序列；(aa)該VH含有SEQ ID NO:64之胺基酸序列，且該VL含有SEQ ID NO:54之胺基酸序列；(bb)該VH含有SEQ ID NO:63之胺基酸序列，且該VL含有SEQ ID NO:52之胺基酸序列；(cc)該VH含有SEQ ID NO:188之胺基酸序列，且該VL含有SEQ ID NO:189之胺基酸序列；或(dd)該VH含有SEQ ID NO:188之胺基酸序列，且該VL含有SEQ ID NO:190之胺基酸序列。In some embodiments according to any of the above-isolated anti-TSLP antibody constructs, (a) the VH contains the amino acid sequence of SEQ ID NO: 1, and the VL contains the amino acid sequence of SEQ ID NO: 5; (b) the VH contains the amino acid sequence of SEQ ID NO: 9, and the VL contains the amino acid sequence of SEQ ID NO: 10; (c) the VH contains the amino acid sequence of SEQ ID NO: 11, and the VL contains the amino acid sequence of SEQ ID NO: 15; (d) the VH contains the amino acid sequence of SEQ ID NO: 19, and the VL contains the amino acid sequence of SEQ ID NO: 23; (e) the VH contains the amino acid sequence of SEQ ID NO: 27, and the VL contains the amino acid sequence of SEQ ID NO: 31; (f) the VH contains the amino acid sequence of SEQ ID NO: 35, and the VL contains the amino acid sequence of SEQ ID NO: NO:39; (g) the VH contains the amino acid sequence of SEQ ID NO:43, and the VL contains the amino acid sequence of SEQ ID NO:47; (h) the VH contains the amino acid sequence of SEQ ID NO:63, and the VL contains the amino acid sequence of SEQ ID NO:53; (i) the VH contains the amino acid sequence of SEQ ID NO:63, and the VL contains the amino acid sequence of SEQ ID NO:54; (j) the VH contains the amino acid sequence of SEQ ID NO:56, and the VL contains the amino acid sequence of SEQ ID NO:52; (k) the VH contains the amino acid sequence of SEQ ID NO:55, and the VL contains the amino acid sequence of SEQ ID NO:51; (l) the VH contains the amino acid sequence of SEQ ID NO:56, and the VL contains the amino acid sequence of SEQ ID NO:51; (m) the VH contains the amino acid sequence of SEQ ID NO: NO:63, and the VL contains the amino acid sequence of SEQ ID NO:51; (n) the VH contains the amino acid sequence of SEQ ID NO:64, and the VL contains the amino acid sequence of SEQ ID NO:51; (o) the VH contains the amino acid sequence of SEQ ID NO:55, and the VL contains the amino acid sequence of SEQ ID NO:52; (p) the VH contains the amino acid sequence of SEQ ID NO:58, and the VL contains the amino acid sequence of SEQ ID NO:52; (q) the VH contains the amino acid sequence of SEQ ID NO:60, and the VL contains the amino acid sequence of SEQ ID NO:52; (r) the VH contains the amino acid sequence of SEQ ID NO:64, and the VL contains the amino acid sequence of SEQ ID NO:52; (s) the VH contains the amino acid sequence of SEQ ID NO:55, and the VL contains the amino acid sequence of SEQ ID NO: NO:53; (t) the VH contains the amino acid sequence of SEQ ID NO:57, and the VL contains the amino acid sequence of SEQ ID NO:53; (u) the VH contains the amino acid sequence of SEQ ID NO:58, and the VL contains the amino acid sequence of SEQ ID NO:53; (v) the VH contains the amino acid sequence of SEQ ID NO:62, and the VL contains the amino acid sequence of SEQ ID NO:53; (w) the VH contains the amino acid sequence of SEQ ID NO:64, and the VL contains the amino acid sequence of SEQ ID NO:53; (x) the VH contains the amino acid sequence of SEQ ID NO:55, and the VL contains the amino acid sequence of SEQ ID NO:54; (y) the VH contains the amino acid sequence of SEQ ID NO:61, and the VL contains the amino acid sequence of SEQ ID NO:54; (z) the VH contains the amino acid sequence of SEQ ID NO:62 NO:62, and the VL contains the amino acid sequence of SEQ ID NO:54; (aa) the VH contains the amino acid sequence of SEQ ID NO:64, and the VL contains the amino acid sequence of SEQ ID NO:54; (bb) the VH contains the amino acid sequence of SEQ ID NO:63, and the VL contains the amino acid sequence of SEQ ID NO:52; (cc) the VH contains the amino acid sequence of SEQ ID NO:188, and the VL contains the amino acid sequence of SEQ ID NO:189; or (dd) the VH contains the amino acid sequence of SEQ ID NO:188, and the VL contains the amino acid sequence of SEQ ID NO:190.
在根據上述分離的抗TSLP抗體構築體之任一者的一些實施例中，該抗TSLP抗體部分係選自由以下組成之群組：全長抗體、Fab、Fab’、F(ab’)2、雙抗體及scFv。In some embodiments according to any of the isolated anti-TSLP antibody constructs described above, the anti-TSLP antibody portion is selected from the group consisting of: a full-length antibody, a Fab, a Fab', a F(ab')2 , a diabody, and a scFv.
在根據上述分離的抗TSLP抗體構築體之任一者的一些實施例中，該抗TSLP抗體部分為全長抗體(「抗TSLP全長抗體」)。在一些實施例中，該抗TSLP全長抗體包含含有選自由SEQ ID NO:73-75所組成群組之胺基酸序列的CL。在一些實施例中，該抗TSLP全長抗體包含衍生自人類IgG (例如，人類IgG1、IgG2或IgG4)的Fc域。在一些實施例中，該Fc域係衍生自人類IgG1，其中：i)該Fc域之第一次單元及第二次單元各自含有SEQ ID NO:76-79之任一者之胺基酸序列；ii)該Fc域之第一次單元含有SEQ ID NO:80之胺基酸序列，且該Fc域之第二次單元含有SEQ ID NO:81之胺基酸序列；iii)該Fc域之第一次單元含有SEQ ID NO:81之胺基酸序列，且該Fc域之第二次單元含有SEQ ID NO:80之胺基酸序列；iv)該Fc域之第一次單元含有SEQ ID NO:95之胺基酸序列，且該Fc域之第二次單元含有SEQ ID NO:96之胺基酸序列；或v)該Fc域之第一次單元含有SEQ ID NO:96之胺基酸序列，且該Fc域之第二次單元含有SEQ ID NO:95之胺基酸序列。在一些實施例中，該Fc域之第一次單元及第二次單元各自含有SEQ ID NO:77或79之胺基酸序列。在一些實施例中，該抗TSLP全長抗體包含：i)兩條各自含有SEQ ID NO:104之胺基酸序列的重鏈及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈；ii)兩條各自含有SEQ ID NO:105之胺基酸序列的重鏈及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈；或iii)含有SEQ ID NO:136之胺基酸序列的第一重鏈、含有SEQ ID NO:137之胺基酸序列的第二重鏈及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈。In some embodiments according to any of the anti-TSLP antibody constructs isolated above, the anti-TSLP antibody portion is a full-length antibody ("anti-TSLP full-length antibody"). In some embodiments, the anti-TSLP full-length antibody comprises a CL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-75. In some embodiments, the anti-TSLP full-length antibody comprises an Fc domain derived from human IgG (e.g., human IgG1, IgG2, or IgG4). In some embodiments, the Fc domain is derived from human IgG1, wherein: i) the first unit and the second unit of the Fc domain each contain the amino acid sequence of any one of SEQ ID NOs: 76-79; ii) the first unit of the Fc domain contains the amino acid sequence of SEQ ID NO: 80, and the second unit of the Fc domain contains the amino acid sequence of SEQ ID NO: 81; iii) the first unit of the Fc domain contains the amino acid sequence of SEQ ID NO: 81, and the second unit of the Fc domain contains the amino acid sequence of SEQ ID NO: 80; iv) the first unit of the Fc domain contains the amino acid sequence of SEQ ID NO: 95, and the second unit of the Fc domain contains the amino acid sequence of SEQ ID NO: 96; or v) the first unit of the Fc domain contains the amino acid sequence of SEQ ID NO: 96, and the second unit of the Fc domain contains the amino acid sequence of SEQ ID NO: 95. In some embodiments, the first and second units of the Fc domain each contain an amino acid sequence of SEQ ID NO: 77 or 79. In some embodiments, the anti-TSLP full-length antibody comprises: i) two heavy chains each containing an amino acid sequence of SEQ ID NO: 104 and two heavy chains each containing an amino acid sequence of SEQ ID NO: 103; ii) two heavy chains each containing an amino acid sequence of SEQ ID NO: 105 and two heavy chains each containing an amino acid sequence of SEQ ID NO: 103; or iii) a first heavy chain containing an amino acid sequence of SEQ ID NO: 136, a second heavy chain containing an amino acid sequence of SEQ ID NO: 137, and two heavy chains each containing an amino acid sequence of SEQ ID NO: 103.
在根據上述分離的抗TSLP抗體構築體之任一者的一些實施例中，該抗TSLP抗體部分為scFv (「抗TSLP scFv」)。在一些實施例中，該抗TSLP scFv含有SEQ ID NO:106、107、191及192之任一者之胺基酸序列。In some embodiments according to any of the isolated anti-TSLP antibody constructs described above, the anti-TSLP antibody portion is a scFv ("anti-TSLP scFv"). In some embodiments, the anti-TSLP scFv contains the amino acid sequence of any one of SEQ ID NOs: 106, 107, 191 and 192.
在根據上述分離的抗TSLP抗體構築體之任一者的一些實施例中，該抗TSLP抗體部分為Fab (「抗TSLP Fab」)。在一些實施例中，該抗TSLP Fab包含含有SEQ ID NO:109之胺基酸序列的第一多肽及含有SEQ ID NO:103之胺基酸序列的第二多肽。In some embodiments according to any of the isolated anti-TSLP antibody constructs described above, the anti-TSLP antibody portion is Fab ("anti-TSLP Fab"). In some embodiments, the anti-TSLP Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 109 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 103.
在根據上述分離的抗TSLP抗體構築體之任一者的一些實施例中，該分離的抗TSLP抗體構築體係單特異性。In some embodiments according to any of the above isolated anti-TSLP antibody constructs, the isolated anti-TSLP antibody construct is monospecific.
在根據上述分離的抗TSLP抗體構築體之任一者的一些實施例中，該分離的抗TSLP抗體構築體為多特異性抗TSLP抗體構築體，其包含特異性地辨識第二標靶抗原(例如，IL-13)的第二抗體部分。在一些實施例中，該第二抗體部分係選自由以下組成之群組：全長抗體、Fab、Fab’、F(ab’)2、sdAb、雙抗體及scFv。在一些實施例中，該第二抗體部分為scFv。在一些實施例中，該第二抗體部分為Fab。在一些實施例中，該第二抗體部分為全長抗體。在一些實施例中，該抗TSLP抗體部分與該第二抗體部分係經由連接子彼此融合，諸如含有GG及SEQ ID NO:14、16-18、97-99及163-172之任一者(例如GG及SEQ ID NO:98-99之任一者)之胺基酸序列的連接子。In some embodiments according to any of the above-described isolated anti-TSLP antibody constructs, the isolated anti-TSLP antibody construct is a multispecific anti-TSLP antibody construct comprising a second antibody portion that specifically recognizes a second target antigen (e.g., IL-13). In some embodiments, the second antibody portion is selected from the group consisting of a full-length antibody, Fab, Fab', F(ab')2 , sdAb, a diabody, and a scFv. In some embodiments, the second antibody portion is a scFv. In some embodiments, the second antibody portion is a Fab. In some embodiments, the second antibody portion is a full-length antibody. In some embodiments, the anti-TSLP antibody portion and the second antibody portion are fused to each other via a linker, such as a linker containing an amino acid sequence of GG and any one of SEQ ID NOs: 14, 16-18, 97-99, and 163-172 (e.g., GG and any one of SEQ ID NOs: 98-99).
亦提供醫藥組成物，其包含本文所述之分離的抗TSLP抗體構築體之任一者及醫藥上可接受之載體。Also provided are pharmaceutical compositions comprising any of the isolated anti-TSLP antibody constructs described herein and a pharmaceutically acceptable carrier.
在本發明之另一態樣中，提供一種治療個體(例如，人類)之發炎性疾病的方法，其包含向個體投予有效量之本文所述之分離的抗TSLP抗體構築體之任一者或其醫藥組成物(例如，本文所述之醫藥組成物之任一者)。在一些實施例中，該發炎性疾病為氣喘、異位性皮膚炎或慢性阻塞性肺病(chronic obstructive pulmonary disease，COPD)。In another aspect of the present invention, a method for treating an inflammatory disease in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of any of the isolated anti-TSLP antibody constructs described herein or a pharmaceutical composition thereof (e.g., any of the pharmaceutical compositions described herein). In some embodiments, the inflammatory disease is asthma, atopic dermatitis, or chronic obstructive pulmonary disease (COPD).
亦提供編碼上述分離的抗TSLP抗體構築體之任一者之多肽部分的分離的核酸、含有此類分離的核酸的載體及含有此類分離的核酸或載體的宿主細胞。Also provided are isolated nucleic acids encoding the polypeptide portion of any of the above-described isolated anti-TSLP antibody constructs, vectors containing such isolated nucleic acids, and host cells containing such isolated nucleic acids or vectors.
亦提供製造抗TSLP抗體構築體的方法，其包含：i)在適合表現該抗TSLP抗體構築體的條件下培養含有編碼上述抗TSLP抗體構築體之任一者之多肽部分的分離的核酸或載體的宿主細胞或編碼上述抗TSLP抗體構築體之任一者的宿主細胞之任一者；及ii)從該宿主細胞或從細胞培養物(例如，從細胞培養基)中獲得表現的抗TSLP抗體構築體。Also provided is a method for producing an anti-TSLP antibody construct comprising: i) culturing a host cell containing an isolated nucleic acid or vector encoding a polypeptide portion of any of the above-described anti-TSLP antibody constructs or any of the host cells encoding any of the above-described anti-TSLP antibody constructs under conditions suitable for expressing the anti-TSLP antibody construct; and ii) obtaining the expressed anti-TSLP antibody construct from the host cell or from a cell culture (e.g., from a cell culture medium).
本發明之此等及其他態樣及優點將由後續實施方式及所附申請專利範圍而變得顯而易見。應理解，可組合本文所述之各種實施例的一者、一些或所有性質以形成本發明之其他實施例。These and other aspects and advantages of the present invention will become apparent from the subsequent implementation and the appended patent claims. It should be understood that one, some or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention.
本文提及的所有出版品、專利、專利申請案及公開的專利申請案的揭露內容皆通過引用整體併入本文中。The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety.
本申請案提供新穎的抗TSLP抗體構築體，其包括單特異性及多特異性抗TSLP抗體構築體。此等新穎的抗TSLP抗體構築體具有幾個優勢。首先，與US-FDA批准的參考抗TSLP抗體相比，該新穎的抗TSLP抗體構築體表現出提高的效力及完全阻斷TSLP傳訊的能力，其可僅部分地阻斷TSLP傳訊。舉例而言，本文所述之抗TSLP抗體構築體已證實具有完全阻斷免疫細胞(諸如週邊血液單核細胞(PBMC)及樹突細胞(DC))分泌CCL17的活性，其表明完全終止TSLP傳訊。不受理論的束縛，據信本文所述之抗TSLP抗體構築體與參考抗TSLP抗體所結合的表位不同。不同於僅阻斷TSLP/TSLPR相互作用的參考抗TSLP抗體，據信本文所述之抗TSLP抗體構築體可阻斷TSLP與TSLPR及IL-7Rα的相互作用，從而提供更強、更理想的TSLP阻斷特徵。其次，本文所述之抗TSLP抗體構築體對人類TSLP表現出非常強的結合親和力，其與US-FDA批准的參考抗TSLP抗體相當，且甚至更強。再者，本文所述之抗TSLP抗體構築體與食蟹獼猴TSLP具有交叉反應性及強結合親和力，其可促進將食蟹獼猴毒性及功效研究的結果推斷至人類臨床研究。The present application provides novel anti-TSLP antibody constructs, including monospecific and multispecific anti-TSLP antibody constructs. These novel anti-TSLP antibody constructs have several advantages. First, compared with the reference anti-TSLP antibodies approved by the US-FDA, the novel anti-TSLP antibody constructs show improved efficacy and the ability to completely block TSLP signaling, which can only partially block TSLP signaling. For example, the anti-TSLP antibody constructs described herein have been shown to have the activity of completely blocking the secretion of CCL17 by immune cells (such as peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs), which indicates complete termination of TSLP signaling. Without being bound by theory, it is believed that the anti-TSLP antibody constructs described herein bind to different epitopes than the reference anti-TSLP antibodies. Unlike the reference anti-TSLP antibodies that only block the TSLP/TSLPR interaction, it is believed that the anti-TSLP antibody constructs described herein can block the interaction of TSLP with TSLPR and IL-7Rα, thereby providing a stronger and more ideal TSLP blocking feature. Secondly, the anti-TSLP antibody constructs described herein exhibit very strong binding affinity for human TSLP, which is comparable to and even stronger than the reference anti-TSLP antibody approved by the US-FDA. Furthermore, the anti-TSLP antibody constructs described herein have cross-reactivity and strong binding affinity with cynomolgus macaque TSLP, which can facilitate the extrapolation of the results of cynomolgus macaque toxicity and efficacy studies to human clinical studies.
因此，在一態樣中，本發明提供一種分離的抗體構築體(「抗TSLP抗體構築體」)，其包含特異性地辨識TSLP的抗體部分(「抗TSLP抗體部分」)。在一些實施例中，該抗TSLP抗體部分為全長抗體(「抗TSLP全長抗體」)。在一些實施例中，該抗TSLP抗體部分為scFv (「抗TSLP scFv」)。在一些實施例中，該抗TSLP抗體部分為Fab (「抗TSLP Fab」)。在一些實施例中，該分離的抗TSLP抗體構築體係單特異性。在一些實施例中，該分離的抗TSLP抗體構築體為多特異性抗TSLP抗體構築體，其進一步包含特異性地辨識第二標靶抗原(例如，IL-13)的第二抗體部分。亦提供分離的抗TSLP抗體構築體，其可與分離的抗TSLP抗體構築體之任一者及/或本文所述之抗TSLP抗體部分競爭結合至TSLP。Thus, in one aspect, the present invention provides an isolated antibody construct ("anti-TSLP antibody construct") comprising an antibody portion that specifically recognizes TSLP ("anti-TSLP antibody portion"). In some embodiments, the anti-TSLP antibody portion is a full-length antibody ("anti-TSLP full-length antibody"). In some embodiments, the anti-TSLP antibody portion is a scFv ("anti-TSLP scFv"). In some embodiments, the anti-TSLP antibody portion is a Fab ("anti-TSLP Fab"). In some embodiments, the isolated anti-TSLP antibody construct is monospecific. In some embodiments, the isolated anti-TSLP antibody construct is a multispecific anti-TSLP antibody construct, which further comprises a second antibody portion that specifically recognizes a second target antigen (e.g., IL-13). Also provided are isolated anti-TSLP antibody constructs that can compete for binding to TSLP with any of the isolated anti-TSLP antibody constructs and/or anti-TSLP antibody portions described herein.
進一步提供包含本文所述之分離的抗TSLP抗體構築體之任一者的醫藥組成物及套組，以及本文所述之分離的抗TSLP抗體構築體之任一者或其醫藥組成物的使用方法，諸如用於治療發炎性疾病，例如氣喘、異位性皮膚炎或COPD。I.定義Further provided are pharmaceutical compositions and kits comprising any of the isolated anti-TSLP antibody constructs described herein, and methods of using any of the isolated anti-TSLP antibody constructs described herein or their pharmaceutical compositions, such as for treating inflammatory diseases, such as asthma, atopic dermatitis, or COPD.I.Definitions
如本文所用，術語「治療」意指經設計以改變所治療之個體或細胞在臨床病理學之病程期間的天然過程的臨床介入。所需治療效果包括降低疾病進展速率、改善或緩和疾病狀態及緩解或改善預後。舉例而言，若與發炎性疾病相關之一或多種症狀得到減輕或消除，包括但不限於降低局部或全身發炎、減少由疾病引起之症狀、提高罹患疾病者之生活品質、降低治療疾病所需之其他藥物的劑量等，則個體為成功「治療的」。As used herein, the term "treatment" means a clinical intervention designed to alter the natural course of the treated individual or cell during the course of a clinical pathology. Desired therapeutic effects include a reduction in the rate of disease progression, an improvement or alleviation of the disease state, and a relief or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with an inflammatory disease are reduced or eliminated, including but not limited to a reduction in local or systemic inflammation, a reduction in symptoms caused by the disease, an improvement in the quality of life of a person suffering from the disease, a reduction in the dosage of other drugs required to treat the disease, etc.
如本文所用，「有效量」意指有效治療受試者(諸如個體，例如人類)之疾病或病症的藥劑或藥物的量。在發炎性疾病的情況下，該藥劑的有效量可減少活性免疫細胞的數量；減少促發炎性細胞激素的量；局部性地及/或全身性地抑制(亦即，在一定程度上減緩，較佳地停止)發炎性免疫細胞活性；及/或在一定程度上緩解與發炎性疾病相關的一或多種症狀。如在臨床背景中所理解的，藥物、化合物或藥物組成物之有效量可與或不與另一藥物、化合物或醫藥組成物聯合而達成。因此，在投予一或多種治療劑之上下文中可考慮「有效量」，且若單一藥劑與一或多種其他藥劑聯合而可達到或已達到所需結果，則該單一藥劑可視為以有效量給出。As used herein, "effective amount" means an amount of an agent or drug that is effective in treating a disease or condition in a subject (e.g., an individual, such as a human). In the case of an inflammatory disease, an effective amount of the agent may reduce the number of active immune cells; reduce the amount of pro-inflammatory cytokines; inhibit (i.e., reduce to some extent, preferably stop) inflammatory immune cell activity locally and/or systemically; and/or alleviate to some extent one or more symptoms associated with the inflammatory disease. As understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may be achieved with or without combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if it, in combination with one or more other agents, can achieve or have achieved the desired result.
如本文所用，「個體」或「受試者」意指哺乳動物，包括但不限於人類、牛、馬、貓、犬、囓齒動物或靈長類動物。在一些實施例中，該個體為人類。As used herein, "individual" or "subject" refers to mammals, including but not limited to humans, cows, horses, cats, dogs, rodents or primates. In some embodiments, the individual is a human.
術語「抗體」係以最廣泛意義使用且具體地涵蓋單株抗體(包括全長單株抗體)、多特異性抗體(例如，雙特異性抗體)及抗體片段，只要其等展現出所需生物活性或功能。如本文所用，術語「免疫球蛋白(Ig)」及「抗體」可互換使用。The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity or function. As used herein, the terms "immunoglobulin (Ig)" and "antibody" can be used interchangeably.
如本文所用，術語「全長抗體」意指呈其實質上完整形式、不為如下文所定義之抗體片段的抗體。特別地，該等術語意指具有含Fc區之重鏈的抗體。全長抗體通常為約150,000道耳吞的異四聚化醣蛋白，其由兩條相同的輕(L)鏈及兩條相同的重(H)鏈組成。As used herein, the term "full-length antibody" refers to an antibody in its substantially complete form, not as an antibody fragment as defined below. In particular, the term refers to an antibody having a heavy chain containing an Fc region. A full-length antibody is typically a heterotetrameric glycoprotein of about 150,000 daltons, which consists of two identical light (L) chains and two identical heavy (H) chains.
術語「恆定域」意指具有相對於含有抗原結合位點之免疫球蛋白之其他部分(可變域)更保留之胺基酸序列的免疫球蛋白分子之部分。恆定域含有重鏈之CH1、CH2及CH3域(統稱為CH)及輕鏈之CHL (或CL)域。The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to the rest of the immunoglobulin (variable domain) that contains the antigen binding site. The constant domain contains theCH1 ,CH2 , and CH3 domains (collectively referred to as CH) of the heavy chain and theCHL (or CL) domain of the light chain.
抗體之「可變區」或「可變域」意指抗體之重鏈或輕鏈的胺基末端域。重鏈之可變域可稱為「VH」。輕鏈之可變域可稱為「VL」。此等域通常為抗體之最可變部分且含有抗原結合位點。The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are generally the most variable parts of the antibody and contain the antigen binding site.
術語「可變」意指以下事實：可變域之某些部分在抗體當中在序列方面廣泛地不同，且用於各特定抗體對於其特定抗原之結合及特異性。然而，可變性並非均勻分佈於抗體的整個可變域中。其集中在輕鏈及重鏈可變域中之三個稱作高度可變區(HVR，亦稱為CDR)的區段中。可變域中保留性較高之部分稱為框架區(FR)。天然重鏈及輕鏈之可變域各自包含四個 FR，主要採用 β-折疊構型，藉由三個HVR連接，其形成連接β-折疊結構之環並在一些情況下形成β-折疊結構之一部分。各鏈中之HVR係藉由FR緊密地結合在一起，且與另一鏈之HVR一起，有助於形成抗體之抗原結合位點(參見Kabat等人，Sequences of Proteins of Immunological Interest，第五版，National Institute of Health，Bethesda，Md. (1991))。恆定域不直接參與抗體與抗原的結合，但展現出各種效應功能，諸如抗體依賴性細胞毒性中抗體的參與。除非另有說明，本文所述之恆定域的殘基係根據EU編號。The term "variable" refers to the fact that certain parts of the variable domain differ widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its specific antigen. However, the variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called highly variable regions (HVRs, also called CDRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of the native heavy and light chains each contain four FRs, which mainly adopt a β-sheet configuration, connected by three HVRs, which form loops connecting the β-sheet structure and in some cases form part of the β-sheet structure. The HVRs in each chain are tightly bound together by the FRs and, together with the HVRs of the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The homeodomain is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity. Unless otherwise indicated, the homeodomain residues described herein are according to the EU numbering.
當在本文中使用時，術語「高度可變區」、「HVR」或「HV」意指抗體可變域的序列高度變異及/或形成結構上定義的環圈的區域。一般而言，抗體包含六個HVR；三個位於VH中(HVR-H1、HVR-H2、HVR-H3)，且三個位於VL中(HVR-L1、HVR-L2、HVR-L3)。在天然抗體中，HVR-H3及HVR-L3在六個HVR中表現出最多的多樣性，特別是據信H3在賦予抗體優異特異性方面發揮獨特的作用。參見，例如Xu等人，Immunity 13:37-45 (2000)；Johnson及Wu，在Methods in Molecular Biology 248:1-25 (Lo，編輯，Human Press，Totowa，N.J.，2003)中。實際上，在不存在輕鏈的情況下，僅由重鏈組成的天然駱駝科抗體具有功能及穩定性。參見，例如Hamers-Casterman等人，Nature 363:446-448 (1993)；Sheriff等人，Nature Struct. Biol. 3:733-736 (1996)。HVR亦稱為「CDR」或「互補決定區」。As used herein, the term "hypervariable region", "HVR" or "HV" refers to the region of the antibody variable domain that is highly variable in sequence and/or forms a structurally defined loop. Generally, an antibody comprises six HVRs; three located in VH (HVR-H1, HVR-H2, HVR-H3) and three located in VL (HVR-L1, HVR-L2, HVR-L3). In natural antibodies, HVR-H3 and HVR-L3 show the most diversity among the six HVRs, and H3 in particular is believed to play a unique role in conferring superior specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). In fact, in the absence of the light chain, natural camel family antibodies composed only of the heavy chain are functional and stable. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). HVRs are also called "CDRs" or "complementary determining regions."
免疫球蛋白可變區之結構及位置可參考Kabat, E. A.等人，Sequences of Proteins of Immunological Interest. 第4版，US Department of Health and Human Services. 1987及現可於網際網路(immuno.bme.nwu.edu)上獲得之其更新來確定。可以使用任何免疫球蛋白可變區定義方案，包括但不限於 Kabat、Chothia、AbM、Contact或IMGT。The structure and location of immunoglobulin variable regions can be determined by reference to Kabat, E. A. et al., Sequences of Proteins of Immunological Interest. 4th edition, US Department of Health and Human Services. 1987 and updates thereof currently available on the Internet (immuno.bme.nwu.edu). Any immunoglobulin variable region definition scheme may be used, including but not limited to Kabat, Chothia, AbM, Contact or IMGT.
如本文所用，術語「CDR」或「互補決定區」意指重鏈及輕鏈多肽之可變區內發現的非連續抗原結合位點。在文獻Kabat等人，J. Biol. Chem. 252:6609-6616 (1977)；Kabat等人，U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991)；Chothia等人，J. Mol. Biol. 196:901-917 (1987)；Al-Lazikani B.等人，J. Mol. Biol., 273: 927-948 (1997)；MacCallum等人，J. Mol. Biol. 262:732-745 (1996)；Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008)；Lefranc M.P.等人，Dev. Comp. Immunol., 27: 55-77 (2003)；及Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001)中已描述此等特定區域，其中當彼此之間互相比較時，此等定義包括胺基酸殘基的重合或子集。儘管如此，應用任何一種定義方式來指示抗體或移植抗體或其變體之CDR，皆包括在本文所定義及使用的術語範疇內。表A中列出由上述引用的各篇參考文獻所定義的CDR所包括的胺基酸殘基，以作為比較。CDR預測的演算法及結合介面在本領域為已知的，包括例如：Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008)；Ehrenmann F.等人，Nucleic Acids Res., 38: D301-D307 (2010)；及Adolf-Bryfogle J.等人，Nucleic Acids Res., 43: D432-D438 (2015)。本段中所引用的參考文獻的內容以其整體引用併入本文中，以用於本申請案及可能包含在本文的一或多個請求項中。除非另有說明，本文提供的CDR的胺基酸殘基係基於Kabat進行描述。表A. CDR定義
「框架」或「FR」殘基是除了本文定義的HVR或CDR殘基之外的那些可變域殘基。除非另有說明，本文中FR的胺基酸殘基是基於Kabat編號描述的。"Framework" or "FR" residues are those variable domain residues other than HVR or CDR residues as defined herein. Unless otherwise indicated, the amino acid residues of FRs herein are described based on Kabat numbering.
任何哺乳動物物種之抗體(免疫球蛋白)的「輕鏈」皆可根據其恆定域之胺基酸序列被歸類為兩種截然不同的類型(稱為卡帕(「κ」)及拉目達(「λ」))之一。The "light chains" of antibodies (immunoglobulins) of any mammalian species can be classified into one of two distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their homeodomains.
如本文所用，術語IgG 「同型」或「子類」意指由其恆定區之化學及抗原特徵所定義之免疫球蛋白子類之任一者。As used herein, the term IgG "isotype" or "subclass" refers to any of the immunoglobulin subclasses defined by the chemical and antigenic characteristics of its constant regions.
根據其重鏈恆定域之胺基酸序列，抗體(免疫球蛋白)可歸類為不同的類別。有五大類免疫球蛋白：IgA、IgD、IgE、IgG及IgM，且彼等中之數種可進一步分為子類(同型)，例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。對應於不同類別之免疫球蛋白的重鏈恆定域分別稱為 α、δ、ɛ、γ 及 μ。不同類別的免疫球蛋白之次單位結構及三維構型為熟習的，且一般描述於例如Abbas等人之Cellular and Mol. Immunology，第 4 版(W.B.Saunders, Co., 2000)。抗體可為較大融合分子之一部分，其藉由抗體與一或多種其他蛋白質或肽的共價或非共價締合形成。Antibodies (immunoglobulins) can be classified into different classes according to the amino acid sequences of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ɛ, γ and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are familiar and generally described in, for example, Abbas et al., Cellular and Mol. Immunology, 4th edition (W.B.Saunders, Co., 2000). An antibody may be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
「抗體片段」包含完整抗體之一部分，較佳地包含其抗原結合區。在一些實施例中，本文所述之抗體片段為抗原結合片段。抗體片段或抗原結合片段之實例包括Fab、Fab'、F(ab')2及Fv片段(諸如單鏈可變片段，scFv)；雙功能抗體；線性抗體；單鏈抗體(sdAb)分子；及由抗體片段形成之多特異性抗體。"Antibody fragments" include a portion of an intact antibody, preferably including its antigen binding region. In some embodiments, the antibody fragments described herein are antigen binding fragments. Examples of antibody fragments or antigen binding fragments include Fab, Fab', F(ab')2 and Fv fragments (such as single-chain variable fragments, scFv); bifunctional antibodies; linear antibodies; single-chain antibody (sdAb) molecules; and multispecific antibodies formed from antibody fragments.
抗體之番木瓜蛋白酶(papain)消化產生兩個相同的抗原結合片段，稱為「Fab」片段，各自具有單一抗原結合位點，以及殘餘「Fc」片段，其名稱反映出其容易結晶的能力。胃蛋白酶(pepsin)處理產生F(ab')2片段，其具有兩個抗原組合位點且仍能夠與抗原交聯。Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking to antigen.
「Fv」為含有整個抗原結合位點的最小抗體片段。在一實施例中，雙鏈Fv物種由一個重鏈及一個輕鏈可變域以緊密、非共價結合之二聚體組成。在單鏈Fv (scFv)物種中，一個重鏈及一個輕鏈可變域可藉由撓性肽連接子共價連接，使得輕鏈及重鏈可結合於類似於雙鏈Fv物種中之結構的「二聚體」結構中。在此組態中，各可變域之三個HVR相互作用以界定VH-VL二聚體表面上之抗原結合位點。六個HVR共同地賦予抗體以抗原結合特異性。然而，即使單一可變域 (或僅包含對抗原具有特異性之三個HVR的Fv的一半)亦具有辨識及結合抗原的能力，儘管其親和力低於整個結合位點。"Fv" is the smallest antibody fragment that contains the entire antigen binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy chain and one light chain variable domain can be covalently linked by a flexible peptide linker so that the light chain and the heavy chain can be combined in a "dimer" structure similar to the structure in the two-chain Fv species. In this configuration, the three HVRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. The six HVRs collectively give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
Fab片段具有兩條多肽鏈，其含有重鏈及輕鏈可變域(VH、VL)，且亦含有輕鏈(CL)之恆定域及重鏈之第一恆定域 (CH1)。Fab’片段與Fab片段不同之處在於，在重鏈CH1域之羧基端添加幾個殘基，包括來自抗體鉸鏈區之一或多個半胱胺酸。Fab’-SH為其中恆定域之半胱胺酸殘基攜有游離硫醇基之Fab’在本文中的名稱。F(ab’)2抗體片段最初係以其間具有鉸鏈半胱胺酸之Fab’片段對形式產生。抗體片段之其他化學耦聯亦為已知的。Fab fragments have two polypeptide chains containing heavy and light chain variable domains (VH, VL), and also contain the homeostatic domain of the light chain (CL) and the first homeostatic domain of the heavy chain (CH1 ). Fab' fragments differ from Fab fragments in that several residues are added to the carboxyl terminus of the heavy chainCH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residues of the homeostatic domains carry a free thiol group. F(ab')2 antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
「單鏈Fv」或「scFv」抗體片段包含抗體之VH域及VL域，其中此等域存在於單一多肽鏈中。一般而言，scFv多肽在VH域及VL域之間進一步包含多肽連接子，其使得scFv能夠形成用於抗原結合之所需結構。關於scFv之回顧，參見例如Plückthun，The Pharmacology of Monoclonal Antibodies，Springer Berlin Heidelberg，1994. 269-315。"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Plückthun, The Pharmacology of Monoclonal Antibodies, Springer Berlin Heidelberg, 1994. 269-315.
「Fc」片段包含兩條重鏈的羧基端部分由雙硫鍵緊密連接在一起。抗體之效應功能由Fc區中的序列決定，該區域亦為某些類型細胞上發現的Fc受體(FcR)所辨識的區域。The "Fc" fragment consists of the carboxyl-terminal portions of the two heavy chains tightly linked together by disulfide bonds. The effector function of the antibody is determined by the sequence in the Fc region, which is also the region recognized by the Fc receptor (FcR) found on certain types of cells.
如本文所用，術語「單株抗體」意指獲自實質上同質之抗體群體的抗體，例如構成該群體之個別抗體為相同的，除了可少量存在之可能的突變，例如天然存在之突變。因此，修飾語「單株」表明抗體不為不同抗體之混合物的特徵。在一些實施例中，此類單株抗體通常包括含有結合標靶之多肽序列的抗體，其中該標靶結合多肽序列係藉由包括自複數個多肽序列選擇單一標靶結合多肽序列之方法獲得。舉例而言，選擇方法可為從複數個殖株(諸如一組融合瘤殖株、噬菌體殖株或重組DNA殖株)中選擇獨特的殖株。應理解，所選標靶結合序列可經進一步改變以例如改善針對標靶之親和力、人源化標靶結合序列、改善其於細胞培養物中之產生、降低其活體內免疫原性、產生多特異性抗體等，且包含經改變標靶結合序列之抗體亦為本發明之單株抗體。與通常包括針對不同決定位(表位)之不同抗體的多株抗體製備物相反，單株抗體製備物之各個單株抗體係針對於抗原上的單一決定位。除其特異性以外，單株抗體製備物亦為有利的，係因其通常未被其他免疫球蛋白污染。As used herein, the term "monoclonal antibody" means an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations that may be present in small amounts, such as naturally occurring mutations. Thus, the modifier "monoclonal" indicates the characteristic that the antibody is not a mixture of different antibodies. In some embodiments, such monoclonal antibodies generally include antibodies comprising a polypeptide sequence that binds a target, wherein the target binding polypeptide sequence is obtained by a method that includes selecting a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection method can be to select a unique strain from a plurality of strains (such as a set of fusion tumor strains, phage strains, or recombinant DNA strains). It should be understood that the selected target binding sequence can be further altered to, for example, improve affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its in vivo immunogenicity, generate multispecific antibodies, etc., and antibodies comprising the altered target binding sequence are also monoclonal antibodies of the present invention. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibody preparations are also advantageous because they are generally not contaminated by other immunoglobulins.
修飾詞「單株」表明抗體之特徵係獲自實質上同源之抗體群體，且不應解釋為需要藉由任何特定方法產生該抗體。舉例而言，待根據本發明使用的單株抗體可藉由多種技術製備，包括例如：融合瘤方法(例如，Kohler及Milstein，Nature 256:495-97 (1975)；Hongo等人，Hybridoma 14 (3): 253-260 (1995)，Harlow等人，Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press，第2版，1988)；Hammerling等人，Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier，N.Y.，1981))、重組DNA方法(參見例如，美國專利號4,816,567)、噬菌體展示技術(參見例如Clackson等人，Nature 352: 624-628 (1991)；Marks等人，J. Mol. Biol. 222: 581-597 (1992)；Sidhu等人，J. Mol. Biol. 338(2): 299-310 (2004)；Lee等人，J. Mol. Biol. 340(5): 1073-1093 (2004)；Fellouse，Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004)；及Lee等人，J. Immunol. Methods 284(1-2): 119-132 (2004))，以及用於在具有人類免疫球蛋白基因座或編碼人類免疫球蛋白序列的基因之一部分或全部的動物中產生人類或人類樣抗體的技術(參見例如WO 1998/24893；WO 1996/34096；WO 1996/33735；WO 1991/10741；Jakobovits等人，Proc. Natl. Acad. Sci. USA 90: 2551 (1993)；Jakobovits等人，Nature 362: 255-258 (1993)；Bruggemann等人，Year in Immunol. 7:33 (1993)；美國專利號5,545,807；5,545,806；5,569,825；5,625,126；5,633,425；及5,661,016；Marks等人，Bio/Technology 10: 779-783 (1992)；Lonberg等人，Nature 368: 856-859 (1994)；Morrison，Nature 368: 812-813 (1994)；Fishwild等人，Nature Biotechnol. 14: 845-851 (1996)；Neuberger，Nature Biotechnol. 14: 826 (1996)；及Lonberg及Huszar，Intern. Rev. Immunol. 13: 65-93 (1995))。The modifier "monoclonal" indicates that the antibody is characterized as being derived from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the present invention can be prepared by a variety of techniques, including, for example, the hypoderma method (e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hongo et al., Hybridoma 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004)), and techniques for producing human or human-like antibodies in animals having a portion or all of a human immunoglobulin locus or a gene encoding a human immunoglobulin sequence (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995)).
本文中之單株抗體具體地包括「嵌合」抗體，其中重鏈及/或輕鏈之一部分與衍生自特定物種或隸屬特定抗體類別或子類之抗體的對應序列一致或同源，而各鏈之其餘部分與衍生自另一物種或隸屬另一抗體類別或子類之抗體以及此類抗體之片段的對應序列一致或同源，只要其展現出所需之生物活性即可(參見例如，美國專利號4,816,567；及Morrison等人，Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984))。嵌合抗體包括PRIMATIZED®抗體，其中該抗體之抗原結合區係衍生自藉由例如利用感興趣之抗原使獼猴免疫而產生的抗體。Monoclonal antibodies herein specifically include "chimeric" antibodies, in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of each chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (see, e.g., U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies, in which the antigen binding region of the antibody is derived from an antibody generated by, for example, immunizing a macaque with an antigen of interest.
非人類(例如，鼠類)抗體之「人源化」形式為含有衍生自非人類免疫球蛋白之最小序列的嵌合抗體。在一實施例中，人源化抗體為人類免疫球蛋白(受體抗體)，其中來自受體HVR的殘基經來自具有所需特異性、親和力及/或容量之非人類物種(供體抗體)，諸如小鼠、大鼠、兔或非人類靈長類動物之HVR的殘基置換。在一些情況下，人類免疫球蛋白之FR殘基經相應之非人類殘基置換。此外，人源化抗體可包含不存在於受體抗體或供體抗體中的殘基。可進行此等修飾以進一步優化抗體效能。一般而言，人源化抗體將包含至少一個且通常兩個可變域中之實質上所有可變域，其中所有或實質上所有高變環對應於非人類免疫球蛋白之高變環，且所有或實質上所有FR為人類免疫球蛋白序列之FR。人源化抗體可選地亦將包含免疫球蛋白恆定區(Fc)之至少一部分，該恆定區通常為人免疫球蛋白之恆定區。關於更多細節，參見例如Jones等人，Nature 321:522-525 (1986)；Riechmann等人，Nature 332:323-329 (1988)；及Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)。亦參見例如Vaswani及Hamilton，Ann. Allergy, Asthma & Immunol. 1:105-115 (1998)；Harris，Biochem. Soc. Transactions 23:1035-1038 (1995)；Hurle及Gross，Curr. Op. Biotech. 5:428-433 (1994)；及美國專利號6,982,321及7,087,409。"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies containing minimal sequence derived from non-human immunoglobulins. In one embodiment, a humanized antibody is a human immunoglobulin (acceptor antibody) in which residues from the acceptor HVR are replaced with residues from HVRs of a non-human species (donor antibody) having the desired specificity, affinity, and/or capacity, such as mouse, rat, rabbit, or non-human primate. In some cases, FR residues of human immunoglobulins are replaced with corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in the acceptor antibody or the donor antibody. Such modifications may be performed to further optimize antibody performance. In general, humanized antibodies will comprise substantially all of the variable domains in at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FRs are FRs of human immunoglobulin sequences. Humanized antibodies may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), which is typically a constant region of a human immunoglobulin. For more details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.
「人類抗體」為具有以下胺基酸序列之抗體：對應於由人類所產生及/或已使用製造如本文所揭露之人類抗體之技術中之任一者所製造之抗體的胺基酸序列。該人類抗體的定義具體地排除包含非人類抗原結合殘基的人源化抗體。人類抗體可使用本領域中已知之各種技術(包括噬菌體顯示庫)來產生。Hoogenboom及Winter，J. Mol. Biol. 227:381 (1991)；Marks等人，J. Mol. Biol. 222:581 (1991)。Cole等人，Monoclonal Antibodies and Cancer Therapy，Alan R. Liss，77 (1985)；Boerner等人，J. Immunol. 147(1):86-95 (1991)中所述之方法亦可用於製備人類單株抗體。亦參見van Dijk及van de Winkel，Curr. Opin. Pharmacol. 5: 368-74 (2001)。可藉由將抗原投予轉基因動物(例如，經免疫之異源小鼠)來製備人類抗體，該轉基因動物已被改造以回應於抗原攻擊而產生此類抗體，但其內源性基因座已失去功能(參見例如，美國專利號6,075,181及6,150,584，其等係關於 XENOMOUSETM技術)。亦參見例如，Li等人，Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006)，其係關於經由人類B細胞融合瘤技術生成之人類抗體。A "human antibody" is an antibody having an amino acid sequence that corresponds to an antibody produced by a human and/or that has been produced using any of the techniques for producing human antibodies as disclosed herein. The definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 77 (1985); Boerner et al., J. Immunol. 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal (e.g., an immunized xenogeneic mouse) that has been engineered to produce such antibodies in response to antigenic challenge but in which the endogenous loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, etc., regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006) regarding human antibodies generated by human B cell fusion tumor technology.
如本文所用，術語「共價連接」意指 經由一或多個化學鍵直接連接或經由一或多個連接子間接連接。任何合適的化學鍵皆可用於產生直接連接，包括但不限於共價鍵(諸如肽鍵及雙硫鍵)或非共價鍵(諸如氫鍵、疏水鍵、離子鍵或凡得瓦鍵)。As used herein, the term "covalently linked" means directly linked via one or more chemical bonds or indirectly linked via one or more linkers. Any suitable chemical bond can be used to create a direct link, including but not limited to covalent bonds (such as peptide bonds and disulfide bonds) or non-covalent bonds (such as hydrogen bonds, hydrophobic bonds, ionic bonds or Van der Waals bonds).
如本文所用，「共價鍵」意指兩個原子之間共用一或多個電子的穩定的鍵。共價鍵之實例包括但不限於肽鍵及雙硫鍵。如本文所用，「肽鍵」意指胺基酸的羧基與相鄰胺基酸的胺基之間形成的共價鍵。如本文所用，「雙硫鍵」意指兩個硫原子之間形成的共價鍵，諸如重鏈片段CH1與輕鏈片段CL藉由一或多個雙硫鍵結合。藉由連接兩個片段中的硫醇基，兩個片段之間可形成一或多個雙硫鍵。在一些實施例中，重鏈片段與輕鏈片段的一或多個半胱胺酸之間可分別形成一或多個雙硫鍵。可藉由兩個硫醇基氧化而形成雙硫鍵。在一些實施例中，該共價連接係藉由共價鍵直接連接。在一些實施例中，該共價連接係藉由肽鍵或雙硫鍵直接連接。As used herein, "covalent bond" means a stable bond between two atoms that share one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, "peptide bond" means a covalent bond formed between the carboxyl group of an amino acid and the amine group of an adjacent amino acid. As used herein, "disulfide bond" means a covalent bond formed between two sulfur atoms, such as the heavy chain fragmentCH1 and the light chain fragment CL are bound by one or more disulfide bonds. One or more disulfide bonds can be formed between the two fragments by connecting the thiol groups in the two fragments. In some embodiments, one or more disulfide bonds can be formed between one or more cysteine amino acids in the heavy chain fragment and the light chain fragment, respectively. A disulfide bond may be formed by oxidation of two thiol groups. In some embodiments, the covalent link is directly linked via a covalent bond. In some embodiments, the covalent link is directly linked via a peptide bond or a disulfide bond.
如本文所用，術語「結合」、「特異性地結合至」、「特異性地辨識」或「對~具有特異性」意指可測量及可再現之相互作用，諸如標靶與抗體之間的結合，其在分子(包括生物分子)之異質群體存在下由標靶之存在所決定。舉例而言，結合至或特異性地辨識標靶(其可為表位)之抗體為結合此標靶之親和力、親合力、容易性及/或持續時間強於其結合至其他標靶的抗體。在一實施例中，抗體與無關靶標之結合的程度小於抗體與標靶之結合的約10%，例如藉由放射免疫試驗(RIA)的測量。在一些實施例中，特異性地辨識標靶之抗體的解離常數(Kd)為≤ 1 μM、≤100 nM、≤ 10 nM、≤ 1 nM或≤ 0.1 nM。在一些實施例中，抗體特異性地辨識不同物種蛋白質中保守的蛋白質上之表位。在另一實施例中，特異性結合可包括但不要求專一性結合。As used herein, the terms "bind,""specifically bind to,""specificallyrecognize," or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, which is determined by the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that binds to or specifically recognizes a target (which may be an epitope) is one that binds to that target with greater affinity, avidity, ease, and/or duration than it binds to other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, for example as measured by a radioimmunoassay (RIA). In some embodiments, the dissociation constant (Kd ) of an antibody that specifically recognizes a target is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM. In some embodiments, the antibody specifically recognizes an epitope on a protein that is conserved among proteins of different species. In another embodiment, specific binding may include but does not require exclusive binding.
如本文所用，肽、多肽或抗體序列之「胺基酸序列同一性百分比(%)」及「同源性」係定義為：在比對序列及導入缺口(若需要)以獲得最大序列同一性百分比，且不將任何保留性取代視為序列同一性之一部分之後，候選序列中與特定肽或多肽序列中胺基酸殘基一致之胺基酸殘基的百分比。為測定胺基酸序列同一性百分比而進行之比對可以此項技術中熟知之多種方式達成，例如使用公用電腦軟體，諸如BLAST、BLAST-2、ALIGN或MEGALIGNTM(DNASTAR)軟體。具有通常知識者可確定用於量測比對之適當參數，包括為獲得與所比較序列全長範圍內之最大比對所需的任何演算法。As used herein, "percentage (%) of amino acid sequence identity" and "homology" of a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to obtain the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in a variety of ways well known in the art, such as using public computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. One of ordinary skill can determine appropriate parameters for measuring alignment, including any algorithm required to obtain maximum alignment over the full length of the compared sequences.
胺基酸取代可包括但不限於利用一個胺基酸置換多肽中的另一個胺基酸。示例性取代如表1所示。可將胺基酸取代導入感興趣之抗體中，並篩選產物以獲得所需的活性，例如保留/改善抗原結合、降低免疫原性，或改善ADCC或CDC。表1. 示例性胺基酸取代。
可根據常規側鏈性質將胺基酸分組：(1)疏水性：正白胺酸、Met、Ala、Val、Leu、Ile；(2)中性親水性：Cys、Ser、Thr、Asn、Gln；(3)酸性：Asp、Glu；(4)鹼性：His、Lys、Arg；(5)影響鏈位向的殘基：Gly、Pro；及(6)芳香性：Trp、Tyr、Phe。非保留性取代將需要將此等類別之一的成員置換為另一類別的成員。Amino acids can be grouped according to conventional side-chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions would require replacing a member of one of these classes with a member of another class.
當結合抗體或抗原結合蛋白使用時，術語「多特異性」意指具有多表位特異性的抗體或抗原結合蛋白(亦即，能夠特異性地辨識一個生物分子上的二、三或多個不同表位，或能夠特異性地辨識不同生物分子上的二、三或多個表位)。除非另有說明，否則由多特異性抗體結合之抗原的順序係於多特異性抗體名稱中任意列出。When used in conjunction with an antibody or antigen-binding protein, the term "multispecific" refers to an antibody or antigen-binding protein with multiple epitope specificity (i.e., capable of specifically recognizing two, three or more different epitopes on one biological molecule, or capable of specifically recognizing two, three or more epitopes on different biological molecules). Unless otherwise specified, the order of the antigens bound by the multispecific antibody is arbitrarily listed in the name of the multispecific antibody.
當結合抗體或抗原結合蛋白使用時，術語「雙特異性」意指能夠特異性地辨識一個生物分子上的二個不同表位的抗體或抗原結合蛋白，或能夠特異性地辨識兩個不同生物分子上的表位的抗體或抗原結合蛋白。除非另有說明，否則由雙特異性抗體結合之抗原的順序係於雙特異性抗體名稱中任意列出。When used in conjunction with an antibody or antigen-binding protein, the term "bispecific" means an antibody or antigen-binding protein that can specifically recognize two different epitopes on one biomolecule, or an antibody or antigen-binding protein that can specifically recognize epitopes on two different biomolecules. Unless otherwise specified, the order of the antigens bound by the bispecific antibody is arbitrarily listed in the bispecific antibody name.
「杵臼」策略(參見例如PCT國際公開號WO 2006/028936)在說明書的解讀中具有其平常的含義，意指可用於產生全長雙特異性抗體的策略。簡言之，可在影響CH3域相互作用的位置處將形成人類IgG中 CH3域界面的選定胺基酸進行突變以促進異二聚體形成。將具有小型側鏈(臼)的胺基酸導入特異性地辨識第一抗原之抗體的重鏈中，且將具有大型側鏈(杵)的胺基酸導入特異性地辨識第二抗原之抗體的重鏈中。在共表現兩種抗體之後，由於帶有「臼」的重鏈與帶有「杵」的重鏈優先相互作用而形成異二聚體。The "knob-and-hole" strategy (see, e.g., PCT International Publication No. WO 2006/028936) has its ordinary meaning in the interpretation of the specification, meaning a strategy that can be used to generate full-length bispecific antibodies. In brief, selected amino acids that form the CH3 domain interface in human IgG can be mutated at positions that affect the interaction of the CH3 domains to promote heterodimer formation. Amino acids with small side chains (holes) are introduced into the heavy chain of antibodies that specifically recognize the first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of antibodies that specifically recognize the second antigen. After co-expression of the two antibodies, heterodimers are formed due to the preferential interaction of the heavy chain with the "hole" with the heavy chain with the "knob".
如本文所用，術語「載體」意指能夠增殖與其連接之另一核酸的核酸分子。該術語包括作為自我複製之核酸結構的載體，以及併入宿主細胞基因體中的載體，其中該載體已被導入。某些載體能夠導引與其可操作地連接之核酸的表現。此類載體在本文中稱為「表現載體」。As used herein, the term "vector" means a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."
應理解，本文所述之本發明的實施例包括「由……組成」及/或「基本上由……組成」的實施例。It should be understood that the embodiments of the present invention described herein include "consisting of" and/or "consisting essentially of" embodiments.
本文中提及的「約」的值或參數包括(及描述)針對該值或參數本身的變異。舉例而言，提及「約X」的描述包括「X」的描述。The value or parameter mentioned herein as "about" includes (and describes) variations with respect to the value or parameter itself. For example, description referring to "about X" includes description of "X".
術語「約」及「大約」意指在20%之內、15%之內、10%之內、9%之內、8%之內、7%之內、6%之內、5%之內、4%之內、3%之內、2%之內、1%之內或更小的給定值或範圍。The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1% or less of a given value or range.
如本文所用，提及「非」的值或參數通常意指並描述「除外」的值或參數。舉例而言，該方法不用於治療X型發炎性疾病意指該方法用於治療X型以外的其他類型的發炎性疾病。As used herein, reference to a value or parameter that is "other than" generally means and describes a value or parameter that is "other than." For example, the method is not useful for treating inflammatory diseases of type X, which means that the method is useful for treating inflammatory diseases of other types other than type X.
本文中使用的術語「約X-Y」與「約X至約Y」具有相同的意義。As used herein, the term "about X-Y" has the same meaning as "about X to about Y".
如本文及所附請求項中所用，單數形式「一」、「或」及「該」包括複數參考對象，除非上下文中另有明確規定。II.分離的抗TSLP抗體構築體As used herein and in the appended claims, the singular forms "a,""an,""or," and "the" include plural references unless the context clearly dictates otherwise.II.Isolated anti-TSLPantibody constructs
TSLP為由腸道及肺上皮細胞、皮膚角質細胞及樹突細胞產生的細胞激素，但其亦可由(例如)呼吸道平滑肌細胞、肥大細胞、單核球、巨噬細胞、顆粒細胞、滑液膜纖維母細胞等產生。TSLP涉及輔助2型T細胞(Th2)發炎反應，且已被確定為治療氣喘的可能的治療標靶。此抗原可用作特定發炎性疾病(例如，過敏或自體免疫疾病)發展的生物標記、預後指標及抗原表現發炎性疾病的免疫治療標靶。TSLP is a cytokine produced by intestinal and lung epithelial cells, skin keratinocytes and dendritic cells, but it can also be produced by, for example, airway smooth muscle cells, mast cells, monocytes, macrophages, granulocytes, synovial membrane fibroblasts, etc. TSLP is involved in the type 2 T helper cell (Th2) inflammatory response and has been identified as a possible therapeutic target for the treatment of asthma. This antigen can be used as a biomarker for the development of specific inflammatory diseases (e.g., allergic or autoimmune diseases), a prognostic indicator, and an immunotherapy target for antigen-expressing inflammatory diseases.
在一態樣中，本申請案提供一種分離的抗體構築體(抗TSLP抗體構築體)，其包含抗TSLP抗體部分(或由其組成，或基本上由其組成)，諸如本文所述之抗TSLP抗體部分之任一者。在一些實施例中，該分離的抗TSLP抗體構築體由一或多個抗TSLP抗體部分組成。因此，在一些實施例中，本申請案亦提供抗TSLP抗體部分，諸如本文所述之抗TSLP抗體部分之任一者。在一些實施例中，該分離的抗TSLP抗體構築體係單特異性。在一些實施例中，該分離的抗TSLP抗體構築體係多特異性(例如，雙特異性)。在一些實施例中，提供一種多特異性 抗TSLP抗體構築體，其包含一或多個本文所述之抗TSLP抗體部分(例如，抗TSLP全長抗體、抗TSLP scFv或抗TSLP Fab)之任一者，以及特異性地辨識一或多個額外標靶抗原或表位的一或多個額外抗體部分。亦提供分離的抗TSLP抗體構築體及分離的抗TSLP抗體部分，其與本文所述之分離的抗TSLP抗體構築體或分離的抗TSLP抗體部分之任一者競爭結合至TSLP。在一些實施例中，提供一種含有特異性地辨識TSLP之工具的分離的抗TSLP抗體構築體。In one aspect, the present application provides an isolated antibody construct (anti-TSLP antibody construct) comprising (or consisting of, or consisting essentially of) an anti-TSLP antibody portion, such as any of the anti-TSLP antibody portions described herein. In some embodiments, the isolated anti-TSLP antibody construct is composed of one or more anti-TSLP antibody portions. Therefore, in some embodiments, the present application also provides an anti-TSLP antibody portion, such as any of the anti-TSLP antibody portions described herein. In some embodiments, the isolated anti-TSLP antibody construct is monospecific. In some embodiments, the isolated anti-TSLP antibody construct is multispecific (e.g., bispecific). In some embodiments, a multispecific anti-TSLP antibody construct is provided that includes any of one or more anti-TSLP antibody portions described herein (e.g., anti-TSLP full-length antibodies, anti-TSLP scFv, or anti-TSLP Fab) and one or more additional antibody portions that specifically recognize one or more additional target antigens or epitopes. Also provided are isolated anti-TSLP antibody constructs and isolated anti-TSLP antibody portions that compete with any of the isolated anti-TSLP antibody constructs or isolated anti-TSLP antibody portions described herein for binding to TSLP. In some embodiments, an isolated anti-TSLP antibody construct containing means for specifically recognizing TSLP is provided.
在一些實施例中，該分離的抗TSLP抗體構築體包含抗TSLP全長抗體、Fab、Fab’、Fab’-SH、F(ab’)2、Fv、scFv或其組合(或由其組成，或基本上由其組成)。在一些實施例中，該分離的抗TSLP抗體構築體包含抗TSLP Fab片段 (或由其組成)。在一些實施例中，該分離的抗TSLP抗體構築體包含抗TSLP scFv (或由其組成)。在一些實施例中，該分離的抗TSLP抗體構築體包含抗TSLP全長抗體(或由其組成)。In some embodiments, the isolated anti-TSLP antibody construct comprises (or consists of, or consists essentially of) an anti-TSLP full-length antibody, Fab, Fab', Fab'-SH, F(ab')2 , Fv, scFv, or a combination thereof. In some embodiments, the isolated anti-TSLP antibody construct comprises (or consists of) an anti-TSLP Fab fragment. In some embodiments, the isolated anti-TSLP antibody construct comprises (or consists of) an anti-TSLP scFv. In some embodiments, the isolated anti-TSLP antibody construct comprises (or consists of) an anti-TSLP full-length antibody.
在一些實施例中，該分離的抗TSLP抗體構築體為單價。在一些實施例中，該分離的抗TSLP抗體構築體為多價。在一些實施例中，該分離的抗TSLP抗體構築體係多價及單特異性(亦即，包含二或多個結合至相同TSLP表位的抗體部分)。在一些實施例中，該分離的抗TSLP抗體構築體係多價及多特異性。In some embodiments, the isolated anti-TSLP antibody construct is monovalent. In some embodiments, the isolated anti-TSLP antibody construct is multivalent. In some embodiments, the isolated anti-TSLP antibody construct is multivalent and monospecific (i.e., comprises two or more antibody moieties that bind to the same TSLP epitope). In some embodiments, the isolated anti-TSLP antibody construct is multivalent and multispecific.
在一些實施例中，該分離的抗TSLP抗體構築體係選自由以下組成之群組：單特異性抗體構築體、多特異性抗體構築體(例如，雙特異性抗體構築體)及抗體檢測標籤接合物。In some embodiments, the isolated anti-TSLP antibody construct is selected from the group consisting of a monospecific antibody construct, a multispecific antibody construct (e.g., a bispecific antibody construct), and an antibody detection tag conjugate.
本發明進一步提供含有本文所述之抗TSLP抗體部分(例如，全長抗體、scFv或Fab)之任一者的融合蛋白，以及含有本文所述之分離的抗TSLP抗體構築體或分離的抗TSLP抗體部分之任一者的接合物(例如，檢測標籤接合物)。亦提供含有抗TSLP抗體部分(例如，全長抗體、scFv或Fab)之任一者的分離的抗TSLP抗體構築體，以及融合配偶體(例如，另一蛋白質、表現標籤、純化標籤或用於增強半衰期的部分)。The present invention further provides fusion proteins containing any of the anti-TSLP antibody portions described herein (e.g., full-length antibodies, scFvs, or Fabs), and conjugates containing any of the isolated anti-TSLP antibody constructs or isolated anti-TSLP antibody portions described herein (e.g., detection tag conjugates). Also provided are isolated anti-TSLP antibody constructs containing any of the anti-TSLP antibody portions (e.g., full-length antibodies, scFvs, or Fabs), and fusion partners (e.g., another protein, expression tags, purification tags, or moieties for enhancing half-life).
在一些實施例中，該分離的抗TSLP抗體構築體之一或多個多肽的N端係額外與訊號肽融合以更好地表現。In some embodiments, the N-terminus of one or more polypeptides of the isolated anti-TSLP antibody construct is additionally fused to a signal peptide for better expression.
在一些實施例中，該分離的抗TSLP抗體構築體之一或多個多肽的N端及/或C端可含有組胺酸標籤(HIS-標籤)以進行蛋白質純化。舉例而言，該抗TSLP全長抗體之一或多個多肽的C端可進一步包含組胺酸標籤。抗TSLP抗體部分In some embodiments, the N-terminus and/or C-terminus of one or more polypeptides of the isolated anti-TSLP antibody construct may contain a histidine tag (HIS-tag) for protein purification. For example, the C-terminus of one or more polypeptides of the anti-TSLP full-lengthantibody may further contain ahistidinetag .
本文所述之抗TSLP抗體部分之任一者可用於本文所述之分離的抗TSLP抗體構築體。Any of the anti-TSLP antibody portions described herein can be used in the isolated anti-TSLP antibody constructs described herein.
TSLP抗原可來自各種動物物種，包括人類、非人類靈長類動物、小鼠、大鼠、兔或其他哺乳動物。在一些實施例中，該TSLP分子為人類TSLP。在一些實施例中，該TSLP分子為小鼠TSLP。在一些實施例中，該TSLP分子為食蟹獼猴TSLP。在一些實施例中，該TSLP分子為野生型。在一些實施例中，該TSLP分子為突變體(例如，與野生型參考TSLP分子相比，包含一或多個插入、缺失及/或胺基酸取代)。TSLP antigens can be from various animal species, including humans, non-human primates, mice, rats, rabbits or other mammals. In some embodiments, the TSLP molecule is human TSLP. In some embodiments, the TSLP molecule is mouse TSLP. In some embodiments, the TSLP molecule is cynomolgus macaque TSLP. In some embodiments, the TSLP molecule is wild-type. In some embodiments, the TSLP molecule is a mutant (e.g., comprising one or more insertions, deletions and/or amino acid substitutions compared to a wild-type reference TSLP molecule).
在一些實施例中，該抗TSLP抗體部分以≤ 1 μM，諸如≤100 nM，較佳為≤10 nM，更佳為≤1 nM的KD結合至TSLP抗原。在一些實施例中，該抗TSLP抗體部分以約1×10-14M至約1×10-7M (諸如約1×10-14M至約1×10-10M、約1×10-14M至約1×10-11M、約1×10-14M至約1×10-12M、約1×10-13M至約1×10-10M、約1×10-13M至約1×10-11M、約1×10-13M至約1×10-12M、約1×10-12M至約1×10-10M、約1×10-11M至約1×10-10M、約1×10-11M至約1×10-9M、約1×10-11M至約1×10-8M、約1×10-10M至約1×10-9M、約1×10-10M至約1×10-8M、約1×10-10M至約1×10-7M、約1×10-9M至約1×10-7M、約1×10-9M至約1×10-8M或約1×10-8M至約1×10-7M)的KD結合至人類TSLP抗原及/或猴(例如，食蟹獼猴) TSLP抗原。在一些實施例中，該抗TSLP抗體部分以約1×10-14M至約1×10-10M的KD結合至人類TSLP。在一些實施例中，該抗TSLP抗體部分以約1×10-12M至約1×10-9M的KD結合至食蟹獼猴TSLP。In some embodiments, the anti-TSLP antibody portion binds to the TSLP antigen with aKD of ≤ 1 μM, such as ≤ 100 nM, preferably ≤ 10 nM, and more preferably ≤ 1 nM. In some embodiments, the anti-TSLP antibody portion is present in an amount of about 1×10-14 M to about 1×10-7 M (e.g., about 1×10-14 M to about 1×10-10 M, about 1×10-14 M to about 1×10-11 M, about 1×10-14 M to about 1×10-12 M, about 1×10-13 M to about 1×10-10 M, about 1×10-13 M to about 1×10-11 M, about 1×10-13 M to about 1×10-12 M, about 1×10-12 M to about 1×10-10 M, about 1×10-11 M to about 1×10-10 M, about 1×10-11 M to about 1×10-9 M, about 1×10-11 M to about 1×10-8 M, about 1×10-10 Insome embodiments,the anti-TSLP antibody portion bindsto a human TSLP antigen and/ or a monkey( e.g., cynomolgusmacaque ) TSLP antigen withaK of about 1×10-14 M to about 1×10-10M. In some embodiments, the anti-TSLP antibody portion binds to a human TSLP antigen and/or a monkey( e.g., cynomolgus macaque) TSLPantigen with aK of about1 ×10-12 M to about 1×10-9 M.
在一些實施例中，與參考抗TSLP抗體(諸如本文所述之US-FDA批准的抗TSLP參考抗體#1 (參考Ab. #1))相比，該抗TSLP抗體部分以類似的親和力(例如，KD差異在2倍以內)結合至TSLP抗原(例如，人類TSLP及/或食蟹獼猴TSLP)。在一些實施例中，與參考抗TSLP抗體(諸如本文所述之US-FDA批准的抗TSLP參考Ab. #1)相比，該抗TSLP抗體部分以更強的親和力(至少約2、5、10、20、100、1000或更多倍之任一者)結合至TSLP抗原(例如，人類TSLP及/或食蟹獼猴TSLP)。舉例而言，在一些實施例中，參考抗TSLP抗體(例如，US-FDA批准的抗TSLP參考Ab. #1)結合至TSLP抗原(例如，人類TSLP及/或食蟹獼猴TSLP)的KD為本文所述之抗TSLP抗體部分的KD的至少約2、5、10、20、100、1000或更多倍之任一者。In some embodiments, the anti-TSLP antibody portion binds to a TSLP antigen (e.g., human TSLP and/or cynomolgus macaque TSLP) with a similar affinity (e.g., aKD difference of within 2-fold) as compared to a reference anti-TSLP antibody (e.g., US-FDA approved anti-TSLP Reference Antibody #1 (Reference Ab. #1) described herein). In some embodiments, the anti-TSLP antibody portion binds to a TSLP antigen (e.g., human TSLP and/or cynomolgus macaque TSLP) with a stronger affinity (at least about any of 2, 5, 10, 20, 100, 1000, or more times) as compared to a reference anti-TSLP antibody (e.g., US-FDA approved anti-TSLP Reference Ab. #1 described herein). For example, in some embodiments, a reference anti-TSLP antibody (e.g., US-FDA approved anti-TSLP reference Ab. #1) binds to a TSLP antigen (e.g., human TSLP and/or cynomolgus macaque TSLP) with aKD that is at least about any of 2, 5, 10, 20, 100, 1000, or more times greater than theKD of the anti-TSLP antibody portion described herein.
在一些實施例中，在TSLP (例如，人類TSLP)存在下，該抗TSLP抗體部分抑制(例如，至少約30%、40%、50%、60%、70%、80%、90%、95%或100%之任一者) TSLP誘導表現TSLP受體之促發炎細胞(例如，PBMC或CD1c+樹突細胞)的CCL17分泌。在一些實施例中，藉由本文所述之抗TSLP抗體部分的TSLP誘導表現TSLP受體之促發炎細胞(例如，PBMC或CD1c+樹突細胞)的CCL17分泌的抑制為藉由參考抗TSLP抗體(例如，US-FDA批准的抗TSLP參考Ab. #1)的至少約1.2倍(例如，至少約1.5、2、5、10、50或更多倍之任一者)。In some embodiments, the anti-TSLP antibody portion inhibits (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) TSLP-induced CCL17 secretion from pro-inflammatory cells (e.g., PBMCs or CD1c+ dendritic cells) expressing a TSLP receptor in the presence of TSLP (e.g., human TSLP). In some embodiments, TSLP-induced CCL17 secretion from pro-inflammatory cells (e.g., PBMCs or CD1c+ dendritic cells) expressing a TSLP receptor is inhibited by the anti-TSLP antibody portion described herein by at least about 1.2 times (e.g., at least about any of 1.5, 2, 5, 10, 50, or more times) that by a reference anti-TSLP antibody (e.g., US-FDA approved anti-TSLP reference Ab. #1).
在一些實施例中，該抗TSLP抗體部分特異性地辨識人類及非人類類靈長類動物(諸如食蟹獼猴) TSLP。包含此類與猴TSLP具有交叉反應性之抗TSLP抗體部分的分離的抗TSLP抗體構築體可促進非人類靈長類動物的毒性研究，其可為人類臨床試驗候選物提供更相關的安全性評估，而無需在黑猩猩中或使用替代分子進行毒性研究。在一些實施例中，該抗TSLP抗體部分僅結合至人類TSLP。在一些實施例中，該抗TSLP抗體部分與人類TSLP的結合比非人類(例如，食蟹獼猴) TSLP的結合更強(諸如至少約1.5、2、5、10、20、50、100、1000或更多倍之任一者)。In some embodiments, the anti-TSLP antibody portion specifically recognizes human and non-human primate (e.g., cynomolgus macaque) TSLP. Isolated anti-TSLP antibody constructs comprising such anti-TSLP antibody portions that are cross-reactive with monkey TSLP can facilitate toxicity studies in non-human primates, which can provide more relevant safety assessments for human clinical trial candidates without the need for toxicity studies in chimpanzees or using surrogate molecules. In some embodiments, the anti-TSLP antibody portion binds only to human TSLP. In some embodiments, the anti-TSLP antibody portion binds human TSLP more strongly (e.g., at least about any of 1.5, 2, 5, 10, 20, 50, 100, 1000, or more) than non-human (e.g., cynomolgus macaque) TSLP.
抗TSLP抗體部分可完全或部分調節、阻斷、抑制、降低、拮抗、中和或干擾TSLP的功能活性(例如，結合至TSLPR複合體及/或誘導傳訊)。在抗TSLP抗體部分存在下，當TSLP的功能活性降低至少約95% (諸如至少約96%、97%、98%、99%或100%之任一者)時，與不被抗TSLP抗體部分結合時相比，該抗TSLP抗體部分被視為能夠完全調控、阻斷、抑制、降低、拮抗、中和或干擾TSLP的功能活性。在抗TSLP抗體部分存在下，當TSLP的功能活性降低至少約50% (諸如至少約55%、60%、65%、70%、75%、80%、85%或90%之任一者)時，與不被抗TSLP抗體部分結合時相比，該抗TSLP抗體部分被視為能夠顯著調控、阻斷、抑制、降低、拮抗、中和或干擾TSLP的功能活性。在抗TSLP抗體部分存在下，當標靶抗原的功能活性降低小於約50% (諸如降低小於約10%、15%、20%、25%、30%、35%、或40%或45%之任一者)時，與不被抗TSLP抗體部分結合時相比，該抗TSLP抗體部分被視為能夠部分調控、阻斷、抑制、降低、拮抗、中和或干擾TSLP的功能活性。The anti-TSLP antibody portion can fully or partially modulate, block, inhibit, reduce, antagonize, neutralize or interfere with the functional activity of TSLP (e.g., binding to the TSLPR complex and/or inducing signaling). When the functional activity of TSLP is reduced by at least about 95% (e.g., at least about 96%, 97%, 98%, 99% or 100%) in the presence of the anti-TSLP antibody portion compared to when not bound by the anti-TSLP antibody portion, the anti-TSLP antibody portion is considered to be able to fully modulate, block, inhibit, reduce, antagonize, neutralize or interfere with the functional activity of TSLP. In the presence of an anti-TSLP antibody portion, when the functional activity of TSLP is reduced by at least about 50% (e.g., at least about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%), compared to when not bound by the anti-TSLP antibody portion, the anti-TSLP antibody portion is considered to be able to significantly modulate, block, inhibit, reduce, antagonize, neutralize, or interfere with the functional activity of TSLP. In the presence of an anti-TSLP antibody portion, when the functional activity of the target antigen is reduced by less than about 50% (e.g., by less than about 10%, 15%, 20%, 25%, 30%, 35%, or any of 40% or 45%), compared to when not bound by the anti-TSLP antibody portion, the anti-TSLP antibody portion is considered to be able to partially modulate, block, inhibit, reduce, antagonize, neutralize, or interfere with the functional activity of TSLP.
在一些實施例中，藉由該抗TSLP抗體部分抑制或降低TSLP活性包含抑制或降低(例如，至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或更多之任一者)以下一或多者：i) TSLP與其受體的結合；i)表現TSLPR的細胞在TSLP存在下的增生、活化及/或分化；iii)在TSLP存在下的極化試驗中產生的Th2細胞激素(亦即，由Th2細胞表現的細胞激素；例如，IL-4、IL-5、IL-13及IL-10)；iv)在TSLP存在下的樹突細胞活化及/或成熟；及v)在TSLP存在下的肥大細胞細胞激素釋放。In some embodiments, partially inhibiting or reducing TSLP activity by the anti-TSLP antibody comprises inhibiting or reducing (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) one or more of: i) binding of TSLP to its receptor; i) proliferation, activation and/or differentiation of cells expressing the TSLPR in the presence of TSLP; iii) Th2 cytokines (i.e., cytokines expressed by Th2 cells; e.g., IL-4, IL-5, IL-13, and IL-10) produced in a polarization assay in the presence of TSLP; iv) dendritic cell activation and/or maturation in the presence of TSLP; and v) mast cell cytokine release in the presence of TSLP.
在一些實施例中，該抗TSLP抗體部分在序列中含有一或多個變異。在一些實施例中，該變體序列中的胺基酸變異(例如，取代，諸如保留性取代)不會實質上降低(例如，降低至多約50%、40%、30%、20%、10%、5%、1%或更少之任一者)抗TSLP抗體部分結合至TSLP的能力。亦考慮實質上改善(例如，改善至少約1.2、1.5、2、5、10、20、50或更多倍之任一者)抗TSLP抗體部分與TSLP之結合親和力或其他性質(諸如特異性、免疫原性及/或與TSLP表位變體的交叉反應)的修飾。In some embodiments, the anti-TSLP antibody portion contains one or more variations in sequence. In some embodiments, the amino acid variations (e.g., substitutions, such as conservative substitutions) in the variant sequence do not substantially reduce (e.g., reduce by at most about any of 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) the ability of the anti-TSLP antibody portion to bind to TSLP. Modifications that substantially improve (e.g., improve by at least about any of 1.2, 1.5, 2, 5, 10, 20, 50 or more fold) the binding affinity or other properties (such as specificity, immunogenicity and/or cross-reactivity with TSLP epitope variants) of the anti-TSLP antibody portion to TSLP are also contemplated.
在一些實施例中，提供一種包含VH及VL的抗TSLP抗體部分，其中該VH包含(i)含有SEQ ID NO:2之胺基酸序列的CDR-H1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:3之胺基酸序列的CDR-H2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:4之胺基酸序列的CDR-H3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；且該VL包含(i)含有SEQ ID NO:6之胺基酸序列的CDR-L1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:7之胺基酸序列的CDR-L2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:8之胺基酸序列的CDR-L3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體。在一些實施例中，該抗TSLP抗體部分包含VH，其包含(i)含有SEQ ID NO:2之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:3之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:4之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:6之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:7之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:8之胺基酸序列的CDR-L3。在一些實施例中，該抗TSLP抗體部分包含含有SEQ ID NO:1之胺基酸序列的VH的CDR-H1、CDR-H2及CDR-H3，以及含有SEQ ID NO:5之胺基酸序列的VL的CDR-L1、CDR-L2及CDR-L3。在一些實施例中，該抗TSLP抗體部分包含(a)含有與SEQ ID NO:1具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:5具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；或(b)含有與SEQ ID NO:9具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:10具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL。在一些實施例中，該抗TSLP抗體部分包含：(a)含有SEQ ID NO:1之胺基酸序列的VH，以及含有SEQ ID NO:5之胺基酸序列的VL；或(b)含有SEQ ID NO:9之胺基酸序列的VH，以及含有SEQ ID NO:10之胺基酸序列的VL。In some embodiments, an anti-TSLP antibody portion comprising a VH and a VL is provided, wherein the VH comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and the VL comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); NO:6 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); (ii) a CDR-L2 containing the amino acid sequence of SEQ ID NO:7 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); and (iii) a CDR-L3 containing the amino acid sequence of SEQ ID NO:8 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions). In some embodiments, the anti-TSLP antibody portion comprises a VH comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4; and a VL comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-TSLP antibody portion comprises CDR-H1, CDR-H2, and CDR-H3 of a VH comprising the amino acid sequence of SEQ ID NO: 1, and CDR-L1, CDR-L2, and CDR-L3 of a VL comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-TSLP antibody portion comprises (a) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:1, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:5; or (b) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:9, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:10. In some embodiments, the anti-TSLP antibody portion comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 5; or (b) a VH comprising the amino acid sequence of SEQ ID NO: 9, and a VL comprising the amino acid sequence of SEQ ID NO: 10.
在一些實施例中，提供一種含有VH及VL的抗TSLP抗體部分，其中該VH包含(i)含有SEQ ID NO:12之胺基酸序列的CDR-H1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:13之胺基酸序列的CDR-H2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:4之胺基酸序列的CDR-H3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；且該VL包含(i)含有SEQ ID NO:6之胺基酸序列的CDR-L1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:7之胺基酸序列的CDR-L2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:8之胺基酸序列的CDR-L3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體。在一些實施例中，該抗TSLP抗體部分包含VH，其包含(i)含有SEQ ID NO:12之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:13之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:4之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:6之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:7之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:8之胺基酸序列的CDR-L3。在一些實施例中，該抗TSLP抗體部分包含含有SEQ ID NO:11之胺基酸序列的VH的CDR-H1、CDR-H2及CDR-H3，以及含有SEQ ID NO:15之胺基酸序列的VL的CDR-L1、CDR-L2及CDR-L3。在一些實施例中，該抗TSLP抗體部分包含含有與SEQ ID NO:11具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:15具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL。在一些實施例中，該抗TSLP抗體部分包含含有SEQ ID NO:11之胺基酸序列的VH，以及含有SEQ ID NO:15之胺基酸序列的VL。In some embodiments, an anti-TSLP antibody portion comprising a VH and a VL is provided, wherein the VH comprises (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); (ii) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and (iii) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and the VL comprises (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); NO:6 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); (ii) a CDR-L2 containing the amino acid sequence of SEQ ID NO:7 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); and (iii) a CDR-L3 containing the amino acid sequence of SEQ ID NO:8 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions). In some embodiments, the anti-TSLP antibody portion comprises a VH comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4; and a VL comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-TSLP antibody portion comprises CDR-H1, CDR-H2, and CDR-H3 of a VH comprising the amino acid sequence of SEQ ID NO: 11, and CDR-L1, CDR-L2, and CDR-L3 of a VL comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-TSLP antibody portion comprises a VH comprising an amino acid sequence having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 11, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 15. In some embodiments, the anti-TSLP antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 11, and a VL comprising the amino acid sequence of SEQ ID NO: 15.
在一些實施例中，提供一種含有VH及VL的抗TSLP抗體部分，其中該VH包含(i)含有SEQ ID NO:28之胺基酸序列的CDR-H1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:29之胺基酸序列的CDR-H2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:30之胺基酸序列的CDR-H3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；且該VL包含(i)含有SEQ ID NO:32之胺基酸序列的CDR-L1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:33之胺基酸序列的CDR-L2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:34之胺基酸序列的CDR-L3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體。在一些實施例中，該抗TSLP抗體部分包含VH，其包含(i)含有SEQ ID NO:28之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:29之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:30之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:32之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:33之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:34之胺基酸序列的CDR-L3。在一些實施例中，該抗TSLP抗體部分包含含有SEQ ID NO:27之胺基酸序列的VH的CDR-H1、CDR-H2及CDR-H3，以及含有SEQ ID NO:31之胺基酸序列的VL的CDR-L1、CDR-L2及CDR-L3。在一些實施例中，該抗TSLP抗體部分包含含有與SEQ ID NO:27具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:31具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL。在一些實施例中，該抗TSLP抗體部分包含含有SEQ ID NO:27之胺基酸序列的VH，以及含有SEQ ID NO:31之胺基酸序列的VL。In some embodiments, an anti-TSLP antibody portion comprising a VH and a VL is provided, wherein the VH comprises (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 28, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); (ii) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and (iii) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 30, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and the VL comprises (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 28, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); NO:32 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); (ii) a CDR-L2 containing the amino acid sequence of SEQ ID NO:33 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); and (iii) a CDR-L3 containing the amino acid sequence of SEQ ID NO:34 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions). In some embodiments, the anti-TSLP antibody portion comprises a VH comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; and a VL comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-TSLP antibody portion comprises CDR-H1, CDR-H2, and CDR-H3 of a VH comprising the amino acid sequence of SEQ ID NO: 27, and CDR-L1, CDR-L2, and CDR-L3 of a VL comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the anti-TSLP antibody portion comprises a VH comprising an amino acid sequence having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 27, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 31. In some embodiments, the anti-TSLP antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 27, and a VL comprising the amino acid sequence of SEQ ID NO: 31.
在一些實施例中，提供一種含有VH及VL的抗TSLP抗體部分，其中該VH包含(i)含有SEQ ID NO:36之胺基酸序列的CDR-H1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:37之胺基酸序列的CDR-H2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:38之胺基酸序列的CDR-H3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；且該VL包含(i)含有SEQ ID NO:40之胺基酸序列的CDR-L1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:41之胺基酸序列的CDR-L2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:42之胺基酸序列的CDR-L3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體。在一些實施例中，該抗TSLP抗體部分包含VH，其包含(i)含有SEQ ID NO:36之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:37之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:38之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:40之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:41之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:42之胺基酸序列的CDR-L3。在一些實施例中，該抗TSLP抗體部分包含含有SEQ ID NO:35之胺基酸序列的VH的CDR-H1、CDR-H2及CDR-H3，以及含有SEQ ID NO:39之胺基酸序列的VL的CDR-L1、CDR-L2及CDR-L3。在一些實施例中，該抗TSLP抗體部分包含含有與SEQ ID NO:35具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:39具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL。在一些實施例中，該抗TSLP抗體部分包含含有SEQ ID NO:35之胺基酸序列的VH，以及含有SEQ ID NO:39之胺基酸序列的VL。In some embodiments, an anti-TSLP antibody portion comprising a VH and a VL is provided, wherein the VH comprises (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); (ii) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and (iii) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and the VL comprises (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); NO:40 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); (ii) a CDR-L2 containing the amino acid sequence of SEQ ID NO:41 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); and (iii) a CDR-L3 containing the amino acid sequence of SEQ ID NO:42 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions). In some embodiments, the anti-TSLP antibody portion comprises a VH comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38; and a VL comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the anti-TSLP antibody portion comprises CDR-H1, CDR-H2, and CDR-H3 of a VH comprising the amino acid sequence of SEQ ID NO: 35, and CDR-L1, CDR-L2, and CDR-L3 of a VL comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the anti-TSLP antibody portion comprises a VH comprising an amino acid sequence having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 35, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 39. In some embodiments, the anti-TSLP antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 35, and a VL comprising the amino acid sequence of SEQ ID NO: 39.
在一些實施例中，提供一種含有VH及VL的抗TSLP抗體部分，其中該VH包含(i)含有SEQ ID NO:44之胺基酸序列的CDR-H1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:45之胺基酸序列的CDR-H2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:46之胺基酸序列的CDR-H3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；且該VL包含(i)含有SEQ ID NO:48之胺基酸序列的CDR-L1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:49之胺基酸序列的CDR-L2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:50之胺基酸序列的CDR-L3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體。在一些實施例中，該抗TSLP抗體部分包含VH，其包含(i)含有SEQ ID NO:44之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:45之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:46之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:48之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:49之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:50之胺基酸序列的CDR-L3。在一些實施例中，該抗TSLP抗體部分包含含有SEQ ID NO:43之胺基酸序列的VH的CDR-H1、CDR-H2及CDR-H3，以及含有SEQ ID NO:47之胺基酸序列的VL的CDR-L1、CDR-L2及CDR-L3。在一些實施例中，該抗TSLP抗體部分包含含有與SEQ ID NO:43具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:47具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL。在一些實施例中，該抗TSLP抗體部分包含含有SEQ ID NO:43之胺基酸序列的VH，以及含有SEQ ID NO:47之胺基酸序列的VL。In some embodiments, an anti-TSLP antibody portion comprising a VH and a VL is provided, wherein the VH comprises (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); (ii) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 45, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and (iii) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 46, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and the VL comprises (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions). NO:48 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); (ii) a CDR-L2 containing the amino acid sequence of SEQ ID NO:49 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); and (iii) a CDR-L3 containing the amino acid sequence of SEQ ID NO:50 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions). In some embodiments, the anti-TSLP antibody portion comprises a VH comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46; and a VL comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 49; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the anti-TSLP antibody portion comprises CDR-H1, CDR-H2, and CDR-H3 of a VH comprising the amino acid sequence of SEQ ID NO: 43, and CDR-L1, CDR-L2, and CDR-L3 of a VL comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the anti-TSLP antibody portion comprises a VH comprising an amino acid sequence having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 43, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 47. In some embodiments, the anti-TSLP antibody portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 43, and a VL comprising the amino acid sequence of SEQ ID NO: 47.
在一些實施例中，該抗TSLP抗體部分包含VH及VL，其中該VH包含(i)含有SEQ ID NO:20之胺基酸序列的CDR-H1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:21之胺基酸序列的CDR-H2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:22之胺基酸序列的CDR-H3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；且該VL包含(i)含有SEQ ID NO:24之胺基酸序列的CDR-L1或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；(ii)含有SEQ ID NO:25之胺基酸序列的CDR-L2或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體；及(iii)含有SEQ ID NO:26之胺基酸序列的CDR-L3或其含有至多約3個(例如，1、2或3個)胺基酸變異(例如，插入、缺失及/或取代，諸如保留性取代)的變體。在一些實施例中，該抗TSLP抗體部分包含VH，其包含(i)含有SEQ ID NO:20之胺基酸序列的CDR-H1；(ii)含有SEQ ID NO:21之胺基酸序列的CDR-H2；及(iii)含有SEQ ID NO:22之胺基酸序列的CDR-H3；及VL，其包含(i)含有SEQ ID NO:24之胺基酸序列的CDR-L1；(ii)含有SEQ ID NO:25之胺基酸序列的CDR-L2；及(iii)含有SEQ ID NO:26之胺基酸序列的CDR-L3。在一些實施例中，該抗TSLP抗體部分包含含有SEQ ID NO:19之胺基酸序列的VH的CDR-H1、CDR-H2及CDR-H3，以及含有SEQ ID NO:23之胺基酸序列的VL的CDR-L1、CDR-L2及CDR-L3。在一些實施例中，該抗TSLP抗體部分包含：(a)含有與SEQ ID NO:19具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:23具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(b)含有與SEQ ID NO:63具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:53具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(c)含有與SEQ ID NO:63具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:54具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(d)含有與SEQ ID NO:56具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:52具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(e)含有與SEQ ID NO:55具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:51具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(f)含有與SEQ ID NO:56具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:51具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(g)含有與SEQ ID NO:63具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:51具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(h)含有與SEQ ID NO:64具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:51具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(i)含有與SEQ ID NO:55具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:52具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(j)含有與SEQ ID NO:58具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:52具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(k)含有與SEQ ID NO:60具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:52具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(l)含有與SEQ ID NO:64具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:52具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(m)含有與SEQ ID NO:55具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:53具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(n)含有與SEQ ID NO:57具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:53具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(o)含有與SEQ ID NO:58具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:53具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(p)含有與SEQ ID NO:62具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:53具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(q)含有與SEQ ID NO:64具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:53具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(r)含有與SEQ ID NO:55具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:54具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(s)含有與SEQ ID NO:61具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:54具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(t)含有與SEQ ID NO:62具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:54具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(u)含有與SEQ ID NO:64具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:54具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(v)含有與SEQ ID NO:63具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:52具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；(w)含有與SEQ ID NO:188具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:189具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL；或(x)含有與SEQ ID NO:188具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VH，以及含有與SEQ ID NO:190具有至少約80% (諸如至少約85%、90%、95%、96%、97%、98%、99%或更多之任一者)序列一致性之胺基酸序列的VL。在一些實施例中，該抗TSLP抗體部分包含：(a)含有SEQ ID NO:19之胺基酸序列的VH，以及含有SEQ ID NO:23之胺基酸序列的VL；(b)含有SEQ ID NO:63之胺基酸序列的VH，以及含有SEQ ID NO:53之胺基酸序列的VL；(c)含有SEQ ID NO:63之胺基酸序列的VH，以及含有SEQ ID NO:54之胺基酸序列的VL；(d)含有SEQ ID NO:56之胺基酸序列的VH，以及含有SEQ ID NO:52之胺基酸序列的VL；(e)含有SEQ ID NO:55之胺基酸序列的VH，以及含有SEQ ID NO:51之胺基酸序列的VL；(f)含有SEQ ID NO:56之胺基酸序列的VH，以及含有SEQ ID NO:51之胺基酸序列的VL；(g)含有SEQ ID NO:63之胺基酸序列的VH，以及含有SEQ ID NO:51之胺基酸序列的VL；(h)含有SEQ ID NO:64之胺基酸序列的VH，以及含有SEQ ID NO:51之胺基酸序列的VL；(i)含有SEQ ID NO:55之胺基酸序列的VH，以及含有SEQ ID NO:52之胺基酸序列的VL；(j)含有SEQ ID NO:58之胺基酸序列的VH，以及含有SEQ ID NO:52之胺基酸序列的VL；(k)含有SEQ ID NO:60之胺基酸序列的VH，以及含有SEQ ID NO:52之胺基酸序列的VL；(l)含有SEQ ID NO:64之胺基酸序列的VH，以及含有SEQ ID NO:52之胺基酸序列的VL；(m)含有SEQ ID NO:55之胺基酸序列的VH，以及含有SEQ ID NO:53之胺基酸序列的VL；(n)含有SEQ ID NO:57之胺基酸序列的VH，以及含有SEQ ID NO:53之胺基酸序列的VL；(o)含有SEQ ID NO:58之胺基酸序列的VH，以及含有SEQ ID NO:53之胺基酸序列的VL；(p)含有SEQ ID NO:62之胺基酸序列的VH，以及含有SEQ ID NO:53之胺基酸序列的VL；(q)含有SEQ ID NO:64之胺基酸序列的VH，以及含有SEQ ID NO:53之胺基酸序列的VL；(r)含有SEQ ID NO:55之胺基酸序列的VH，以及含有SEQ ID NO:54之胺基酸序列的VL；(s)含有SEQ ID NO:61之胺基酸序列的VH，以及含有SEQ ID NO:54之胺基酸序列的VL；(t)含有SEQ ID NO:62之胺基酸序列的VH，以及含有SEQ ID NO:54之胺基酸序列的VL；(u)含有SEQ ID NO:64之胺基酸序列的VH，以及含有SEQ ID NO:54之胺基酸序列的VL；(v)含有SEQ ID NO:63之胺基酸序列的VH，以及含有SEQ ID NO:52之胺基酸序列的VL；(w)含有SEQ ID NO:188之胺基酸序列的VH，以及含有SEQ ID NO:189之胺基酸序列的VL；或(x)含有SEQ ID NO:188之胺基酸序列的VH，以及含有SEQ ID NO:190之胺基酸序列的VL。In some embodiments, the anti-TSLP antibody portion comprises a VH and a VL, wherein the VH comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and the VL comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions). NO:24 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); (ii) a CDR-L2 containing the amino acid sequence of SEQ ID NO:25 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); and (iii) a CDR-L3 containing the amino acid sequence of SEQ ID NO:26 or a variant thereof containing up to about 3 (e.g., 1, 2 or 3) amino acid variations (e.g., insertions, deletions and/or substitutions, such as conservative substitutions). In some embodiments, the anti-TSLP antibody portion comprises a VH comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 22; and a VL comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-TSLP antibody portion comprises CDR-H1, CDR-H2, and CDR-H3 of a VH comprising the amino acid sequence of SEQ ID NO: 19, and CDR-L1, CDR-L2, and CDR-L3 of a VL comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-TSLP antibody portion comprises: (a) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 19, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 23; (b) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 63, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 53. (c) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:63, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:54; (d) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:56, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:52. (e) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:55, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:51; (f) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:56, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:51. (g) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:63, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:51; (h) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:64, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:51. (i) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:55, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:52; (j) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:58, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:52. (k) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:60, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:52; (l) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:64, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:52. (m) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:55, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:53; (n) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:57, and a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:53. (o) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:58, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:53; (p) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:62, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:53. (a) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:64, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:53; (b) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:55, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:54. (s) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:61, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:54; (t) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:62, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:54. (i) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:64, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:54; (ii) a VH comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:63, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:52. (w) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 188, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 189; or (x) a VH comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 188, and a VL comprising an amino acid sequence having at least about 80% (such as at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 190. (e.g., any of at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to a VL of an amino acid sequence. In some embodiments, the anti-TSLP antibody portion comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 23; (b) a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 53; (c) a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 54; (d) a VH comprising the amino acid sequence of SEQ ID NO: 56, and a VL comprising the amino acid sequence of SEQ ID NO: 52; (e) a VH comprising the amino acid sequence of SEQ ID NO: 55, and a VL comprising the amino acid sequence of SEQ ID NO: 51; (f) a VH comprising the amino acid sequence of SEQ ID NO: 56, and a VL comprising the amino acid sequence of SEQ ID NO: 51; (g) a VH comprising the amino acid sequence of SEQ ID NO: (i) a VH comprising the amino acid sequence of SEQ ID NO:55, and a VL comprising the amino acid sequence of SEQ ID NO:52; (j) a VH comprising the amino acid sequence of SEQ ID NO:58, and a VL comprising the amino acid sequence of SEQ ID NO:52; (k) a VH comprising the amino acid sequence of SEQ ID NO:60, and a VL comprising the amino acid sequence of SEQ ID NO:52; (l) a VH comprising the amino acid sequence of SEQ ID NO:64, and a VL comprising the amino acid sequence of SEQ ID NO:52; (m) a VH comprising the amino acid sequence of SEQ ID NO:55, and a VL comprising the amino acid sequence of SEQ ID NO: NO:53; (n) a VH comprising the amino acid sequence of SEQ ID NO:57, and a VL comprising the amino acid sequence of SEQ ID NO:53; (o) a VH comprising the amino acid sequence of SEQ ID NO:58, and a VL comprising the amino acid sequence of SEQ ID NO:53; (p) a VH comprising the amino acid sequence of SEQ ID NO:62, and a VL comprising the amino acid sequence of SEQ ID NO:53; (q) a VH comprising the amino acid sequence of SEQ ID NO:64, and a VL comprising the amino acid sequence of SEQ ID NO:53; (r) a VH comprising the amino acid sequence of SEQ ID NO:55, and a VL comprising the amino acid sequence of SEQ ID NO:54; (s) a VH comprising the amino acid sequence of SEQ ID NO:61, and a VL comprising the amino acid sequence of SEQ ID NO:54; (t) a VH comprising the amino acid sequence of SEQ ID NO:62 (i) a VH comprising the amino acid sequence of SEQ ID NO: 62, and a VL comprising the amino acid sequence of SEQ ID NO: 54; (u) a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL comprising the amino acid sequence of SEQ ID NO: 54; (v) a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 52; (w) a VH comprising the amino acid sequence of SEQ ID NO: 188, and a VL comprising the amino acid sequence of SEQ ID NO: 189; or (x) a VH comprising the amino acid sequence of SEQ ID NO: 188, and a VL comprising the amino acid sequence of SEQ ID NO: 190.
在一些實施例中，提供一種抗TSLP抗體部分，其與參考抗TSLP抗體競爭結合(例如，競爭結合至少約50%或更多，諸如至少約55%、60%、70%、80%、90%、95%、96%、97%、98%、99%或更多之一者)至TSLP，該參考抗TSLP抗體包含：(a)含有SEQ ID NO:1之胺基酸序列的參考VH及含有SEQ ID NO:5之胺基酸序列的參考VL；(b)含有SEQ ID NO:9之胺基酸序列的參考VH及含有SEQ ID NO:10之胺基酸序列的參考VL；(c)含有SEQ ID NO:11之胺基酸序列的參考VH及含有SEQ ID NO:15之胺基酸序列的參考VL；(d)含有SEQ ID NO:19之胺基酸序列的參考VH及含有SEQ ID NO:23之胺基酸序列的參考VL；(e)含有SEQ ID NO:27之胺基酸序列的參考VH及含有SEQ ID NO:31之胺基酸序列的參考VL；(f)含有SEQ ID NO:35之胺基酸序列的參考VH及含有SEQ ID NO:39之胺基酸序列的參考VL；(g)含有SEQ ID NO:43之胺基酸序列的參考VH及含有SEQ ID NO:47之胺基酸序列的參考VL；(h)含有SEQ ID NO:63之胺基酸序列的參考VH及含有SEQ ID NO:53之胺基酸序列的參考VL；(i)含有SEQ ID NO:63之胺基酸序列的參考VH及含有SEQ ID NO:54之胺基酸序列的參考VL；(j)含有SEQ ID NO:56之胺基酸序列的參考VH及含有SEQ ID NO:52之胺基酸序列的參考VL；(k)含有SEQ ID NO:55之胺基酸序列的參考VH及含有SEQ ID NO:51之胺基酸序列的參考VL；(l)含有SEQ ID NO:56之胺基酸序列的參考VH及含有SEQ ID NO:51之胺基酸序列的參考VL；(m)含有SEQ ID NO:63之胺基酸序列的參考VH及含有SEQ ID NO:51之胺基酸序列的參考VL；(n)含有SEQ ID NO:64之胺基酸序列的參考VH及含有SEQ ID NO:51之胺基酸序列的參考VL；(o)含有SEQ ID NO:55之胺基酸序列的參考VH及含有SEQ ID NO:52之胺基酸序列的參考VL；(p)含有SEQ ID NO:58之胺基酸序列的參考VH及含有SEQ ID NO:52之胺基酸序列的參考VL；(q)含有SEQ ID NO:60之胺基酸序列的參考VH及含有SEQ ID NO:52之胺基酸序列的參考VL；(r)含有SEQ ID NO:64之胺基酸序列的參考VH及含有SEQ ID NO:52之胺基酸序列的參考VL；(s)含有SEQ ID NO:55之胺基酸序列的參考VH及含有SEQ ID NO:53之胺基酸序列的參考VL；(t)含有SEQ ID NO:57之胺基酸序列的參考VH及含有SEQ ID NO:53之胺基酸序列的參考VL；(u)含有SEQ ID NO:58之胺基酸序列的參考VH及含有SEQ ID NO:53之胺基酸序列的參考VL；(v)含有SEQ ID NO:62之胺基酸序列的參考VH及含有SEQ ID NO:53之胺基酸序列的參考VL；(w)含有SEQ ID NO:64之胺基酸序列的參考VH及含有SEQ ID NO:53之胺基酸序列的參考VL；(x)含有SEQ ID NO:55之胺基酸序列的參考VH及含有SEQ ID NO:54之胺基酸序列的參考VL；(y)含有SEQ ID NO:61之胺基酸序列的參考VH及含有SEQ ID NO:54之胺基酸序列的參考VL；(z)含有SEQ ID NO:62之胺基酸序列的參考VH及含有SEQ ID NO:54之胺基酸序列的參考VL；(aa)含有SEQ ID NO:64之胺基酸序列的參考VH及含有SEQ ID NO:54之胺基酸序列的參考VL；(bb)含有SEQ ID NO:63之胺基酸序列的參考VH及含有SEQ ID NO:52之胺基酸序列的參考VL；(cc)含有SEQ ID NO:188之胺基酸序列的參考VH及含有SEQ ID NO:189之胺基酸序列的參考VL；或(dd)含有SEQ ID NO:188之胺基酸序列的參考VH及含有SEQ ID NO:190之胺基酸序列的參考VL。In some embodiments, an anti-TSLP antibody portion is provided that competes for binding to TSLP (e.g., competes for binding at least about 50% or more, such as at least about 55%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more) with a reference anti-TSLP antibody comprising: (a) a reference VH comprising the amino acid sequence of SEQ ID NO: 1 and a reference VL comprising the amino acid sequence of SEQ ID NO: 5; (b) a reference VH comprising the amino acid sequence of SEQ ID NO: 9 and a reference VL comprising the amino acid sequence of SEQ ID NO: 10; (c) a reference VH comprising the amino acid sequence of SEQ ID NO: 11 and a reference VL comprising the amino acid sequence of SEQ ID NO: 15; (d) a reference VH comprising the amino acid sequence of SEQ ID NO: 19 and a reference VL comprising the amino acid sequence of SEQ ID NO: 20; (e) a reference VH containing the amino acid sequence of SEQ ID NO:27 and a reference VL containing the amino acid sequence of SEQ ID NO:31; (f) a reference VH containing the amino acid sequence of SEQ ID NO:35 and a reference VL containing the amino acid sequence of SEQ ID NO:39; (g) a reference VH containing the amino acid sequence of SEQ ID NO:43 and a reference VL containing the amino acid sequence of SEQ ID NO:47; (h) a reference VH containing the amino acid sequence of SEQ ID NO:63 and a reference VL containing the amino acid sequence of SEQ ID NO:53; (i) a reference VH containing the amino acid sequence of SEQ ID NO:63 and a reference VL containing the amino acid sequence of SEQ ID NO:54; (j) a reference VH containing the amino acid sequence of SEQ ID NO:56 and a reference VL containing the amino acid sequence of SEQ ID NO:57; NO:52; (k) a reference VH containing the amino acid sequence of SEQ ID NO:55 and a reference VL containing the amino acid sequence of SEQ ID NO:51; (l) a reference VH containing the amino acid sequence of SEQ ID NO:56 and a reference VL containing the amino acid sequence of SEQ ID NO:51; (m) a reference VH containing the amino acid sequence of SEQ ID NO:63 and a reference VL containing the amino acid sequence of SEQ ID NO:51; (n) a reference VH containing the amino acid sequence of SEQ ID NO:64 and a reference VL containing the amino acid sequence of SEQ ID NO:51; (o) a reference VH containing the amino acid sequence of SEQ ID NO:55 and a reference VL containing the amino acid sequence of SEQ ID NO:52; (p) a reference VH containing the amino acid sequence of SEQ ID NO:58 and a reference VL containing the amino acid sequence of SEQ ID NO: NO:52; (q) a reference VH containing the amino acid sequence of SEQ ID NO:60 and a reference VL containing the amino acid sequence of SEQ ID NO:52; (r) a reference VH containing the amino acid sequence of SEQ ID NO:64 and a reference VL containing the amino acid sequence of SEQ ID NO:52; (s) a reference VH containing the amino acid sequence of SEQ ID NO:55 and a reference VL containing the amino acid sequence of SEQ ID NO:53; (t) a reference VH containing the amino acid sequence of SEQ ID NO:57 and a reference VL containing the amino acid sequence of SEQ ID NO:53; (u) a reference VH containing the amino acid sequence of SEQ ID NO:58 and a reference VL containing the amino acid sequence of SEQ ID NO:53; (v) a reference VH containing the amino acid sequence of SEQ ID NO:62 and a reference VL containing the amino acid sequence of SEQ ID NO: NO:53; (w) a reference VH containing the amino acid sequence of SEQ ID NO:64 and a reference VL containing the amino acid sequence of SEQ ID NO:53; (x) a reference VH containing the amino acid sequence of SEQ ID NO:55 and a reference VL containing the amino acid sequence of SEQ ID NO:54; (y) a reference VH containing the amino acid sequence of SEQ ID NO:61 and a reference VL containing the amino acid sequence of SEQ ID NO:54; (z) a reference VH containing the amino acid sequence of SEQ ID NO:62 and a reference VL containing the amino acid sequence of SEQ ID NO:54; (aa) a reference VH containing the amino acid sequence of SEQ ID NO:64 and a reference VL containing the amino acid sequence of SEQ ID NO:54; (bb) a reference VH containing the amino acid sequence of SEQ ID NO:63 and a reference VL containing the amino acid sequence of SEQ ID NO: (cc) a reference VH comprising the amino acid sequence of SEQ ID NO: 188 and a reference VL comprising the amino acid sequence of SEQ ID NO: 189; or (dd) a reference VH comprising the amino acid sequence of SEQ ID NO: 188 and a reference VL comprising the amino acid sequence of SEQ ID NO: 190.
在一些實施例中，該抗TSLP抗體部分係選自由以下組成之群組：全長抗體、Fab、Fab’、F(ab’)2、雙抗體及scFv。In some embodiments, the anti-TSLP antibody portion is selected from the group consisting of a full length antibody, Fab, Fab', F(ab')2 , a diabody, and a scFv.
在一些實施例中，該抗TSLP抗體部分為全長抗體(抗TSLP全長抗體)。在一些實施例中，該抗TSLP全長抗體包含人類κ CL或人類λ CL，諸如含有SEQ ID NO:73-75之任一者之胺基酸序列的CL。在一些實施例中，該抗TSLP全長抗體包含衍生自人類IgG (諸如人類IgG1、IgG2、IgG3或IgG4，例如人類IgG1)的Fc域。以下「Fc域」小節中所述之任何Fc域皆可在此使用。在一些實施例中，該Fc域包含：i) L234A+L235A (「LALA」)突變，ii) M252Y+S254T+T256E (「YTE」)突變，及/或 iii) M428L+N434S (「LS」)突變，其係根據EU編號。在一些實施例中，該Fc域進一步包含杵臼突變，諸如在一個Fc次單元中的T366W 「杵」突變，以及在另一個Fc次單元中的T366S+L368A+Y407V 「臼」突變。在一些實施例中，i)該Fc域之第一次單元及第二次單元各自含有SEQ ID NO:76-79之任一者的胺基酸序列；ii)該Fc域之第一次單元含有SEQ ID NO: 80之胺基酸序列，且該Fc域之第二次單元含有SEQ ID NO: 81之胺基酸序列；或iii)該Fc域之第一次單元含有SEQ ID NO: 81之胺基酸序列，且該Fc域之第二次單元含有SEQ ID NO: 80之胺基酸序列。在一些實施例中，該Fc域之第一次單元及第二次單元各自含有SEQ ID NO:77或79之胺基酸序列。在一些實施例中，該抗TSLP全長抗體包含：i)兩條各自含有SEQ ID NO:104之胺基酸序列的重鏈或其與SEQ ID NO:104之胺基酸序列具有至少約80% (諸如至少約85%、90%、95%、99%或更多之任一者)序列一致性的變體及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈或其與SEQ ID NO:103之胺基酸序列具有至少約80% (諸如至少約85%、90%、95%、99%或更多之任一者)序列一致性的變體；ii)兩條各自含有SEQ ID NO:105之胺基酸序列的重鏈或其與SEQ ID NO:105之胺基酸序列具有至少約80% (諸如至少約85%、90%、95%、99%或更多之任一者)序列一致性的變體及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈或其與SEQ ID NO:103之胺基酸序列具有至少約80% (諸如至少約85%、90%、95%、99%或更多之任一者)序列一致性的變體；或iii)含有SEQ ID NO:136之胺基酸序列的第一重鏈(或其與SEQ ID NO:136之胺基酸序列具有至少約80% (諸如至少約85%、90%、95%、99%或更多之任一者)序列一致性的變體)、含有SEQ ID NO:137之胺基酸序列的第二重鏈(或其與SEQ ID NO:137之胺基酸序列具有至少約80% (諸如至少約85%、90%、95%、99%或更多之任一者)序列一致性的變體)及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈(或其與SEQ ID NO:103之胺基酸序列具有至少約80% (諸如至少約85%、90%、95%、99%或更多之任一者)序列一致性的變體)。在一些實施例中，該抗TSLP全長抗體包含：i)兩條各自含有SEQ ID NO:104之胺基酸序列的重鏈及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈；ii)兩條各自含有SEQ ID NO:105之胺基酸序列的重鏈及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈；或iii)含有SEQ ID NO:136之胺基酸序列的第一重鏈、含有SEQ ID NO:137之胺基酸序列的第二重鏈及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈。In some embodiments, the anti-TSLP antibody portion is a full-length antibody (anti-TSLP full-length antibody). In some embodiments, the anti-TSLP full-length antibody comprises human κ CL or human λ CL, such as a CL comprising the amino acid sequence of any one of SEQ ID NOs: 73-75. In some embodiments, the anti-TSLP full-length antibody comprises an Fc domain derived from human IgG (such as human IgG1, IgG2, IgG3 or IgG4, for example, human IgG1). Any Fc domain described in the "Fc domain" section below can be used here. In some embodiments, the Fc domain comprises: i) L234A+L235A ("LALA") mutations, ii) M252Y+S254T+T256E ("YTE") mutations, and/or iii) M428L+N434S ("LS") mutations, according to EU numbering. In some embodiments, the Fc domain further comprises a knob-hole mutation, such as a T366W "knob" mutation in one Fc subunit and a T366S+L368A+Y407V "hole" mutation in another Fc subunit. In some embodiments, i) the first and second subunits of the Fc domain each contain an amino acid sequence of any one of SEQ ID NOs: 76-79; ii) the first subunit of the Fc domain contains an amino acid sequence of SEQ ID NO: 80, and the second subunit of the Fc domain contains an amino acid sequence of SEQ ID NO: 81; or iii) the first subunit of the Fc domain contains an amino acid sequence of SEQ ID NO: 81, and the second subunit of the Fc domain contains an amino acid sequence of SEQ ID NO: 80. In some embodiments, the first and second subunits of the Fc domain each contain an amino acid sequence of SEQ ID NO: 77 or 79. In some embodiments, the anti-TSLP full-length antibody comprises: i) two heavy chains each comprising the amino acid sequence of SEQ ID NO: 104 or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 104 and two heavy chains each comprising the amino acid sequence of SEQ ID NO: 103 or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 103; ii) two heavy chains each comprising the amino acid sequence of SEQ ID NO: 105 or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 105 and two heavy chains each comprising the amino acid sequence of SEQ ID NO: 103 or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 103; NO:103 or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:103; or iii) a first heavy chain containing the amino acid sequence of SEQ ID NO:136 (or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:136), a second heavy chain containing the amino acid sequence of SEQ ID NO:137 (or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:137), and two heavy chains each containing the amino acid sequence of SEQ ID NO:103 (or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:137); In some embodiments, the anti-TSLP full-length antibody comprises: i) two heavy chains each containing the amino acid sequence of SEQ ID NO: 104 and two heavy chains each containing the amino acid sequence of SEQ ID NO: 103; ii) two heavy chains each containing the amino acid sequence of SEQ ID NO: 105 and two heavy chains each containing the amino acid sequence of SEQ ID NO: 103; or iii) a first heavy chain containing the amino acid sequence of SEQ ID NO: 136, a second heavy chain containing the amino acid sequence of SEQ ID NO: 137 and two heavy chains each containing the amino acid sequence of SEQ ID NO: 103.
在一些實施例中，該抗TSLP抗體部分為scFv (抗TSLP scFv)。在一些實施例中，該抗TSLP scFv包含經由可選的連接子彼此融合的VH及VL。在一些實施例中，該抗TSLP scFv之N端至C端包含：VH-可選的連接子-VL，或VL-可選的連接子-VH。以下「連接子」小節中的任何連接子皆可用於連接抗TSLP scFv的VH及VL。在一些實施例中，該可選的連接子含有選自由以下所組成群組之胺基酸序列：GG及SEQ ID NO:14、16-18、97-99及163-172，諸如GG及SEQ ID NO:98-99之任一者。在一些實施例中，該抗TSLP scFv含有SEQ ID NO:106、107、191及192之任一者之胺基酸序列。In some embodiments, the anti-TSLP antibody portion is a scFv (anti-TSLP scFv). In some embodiments, the anti-TSLP scFv comprises VH and VL fused to each other via an optional linker. In some embodiments, the anti-TSLP scFv comprises from the N-terminus to the C-terminus: VH-optional linker-VL, or VL-optional linker-VH. Any linker in the following "Linker" subsection can be used to connect the VH and VL of the anti-TSLP scFv. In some embodiments, the optional linker contains an amino acid sequence selected from the group consisting of: GG and SEQ ID NO: 14, 16-18, 97-99 and 163-172, such as GG and any one of SEQ ID NO: 98-99. In some embodiments, the anti-TSLP scFv contains the amino acid sequence of any one of SEQ ID NOs: 106, 107, 191, and 192.
在一些實施例中，該抗TSLP抗體部分為Fab片段(抗TSLP Fab片段)，其包含含有VH及CH1的第一多肽，以及含有VL及CL的第二多肽。在一些實施例中，該抗TSLP Fab片段內之可變區及恆定區的配置可能與天然抗TSLP Fab片段中發現的不同。舉例而言，在一些實施例中，該抗TSLP Fab片段包含含有VH及CL的第一多肽，以及含有VL及CH1的第二多肽(參見例如Shaefer等人(2011)，PNAS，108:111870-92，其內容通過引用整體併入本文)。In some embodiments, the anti-TSLP antibody portion is a Fab fragment (anti-TSLP Fab fragment) comprising a first polypeptide comprising VH and CH1, and a second polypeptide comprising VL and CL. In some embodiments, the configuration of the variable and constant regions within the anti-TSLP Fab fragment may be different from that found in natural anti-TSLP Fab fragments. For example, in some embodiments, the anti-TSLP Fab fragment comprises a first polypeptide comprising VH and CL, and a second polypeptide comprising VL and CH1 (see, e.g., Shaefer et al. (2011), PNAS, 108: 111870-92, the contents of which are incorporated herein by reference in their entirety).
在一些實施例中，該抗TSLP Fab中的CH1及VH與VL及CL進行異二聚化，並藉由重鏈與輕鏈恆定區之間的雙硫鍵共價連接。在一些實施例中，該抗TSLP Fab片段具有基本結構NH2-VL-CL-S-S-CH1-VH-NH2。在一些實施例中，該抗TSLP Fab片段的CH1及CL藉由一或多個雙硫鍵連接。在一些實施例中，該抗TSLP Fab片段之CH1與CL之間的雙硫鍵數量為至少1個，諸如2、3、4、5個或更多。在一些實施例中，該抗TSLP Fab片段(諸如CH1及CL區域)中的半胱胺酸殘基係經工程改造以導入雙硫鍵。In some embodiments, CH1 and VH in the anti-TSLP Fab heterodimerize with VL and CL and are covalently linked by disulfide bonds between the heavy chain and light chain constant regions. In some embodiments, the anti-TSLP Fab fragment has the basic structure NH2 -VL-CL-SS-CH1-VH-NH2 . In some embodiments, CH1 and CL of the anti-TSLP Fab fragment are linked by one or more disulfide bonds. In some embodiments, the number of disulfide bonds between CH1 and CL of the anti-TSLP Fab fragment is at least 1, such as 2, 3, 4, 5 or more. In some embodiments, the cysteine residues in the anti-TSLP Fab fragment (such as the CH1 and CL regions) are engineered to introduce disulfide bonds.
在一些實施例中，該抗TSLP Fab片段在C端處不含雙硫鍵。舉例而言，該抗TSLP Fab片段之重鏈及輕鏈可以此方式進行工程改造，以便穩定相互作用而不需要雙硫鍵。舉例而言，重鏈及輕鏈可以此方式進行工程改造，以便穩定相互作用而不需要雙硫鍵。在一些實施例中，重鏈或輕鏈可經工程改造以移除半胱胺酸殘基，且其中重鏈及輕鏈仍然穩定地相互作用並發揮Fab的功用。在一些實施例中，進行突變以促進重鏈與輕鏈之間的穩定相互作用。舉例而言，「杵臼」工程改造策略可用於促進Fab的重鏈與輕鏈之間的二聚化(參見例如1996 Protein Engineering，9:617 - 621)。本文亦考慮使用針對特定目的而設計的變體Fab片段，例如CH1及/或CL之恆定域的胺基酸變化，以及移除雙硫鍵或添加用於純化的標籤等。在以下「恆定域及Fc域」小節中更詳細地描述此等突變。In some embodiments, the anti-TSLP Fab fragment does not contain a disulfide bond at the C-terminus. For example, the heavy chain and light chain of the anti-TSLP Fab fragment can be engineered in such a way that they can stably interact without a disulfide bond. For example, the heavy chain and light chain can be engineered in such a way that they can stably interact without a disulfide bond. In some embodiments, the heavy chain or the light chain can be engineered to remove a cysteine residue, and wherein the heavy chain and light chain still stably interact and function as a Fab. In some embodiments, mutations are made to promote a stable interaction between the heavy chain and the light chain. For example, the "knob-in-hole" engineering strategy can be used to promote dimerization between the heavy and light chains of Fab (see, e.g., 1996 Protein Engineering, 9: 617-621). The use of variant Fab fragments designed for specific purposes, such as amino acid changes in the constant domains of CH1 and/or CL, and removal of disulfide bonds or addition of tags for purification, etc., is also contemplated herein. These mutations are described in more detail in the "Constant Domain and Fc Domain" section below.
在一些實施例中，該抗TSLP Fab片段之CH1及CL由至少1或2個雙硫鍵連接。在一些實施例中，該抗TSLP Fab片段包含人類免疫球蛋白CH1，例如含有SEQ ID NO:158之胺基酸序列。在一些實施例中，該抗TSLP Fab片段包含人類λ輕鏈恆定區，例如含有SEQ ID NO:74或75之胺基酸序列。在一些實施例中，該抗TSLP Fab片段包含人類κ輕鏈恆定區，例如含有SEQ ID NO:73之胺基酸序列。In some embodiments, the CH1 and CL of the anti-TSLP Fab fragment are linked by at least 1 or 2 disulfide bonds. In some embodiments, the anti-TSLP Fab fragment comprises a human immunoglobulin CH1, for example, comprising the amino acid sequence of SEQ ID NO: 158. In some embodiments, the anti-TSLP Fab fragment comprises a human λ light chain constant region, for example, comprising the amino acid sequence of SEQ ID NO: 74 or 75. In some embodiments, the anti-TSLP Fab fragment comprises a human κ light chain constant region, for example, comprising the amino acid sequence of SEQ ID NO: 73.
在一些實施例中，該抗TSLP Fab片段包含含有SEQ ID NO:109之胺基酸序列的第一多肽及含有SEQ ID NO:103之胺基酸序列的第二多肽。多特異性抗TSLP抗體構築體In some embodiments, the anti-TSLP Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 109 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 103.Multispecific anti-TSLPantibody constructs
在一些實施例中，本文所述之分離的抗TSLP抗體構築體為多特異性抗TSLP抗體構築體，其包含抗TSLP抗體部分(例如，本文所述之抗TSLP抗體部分之任一者，諸如抗TSLP全長抗體、抗TSLP Fab及/或 抗TSLP scFv)，以及特異性地辨識第二標靶抗原(例如，TSLP或IL-13)的第二抗體部分。在一些實施例中，該分離的抗TSLP抗體構築體包含一或多個抗TSLP抗體部分(例如，本文所述之抗TSLP抗體部分之任一者)，以及特異性地辨識一或多個標靶抗原(例如，TSLP或IL-13)的一或多個抗體部分。在一些實施例中，該分離的抗TSLP抗體構築體係多價但單特異性，亦即其中所含的所有抗體部分特異性地辨識相同TSLP表位。在一些實施例中，該分離的抗TSLP抗體構築體係多價及多特異性(例如，雙特異性)。在一些實施例中，該分離的抗TSLP抗體構築體特異性地辨識二或多個TSLP表位。在一些實施例中，該分離的抗TSLP抗體構築體經由其一或多個抗TSLP抗體部分(例如，本文所述之抗TSLP抗體部分之任一者)特異性地辨識TSLP，且一或多個標靶抗原不為TSLP。在一些實施例中，該第二標靶抗原為IL-13。在一些實施例中，該二或多個抗TSLP抗體部分(例如，scFv、Fab)具有相同的胺酸序列。在一些實施例中，該二或多個抗TSLP抗體部分(例如，scFv、Fab) 具有不同的胺酸序列。在一些實施例中，該二或多個抗TSLP抗體部分結合至相同的TSLP表位。在一些實施例中，該二或多個抗TSLP抗體部分結合至不同的TSLP表位。在一些實施例中，該第二抗體部分係選自由以下組成之群組：全長抗體、Fab、Fab’、F(ab’)2、sdAb、雙抗體及scFv。在一些實施例中，該第二抗體部分為scFv (例如，抗IL-13 scFv)。在一些實施例中，該第二抗體部分為Fab (例如，抗IL-13 Fab)。在一些實施例中，該第二抗體部分為全長抗體(例如，抗IL-13 全長抗體)。在一些實施例中，該一或多個抗TSLP抗體部分(例如，本文所述之抗TSLP抗體部分之任一者)及特異性地辨識一或多個標靶抗原(例如，TSLP或IL-13)的一或多個抗體部分係經由連接子彼此融合。以下「連接子」小節中所述之任何連接子皆可在此使用。在一些實施例中，該連接子包含選自以下之任一者的胺基酸序列：GG及SEQ ID NO:14、16-18、97-99及163-172，諸如GG及SEQ ID NO:98-99之任一者。當該分離的抗TSLP抗體構築體包含Fc域時，以下「Fc域」小節中所述之任何Fc域皆可在此使用。Fc域In some embodiments, the isolated anti-TSLP antibody constructs described herein are multispecific anti-TSLP antibody constructs, which include an anti-TSLP antibody portion (e.g., any of the anti-TSLP antibody portions described herein, such as anti-TSLP full-length antibodies, anti-TSLP Fabs, and/or anti-TSLP scFvs), and a second antibody portion that specifically recognizes a second target antigen (e.g., TSLP or IL-13). In some embodiments, the isolated anti-TSLP antibody constructs include one or more anti-TSLP antibody portions (e.g., any of the anti-TSLP antibody portions described herein), and one or more antibody portions that specifically recognize one or more target antigens (e.g., TSLP or IL-13). In some embodiments, the isolated anti-TSLP antibody construct is multivalent but monospecific, i.e., all antibody moieties contained therein specifically recognize the same TSLP epitope. In some embodiments, the isolated anti-TSLP antibody construct is multivalent and multispecific (e.g., bispecific). In some embodiments, the isolated anti-TSLP antibody construct specifically recognizes two or more TSLP epitopes. In some embodiments, the isolated anti-TSLP antibody construct specifically recognizes TSLP via one or more anti-TSLP antibody moieties thereof (e.g., any of the anti-TSLP antibody moieties described herein), and one or more target antigens are not TSLP. In some embodiments, the second target antigen is IL-13. In some embodiments, the two or more anti-TSLP antibody portions (e.g., scFv, Fab) have the same amino acid sequence. In some embodiments, the two or more anti-TSLP antibody portions (e.g., scFv, Fab) have different amino acid sequences. In some embodiments, the two or more anti-TSLP antibody portions bind to the same TSLP epitope. In some embodiments, the two or more anti-TSLP antibody portions bind to different TSLP epitopes. In some embodiments, the second antibody portion is selected from the group consisting of: full-length antibody, Fab, Fab', F(ab')2 , sdAb, bispecific antibody, and scFv. In some embodiments, the second antibody portion is scFv (e.g., anti-IL-13 scFv). In some embodiments, the second antibody portion is Fab (e.g., anti-IL-13 Fab). In some embodiments, the second antibody portion is a full-length antibody (e.g., an anti-IL-13 full-length antibody). In some embodiments, the one or more anti-TSLP antibody portions (e.g., any of the anti-TSLP antibody portions described herein) and the one or more antibody portions that specifically recognize one or more target antigens (e.g., TSLP or IL-13) are fused to each other via a linker. Any linker described in the "Linker" subsection below can be used herein. In some embodiments, the linker comprises an amino acid sequence selected from any of the following: GG and SEQ ID NO: 14, 16-18, 97-99, and 163-172, such as GG and any of SEQ ID NO: 98-99. When the isolated anti-TSLP antibody construct comprises an Fc domain, any Fc domain described in the "Fc domain" subsection below can be used herein.Fcdomain
本文進一步描述Fc域(例如，兩個Fc次單元之一者)及其變體，其可包括在本文所述之分離的抗TSLP抗體構築體之任一者(例如，抗TSLP全長抗體)中。Further described herein are Fc domains (e.g., one of the two Fc subunits) and variants thereof, which can be included in any of the isolated anti-TSLP antibody constructs described herein (e.g., an anti-TSLP full-length antibody).
Fc (片段、可結晶)域為抗體的尾部區域，其與稱為Fc受體的細胞表面受體及補體系統的一些蛋白質相互作用。抗體之Fc媒介的效應功能包括抗體依賴性細胞毒性(ADCC)、抗體依賴性細胞吞噬作用(ADCP)及補體依賴性細胞毒性(CDC)。此性質為抗體活化免疫系統的關鍵。此Fc域由二或三個恆定域組成，該些恆定域附接至兩條重鏈之每一者上的CH域，取決於抗體的類別。當組裝兩條抗體多肽時，兩條重鏈上的Fc域之CH3次單元的突變(稱為「杵臼」突變)可進一步改善異二聚合作用。舉例而言，當以下的「臼」取代被導入第二重鏈上的Fc域之CH3域的位置366、368及407處：T366S、L368A及Y407V時，第一重鏈上的Fc域之CH3域可包含「杵」突變(例如，T366W)。杵與臼突變的位置可轉換，例如該杵突變可位於第二重鏈上，且該臼突變可位於第一重鏈上。可導入額外的Fc域突變，諸如LALA突變(例如，Fc域之CH2域的L234A及L235A取代)，以降低FcγR活化及Fc媒介的毒性，其可在投予該抗體之個體中引發副作用，諸如細胞激素釋放症候群。此外，可導入Fc域突變，諸如LS突變(例如，Fc域之CH3域的M428L及N434S取代)，以延長抗體半衰期而不影響效力。本文亦可使用FcRn親和力增強Fc突變體，諸如M252Y/S254T/T256E (YTE)突變，其在導入Fc域中時，能夠延長血清半衰期。因此，所述之突變可單獨或組合使用，以改善抗體功能及降低對患者的毒性。在一些實施例中，該Fc域包含LALA突變。在一些實施例中，該Fc域包含LS或YTE突變。在一些實施例中，該Fc域包含LALA突變及LS 突變。在一些實施例中，該Fc域包含LALA突變及YTE突變。在一些實施例中，i)該Fc域之第一次單元及第二次單元各自含有SEQ ID NO:76-79之任一者的胺基酸序列；ii)該Fc域之第一次單元含有SEQ ID NO:80之胺基酸序列，且該Fc域之第二次單元含有SEQ ID NO:81之胺基酸序列；或iii)該Fc域之第一次單元含有SEQ ID NO:81之胺基酸序列，且該Fc域之第二次單元含有SEQ ID NO:80之胺基酸序列。連接子The Fc (fragment, crystallizable) domain is the tail region of the antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. The Fc-mediated effector functions of antibodies include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). This property is key to the immune system activation of antibodies. This Fc domain consists of two or three homeostatic domains attached to the CH domain on each of the two heavy chains, depending on the class of the antibody. When two antibody polypeptides are assembled, mutations in the CH3 subunits of the Fc domains on both heavy chains (called "knob-in-hole" mutations) can further improve heterodimerization. For example, the CH3 domain of the Fc domain on the first heavy chain may comprise a "knob" mutation (e.g., T366W) when the following "hole" substitutions are introduced into the CH3 domain of the Fc domain on the second heavy chain at positions 366, 368, and 407: T366S, L368A, and Y407V. The locations of the knob and hole mutations may be switched, e.g., the knob mutation may be located on the second heavy chain and the hole mutation may be located on the first heavy chain. Additional Fc domain mutations, such as LALA mutations (e.g., L234A and L235A substitutions of the CH2 domain of the Fc domain), may be introduced to reduce FcγR activation and Fc-mediated toxicity, which may induce side effects, such as cytokine release syndrome, in a subject to which the antibody is administered. In addition, Fc domain mutations, such as LS mutations (e.g., M428L and N434S substitutions of the CH3 domain of the Fc domain), can be introduced to extend the antibody half-life without affecting efficacy. FcRn affinity enhancing Fc mutants, such as M252Y/S254T/T256E (YTE) mutations, can also be used herein, which, when introduced into the Fc domain, can extend the serum half-life. Therefore, the mutations described can be used alone or in combination to improve antibody function and reduce toxicity to patients. In some embodiments, the Fc domain comprises a LALA mutation. In some embodiments, the Fc domain comprises a LS or YTE mutation. In some embodiments, the Fc domain comprises a LALA mutation and a LS mutation. In some embodiments, the Fc domain comprises a LALA mutation and a YTE mutation. In some embodiments, i) the first and second subunits of the Fc domain each contain an amino acid sequence of any one of SEQ ID NOs: 76-79; ii) the first subunit of the Fc domain contains an amino acid sequence of SEQ ID NO: 80, and the second subunit of the Fc domain contains an amino acid sequence of SEQ ID NO: 81; or iii) the first subunit of the Fc domain contains an amino acid sequence of SEQ ID NO: 81, and the second subunit of the Fc domain contains an amino acid sequence of SEQ ID NO: 80.Linker
本文所述之分離的抗TSLP抗體構築體(例如，多特異性抗TSLP抗體構築體、抗TSLP scFv)可包含一或多個連接子(諸如肽連接子)，該連接子連接其中所含的二或多個域或部分，例如介於VH與VL域之間(例如，scFv之內)、介於抗TSLP抗體部分與其融合配偶體或特異性地辨識第二標靶抗原的第二抗體部分(例如，scFv、Fab或全長抗體)之間，或介於二或多個抗TSLP抗體部分之間。在一些實施例中，該二或多個連接子相同。在一些實施例中，該二或多個連接子不同。在一些實施例中，該分離的抗TSLP抗體構築體不包含連接子，亦即其中所含的二或多個域或部分彼此直接連接。The isolated anti-TSLP antibody constructs described herein (e.g., multispecific anti-TSLP antibody constructs, anti-TSLP scFv) may comprise one or more linkers (e.g., peptide linkers) linking two or more domains or portions contained therein, such as between the VH and VL domains (e.g., within an scFv), between an anti-TSLP antibody portion and its fusion partner or a second antibody portion that specifically recognizes a second target antigen (e.g., scFv, Fab, or full-length antibody), or between two or more anti-TSLP antibody portions. In some embodiments, the two or more linkers are the same. In some embodiments, the two or more linkers are different. In some embodiments, the isolated anti-TSLP antibody construct does not comprise a linker, i.e., the two or more domains or portions contained therein are directly linked to each other.
該連接子可為任何長度的肽連接子。在一些實施例中，該肽連接子長度約1至約10個胺基酸(aa)、長度約2至約15個胺基酸、長度約3至約12個胺基酸、長度約4至約10個胺基酸、長度約5至約9個胺基酸、長度約6至約8個胺基酸、長度約1至約20個胺基酸、長度約21至約30個胺基酸、長度約1至約30個胺基酸、長度約10至約30個胺基酸或長度約2至約5個胺基酸。在一些實施例中，該肽連接子為長度1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20個胺基酸之任一者。在一些實施例中，該肽連接子為長度21、22、23、24、25、26、27、28、29或30個胺基酸之任一者。在一些實施例中，該肽連接子長度約2至約15個胺基酸。The linker can be a peptide linker of any length. In some embodiments, the peptide linker is about 1 to about 10 amino acids (aa), about 2 to about 15 amino acids, about 3 to about 12 amino acids, about 4 to about 10 amino acids, about 5 to about 9 amino acids, about 6 to about 8 amino acids, about 1 to about 20 amino acids, about 21 to about 30 amino acids, about 1 to about 30 amino acids, about 10 to about 30 amino acids, or about 2 to about 5 amino acids. In some embodiments, the peptide linker is any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the peptide linker is any of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In some embodiments, the peptide linker is about 2 to about 15 amino acids in length.
肽連接子可具有天然存在的序列或非天然存在的序列。舉例而言，衍生自僅抗體重鏈鉸鏈區之序列可用作連接子。參見例如WO1996/34103。在一些實施例中，該肽連接子為人類IgG1或IgG4鉸鏈。在一些實施例中，該連接子為撓性連接子。示例性撓性連接子包括甘胺酸聚合物(G)n (例如，GG)、甘胺酸-絲胺酸聚合物(包括例如：(GS)n、(GSGGS)n (SEQ ID NO:14)、(GGGS)n (SEQ ID NO:16)或(GGGGS)n (SEQ ID NO:17)，其中n為至少1的整數)、甘胺酸-丙胺酸聚合物、丙胺酸-絲胺酸聚合物及本領域已知之其他撓性連接子。甘胺酸及甘胺酸-絲胺酸聚合物相對非結構化，從而可充當各組分之間的中性栓鏈(tether)。甘胺酸比丙胺酸明顯擷取更多的phi-psi空間，且比側鏈較長的殘基更不受限制(參見Scheraga，Rev. Computational Chem. 11 173-142 (1992))。在一些實施例中，該連接子包含選自由以下所組成群組之胺基酸殘基：甘胺酸、絲胺酸、精胺酸及丙胺酸。示例性撓性連接子包括但不限於：Gly-Gly、GGSG (SEQ ID NO:18)、GGSGG (SEQ ID NO:163)、GSGSG (SEQ ID NO:164)、GSGGG (SEQ ID NO:165)、GGGSG (SEQ ID NO:166)、GSSSG (SEQ ID NO:167)、GGSGGS (SEQ ID NO:168)、SGGGGS (SEQ ID NO:169)、GRAGGGGAGGGG (SEQ ID NO:170)、GRAGGG (SEQ ID NO:171)、GGGGSGGGGSGGGGS (SEQ ID NO:99)、GGGG (SEQ ID NO: 98)、GGGGS (SEQ ID NO:172)及其類似物。在一些實施例中，介於scFv 的VH與VL之間的連接子含有SEQ ID NO:99之序列。具有通常知識者將理解，分離的抗TSLP抗體構築體或抗TSLP抗體部分之設計可包括全部或部分撓性的連接子，使得該連接子可包括撓性連接子部分以及一或多個賦予較少撓性結構的部分，以提供所需之抗TSLP抗體構築體結構。The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from only the hinge region of the antibody heavy chain can be used as a linker. See, for example, WO1996/34103. In some embodiments, the peptide linker is a human IgG1 or IgG4 hinge. In some embodiments, the linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G)n (e.g., GG), glycine-serine polymers (including, for example: (GS)n, (GSGGS)n (SEQ ID NO: 14), (GGGS)n (SEQ ID NO: 16) or (GGGGS)n (SEQ ID NO: 17), wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and thus can act as neutral tethers between components. Glycine captures significantly more phi-psi space than alanine and is less constrained than longer side chain residues (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). In some embodiments, the linker comprises an amino acid residue selected from the group consisting of glycine, serine, arginine, and alanine. Exemplary flexible linkers include, but are not limited to, Gly-Gly, GGSG (SEQ ID NO: 18), GGSGG (SEQ ID NO: 163), GSGSG (SEQ ID NO: 164), GSGGG (SEQ ID NO: 165), GGGSG (SEQ ID NO: 166), GSSSG (SEQ ID NO: 167), GGSGGS (SEQ ID NO: 168), SGGGGS (SEQ ID NO: 169), GRAGGGGAGGGG (SEQ ID NO: 170), GRAGGG (SEQ ID NO: 171), GGGGSGGGGSGGGGS (SEQ ID NO: 99), GGGG (SEQ ID NO: 98), GGGGS (SEQ ID NO: 172), and the like. In some embodiments, the linker between the VH and VL of the scFv contains the sequence of SEQ ID NO: 99. One of ordinary skill in the art will appreciate that the design of an isolated anti-TSLP antibody construct or anti-TSLP antibody portion may include a fully or partially flexible linker such that the linker may include a flexible linker portion and one or more portions that impart a less flexible structure to provide the desired anti-TSLP antibody construct structure.
在一些實施例中，該抗TSLP抗體部分與該第二抗體部分之間的連接子，或該抗TSLP抗體部分之內的連接子為穩定的連接子(不被蛋白酶(尤其是MMP)裂解)。In some embodiments, the linker between the anti-TSLP antibody portion and the second antibody portion, or the linker within the anti-TSLP antibody portion, is a stable linker (not cleaved by proteases, particularly MMPs).
在一些實施例中，該連接子可裂解的連接子。可被MMP裂解的受質序列已被廣泛研究。舉例而言，PLGLAG之序列(SEQ ID NO:97)可被大多數的MMP裂解。III.製備方法In some embodiments, the linker is a cleavable linker. Substrate sequences that can be cleaved by MMPs have been widely studied. For example, the sequence of PLGLAG (SEQ ID NO: 97) can be cleaved by most MMPs.III.Preparation Methods
亦提供製造本文所述之分離的抗TSLP抗體構築體或抗TSLP抗體部分之任一者的方法。Also provided are methods of making any of the isolated anti-TSLP antibody constructs or anti-TSLP antibody portions described herein.
本文所述之分離的抗TSLP抗體構築體或抗TSLP抗體部分可由本領域已知之任何蛋白質表現及純化方法來製備。可完全合成編碼分離的抗TSLP抗體構築體或抗TSLP抗體部分的DNA序列。在獲得此類序列之後，將其選殖至適合的表現載體中，隨後轉染至適合的宿主細胞中。培養轉染的宿主細胞，收穫並純化上清液，以獲得本文所述之抗TSLP抗體構築體或抗TSLP抗體部分。在一些實施例中，將宿主細胞溶解，以獲得表現的抗TSLP抗體構築體或抗TSLP抗體部分。The isolated anti-TSLP antibody constructs or anti-TSLP antibody portions described herein can be prepared by any protein expression and purification method known in the art. DNA sequences encoding isolated anti-TSLP antibody constructs or anti-TSLP antibody portions can be fully synthesized. After obtaining such sequences, they are cloned into suitable expression vectors and then transfected into suitable host cells. The transfected host cells are cultured, and the supernatant is harvested and purified to obtain the anti-TSLP antibody constructs or anti-TSLP antibody portions described herein. In some embodiments, the host cells are lysed to obtain the expressed anti-TSLP antibody constructs or anti-TSLP antibody portions.
在一些實施例中本申請案提供分離的核酸，其編碼本文所述之分離的抗TSLP抗體構築體之任一者的一或多種多肽。分離的核酸可為DNA或RNA。In some embodiments, the present application provides an isolated nucleic acid encoding one or more polypeptides of any of the isolated anti-TSLP antibody constructs described herein. The isolated nucleic acid can be DNA or RNA.
在一些實施例中，該分離的核酸被插入載體中，諸如表現載體、病毒載體或選殖載體。因此，亦提供含有本文所述之任何分離核酸的載體。為了表現核酸，可將載體導入宿主細胞中以允許核酸在宿主細胞內表現。表現載體可含有用於控制表現的多種元件，包括但不限於啟動子序列、轉錄起始序列、增強子序列、選擇標記及訊號序列。本領域之具有通常知識者可適當地選擇此等元件。舉例而言，可選擇啟動子序列以促進載體中多核苷酸的轉錄。合適的啟動子序列包括但不限於：T7啟動子、T3啟動子、SP6啟動子、β-肌動蛋白啟動子、EF1a啟動子、CMV啟動子及SV40啟動子。可選擇增強子序列以增強核酸的轉錄。可選擇可擇的標記以允許從缺乏載體的彼等細胞中選擇插入載體的宿主細胞，例如，可選擇的標記可為賦予抗生素抗性的基因。可選擇訊號序列以允許表現的多肽被運輸至宿主細胞外部。In some embodiments, the isolated nucleic acid is inserted into a vector, such as an expression vector, a viral vector, or a selection vector. Therefore, a vector containing any isolated nucleic acid described herein is also provided. In order to express the nucleic acid, the vector can be introduced into a host cell to allow the nucleic acid to be expressed in the host cell. The expression vector can contain a variety of elements for controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection marker, and a signal sequence. Those with ordinary knowledge in the art can appropriately select these elements. For example, a promoter sequence can be selected to promote the transcription of a polynucleotide in a vector. Suitable promoter sequences include, but are not limited to, T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. Enhancer sequences may be selected to enhance transcription of nucleic acids. Selectable markers may be selected to allow host cells into which the vector is inserted to be selected from those cells lacking the vector, for example, selectable markers may be genes that confer antibiotic resistance. Signal sequences may be selected to allow the expressed polypeptide to be transported to the outside of the host cell.
在一些實施例中，提供一種分離的宿主細胞，其含有編碼本文所述之分離的抗TSLP抗體構築體之任一者之多肽部分的任何分離的核酸或載體。在一些實施例中，提供一種分離的宿主細胞，其表現本文所述之分離的抗TSLP抗體構築體之任一者。在一些實施例中，本文所述之分離的抗TSLP抗體構築體(例如，全長抗體、Fab片段或多特異性抗TSLP抗體構築體)之任一者的二或多種多肽由單一載體編碼。在一些實施例中，本文所述之分離的抗TSLP抗體構築體(例如，全長抗體、Fab片段或多特異性分離的抗TSLP抗體構築體)之任一者的二或多種多肽由二或多種載體編碼。含有載體的宿主細胞可用於表現或選殖分離的核酸。合適的宿主細胞可包括但不限於：原核細胞、真菌細胞、酵母細胞或高等真核細胞，諸如哺乳動物細胞。抗體及抗原結合片段在原核細胞(諸如大腸桿菌)中的表現為本領域中充分證實的。相關回顧文獻，參見例如Pluckthun, A. BioTechnology 9: 545-551 (1991)。本領域具有通常知識者亦可使用真核細胞培養表現來作為生產抗體或其抗原結合片段的選項，參見回顧文獻，例如Ref, M. E. (1993)Curr. Opinion Biotech.4: 573-576；Trill J. J.等人(1995) Curr. Opinion Biotech 6: 553-560。高等真核細胞，特別是源自多細胞生物的細胞可用於表現醣基化多肽。合適的高等真核細胞包括但不限於無脊椎動物細胞與昆蟲細胞以及脊椎動物細胞。在一些實施例中，該宿主細胞為大腸桿菌。在一些實施例中，該宿主細胞為中國倉鼠卵巢(CHO)細胞或HEK293細胞。In some embodiments, an isolated host cell is provided that contains any isolated nucleic acid or vector encoding a polypeptide portion of any of the isolated anti-TSLP antibody constructs described herein. In some embodiments, an isolated host cell is provided that expresses any of the isolated anti-TSLP antibody constructs described herein. In some embodiments, two or more polypeptides of any of the isolated anti-TSLP antibody constructs (e.g., full-length antibodies, Fab fragments, or multispecific anti-TSLP antibody constructs) described herein are encoded by a single vector. In some embodiments, two or more polypeptides of any of the isolated anti-TSLP antibody constructs (e.g., full-length antibodies, Fab fragments, or multispecific isolated anti-TSLP antibody constructs) described herein are encoded by two or more vectors. Host cells containing the vector can be used to express or clone the isolated nucleic acid. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. The expression of antibodies and antigen-binding fragments in prokaryotic cells (such as E. coli) is well established in the art. For a review of the literature, see, for example, Pluckthun, A. BioTechnology 9: 545-551 (1991). Those skilled in the art may also use eukaryotic cell culture expression as an option for producing antibodies or antigen-binding fragments thereof, see review literature, e.g., Ref, ME (1993)Curr. Opinion Biotech. 4: 573-576; Trill JJ et al. (1995) Curr. Opinion Biotech 6: 553-560. Higher eukaryotic cells, particularly cells derived from multicellular organisms, can be used to express glycosylated polypeptides. Suitable higher eukaryotic cells include, but are not limited to, invertebrate cells and insect cells as well as vertebrate cells. In some embodiments, the host cell is Escherichia coli. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell or a HEK293 cell.
可使用本領域已知之任何合適方法將載體導入宿主細胞，包括但不限於：DEAE-葡聚醣媒介的遞輸、磷酸鈣沉澱法、陽離子脂質媒介的遞輸、微質體媒介的轉染、電穿孔、微粒轟擊、受體媒介的基因遞輸、由聚離胺酸、組蛋白、殼聚醣及肽媒介的遞輸。用於轉染及轉形細胞以表現感興趣載體的標準方法為本領域熟習的。在一些實施例中，該宿主細胞含有各自編碼本文所述之分離的抗TSLP抗體構築體之任一者之多肽的二或多種載體。可按相同比率或不同比率將二或多種載體導入宿主細胞中。在一些實施例中，該宿主細胞包含含有分離核酸的單一載體，該載體編碼本文所述之分離的抗TSLP抗體構築體之任一者的二或多種多肽。編碼分離的抗TSLP抗體構築體之二或多種多肽的二或多種核酸可處於相同啟動子控制下或處於不同啟動子控制下。舉例而言，相同啟動子控制下的二或多種核酸可經由IRES序列或編碼自裂解肽(例如，P2A、T2A)的序列連接。The vectors may be introduced into the host cells using any suitable method known in the art, including but not limited to: DEAE-dextran mediated delivery, calcium phosphate precipitation, cationic lipid mediated delivery, miniplastid mediated transfection, electroporation, microprojectile bombardment, receptor mediated gene delivery, polylysine, histone, chitosan and peptide mediated delivery. Standard methods for transfecting and transforming cells to express vectors of interest are well known in the art. In some embodiments, the host cell contains two or more vectors each encoding a polypeptide of any of the isolated anti-TSLP antibody constructs described herein. The two or more vectors may be introduced into the host cell in the same ratio or in different ratios. In some embodiments, the host cell comprises a single vector containing isolated nucleic acids encoding two or more polypeptides of any of the isolated anti-TSLP antibody constructs described herein. The two or more nucleic acids encoding the two or more polypeptides of the isolated anti-TSLP antibody constructs can be under the control of the same promoter or under the control of different promoters. For example, the two or more nucleic acids under the control of the same promoter can be linked via an IRES sequence or a sequence encoding a self-cleaving peptide (e.g., P2A, T2A).
在一些實施例中，本申請案提供製造本文所述之分離的抗TSLP抗體構築體之任一者的方法，其包含：i)在適合表現該抗TSLP抗體構築體的條件下培養含有本文所述之分離的核酸之任一者或本文所述之載體之任一者的分離的宿主細胞，或本文所述之分離的宿主細胞之任一者(例如，含有本文所述之分離的核酸或載體之任一者的宿主細胞)，以及ii)從該宿主細胞(例如，從細胞培養物或藉由溶解宿主細胞)獲得該表現的抗TSLP抗體構築體。該分離的宿主細胞在允許插入載體之核酸表現的條件下培養。適合多核苷酸表現的條件可包括但不限於：合適的培養基、培養基中合適的宿主細胞密度、必需營養素的存在、補充因子的存在、合適的溫度與濕度及不存在微生物污染物。本領域之具有通常知識者可根據表現目的適當地選擇合適的條件。在一些實施例中，該製造方法進一步包含純化獲得的抗TSLP抗體構築體之任一者。In some embodiments, the present application provides a method of making any of the isolated anti-TSLP antibody constructs described herein, comprising: i) culturing an isolated host cell containing any of the isolated nucleic acids described herein or any of the vectors described herein, or any of the isolated host cells described herein (e.g., a host cell containing any of the isolated nucleic acids or vectors described herein) under conditions suitable for expression of the anti-TSLP antibody construct, and ii) obtaining the expressed anti-TSLP antibody construct from the host cell (e.g., from a cell culture or by lysing the host cell). The isolated host cell is cultured under conditions that allow expression of the nucleic acid inserted into the vector. Suitable conditions for polynucleotide expression may include, but are not limited to, suitable culture medium, suitable host cell density in the culture medium, the presence of essential nutrients, the presence of supplementary factors, suitable temperature and humidity, and the absence of microbial contaminants. A person skilled in the art can appropriately select suitable conditions according to the purpose of expression. In some embodiments, the production method further comprises purifying any one of the anti-TSLP antibody constructs obtained.
在一些實施例中，由宿主細胞表現的多肽可組裝在一起(例如，形成多肽複合體，諸如二聚體)並產生本文所述之分離的抗TSLP抗體構築體之任一者。在一些實施例中，該多肽複合體可形成在宿主細胞內部。舉例而言，該多肽複合體可藉助相關酵素及/或輔因子而形成在宿主細胞內部。在一些實施例中，該多肽複合體可從細胞中分泌出來。在一些實施例中，單獨的多肽可從宿主細胞中分泌出來，隨後在宿主細胞外部形成多肽複合體(諸如本文所述之分離的抗TSLP抗體構築體之任一者 )。In some embodiments, polypeptides expressed by host cells can assemble together (e.g., to form polypeptide complexes, such as dimers) and produce any of the isolated anti-TSLP antibody constructs described herein. In some embodiments, the polypeptide complex can be formed inside the host cell. For example, the polypeptide complex can be formed inside the host cell with the help of associated enzymes and/or cofactors. In some embodiments, the polypeptide complex can be secreted from the cell. In some embodiments, individual polypeptides can be secreted from the host cell and then form a polypeptide complex (such as any of the isolated anti-TSLP antibody constructs described herein) outside the host cell.
在一些實施例中，本文所述之分離的抗TSLP抗體構築體之任一者的二或多種多肽可單獨表現，並允許在合適的條件下形成(例如，二聚化)分離的抗TSLP抗體構築體。舉例而言，可在合適的緩衝液中將第一多肽與第二多肽組合，以使第一蛋白質單體與第二蛋白質單體經由適當的相互作用(諸如疏水性相互作用)進行二聚化。在一些實施例中，可在含有酵素及/或輔因子的合適緩衝液中將第一多肽與第二多肽組合，該酵素及/或輔因子可促進第一多肽與第二多肽的二聚化。在一些實施例中，可在合適的媒劑中將第一多肽與第二多肽組合，以使其等在合適的試劑及/或催化劑存在下彼此反應。In some embodiments, two or more polypeptides of any of the isolated anti-TSLP antibody constructs described herein can be expressed separately and allowed to form (e.g., dimerize) an isolated anti-TSLP antibody construct under appropriate conditions. For example, a first polypeptide can be combined with a second polypeptide in a suitable buffer so that the first protein monomer dimerizes with the second protein monomer via appropriate interactions (e.g., hydrophobic interactions). In some embodiments, a first polypeptide can be combined with a second polypeptide in a suitable buffer containing an enzyme and/or cofactor that promotes dimerization of the first polypeptide with the second polypeptide. In some embodiments, a first polypeptide can be combined with a second polypeptide in a suitable medium so that they react with each other in the presence of a suitable reagent and/or catalyst.
可使用任何合適的方法收集表現的多肽及/或多肽複合體。多肽及/或多肽複合體可表現在細胞內、週質空間中，或分泌至細胞外的培養基中。若多肽及/或多肽複合體表現在細胞內，則可將含有多肽及/或多肽複合體的宿主細胞溶解，並藉由離心或超濾而移除不需要的碎片，從溶解物中分離多肽及/或多肽複合體。若多肽及/或多肽複合體分泌至大腸桿菌的週質空間中，則可在諸如乙酸鈉(pH 3.5)、EDTA及苯甲基磺醯基氟(PMSF)等試劑存在下將細胞漿糊解凍約30分鐘，並可藉由離心移除細胞碎片(Carter等人，BioTechnology 10:163-167 (1992))。若多肽及/或多肽複合體分泌至培養基中，則可收集細胞培養物的上清液，並使用市售蛋白質濃縮過濾器(例如，Amincon或Millipore Pellicon超濾裝置)進行濃縮。可在收集及濃縮步驟中添加蛋白酶抑制劑及/或抗生素，以抑制蛋白質降解及/或污染性微生物的生長。The expressed polypeptide and/or polypeptide complex can be collected using any suitable method. The polypeptide and/or polypeptide complex can be expressed intracellularly, in the periplasmic space, or secreted into the culture medium outside the cell. If the polypeptide and/or polypeptide complex is expressed intracellularly, the host cells containing the polypeptide and/or polypeptide complex can be lysed and the polypeptide and/or polypeptide complex can be separated from the lysate by removing unwanted debris by centrifugation or ultrafiltration. If the polypeptide and/or polypeptide complex is secreted into the periplasmic space of E. coli, the cytoplasmic paste can be thawed for about 30 minutes in the presence of reagents such as sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF), and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10: 163-167 (1992)). If the polypeptide and/or polypeptide complex is secreted into the culture medium, the supernatant of the cell culture can be collected and concentrated using a commercially available protein concentration filter (e.g., Amincon or Millipore Pellicon ultrafiltration device). Protease inhibitors and/or antibiotics may be added during the collection and concentration steps to inhibit protein degradation and/or the growth of contaminating microorganisms.
可藉由合適的方法進一步純化所表現的多肽及/或多肽複合體，諸如但不限於：親和力層析法、羥基磷灰石層析法、尺寸排阻層析法、凝膠電泳、透析、離子交換管柱上的離子交換分離等、乙醇沉澱、逆相HPLC、矽膠層析法、肝素瓊脂糖層析法、陰離子或陽離子交換樹脂層析法(諸如，聚天門冬胺酸管柱)、聚焦層析法、SDS-PAGE及硫酸銨沉澱(回顧文獻，參見Bonner, P. L., Protein purification，由Taylor & Francis出版，2007；Janson, J. C.,等人，Protein purification: principles, high resolution methods and applications，由Wiley-VCH出版，1998)。The expressed polypeptide and/or polypeptide complex may be further purified by suitable methods, such as, but not limited to, affinity chromatography, hydroxyapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange separation on an ion exchange column, etc., ethanol precipitation, reversed phase HPLC, silica gel chromatography, heparin agarose chromatography, anion or cation exchange resin chromatography (e.g., polyaspartic acid column), focusing chromatography, SDS-PAGE, and ammonium sulfate precipitation (for review, see Bonner, P. L., Protein purification, published by Taylor & Francis, 2007; Janson, J. C., et al., Protein purification: principles, high resolution methods and applications, published by Wiley-VCH, 1998).
在一些實施例中，可藉由親和力層析法來純化多肽及/或多肽複合體。在一些實施例中，蛋白A層析法或蛋白A/G (蛋白A與蛋白G的融合蛋白)層析法可用於純化含有源自抗體CH2域及/或CH3域組分的多肽及/或多肽複合體(Lindmark等人，J. Immunol. Meth. 62:1-13 (1983))；Zettlit, K. A.，Protein Engineering，第V部分，531-535，2010)。在一些實施例中，蛋白G層析法可用於純化含有IgG γ3重鏈的多肽及/或多肽複合體(Guss等人，EMBO J. 5:1567 1575 (1986))。在一些實施例中，蛋白L層析法可用於純化含有κ 輕鏈的多肽及/或多肽複合體(Sudhir, P.，Antigen engineering protocols，第26章，由Humana Press出版，1995；Nilson, B. H. K.等人，J. Biol. Chem., 267, 2234-2239 (1992))。親和配體附接的基質通常為瓊脂糖，但亦可使用其他基質。機械穩定的基質(諸如可控孔徑玻璃或聚(苯乙烯二乙烯基)苯)允許達成比瓊脂糖更快的流速及更短的處理時間。當任何分離的抗TSLP抗體構築體包含額外的CH3域時，可使用Bakerbond ABX樹脂(J. T. Baker，Phillipsburg，N.J.)進行純化。IV.醫藥組成物、單位劑量、製品及套組In some embodiments, the polypeptide and/or polypeptide complex can be purified by affinity chromatography. In some embodiments, protein A chromatography or protein A/G (a fusion protein of protein A and protein G) chromatography can be used to purify polypeptides and/or polypeptide complexes containing components derived from antibody CH2 domains and/or CH3 domains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)); Zettlit, KA, Protein Engineering, Part V, 531-535, 2010). In some embodiments, protein G chromatography can be used to purify polypeptides and/or polypeptide complexes containing IgG γ3 heavy chains (Guss et al., EMBO J. 5:1567 1575 (1986)). In some embodiments, protein L chromatography can be used to purify polypeptides and/or polypeptide complexes containing kappa light chains (Sudhir, P., Antigen engineering protocols, Chapter 26, published by Humana Press, 1995; Nilson, BHK et al., J. Biol. Chem., 267, 2234-2239 (1992)). The matrix to which the affinity ligand is attached is typically agarose, but other matrices can also be used. Mechanically stable matrices (such as controlled pore glass or poly(styrenedivinyl)benzene) allow faster flow rates and shorter processing times than agarose. When any isolated anti-TSLP antibody construct contains an additional CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) can be used for purification.IV.Pharmaceutical compositions, unit doses, preparations and kits
本申請案進一步提供含有本發明之分離的抗TSLP抗體構築體及可選地醫藥上可接受之載體的醫藥組成物。The present application further provides a pharmaceutical composition comprising the isolated anti-TSLP antibody construct of the present invention and optionally a pharmaceutically acceptable carrier.
本文所述之分離的抗TSLP抗體構築體或其醫藥組成物可適合本文所述之多種投予方式，包括例如全身或局部投予。在一些實施例中，該醫藥組成物係調配成靜脈內投予。在一些實施例中，該醫藥組成物係調配成皮下注射。The isolated anti-TSLP antibody constructs or pharmaceutical compositions described herein can be suitable for a variety of administration methods described herein, including, for example, systemic or local administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous injection.
如本文所用，「載劑」包括醫藥上可接受之載體、賦形劑或穩定劑，其在所用劑量及濃度下對暴露於其之細胞或哺乳動物無毒。通常生理上可接受之載劑為pH緩衝水溶液。可接受之載劑、賦形劑或穩定劑在所用劑量及濃度下對接受者無毒，且包括：緩衝液，諸如磷酸鹽、檸檬酸鹽及其他有機酸；抗氧化劑，包括抗壞血酸及甲硫胺酸；防腐劑(諸如十八烷基二甲基苄基氯化銨；六甲氯銨；氯化苄烷銨；氯化苄甲乙氧銨；酚類、丁醇或苄醇；對羥基苯甲酸烷基酯，諸如對羥基苯甲酸甲酯或對羥基苯甲酸丙酯；兒茶酚；間苯二酚；環己醇；3-戊醇；及間甲酚)；低分子量(小於約10個殘基)多肽；蛋白質，諸如血清白蛋白、明膠或免疫球蛋白；親水聚合物，諸如聚乙烯吡咯啶酮；胺基酸，諸如甘胺酸、麩醯胺酸、天冬醯胺酸、組胺酸、精胺酸或離胺酸；單醣、雙醣及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合劑，諸如EDTA；糖，諸如蔗糖、甘露醇、海藻糖或山梨醇；鹽形成反離子，諸如鈉；金屬複合體(例如Zn-蛋白質複合體)；及/或非離子型表面活性劑，諸如TWEEN™、PLURONICS™或聚乙二醇(PEG)。As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient or stabilizer that is non-toxic to cells or mammals exposed thereto at the dosage and concentration used. Typically, a physiologically acceptable carrier is a pH buffered aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethylammonium chloride; benzylammonium chloride; benzylmethylethoxyammonium chloride; phenols, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
在一些實施例中，該醫藥組成物係調配成pH範圍為約4.5至約9.0，包括例如pH範圍為約5.0至約8.0、約6.5至約7.5、約6.5至約7.0或約7.0至約7.5之任一者。在一些實施例中，該醫藥組成物亦可藉由添加合適的張力調節劑(諸如甘油)而使其與血液等張。In some embodiments, the pharmaceutical composition is formulated to have a pH range of about 4.5 to about 9.0, including, for example, a pH range of about 5.0 to about 8.0, about 6.5 to about 7.5, about 6.5 to about 7.0, or about 7.0 to about 7.5. In some embodiments, the pharmaceutical composition can also be made isotonic with blood by adding a suitable tonicity adjuster such as glycerol.
用於體內投予的醫藥組成物通常調配成無菌的、實質上等張的且完全符合美國食品藥品管理局的所有藥品優良製造規範(GMP)規定。經由無菌濾膜過濾即可輕易實現無菌。在一些實施例中，該組成物不含任何病原體。為了注射，該醫藥組成物可呈水溶液形式，例如生理學上相容之緩衝液(諸如漢克氏溶液(Hank's solution)或林格氏溶液(Ringer's solution))。此外，該醫藥組成物可呈固體形式，並在使用前立即重新溶解或懸浮。亦包括冷凍乾燥組成物。Pharmaceutical compositions for in vivo administration are typically formulated to be sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Sterility is easily achieved by filtration through a sterile filter membrane. In some embodiments, the composition does not contain any pathogens. For injection, the pharmaceutical composition may be in the form of an aqueous solution, such as a physiologically compatible buffer (such as Hank's solution or Ringer's solution). In addition, the pharmaceutical composition may be in solid form and re-dissolved or suspended immediately before use. Freeze-dried compositions are also included.
在一些實施例中，該醫藥組成物依照常規程序調配為適應於非經口(例如，靜脈內、肌內或皮下)投予的醫藥組成物。通常，注射用組成物為溶於無菌等張緩衝水溶液中的溶液。當必要時，該組成物亦可包括增溶劑及局部麻醉劑(諸如利多卡因(lidocaine))，以減輕注射部位的疼痛。一般而言，各成分可單獨提供或以單位劑量形式混合在一起，例如以凍乾粉末或無水濃縮物形式置於密封容器(諸如安瓿瓶或小袋)中，並標註活性劑的量。當藉由輸注來投予組成物時，可使用含有無菌醫藥級水或鹽水的輸液瓶進行分配。當藉由注射而投予組成物時，可提供無菌注射用水或鹽水的安瓿瓶，以便在投予前將各成分混合。In some embodiments, the pharmaceutical composition is formulated according to routine procedures as a pharmaceutical composition suitable for non-oral (e.g., intravenous, intramuscular or subcutaneous) administration. Typically, the composition for injection is a solution dissolved in a sterile isotonic buffered aqueous solution. When necessary, the composition may also include a solubilizer and a local anesthetic (such as lidocaine) to reduce pain at the injection site. In general, each component may be provided separately or mixed together in a unit dose form, for example, in a lyophilized powder or anhydrous concentrate form in a sealed container (such as an ampoule or a pouch), and the amount of the active agent is marked. When the composition is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline may be used for distribution. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
在一些實施例中，適合向哺乳動物(諸如人類)投予醫藥組成物。在一些實施例中，適合向囓齒類動物(例如，小鼠、大鼠)或非人類靈長類動物(例如，食蟹獼猴)投予醫藥組成物。在一些實施例中，該醫藥組成物係含於一次性小瓶(諸如一次性密封小瓶)中。在一些實施例中，該醫藥組成物係含於多次性小瓶中。在一些實施例中，該醫藥組成物以散裝形式裝在容器中。在一些實施例中，該醫藥組成物係冷凍保存。In some embodiments, the pharmaceutical composition is suitable for administration to mammals (such as humans). In some embodiments, the pharmaceutical composition is suitable for administration to rodents (e.g., mice, rats) or non-human primates (e.g., cynomolgus macaques). In some embodiments, the pharmaceutical composition is contained in a disposable vial (such as a disposable sealed vial). In some embodiments, the pharmaceutical composition is contained in a multiple-use vial. In some embodiments, the pharmaceutical composition is packaged in a container in bulk form. In some embodiments, the pharmaceutical composition is stored frozen.
亦提供本文所述之分離的抗TSLP抗體構築體或其組成物(諸如醫藥組成物)之任一者的單位劑量形式。術語「單位劑量形式」意指適合作為個體(例如，人類)單位劑量的物理上離散單位，每個單位含有預定量的活性物質，經計算以產生所需治療效果，並結合合適的醫藥載劑、稀釋劑或賦形劑。此等單位劑量形式可以單一或多個單位劑量儲存在合適的包裝中，亦可進一步滅菌及密封。Also provided are unit dosage forms of any of the isolated anti-TSLP antibody constructs or compositions thereof (e.g., pharmaceutical compositions) described herein. The term "unit dosage form" refers to physically discrete units suitable as individual (e.g., human) unit doses, each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect, in combination with a suitable pharmaceutical carrier, diluent, or formulation. Such unit dosage forms may be stored in single or multiple unit doses in suitable packaging, which may be further sterilized and sealed.
本申請案進一步提供含有適用於包裝的本文所述之組成物(諸如醫藥組成物)的製品。本文所述之組成物(諸如醫藥組成物)的適用包裝為本領域已知的，包括例如小瓶(諸如密封小瓶)、容器、安瓿瓶、瓶子、罐子、撓性包裝(例如，密封聚酯樹脂(Mylar)或塑料袋)及其類似物。此等製品可進一步滅菌及/或密封。The present application further provides an article containing a composition described herein (such as a pharmaceutical composition) suitable for packaging. Suitable packaging for the composition described herein (such as a pharmaceutical composition) is known in the art, including, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (such as sealed polyester resin (Mylar) or plastic bags) and the like. Such articles can be further sterilized and/or sealed.
本申請案亦提供含有本文所述之組成物(諸如醫藥組成物)的套組，並可進一步包含使用該組成物之方法(諸如本文所述之用途)的說明書。本文所述之套組可進一步包括從商業及用戶觀點而言所需的其他材料，包括其他緩衝液、稀釋劑、過濾器、針頭、注射器及/或塗藥器以及帶有用於進行本文所述之任何方法的說明書的包裝仿單。V.治療發炎性疾病的方法The present application also provides kits containing the compositions described herein (such as pharmaceutical compositions) and may further include instructions for methods of using the compositions (such as the uses described herein). The kits described herein may further include other materials necessary from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes and/or applicators and packaging leaflets with instructions for performing any of the methods described herein.V.Methods of treating inflammatory diseases
亦提供治療個體(例如，人類)發炎性疾病的方法，其包含向個體投予(例如，靜脈內或皮下)有效量之本文所述之分離的抗TSLP抗體構築體或醫藥組成物之任一者。在一些實施例中，該發炎性疾病為氣喘(例如，嚴重氣喘)、異位性皮膚炎(例如，中度至嚴重異位性皮膚炎)或慢性阻塞性肺病(COPD)。在一些實施例中，該方法進一步包含同時或依序投予有效量之皮質類固醇與該分離的抗TSLP抗體構築體或其醫藥組成物。Also provided are methods of treating an inflammatory disease in a subject (e.g., a human) comprising administering to the subject (e.g., intravenously or subcutaneously) an effective amount of any of the isolated anti-TSLP antibody constructs or pharmaceutical compositions described herein. In some embodiments, the inflammatory disease is asthma (e.g., severe asthma), atopic dermatitis (e.g., moderate to severe atopic dermatitis), or chronic obstructive pulmonary disease (COPD). In some embodiments, the method further comprises administering an effective amount of a corticosteroid concurrently or sequentially with the isolated anti-TSLP antibody construct or pharmaceutical composition thereof.
在一些實施例中，該發炎性疾病為TSLP相關疾病。與TSLP相關的發炎性疾病可為由TSLP之表現引起的或與其相關的疾病，例如與健康狀態相比過度表現，或在不同於健康狀態的位置處(例如，在不同的細胞或組織上)錯誤表現。In some embodiments, the inflammatory disease is a TSLP-related disease. An inflammatory disease associated with TSLP may be a disease caused by or associated with the expression of TSLP, such as overexpression compared to a healthy state, or misexpression at a different location than a healthy state (e.g., on a different cell or tissue).
在一些實施例中，該發炎性疾病為自體免疫疾病或自體免疫病況，諸如異位性皮膚炎、Netherton氏症候群、狼瘡(lupus)、休格連氏症候群(Sjogren's syndrome)、多發性硬化症(multiple sclerosis)、重症肌無力(myasthenia gravis)、乾癬(psoriasis)、乾性關節炎(psoriatic arthritis)、類風濕性關節炎(rheumatoid arthritis)、類肉瘤病(sarcoidosis)、潰瘍性結腸炎(ulcerative colitis)、發炎性腸道疾病等。在一些實施例中，該發炎性疾病為為另一種與免疫功能有關的疾病(例如，氣喘、子宮內膜異位症(endometriosis)、纖維化(fibrosis)、霍奇金氏淋巴瘤(Hodgkin's lymphoma))。在一些實施例中，該發炎性疾病為過敏反應，諸如過敏性氣喘、過敏性鼻炎(allergic rhinitis)、過敏性鼻竇炎(allergic rhinosinusitis)、過敏性結膜炎(allergic conjunctivitis)、異位性皮膚炎、嗜酸性細胞性食道炎(eosinophilic esophagitis)等。在一些實施例中，該發炎性疾病為纖維化、發炎性腸道疾病或COPD。在一些實施例中，該發炎性疾病為氣喘或異位性皮膚炎，諸如任何嚴重程度的氣喘或異位性皮膚炎。In some embodiments, the inflammatory disease is an autoimmune disease or autoimmune condition, such as atopic dermatitis, Netherton's syndrome, lupus, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, ulcerative colitis, inflammatory bowel disease, etc. In some embodiments, the inflammatory disease is another disease related to immune function (e.g., asthma, endometriosis, fibrosis, Hodgkin's lymphoma). In some embodiments, the inflammatory disease is an allergic reaction, such as allergic asthma, allergic rhinitis, allergic rhinosinusitis, allergic conjunctivitis, atopic dermatitis, eosinophilic esophagitis, etc. In some embodiments, the inflammatory disease is fibrosis, inflammatory bowel disease or COPD. In some embodiments, the inflammatory disease is asthma or atopic dermatitis, such as asthma or atopic dermatitis of any severity.
在一些實施例中，提供一種治療個體(例如，人類)發炎性疾病(例如，異位性皮膚炎、COPD或氣喘)的方法，其包含向個體投予(例如，靜脈內或皮下)有效量之分離的抗TSLP抗體構築體(或其醫藥組成物)，其中該分離的抗TSLP抗體構築體包含抗TSLP Fab，其中該抗TSLP Fab包含含有SEQ ID NO:109之胺基酸序列的第一多肽及含有SEQ ID NO:103之胺基酸序列的第二多肽。In some embodiments, a method for treating an inflammatory disease (e.g., atopic dermatitis, COPD, or asthma) in an individual (e.g., a human) is provided, comprising administering (e.g., intravenously or subcutaneously) an effective amount of an isolated anti-TSLP antibody construct (or a pharmaceutical composition thereof) to the individual, wherein the isolated anti-TSLP antibody construct comprises an anti-TSLP Fab, wherein the anti-TSLP Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 109 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 103.
在一些實施例中，提供一種治療個體(例如，人類)發炎性疾病(例如，異位性皮膚炎、COPD或氣喘)的方法，其包含向個體投予(例如，靜脈內或皮下)有效量之分離的抗TSLP抗體構築體(或其醫藥組成物)，其中該分離的抗TSLP抗體構築體包含抗TSLP全長抗體，其中該抗TSLP全長抗體包含：i)兩條各自含有SEQ ID NO:104之胺基酸序列的重鏈及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈；ii)兩條各自含有SEQ ID NO:105之胺基酸序列的重鏈及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈；或iii)含有SEQ ID NO:136之胺基酸序列的第一重鏈、含有SEQ ID NO:137之胺基酸序列的第二重鏈及兩條各自含有SEQ ID NO:103之胺基酸序列的重鏈。In some embodiments, a method for treating an inflammatory disease (e.g., atopic dermatitis, COPD, or asthma) in a subject (e.g., a human) is provided, comprising administering (e.g., intravenously or subcutaneously) an effective amount of an isolated anti-TSLP antibody construct (or a pharmaceutical composition thereof) to the subject, wherein the isolated anti-TSLP antibody construct comprises an anti-TSLP full-length antibody, wherein the anti-TSLP full-length antibody comprises: i) two heavy chains each containing the amino acid sequence of SEQ ID NO: 104 and two heavy chains each containing the amino acid sequence of SEQ ID NO: 103; ii) two heavy chains each containing the amino acid sequence of SEQ ID NO: 105 and two heavy chains each containing the amino acid sequence of SEQ ID NO: 103; or iii) a first heavy chain containing the amino acid sequence of SEQ ID NO: 136, a second heavy chain containing the amino acid sequence of SEQ ID NO: 137, and a third heavy chain containing the amino acid sequence of SEQ ID NO: 138. A second heavy chain containing the amino acid sequence of SEQ ID NO:137 and two heavy chains each containing the amino acid sequence of SEQ ID NO:103.
在一些實施例中，提供一種治療個體(例如，人類)發炎性疾病(例如，異位性皮膚炎、COPD或氣喘)的方法，其包含向個體投予(例如，靜脈內或皮下)有效量之分離的抗TSLP抗體構築體(或其醫藥組成物)，其中該分離的抗TSLP抗體構築體包含含有SEQ ID NO:106、107、191及192之任一者之胺基酸序列的抗TSLP scFv。In some embodiments, a method for treating an inflammatory disease (e.g., atopic dermatitis, COPD, or asthma) in an individual (e.g., a human) is provided, comprising administering (e.g., intravenously or subcutaneously) an effective amount of an isolated anti-TSLP antibody construct (or a pharmaceutical composition thereof) to the individual, wherein the isolated anti-TSLP antibody construct comprises an anti-TSLP scFv comprising the amino acid sequence of any one of SEQ ID NOs: 106, 107, 191, and 192.
在一些實施例中，該治療發炎性疾病的方法可達到一或多個以下生物活性：(1)抑制(例如，至少約10%、20%、30%、40%、50%、60%、70%、80%、90%或100%之任一者)免疫細胞成熟及/或活化(例如，B細胞、肥大細胞、T輔助2型(Th2)細胞或APC (例如，樹突細胞))；(2)抑制(例如，至少約10%、20%、30%、40%、50%、60%、70%、80%、90%或100%之任一者)免疫細胞增生；(3)降低(例如，至少約10%、20%、30%、40%、50%、60%、70%、80%、90%或100%之任一者)全身或局部發炎水平；(4)緩解(例如，至少約10%、20%、30%、40%、50%、60%、70%、80%、90%或100%之任一者)發炎性疾病個體的一或多種症狀；(5)降低(例如，至少約10%、20%、30%、40%、50%、60%、70%、80%、90%或100%之任一者)全身或局部細胞激素水平(諸如Th2細胞激素水平，例如IL-13、IL-4、IL-5)；(6)降低(例如，至少約10%、20%、30%、40%、50%、60%、70%、80%、90%或100%之任一者)發炎性疾病之表現、發生率或負荷；(7)延長發炎性免疫爆發的時間，諸如將個體發炎性疾病症狀消退後直至症狀重新出現的時間延長至少約1、2、4、6、12、18、20或更多小時，1、2、3、4、5、6、7或更多天，1、2、3、4或更多週，1、2、3、4、5、6、7、8、9、10、11、12、18或24個月或更長時間之任一者；(8)防止發炎性疾病的症狀再次出現持續至少約1、2、4、6、12、18、20或更多小時，1、2、3、4、5，6、7或更多天，1週、2週、3週、4週、5週、6週、7週、8週、9週、10週、11週、12週、1個月、2個月、3個月、4個月、5個月、6個月、7個月、8個月、9個月、10個月、11個月、12個月或更長時間之任一者；及(9)預防、抑制或降低(例如，至少約10%、20%、30%、40%、50%、60%、70%、80%、90%或100%之任一者)發炎性疾病復發的可能性。In some embodiments, the method for treating an inflammatory disease can achieve one or more of the following biological activities: (1) inhibiting (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) immune cell maturation and/or activation (e.g., B cells, mast cells, T helper type 2 (Th2) cells, or APCs); (e.g., dendritic cells); (2) inhibit (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) immune cell proliferation; (3) reduce (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) systemic or local inflammation levels; (4) alleviate (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). (5) reducing (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) systemic or local cytokine levels (e.g., Th2 cytokine levels, such as IL-13, IL-4, IL-5); (6) reducing (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) the manifestations, incidence, or severity of an inflammatory disease. or burden; (7) prolonging the duration of an inflammatory immune outbreak, such as prolonging the time from the resolution of symptoms of an inflammatory disease in a subject until the symptoms reappear by at least about 1, 2, 4, 6, 12, 18, 20 or more hours, 1, 2, 3, 4, 5, 6, 7 or more days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months or longer; (8) preventing the reappearance of symptoms of an inflammatory disease for at least about 1, 2, 4, 6, 12, 18, 20 or more hours, 1, 2, 3, 4, 5, 6, 7 or more days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months or longer. , 2, 3, 4, 5, 6, 7 or more days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer; and (9) preventing, inhibiting or reducing (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) the likelihood of a recurrence of an inflammatory disease.
本文所提供之方法可在主要治療環境中實踐，亦即其中所進行之方法為主要/主程性治療。在一些實施例中，該方法可在主要/主程性治法之前或與其結合進行。在一些實施例中，該方法用於治療先前接受過治療的個體(諸如人類)。本文所提供之任何治療方法皆可用於治療先前未接受過治療的個體(諸如人類)。在一些實施例中，該方法係用作一線療法。在一些實施例中，該方法係用作二線療法。The methods provided herein can be practiced in a primary treatment setting, i.e., the method performed therein is a primary/primary treatment. In some embodiments, the method can be performed before or in conjunction with a primary/primary treatment. In some embodiments, the method is used to treat an individual (e.g., a human) who has previously been treated. Any of the treatment methods provided herein can be used to treat an individual (e.g., a human) who has not previously been treated. In some embodiments, the method is used as a first-line treatment. In some embodiments, the method is used as a second-line treatment.
本文所述之方法適合治療多種發炎性疾病。該方法適用於所有階段的發炎性疾病，包括症狀出現後不久或處於緩解期的發炎性疾病。本文所述之方法可用作第一療法、第二療法、第三療法或與本領域已知之其他類型療法的組合療法，諸如抗發炎劑(例如，皮質類固醇)、基因療法、免疫療法、骨髓移植、幹細胞移植、標靶療法、營養療法等。在一些實施例中，該發炎性疾病對先前的療法沒有反應。The methods described herein are suitable for treating a variety of inflammatory diseases. The methods are applicable to inflammatory diseases at all stages, including inflammatory diseases shortly after symptoms appear or in remission. The methods described herein can be used as a primary therapy, a secondary therapy, a tertiary therapy, or a combination therapy with other types of therapy known in the art, such as anti-inflammatory agents (e.g., corticosteroids), gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, nutritional therapy, etc. In some embodiments, the inflammatory disease is unresponsive to previous therapy.
本文所述之分離的抗TSLP抗體構築體(或其醫藥組成物)之任一者的示例性投予途徑包括但不限於：靜脈內、腔內、動脈內、肌內、皮下、非經口、透皮或腹膜內途徑，或可遞輸至受到發炎性疾病影響的淋巴腺、體腔、器官或組織。在一些實施例中，藉由靜脈內(諸如輸注)來投予分離的抗TSLP抗體構築體或其醫藥組成物。Exemplary routes of administration of any of the isolated anti-TSLP antibody constructs (or pharmaceutical compositions thereof) described herein include, but are not limited to, intravenous, intracavitary, intraarterial, intramuscular, subcutaneous, parenteral, transdermal, or intraperitoneal routes, or delivery to a lymph node, body cavity, organ, or tissue affected by an inflammatory disease. In some embodiments, the isolated anti-TSLP antibody construct or pharmaceutical composition thereof is administered intravenously (e.g., by infusion).
在一些實施例中，藉由靜脈內輸注，以任何合適速率投予本文所述之分離的抗TSLP抗體構築體(或其醫藥組成物)。In some embodiments, an isolated anti-TSLP antibody construct described herein (or a pharmaceutical composition thereof) is administered by intravenous infusion at any suitable rate.
向個體(諸如人類)投予的本文所述之分離的抗TSLP抗體構築體或其醫藥組成物的給藥方案可能因特定組成物、投予方法及待治療之發炎性疾病的特定類型而變。在一些實施例中，該分離的抗TSLP抗體構築體之有效量低於引發毒性作用的量(亦即，高於臨床上可接受之毒性水平的作用)或當向個體投予組成物時其潛在副作用可處於可控或耐受的水平。The dosing regimen of the isolated anti-TSLP antibody constructs or pharmaceutical compositions thereof described herein administered to an individual (e.g., a human) may vary depending on the specific composition, the method of administration, and the specific type of inflammatory disease to be treated. In some embodiments, the effective amount of the isolated anti-TSLP antibody construct is below the amount that causes toxic effects (i.e., effects above a clinically acceptable level of toxicity) or the potential side effects are at a manageable or tolerable level when the composition is administered to an individual.
本文所述之分離的抗TSLP抗體構築體(或其醫藥組成物)的有效量可以單劑量或多劑量方式投予。針對包含以多劑量投予分離的抗TSLP抗體構築體(或其醫藥組成物)的方法，示例性給藥頻率包括但不限於：每小時、每天、每天不間斷、每週、每週不間斷、三週中的兩週每週一次、四週中的三週每週一次、每三週一次、每兩週一次、每月、每六個月、每年等。在一些實施例中，該分離的抗TSLP抗體構築體(或其醫藥組成物)的投予為約每2週一次、每3週一次，每4週一次、每6週一次或每8週一次。在一些實施例中，該分離的抗TSLP抗體構築體(或其醫藥組成物)的投予為每週至少約1次、2次、3次、4次、5次、6次或7次(亦即，每天)之任一者。在一些實施例中，每次投予之間的間隔為小於約3年、2年、1年、12個月、11個月、10個月、9個月、8個月、7個月、6個月、5個月、4個月、3個月、2個月、1個月、4週、3週、2週、1週、6天、5天、4天、3天、2天或1天之任一者。在一些實施例中，每次投予之間的間隔為大於約1天、2天、3天、4天、5天、6天、1週、2週、3週、4週、1個月、2個月、3個月、4個月、5個月、6個月、7個月、8個月、9個月、10個月、11個月、12個月、2年或3年之任一者。在一些實施例中，給藥時間表沒有間斷。An effective amount of the isolated anti-TSLP antibody construct (or pharmaceutical composition thereof) described herein can be administered in a single dose or in multiple doses. For methods comprising administering the isolated anti-TSLP antibody construct (or pharmaceutical composition thereof) in multiple doses, exemplary dosing frequencies include, but are not limited to, every hour, every day, every day without interruption, every week, every week without interruption, once a week for two out of three weeks, once a week for three out of four weeks, once every three weeks, once every two weeks, monthly, every six months, annually, etc. In some embodiments, the isolated anti-TSLP antibody construct (or pharmaceutical composition thereof) is administered about once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 8 weeks. In some embodiments, the isolated anti-TSLP antibody construct (or pharmaceutical composition thereof) is administered at least about 1, 2, 3, 4, 5, 6, or 7 times per week (i.e., daily). In some embodiments, the interval between each administration is less than about 3 years, 2 years, 1 year, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the interval between each administration is greater than about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years. In some embodiments, there is no break in the dosing schedule.
本文所述之分離的抗TSLP抗體構築體(或其醫藥組成物)的投予可延長一段較長的時間，諸如1天至約一週、約一週至約一個月、約一個月至約一年、約一年至約幾年。在一些實施例中，該分離的抗TSLP抗體構築體(或其醫藥組成物)係投予持續至少約1天、2天、3天、4天、5天、6天、7天、8天、1週、2週、3週、4週、5週、1個月、2個月、3個月、4個月、5個月、6個月、7個月、8個月、9個月、10個月、11個月、12個月、1年、2年、3年、4年或更多之任一者的時間。 實例The administration of the isolated anti-TSLP antibody constructs (or pharmaceutical compositions thereof) described herein can be extended over a longer period of time, such as 1 day to about 1 week, about 1 week to about 1 month, about 1 month to about 1 year, about 1 year to about several years. In some embodiments, the isolated anti-TSLP antibody construct (or pharmaceutical composition thereof) is administered for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years or more.Examples
以下實施例僅用於示例本發明，因此不應被視為以任何方式限制本發明。以下實例及詳細描述僅用於說明目的而非限制。針對未描述實驗方法細節的實施例，此類方法係根據常規條件進行，諸如在Sambrook等人之Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989)中描述的條件，或按照製造商建議的條件。實例1：示例性抗TSLP抗體之產生。The following examples are intended only to illustrate the present invention and should not be construed as limiting the present invention in any way. The following examples and detailed descriptions are intended for illustrative purposes only and are not intended to be limiting. For examples in which the details of experimental methods are not described, such methods are performed according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.Example1: Generation of exemplary anti-TSLPantibodies
使用人類TSLP及食蟹獼猴(以下稱為「cyno」) TSLP作為免疫原來產生抗TSLP抗體。在確定個別動物具有適合的抗體滴度後，從免疫接種小鼠的脾臟及淋巴結中獲得淋巴球的單細胞懸浮液。藉由標準方法將淋巴球與鼠科骨髓瘤細胞融合以產生融合瘤。藉由ELISA篩選融合瘤上清液與人類TSLP及食蟹獼猴TSLP的結合。藉由報導細胞試驗進一步篩選出與人類及食蟹獼猴TSLP結合的融合瘤，以確定抗TSLP阻斷抗體。Human TSLP and cynomolgus (hereafter "cyno") TSLP were used as immunogens to generate anti-TSLP antibodies. After determining that the individual animals had appropriate antibody titers, single cell suspensions of lymphocytes were obtained from the spleen and lymph nodes of the immunized mice. Lymphocytes were fused with murine myeloma cells by standard methods to generate fusion tumors. Fusion tumor supernatants were screened for binding to human TSLP and cynomolgus TSLP by ELISA. Fusion tumors that bound to human and cynomolgus TSLP were further screened by reporter cell assays to identify anti-TSLP blocking antibodies.
TSLP報導細胞試驗：TSLP報導細胞試驗確定了抗TSLP抗體在共同表現TSLPR/IL-7Rα的STAT5-螢光素酶HEK-293報導細胞中對TSLP的阻斷程度，其中STAT5傳訊增加會誘導螢光素酶產量增加。已知TSLP與TSLP受體複合體的結合可活化STAT5傳訊路徑。因此，藉由測量螢光素酶活性來監控STAT5傳訊路徑的活化，可確定TSLP阻斷程度。將具有TSLPR/IL-7Rα共同表現的STAT5-螢光素酶HEK-293報導細胞與10 ng/mL人類TSLP及本文所述之連續稀釋的抗TSLP抗體同時在37°C下培養5小時。在共同培養5小時之後，測量生物發光訊號。圖1顯示了使用GraphPad Prism分析的報導細胞的螢光素酶抑制百分比(IC90)結果，其證實所有示例性抗TSLP抗體殖株皆能有效阻斷游離TSLP與TSLP受體複合體的結合。實例2：示例性抗TSLP抗體之結合親和力的測量。TSLPreporter cell assay : The TSLP reporter cell assay determines the extent of TSLP blockade by anti-TSLP antibodies in STAT5-luciferase HEK-293 reporter cells co-expressing TSLPR/IL-7Rα, where increased STAT5 signaling induces increased luciferase production. Binding of TSLP to the TSLP receptor complex is known to activate the STAT5 signaling pathway. Therefore, monitoring activation of the STAT5 signaling pathway by measuring luciferase activity can determine the extent of TSLP blockade. STAT5-luciferase HEK-293 reporter cells co-expressing TSLPR/IL-7Rα were incubated with 10 ng/mL human TSLP and serial dilutions of anti-TSLP antibodies as described herein for 5 hours at 37°C. After 5 hours of co-culture, the bioluminescent signal was measured.Figure1 shows the percent luciferase inhibition (IC90 ) results of reporter cells analyzed using GraphPad Prism, which demonstrates that all exemplary anti-TSLP antibody clones can effectively block the binding of free TSLP to the TSLP receptor complex.Example2: Measurement of binding affinity of exemplary anti-TSLPantibodies.
基於示例性小鼠抗TSLP mAb建構出含有人類恆定域的重組嵌合抗TSLP單株抗體(mAb)。純化示例性小鼠抗TSLP mAb及嵌合抗TSLP mAb。A recombinant chimeric anti-TSLP monoclonal antibody (mAb) containing a human constant domain was constructed based on an exemplary mouse anti-TSLP mAb. The exemplary mouse anti-TSLP mAb and the chimeric anti-TSLP mAb were purified.
使用Biacore來進行靶向TSLP之示例性融合瘤抗體之結合親和力的表面電漿共振(SPR)測量。Surface plasmon resonance (SPR) measurements of the binding affinity of exemplary fusion tumor antibodies targeting TSLP were performed using Biacore.
簡言之，利用抗Fc抗體晶片來捕獲純化的抗體(亦即，針對具有人類Fc的重組抗體採用抗人類Fc晶片，且針對融合瘤或重組小鼠抗體採用抗小鼠Fc晶片)。抗原以不同的濃度(以1：3進行連續稀釋，從48 nM至0.2 nM)流過晶片。捕獲的抗原在900至1200秒內從晶片上分離。將感測圖擬合至1：1結合模型。結果總結於下表2至4。Briefly, purified antibodies were captured using an anti-Fc antibody chip (i.e., an anti-human Fc chip for recombinant antibodies with human Fc and an anti-mouse Fc chip for fusion tumor or recombinant mouse antibodies). Antigen was flowed over the chip at different concentrations (serial dilutions 1:3 from 48 nM to 0.2 nM). The captured antigen was separated from the chip within 900 to 1200 seconds. The sensorgrams were fitted to a 1:1 binding model. The results are summarized inTables2to4 below.
從表2至4可看出，所有測試的示例性小鼠抗TSLP mAb與人類及食蟹獼猴TSLP皆表現出非常強的結合(對人類TSLP的結合更佳)。此外，嵌合化不會顯著影響抗TSLP抗體的結合親和力(例如，與51A4及Ch51A4相比)。表2. 來自小鼠融合瘤之純化抗體的抗TSLP結合親和力結果。
與參考抗TSLP抗體相比，確定51B2結合表位及效力：將不同濃度的51B2及US-FDA批准的抗TSLP參考抗體#1 (「參考Ab. #1」)與生物素化的TSLP預先培養2小時，之後添加至預先塗覆有參考Ab. #1的ELISA盤上，以評估參考Ab. #1與51B2之間的交叉競爭。使用惰性陰性對照抗體(Ab)作為非競爭性Ab。在ELISA盤進行短暫培養及清洗之後，藉由HRP-鏈黴親和素來檢測捕獲在盤上的生物素-TSLP。圖2的結果顯示了惰性陰性對照Ab不與TSLP結合，且參考Ab. #1完全與自身競爭TSLP結合。圖2證實了51B2不完全與參考Ab. #1競爭以防止其與TSLP結合。此等結果顯示，51B2具有不同於參考Ab. #1的獨特結合表位。Determination of51B2binding epitope and potencycomparedto a reference anti-TSLP antibody: 51B2 and US-FDA approved anti-TSLP reference antibody #1 ("Reference Ab. #1") at different concentrations were pre-incubated with biotinylated TSLP for 2 hours before being added to an ELISA plate pre-coated with Reference Ab. #1 to assess cross-competition between Reference Ab. #1 and 51B2. An inert negative control antibody (Ab) was used as a non-competing Ab. After a brief incubation and washing of the ELISA plate, biotin-TSLP captured on the plate was detected by HRP-streptavidin. The results inFigure2 show that the inert negative control Ab does not bind to TSLP, and that reference Ab. #1 completely competes with itself for TSLP binding.Figure2 demonstrates that 51B2 does not completely compete with reference Ab. #1 to prevent it from binding to TSLP. These results show that 51B2 has a unique binding epitope that is different from reference Ab. #1.
在人類PBMC及分離的CD1c+樹突狀細胞中測試51B2抑制初代人類細胞TSLP傳訊的效力。簡言之，將人類PBMC及分離的CD1c+樹突細胞接種於96孔盤中，並在多種濃度的51B2或參考Ab #1存在下分別與50 ng/ml或5 ng/ml TSLP一起培養。在48小時(PBMC)或24小時(樹突狀細胞)後收集細胞培養上清液，並按製造商的方案使用市售ELISA套組來分析TSLP誘導的CCL17分泌。TSLP傳訊增強會誘導CCL17分泌增加，因此，當以TSLP細胞激素處理表現TSLP受體的促發炎細胞時，CCL17分泌的減少或抑制可作為抑制TSLP與TSLPR複合體結合的讀數。使用GraphPad Prism分析及呈現數據。如圖3A(人類PBMC)及圖3B(CD1c+樹突細胞)所示，在人類PBMC及分離的CD1c+樹突細胞中，與參考Ab. #1相比，51B2在每個測試的抗體濃度下皆表現出顯著抑制CCL17。因此，此等結果證實，51B2在阻斷促發炎免疫細胞中的TSLP傳訊上比US-FDA批准的參考Ab. #1更有效。The potency of 51B2 in inhibiting TSLP signaling in primary human cells was tested in human PBMCs and isolated CD1c+ dendritic cells. Briefly, human PBMCs and isolated CD1c+ dendritic cells were seeded in 96-well plates and cultured with 50 ng/ml or 5 ng/ml TSLP in the presence of various concentrations of 51B2 or reference Ab #1. Cell culture supernatants were collected after 48 hours (PBMCs) or 24 hours (dendritic cells) and analyzed for TSLP-induced CCL17 secretion using a commercial ELISA kit according to the manufacturer's protocol. Enhanced TSLP signaling induces increased CCL17 secretion, therefore, when pro-inflammatory cells expressing the TSLP receptor are treated with TSLP cytokines, a decrease or inhibition of CCL17 secretion can be used as a readout for inhibition of TSLP binding to the TSLPR complex. Data were analyzed and presented using GraphPad Prism. As shown inFigure3A (human PBMC) andFigure3B (CD1c+ dendritic cells), 51B2 showed significant inhibition of CCL17 at every antibody concentration tested compared to reference Ab. #1 in both human PBMCs and isolated CD1c+ dendritic cells. Therefore, these results demonstrate that 51B2 is more effective than the US-FDA-approved reference Ab. #1 in blocking TSLP signaling in pro-inflammatory immune cells.
51B2的人源化及人源化51B2變體：藉由將重鏈(HC)及輕鏈(LC)的CDR移植至人類HC及LC框架序列上使親代殖株51B2人源化。簡言之，將嵌合51B2的HC及LC CDR移植至最接近的HC及LC人類種系上。利用目視檢查序列來識別就CDR結合及結構穩定性而言重要的框架殘基。將此等框架殘基進行單獨或組合突變。參見針對VL序列的SEQ ID NO:51-54及針對VH序列的SEQ ID NO:55-64。選擇保留結合性及穩定性的人源化變體作為最終的先導殖株。Humanization of51B2and humanized51B2variants : The parental clone 51B2 was humanized by grafting the CDRs of the heavy chain (HC) and light chain (LC) onto human HC and LC framework sequences. Briefly, the HC and LC CDRs of the chimeric 51B2 were grafted onto the closest HC and LC human germlines. Visual inspection of the sequence was used to identify framework residues important for CDR binding and structural stability. These framework residues were mutated individually or in combination. See SEQ ID NOs: 51-54 for VL sequences and SEQ ID NOs: 55-64 for VH sequences. Humanized variants that retained binding and stability were selected as the final lead clones.
使用Biacore測試人源化51B2抗體的結合親和力，並根據活性(類似於實例1所述之TSLP報導試驗)及表現品質/純化行為來進行排名。基於Biacore結合結果(參見表5至6)及活性試驗(參見圖4及圖5A)，將人源化51B2抗體、L3H9 (亦即，具有VL-3 (SEQ ID NO: 53)及VH-9 (SEQ ID NO: 63)配對)選為先導人源化51B2殖株。The binding affinity of humanized 51B2 antibodies was tested using Biacore and ranked based on activity (similar to the TSLP reporter assay described in Example 1) and performance quality/purification behavior. Based on the Biacore binding results (see Tables5to6 ) and activityassays (seeFigures4 and5A ), the humanized 51B2 antibody, L3H9 (i.e., having a VL-3 (SEQ ID NO: 53) and VH-9 (SEQ ID NO: 63) pairing) was selected as the lead humanized 51B2 clone.
人源化51B2變體在Biacore中與huTSLP結合：利用抗人類Fc抗體晶片捕獲純化的人源化或嵌合抗體，並以不同濃度(以1：3進行連續稀釋，從48 nM至0.2 nM)的抗原(人類TSLP)流過具有捕獲的抗體的晶片，如上所述。隨後，捕獲的抗原在1200秒內從晶片上分離。將感測圖擬合至1：1結合模型並總結於下表5中。Humanized51B2variantsbindtohuTSLP inBiacore : Purified humanized or chimeric antibodies were captured using an anti-human Fc antibody chip, and antigen (human TSLP) at different concentrations (serial dilutions 1:3 from 48 nM to 0.2 nM) was flowed over the chip with captured antibodies as described above. The captured antigen was then separated from the chip within 1200 seconds. The sensorgrams were fitted to a 1:1 binding model and are summarized inTable5 below.
如表5所示，除了L1H2及L1H9變體以外，幾乎所有的人源化51B2變體皆保留與人類TSLP類似或甚至更強的結合親和力。表5. 人源化51B2變體與人類TSLP的結合親和力。
抗TSLP抗體hz51B2 L3H9的活性表徵：在Biacore T200上使用針對抗體捕獲的抗人類Fc捕獲晶片及作為分析物的不同濃度的純化的TSLP來測量hz51B2-L3H9與人類及食蟹獼猴TSLP的結合親和力。將結合感測圖擬合至1：1結合動力學模型。總結於下表5中。結合速率、解離速率及平衡結合KD等結合常數皆總結於下表6中。表6. hz51B2 L3H9抗體與人類及食蟹獼猴TSLP的結合親和力。
表6總結的結果顯示人源化(hz)51B2 L3H9抗體與人類TSLP及食蟹獼猴TSLP皆有強的結合，其有利於將食蟹獼猴毒性及功效研究的結果推斷至人類臨床研究中，以評估抗TSLP抗體或包含其之構築體(諸如本文所述之多特異性抗TSLP抗體構築體之任一者)。The results summarized inTable6 show that the humanized (hz) 51B2 L3H9 antibody has strong binding to both human TSLP and cynomolgus macaque TSLP, which facilitates the extrapolation of the results of cynomolgus macaque toxicity and efficacy studies to human clinical studies to evaluate anti-TSLP antibodies or constructs comprising the same (such as any of the multispecific anti-TSLP antibody constructs described herein).
藉由使用BiacoreT200儀器測量平衡結合反應混合物中之游離TSLP濃度來表徵hz51B2 L3H9與人類TSLP的結合。簡言之，在室溫下，在含有0.1 mg/ml BSA及0.005%聚山梨醇20的PBS溶液中，將1 nM人類TSLP與3×連續稀釋的hz51B2 L3H9溶液(範圍從96 nM至44 pM的Fab臂)培養過夜。藉由將結合溶液注射至胺固定的hz51B2 L3H9表面上方來測量各個結合反應中未結合的TSLP。使用GraphPad將各個反應混合物中的游離TSLP濃度百分比擬合至1:1平衡Ab-Ag結合模型，並從曲線擬合推導出KD(圖5B)。Binding of hz51B2 L3H9 to human TSLP was characterized by measuring the free TSLP concentration in the equilibrium binding reaction mixture using a Biacore T200 instrument. Briefly, 1 nM human TSLP was incubated with 3× serially diluted hz51B2 L3H9 solutions (ranging from 96 nM to 44 pM of Fab arm) in PBS containing 0.1 mg/ml BSA and 0.005% polysorbate 20 at room temperature overnight. Unbound TSLP in each binding reaction was measured by injecting the binding solution over the amine-immobilized hz51B2 L3H9 surface. The percent free TSLP concentration in each reaction mixture was fit to a 1:1 equilibrium Ab-Ag binding model using GraphPad, and theKD was deduced from the curve fit (Fig.5B ).
總之，先導51B2抗體(例如，hz51B2 Ab，諸如hz51B2 L3H9)為人類TSLP的緊密結合抗體，其中KD為＜ 10 pM。不受理論的束縛，此種性質使得在PBMC及樹突細胞試驗中能夠完全中和TSLP誘導的CCL17 (參見圖3A-3B)，而US-FDA批准的參考Ab. #1則不完全抑制TSLP誘導的CCL17。In summary, the lead 51B2 antibody (e.g., hz51B2 Ab, such as hz51B2 L3H9) is a tight binding antibody to human TSLP with aKD of < 10 pM. Without being bound by theory, this property enables complete neutralization of TSLP-induced CCL17 in PBMC and dendritic cell assays (seeFigures3A-3B ), while the US-FDA-approved reference Ab. #1 does not completely inhibit TSLP-induced CCL17.
本發明中提及的所有參考文獻皆通過引用併入本文，如同每一參考文獻皆通過引用單獨併入本文一般。儘管該描述參考特定實施例，但本領域具有通常知識者應明瞭，本發明可利用此等具體細節的變化來實踐。因此，本發明不應理解為限於本文所述之實施例。 序列表
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圖1顯示了當以示例性抗TSLP抗體(亦即，51B2、51A3、51A4、54A2、54B3及54C4)處理時，TSLP結合至表現在STAT5-HEK293受體細胞上的TSLP受體複合體的抑制百分比。將STAT5-HEK293受體細胞與不同濃度的各個抗體及10 ng/mL TSLP一起培養。將TSLP誘導的報導細胞的螢光素酶產生作為TSLP結合活性的讀數，並根據抗體劑量依賴性結合抑制曲線計算各個抗體的IC90值。使用GraphPad分析及呈現數據。Ab，抗體；Conc.，濃度。FIG1 shows the percent inhibition of TSLP binding to the TSLP receptor complex expressed on STAT5-HEK293 receptor cells when treated with exemplary anti- TSLP antibodies (i.e., 51B2, 51A3, 51A4, 54A2, 54B3, and 54C4). STAT5-HEK293 receptor cells were incubated with various concentrations of each antibody and 10 ng/mL TSLP. TSLP-induced luciferase production by reporter cells was used as a readout of TSLP binding activity, and IC90 values for each antibody were calculated based on antibody dose-dependent binding inhibition curves. Data were analyzed and presented using GraphPad. Ab, antibody; Conc., concentration.
圖2顯示了在ELISA競爭實驗中51B2及US-FDA批准的抗TSLP Ab (參考抗體#1或「參考Ab. #1」)結合至TSLP上重合但不同的表位。Ab，抗體；OD，光學密度。FIG.2 shows that 51B2 and US-FDA approved anti-TSLP Ab (reference antibody #1 or “Reference Ab. #1”) bind to overlapping but different epitopes on TSLP in ELISA competition experiments. Ab, antibody; OD, optical density.
圖3A至3B顯示了與US-FDA批准的抗TSLP Ab (參考Ab. #1)相比，51B2完全抑制TSLP誘導的PBMC (圖3A)或分離的樹突細胞(圖3B)的CCL17分泌。圖3A顯示了TSLP誘導的人類PBMC的CCL17分泌的抑制，該人類PBMC接種在96孔盤上並以50 ng/ml TSLP及多個濃度的51B2或參考Ab. #1培養。藉由ELISA分析於48小時收集的細胞培養上清液中分泌的CCL17含量。圖3B顯示了TSLP誘導的分離的CD1c+樹突細胞的CCL17分泌的抑制，該分離的CD1c+樹突細胞接種在96孔盤上並以5 ng/ml TSLP及多個濃度的51B2或參考Ab. #1培養。藉由ELISA分析於24小時收集的細胞培養上清液中分泌的CCL17含量。使用GraphPad Prism分析及呈現數據。Ab，抗體；PBMC，週邊血液單核細胞。Figures3Ato3B show that 51B2 completely inhibits TSLP-induced CCL17 secretion by PBMC (Figure3A ) or isolated dendritic cells (Figure3B ) compared to a US-FDA approved anti-TSLP Ab (reference Ab. #1).Figure3A shows the inhibition of TSLP-induced CCL17 secretion by human PBMCs seeded in 96-well plates and cultured with 50 ng/ml TSLP and various concentrations of 51B2 or reference Ab. #1. The secreted CCL17 levels in the cell culture supernatants collected at 48 hours were analyzed by ELISA.FIG3B shows TSLP-induced inhibition of CCL17 secretion by isolated CD1c+ dendritic cells seeded in 96-well plates and cultured with 5 ng/ml TSLP and various concentrations of 51B2 or reference Ab. #1. The secreted CCL17 levels in the cell culture supernatants collected at 24 hours were analyzed by ELISA. Data were analyzed and presented using GraphPad Prism. Ab, antibody; PBMC, peripheral blood mononuclear cells.
圖4顯示了在使用TSLP受體複合體轉染的HEK293受體細胞進行的人類及食蟹獼猴(「cyno」) TSLP抑制試驗中，與親代Ch51B2殖株相比，各個產生的人源化51B2抗體所得的IC50(nM)及IC90(nM)值。由方框標記的人源化抗抗體殖株表明被選來進行進一步分析的殖株。Figure4 shows the IC50 (nM) and IC90 (nM) values obtained for each of the generated humanized 51B2 antibodies compared to the parental Ch51B2 strain in human andcynomolgus macaque ("cyno") TSLP inhibition assays usingHEK293 receptor cells transfected with the TSLP receptor complex. The humanized antibody strains marked by the boxes indicate the strains selected for further analysis.
圖5A顯示了當以所選的人源化抗TSLP抗體(ch51B2親代嵌合殖株hz51B2 L2H2、hz51B2 L2H9及hz51B2 L3H9)處理時，TSLP結合至表現在STAT5-HEK293受體細胞上的TSLP受體複合體的抑制百分比。將STAT5-HEK293受體細胞與10 ng/mL TSLP及多個濃度的各個抗體及一起培養，並根據抗體劑量依賴性結合抑制曲線計算各個抗體的IC90值。使用GraphPad分析及呈現數據。Ab，抗體；Conc.，濃度。FIG5A shows the percent inhibition of TSLP binding to the TSLP receptor complex expressed on STAT5-HEK293 receptor cells when treated with selected humanized anti- TSLP antibodies (ch51B2 parental chimeric strains hz51B2 L2H2, hz51B2 L2H9, and hz51B2 L3H9). STAT5-HEK293 receptor cells were incubated with 10 ng/mL TSLP and various concentrations of each antibody, and the IC90 value of each antibody was calculated based on the antibody dose-dependent binding inhibition curve. Data were analyzed and presented using GraphPad. Ab, antibody; Conc., concentration.
圖5B顯示了hz51B2 L3H9在溶液中結合至人類TSLP的平衡結合分析。Figure5B shows equilibrium binding analysis of hz51B2 L3H9 binding to human TSLP in solution.
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