TW202413410A

TW202413410A - Multi-domain binding molecules

Info

Publication number: TW202413410A
Application number: TW112131156A
Authority: TW
Inventors: 阿曼丁喬治斯; 史蒂芬哈帝; 勒克韓麥
Original assignee: 英商英美偌科有限公司
Priority date: 2022-08-18
Filing date: 2023-08-18
Publication date: 2024-04-01

Abstract

The present invention relates to multi-domain, single-chain binding molecules. The molecules comprise i) a peptide-major histocompatibility complex (pMHC) binding domain comprising a first variable region linked to a constant region (VC1) and a second variable region linked to a constant region (VC2); ii) a T cell engaging immune effector domain comprising an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH); and iii) a half-life extending domain comprising a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region and FC2 region dimerise to form an Fc domain. The binding molecules can be used to treat diseases such as cancer and infectious diseases.

Description

多域結合分子Multidomain Binding Molecules

本申請案係關於多域單鏈結合分子及相關核酸、表現載體、宿主細胞、製備方法、醫藥組成物及用途。 序列表 This application relates to multi-domain single-chain binding molecules and related nucleic acids, expression vectors, host cells, preparation methods, pharmaceutical compositions and uses. Sequence Listing

本申請案含有序列表，其以全文引用之方式併入本文中。該XML複本創建於2023年8月2日，命名為P206512WO ST.26序列表.xml（P206512WO ST.26 Sequence Listing.xml）且大小為55320位元組。This application contains a sequence listing, which is incorporated herein by reference in its entirety. The XML copy was created on August 2, 2023, is named P206512WO ST.26 Sequence Listing.xml and is 55320 bytes in size.

包括抗體片段及融合蛋白之多種基於蛋白質之治療劑在投予後會迅速從體內清除。其短循環半衰期係通常歸因於其尺寸較小，此允許經由腎過濾而有效清除，以及缺乏針對細胞內降解之保護。在此類情況下，需要頻繁投予或長時間輸注來在較長時段內維持藥物之有效濃度。為改良給藥，已採用若干策略來延長循環半衰期。此等策略包括經由連接可撓性親水性分子，諸如碳水化合物或聚乙二醇（polyethelene glycol；PEG）來增加蛋白質之流體動力學半徑，及經由連接抗體Fc域或血清白蛋白來利用經由新生兒Fc受體（neonatal Fc receptor；FcRn）進行之再循環（Konnteman, Curr Opin Biotechnol. 2011年12月;22(6):868-76）。Many protein-based therapeutics, including antibody fragments and fusion proteins, are rapidly cleared from the body after administration. Their short circulation half-life is generally attributed to their small size, which allows efficient clearance via renal filtration, and lack of protection from intracellular degradation. In such cases, frequent administration or prolonged infusion is required to maintain effective concentrations of the drug over a prolonged period of time. To improve drug administration, several strategies have been employed to extend the circulation half-life. These strategies include increasing the hydrodynamic radius of the protein by attaching flexible hydrophilic molecules such as carbohydrates or polyethylene glycol (PEG) and exploiting recycling via the neonatal Fc receptor (FcRn) by attaching antibody Fc domains or serum albumin (Konnteman, Curr Opin Biotechnol. 2011 Dec;22(6):868-76).

利用FcRn介導之再循環的策略由於活體內誘導免疫原性之風險較低及可實現之較長半衰期延長而尤其具有吸引力。舉例而言，據報告，在連接Fc域之後，BiTE®形式之T細胞接合雙特異性抗體之半衰期超過200小時（Lorenczewski等人，Blood 2017. 130(增刊1), 2815）。類似地，據報告，合併有白蛋白結合域之TriTac®形式之雙特異性抗體具有超過四天之半衰期（Wesche等人，Cancer Res 2018;78(13增刊):摘要nr 3814）。Strategies that exploit FcRn-mediated recycling are particularly attractive due to the lower risk of inducing immunogenicity in vivo and the longer half-life extensions that can be achieved. For example, after attachment of the Fc domain, the half-life of a T cell-engaged bispecific antibody in the BiTE® format has been reported to exceed 200 hours (Lorenczewski et al., Blood 2017. 130(Suppl 1), 2815). Similarly, a bispecific antibody in the TriTac® format incorporating an albumin-binding domain has been reported to have a half-life of more than four days (Wesche et al., Cancer Res 2018;78(13 Suppl):Abstract nr 3814).

包含融合至抗CD3抗體片段之可溶性T細胞受體（T cell receptor；TCR）之融合蛋白為相對較新類別之免疫細胞（例如T細胞）接合雙特異性融合蛋白，其活體內半衰期在6至8小時範圍內（Sato等人，2018 J Clin Onc 2018 36, 第15期, 增刊9521-9521；Middleton等人，J Clin Onc 2016 34, 第15期, 增刊3016-3016）。此遠短於傳統單株抗體，傳統單株抗體通常具有在260至720小時範圍內之半衰期（Ovacik及Lin, 2018 Clin Transl Sci, 11:540）。此外，TCR-抗CD3融合蛋白已展現包括皮莫耳效能之有利治療特性（Lowe等人，2019 Cancer treatment reviews, 第77卷35-43）。因此，需要鑑別適用於延長TCR-免疫細胞接合域融合蛋白，諸如TCR-抗CD3融合蛋白及其他含TCR蛋白之半衰期的方法，以便降低給藥頻率且在較長時段內維持有效濃度，而不影響其他治療特性。Fusion proteins comprising a soluble T cell receptor (TCR) fused to an anti-CD3 antibody fragment are a relatively new class of immune cell (e.g., T cell) engaging bispecific fusion proteins with an in vivo half-life in the range of 6 to 8 hours (Sato et al., 2018 J Clin Onc 2018 36, No. 15, Suppl. 9521-9521; Middleton et al., J Clin Onc 2016 34, No. 15, Suppl. 3016-3016). This is much shorter than traditional monoclonal antibodies, which typically have a half-life in the range of 260 to 720 hours (Ovacik and Lin, 2018 Clin Transl Sci, 11:540). In addition, TCR-anti-CD3 fusion proteins have demonstrated favorable therapeutic properties including picomolar potency (Lowe et al., 2019 Cancer treatment reviews, vol. 77, 35-43). Therefore, there is a need to identify methods for extending the half-life of TCR-immune cell engaging domain fusion proteins, such as TCR-anti-CD3 fusion proteins and other TCR-containing proteins, so as to reduce the frequency of dosing and maintain effective concentrations for a longer period of time without affecting other therapeutic properties.

不同於傳統抗體，TCR經設計以識別源自細胞內抗原且藉由人類白血球抗原（human leukocyte antigen）呈現於細胞表面上之短肽（肽-HLA）。抗原呈現細胞上之肽-HLA複合物與諸如T細胞的免疫細胞上之對應受體之間的有效免疫突觸形成依賴於例如仔細編排之交互作用，其可被膜間距離增加干擾（Choudhuri等人，2005 Nature 7月28日;436(7050):578-82；Holland等人，J Clin Invest. 2020;130(5):2673-2688）。因此，增加含TCR蛋白之半衰期的融合方法，諸如連接抗體Fc域或血清白蛋白，由於干擾TCR結合所需之交互作用幾何結構的風險而具有高度挑戰性。類似挑戰亦適用於含有與肽-HLA複合物結合之抗體的融合蛋白，該等抗體被稱為TCR樣或TCR模擬抗體。Unlike traditional antibodies, TCRs are designed to recognize short peptides (peptide-HLA) derived from intracellular antigens and presented on the cell surface by human leukocyte antigen. Effective immune synapse formation between peptide-HLA complexes on antigen-presenting cells and corresponding receptors on immune cells such as T cells depends on, for example, carefully orchestrated interactions that can be perturbed by increased membrane distance (Choudhuri et al. 2005 Nature Jul 28;436(7050):578-82; Holland et al. J Clin Invest. 2020;130(5):2673-2688). Therefore, fusion approaches to increase the half-life of TCR-containing proteins, such as attachment of antibody Fc domains or serum albumin, are highly challenging due to the risk of interfering with the interaction geometry required for TCR binding. Similar challenges apply to fusion proteins containing antibodies that bind to peptide-HLA complexes, which are referred to as TCR-like or TCR-mimetic antibodies.

WO 2020/157211描述一種藉由使TCR-抗CD3融合蛋白融合至免疫球蛋白Fc域或白蛋白結合域而延長其半衰期之方法。然而，此類多域結合分子為大型且複雜的蛋白質，因此存在多種可能的形式，亦即各域（及各域中之各區）在一或多個多肽鏈上之位置及位向的可能組合。分子中各域（及其區）之位置及位向以及所存在的多肽鏈之數目可影響結合分子之特徵，諸如活性、半衰期及可製造性。因此，仍需要鑑別此類多域結合分子之有利形式。WO 2020/157211 describes a method for extending the half-life of a TCR-anti-CD3 fusion protein by fusing it to an immunoglobulin Fc domain or an albumin binding domain. However, such multi-domain binding molecules are large and complex proteins and therefore have many possible forms, i.e., possible combinations of the position and orientation of the domains (and regions within each domain) on one or more polypeptide chains. The position and orientation of the domains (and regions thereof) in the molecule and the number of polypeptide chains present can affect the characteristics of the binding molecule, such as activity, half-life, and manufacturability. Therefore, there is still a need to identify favorable forms of such multi-domain binding molecules.

本發明大體上係關於多域結合分子。本發明尤其係關於包含以下之多域結合分子：i）肽-主要組織相容性複合物（peptide-major histocompatibility complex；pMHC）結合域，其包含連接至恆定區之第一可變區（VC1）及連接至恆定區之第二可變區（VC2）；ii）免疫細胞接合（immune cell engaging；ICE）域（諸如T細胞接合免疫效應子域），其包含例如抗體輕鏈可變區（TCE-VL）及抗體重鏈可變區（TCE-VH）；及iii）半衰期延長域，其包含第一IgG Fc區（FC1）及第二IgG Fc區（FC2），其中該FC1區與該FC2區二聚形成Fc域。該等結合分子可用於治療諸如癌症、感染性疾病及自體免疫疾病之疾病。The present invention generally relates to multi-domain binding molecules. In particular, the present invention relates to multi-domain binding molecules comprising: i) a peptide-major histocompatibility complex (pMHC) binding domain comprising a first variable region (VC1) connected to a constant region and a second variable region (VC2) connected to a constant region; ii) an immune cell engaging (ICE) domain (such as a T cell engaging immune effector domain) comprising, for example, an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH); and iii) a half-life extension domain comprising a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region dimerizes with the FC2 region to form an Fc domain. These binding molecules can be used to treat diseases such as cancer, infectious diseases and autoimmune diseases.

本發明人測試了超過35種不同形式（亦即多肽中之各域之位向及位置）之包含pMHC結合域、T細胞接合免疫效應子域及半衰期延長域之多域結合分子。藉此，發現在許多形式中，使TCR-抗CD3融合蛋白與Fc域融合引起試管內效能實質上損失。然而，本發明人意外地鑑別出可表現為單一多肽鏈，具有顯著增強的半衰期，且保持原始分子之高效能的此類分子之形式。實施例8進一步展示，所鑑別之形式與結合不同目標之TCR一起，有利地發揮作用。The inventors tested over 35 different formats (i.e., the orientation and location of the domains in the polypeptide) of multi-domain binding molecules comprising a pMHC binding domain, a T cell engaging immune effector domain, and a half-life extension domain. In doing so, it was found that in many formats, fusing the TCR-anti-CD3 fusion protein to the Fc domain resulted in a substantial loss of in vitro potency. However, the inventors unexpectedly identified a format of such a molecule that can be expressed as a single polypeptide chain, has a significantly enhanced half-life, and retains the high potency of the original molecule. Example 8 further demonstrates that the identified format works advantageously with TCRs that bind to different targets.

在第一態樣中，提供一種多域單鏈結合分子，其包含： i）肽-主要組織相容性複合物（pMHC）結合域，其包含連接至恆定區之第一可變區（VC1）及連接至恆定區之第二可變區（VC2），其中VC1與VC2二聚形成該pMHC結合域； ii）免疫細胞接合（ICE）域；及 iii）半衰期延長域，其包含第一IgG Fc區（FC1）及第二IgG Fc區（FC2），其中該FC1區與該FC2區二聚形成Fc域；其中該ICE域連接至VC1之N端，VC1經由其C端連接至該FC1區之N端，該FC1區經由其C端連接至VC2之N端，且VC2經由其C端連接至該FC2區之N端；及其中該pMHC結合域及該T細胞接合免疫效應子域分別能夠結合pMHC複合物及免疫細胞。 In a first embodiment, a multi-domain single-chain binding molecule is provided, comprising: i) a peptide-major histocompatibility complex (pMHC) binding domain, comprising a first variable region (VC1) connected to a constant region and a second variable region (VC2) connected to a constant region, wherein VC1 and VC2 dimerize to form the pMHC binding domain; ii) an immune cell engagement (ICE) domain; and iii) a half-life extension domain, comprising a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region dimerizes with the FC2 region to form an Fc domain; wherein the ICE domain is connected to the N-terminus of VC1, VC1 is connected to the N-terminus of the FC1 region via its C-terminus, the FC1 region is connected to the N-terminus of VC2 via its C-terminus, and VC2 is connected to the N-terminus of the FC2 region via its C-terminus; and The pMHC binding domain and the T cell-engaging immune effector domain are capable of binding to pMHC complexes and immune cells, respectively.

在一個具體實例中，提供一種多域單鏈結合分子，其包含： i）肽-主要組織相容性複合物（pMHC）結合域，其包含連接至恆定區之第一可變區（VC1）及連接至恆定區之第二可變區（VC2），其中VC1與VC2二聚形成該pMHC結合域； ii）T細胞接合免疫效應子域，其包含抗體輕鏈可變區（TCE-VL）及抗體重鏈可變區（TCE-VH）；及 iii）半衰期延長域，其包含第一IgG Fc區（FC1）及第二IgG Fc區（FC2），其中該FC1區與該FC2區二聚形成Fc域；其中該T細胞接合免疫效應子域連接至VC1之N端，VC1經由其C端連接至該FC1區之N端，該FC1區經由其C端連接至VC2之N端，且VC2經由其C端連接至該FC2區之N端；及其中該pMHC結合域及該T細胞接合免疫效應子域分別能夠結合pMHC複合物及T細胞。 In one specific example, a multi-domain single-chain binding molecule is provided, comprising: i) a peptide-major histocompatibility complex (pMHC) binding domain, comprising a first variable region (VC1) connected to a constant region and a second variable region (VC2) connected to a constant region, wherein VC1 and VC2 dimerize to form the pMHC binding domain; ii) a T cell engaging immune effector domain, comprising an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH); and iii) a half-life extension domain, comprising a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region dimerizes with the FC2 region to form an Fc domain; wherein the T cell-engaging immune effector domain is connected to the N-terminus of VC1, VC1 is connected to the N-terminus of the FC1 region via its C-terminus, the FC1 region is connected to the N-terminus of VC2 via its C-terminus, and VC2 is connected to the N-terminus of the FC2 region via its C-terminus; and wherein the pMHC binding domain and the T cell-engaging immune effector domain are capable of binding to a pMHC complex and a T cell, respectively.

在另一態樣中，提供一種多域單鏈結合分子，其包含： i）可溶性TCR，其包含連接至恆定區之第一可變區（VC1）及連接至恆定區之第二可變區（VC2），其中VC1包含具有SEQ ID NO: 16中所提供之胺基酸序列或與其至少90%、至少95%或至少98%一致之序列的TCRβ可變區及恆定區，且VC2包含具有SEQ ID NO: 14中提供之胺基酸序列或與其至少90%、至少95%或至少98%一致之序列的TCRα可變區及恆定區； ii）抗CD3 scFv，其包含具有SEQ ID NO: 31中提供之胺基酸序列或與其至少90%、至少95%或至少98%一致之序列的抗體輕鏈可變區（TCE-VL），及具有SEQ ID NO: 32中提供之胺基酸序列或與其至少90%、至少95%或至少98%一致之序列的抗體重鏈可變區（TCE-VH）；及 iii）半衰期延長域，其包含具有SEQ ID NO: 42中所提供之胺基酸序列或與其至少90%、至少95%或至少98%一致之序列的第一IgG Fc區（FC1），及具有SEQ ID NO: 43中所提供之胺基酸序列或與其至少90%、至少95%或至少98%一致之序列的第二IgG Fc區（FC2），其中該FC1區與該FC2區二聚形成Fc域；其中該T細胞接合免疫效應子域連接至VC1之N端，VC1經由其C端連接至該FC1區之N端，該FC1區經由其C端連接至VC2之N端，且VC2經由其C端連接至該FC2區之N端；及其中該pMHC結合域及該T細胞接合免疫效應子域分別能夠結合pMHC複合物及T細胞。 In another embodiment, a multi-domain single-chain binding molecule is provided, comprising: i) a soluble TCR comprising a first variable region (VC1) connected to a constant region and a second variable region (VC2) connected to a constant region, wherein VC1 comprises a TCRβ variable region and a constant region having an amino acid sequence provided in SEQ ID NO: 16 or a sequence at least 90%, at least 95% or at least 98% identical thereto, and VC2 comprises a TCRα variable region and a constant region having an amino acid sequence provided in SEQ ID NO: 14 or a sequence at least 90%, at least 95% or at least 98% identical thereto; ii) an anti-CD3 scFv comprising an antibody light chain variable region (TCE-VL) having an amino acid sequence provided in SEQ ID NO: 31 or a sequence at least 90%, at least 95% or at least 98% identical thereto, and having SEQ ID NO: 32 or a sequence at least 90%, at least 95% or at least 98% identical thereto; and iii) a half-life extension domain, comprising a first IgG Fc region (FC1) having an amino acid sequence provided in SEQ ID NO: 42 or a sequence at least 90%, at least 95% or at least 98% identical thereto, and a second IgG Fc region (FC2) having an amino acid sequence provided in SEQ ID NO: 43 or a sequence at least 90%, at least 95% or at least 98% identical thereto, wherein the FC1 region dimerizes with the FC2 region to form an Fc domain; wherein the T cell engaging immune effector domain is connected to the N-terminus of VC1, VC1 is connected to the N-terminus of the FC1 region via its C-terminus, the FC1 region is connected to the N-terminus of VC2 via its C-terminus, and VC2 is connected to the N-terminus of the FC2 region via its C-terminus; and The pMHC binding domain and the T cell-engaging immune effector domain are capable of binding to pMHC complexes and T cells, respectively.

在另一態樣中，提供一種包含SEQ ID NO: 45中所提供之胺基酸序列之多域單鏈結合分子。In another aspect, a multi-domain single-chain binding molecule comprising the amino acid sequence provided in SEQ ID NO: 45 is provided.

在又一態樣中，提供一種編碼多域結合分子之核酸。亦提供一種表現載體，其包含此態樣之核酸。另外，提供一種宿主細胞，其包含此態樣之核酸或載體。In another aspect, a nucleic acid encoding a multi-domain binding molecule is provided. Also provided is an expression vector comprising the nucleic acid of this aspect. In addition, provided is a host cell comprising the nucleic acid or vector of this aspect.

在另一態樣中，亦提供一種製造多域結合分子之方法，其包含將上述宿主細胞維持於最佳條件下，該等最佳條件係用於表現核酸的最佳條件，且分離該多域結合分子。In another aspect, a method for producing a multi-domain binding molecule is also provided, comprising maintaining the above-mentioned host cells under optimal conditions, wherein the optimal conditions are optimal conditions for expressing nucleic acids, and isolating the multi-domain binding molecule.

在另一態樣中，提供一種包含多域結合分子之醫藥組成物。In another aspect, a pharmaceutical composition comprising a multi-domain binding molecule is provided.

以上態樣中之任一者之多域結合分子、核酸、載體、宿主細胞或醫藥組成物可用於治療諸如癌症、感染性疾病及自體免疫疾病之疾病。因此，在另一態樣中，亦提供用作藥劑之多域結合分子、核酸、載體、宿主細胞或醫藥組成物。在再另一態樣中，提供一種治療方法，其包含向有需要之患者投予多域結合分子、核酸、載體、宿主細胞或醫藥組成物。肽-主要組織相容性複合物（pMHC）結合域 The multi-domain binding molecules, nucleic acids, vectors, host cells or pharmaceutical compositions of any of the above aspects can be used to treat diseases such as cancer, infectious diseases and autoimmune diseases. Therefore, in another aspect, a multi-domain binding molecule, nucleic acid, vector, host cell or pharmaceutical composition for use as a medicament is also provided. In yet another aspect, a method of treatment is provided, which comprises administering a multi-domain binding molecule, nucleic acid, vector, host cell or pharmaceutical composition to a patient in need. Peptide-major histocompatibility complex (pMHC) binding domain

如本文所用，「pMHC結合域（pMHC binding domain）」為能夠結合肽-MHC複合物之蛋白質域。連接至恆定區之第一可變區（VC1）及連接至恆定區之第二可變區（VC2）二聚形成pMHC結合域。在此上下文中，「VC1」係指包含連接至恆定區之第一可變區的pMHC結合域序列區，且「VC2」係指包含連接至恆定區之第二可變區的區。pMHC結合位在VC1及VC2之可變區內。適合可變區及恆定區序列包括TCR或抗體可變區及恆定區。如本文所用，術語「MHC」及「HLA」可互換使用。As used herein, a "pMHC binding domain" is a protein domain capable of binding to a peptide-MHC complex. A first variable region (VC1) connected to a constant region and a second variable region (VC2) connected to a constant region dimerize to form a pMHC binding domain. In this context, "VC1" refers to a region of pMHC binding domain sequence comprising a first variable region connected to a constant region, and "VC2" refers to a region comprising a second variable region connected to a constant region. The pMHC binding site is within the variable regions of VC1 and VC2. Suitable variable and constant region sequences include TCR or antibody variable and constant regions. As used herein, the terms "MHC" and "HLA" are used interchangeably.

pMHC結合域可包含TCRα及TCRβ鏈之至少一部分。舉例而言，VC1及VC2之可變區可為TCR可變區。VC1可包含TCRα或TCRβ可變區且VC2可包含TCRα及TCRβ可變區中之另一者。舉例而言：（i）VC1可包含（a）TCRα可變區及恆定區或（b）TCRβ可變區及恆定區；及（ii）VC2可包含（a）或（b）中之另一者。較佳地，VC1包含該TCRβ可變區及恆定區且VC2包含該TCRα可變區及恆定區。 The pMHC binding domain may include at least a portion of the TCRα and TCRβ chains. For example, the variable regions of VC1 and VC2 may be TCR variable regions. VC1 may include a TCRα or TCRβ variable region and VC2 may include the other of the TCRα and TCRβ variable regions. For example: (i) VC1 may include (a) a TCRα variable region and a constant region or (b) a TCRβ variable region and a constant region; and (ii) VC2 may include the other of (a) or (b). Preferably, VC1 includes the TCRβ variable region and the constant region and VC2 includes the TCRα variable region and the constant region.

pMHC結合域可為包含TCR可變區及恆定區之T細胞受體（TCR），諸如可溶性TCR。本文所定義之TCR序列參考IMGT命名法描述，該命名法廣泛已知且TCR領域工作人員可獲得。舉例而言，參見：LeFranc及LeFranc, (2001). 「T cell Receptor Factsbook」, Academic Press；Lefranc, (2011), Cold Spring Harb Protoc 2011 (6): 595-603；Lefranc, (2001), Curr Protoc Immunol附錄1：附錄100；及Lefranc, (2003), Leukemia 17(1): 260-266。簡言之，TCR由兩個二硫鍵連接之鏈組成。各鏈（α及β）一般被視為具有兩個細胞外區，亦即可變區及恆定區。短接合區連接可變區及恆定區且通常被視為α可變區之一部分。另外，β鏈通常含有緊鄰接合區之短多樣性區，其通常亦被視為β可變區之一部分。典型TCR之各鏈之可變區位於N端，且包含嵌入構架序列中之三個互補決定區（Complementarity Determining Region；CDR）。CDR包含針對肽-MHC結合之識別位。The pMHC binding domain may be a T cell receptor (TCR) comprising a TCR variable region and a constant region, such as a soluble TCR. TCR sequences as defined herein are described with reference to the IMGT nomenclature, which is widely known and available to those working in the TCR field. For example, see: LeFranc and LeFranc, (2001). "T cell Receptor Factsbook", Academic Press; Lefranc, (2011), Cold Spring Harb Protoc 2011 (6): 595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 100; and Lefranc, (2003), Leukemia 17(1): 260-266. In short, a TCR consists of two disulfide-linked chains. Each chain (α and β) is generally considered to have two extracellular regions, namely a variable region and a constant region. A short joining region connects the variable and constant regions and is usually considered to be part of the α variable region. In addition, the β chain usually contains a short diversity region adjacent to the joining region, which is also usually considered to be part of the β variable region. The variable region of each chain of a typical TCR is located at the N-terminus and contains three complementarity determining regions (CDRs) embedded in the framework sequences. The CDRs contain the recognition sites for peptide-MHC binding.

替代地，pMHC結合域可包含抗體之可變區。VC1及VC2可變區可為抗體重鏈或輕鏈可變區。舉例而言，VC1可包含重鏈或輕鏈抗體可變區且VC2可包含重鏈或輕鏈抗體可變區中之另一者。就此而言，pMHC結合域可為TCR樣抗體，亦稱為「TCR模擬抗體（TCR mimic antibody）」（TCRm-Ab）。舉例而言，pMHC結合域可包含TCR樣抗體之可變區。抗體並不天然識別pMHC複合物。然而，已知可經工程改造得到對pMHC具有特異性之抗體，如Chang等人，Expert Opin Biol Ther. 2016年8月;16(8):979-87及Dahan等人，Expert Rev Mol Med. 2012年2月24日;14:e6中所描述。Alternatively, the pMHC binding domain may comprise the variable region of an antibody. The VC1 and VC2 variable regions may be antibody heavy chain or light chain variable regions. For example, VC1 may comprise a heavy chain or light chain antibody variable region and VC2 may comprise the other of the heavy chain or light chain antibody variable regions. In this regard, the pMHC binding domain may be a TCR-like antibody, also known as a "TCR mimic antibody" (TCRm-Ab). For example, the pMHC binding domain may comprise the variable region of a TCR-like antibody. Antibodies do not naturally recognize pMHC complexes. However, it is known that antibodies specific for pMHC can be engineered, as described in Chang et al., Expert Opin Biol Ther. 2016 Aug;16(8):979-87 and Dahan et al., Expert Rev Mol Med. 2012 Feb 24;14:e6.

pMHC結合域可包含至少一個免疫球蛋白恆定區。舉例而言，VC1及VC2中之恆定區可為免疫球蛋白恆定區。恆定區可對應於來自TCRα鏈或TCRβ鏈之恆定區（分別為TRAC或TRBC）。替代地，pMHC結合域之恆定區可為來自抗體輕鏈或重鏈之恆定區（CL、CH1、CH2、CH3或CH4）。恆定區可為全長的或可經截短。可截短TCR恆定區以移除跨膜域及胞質尾區。在恆定區經截短之情況下，較佳地僅自C端移除膜相關及細胞質部分。在pMHC結合域包含TCRα或TCRβ鏈序列的情況下，VC1及VC2可各自包含TCR可變區及TCR恆定區。較佳地，VC1及VC2不包含跨膜或細胞質域，亦即，較佳地，pMHC結合域為可溶性的。相對於天然恆定區，可將額外突變引入恆定區之胺基酸序列中。恆定區亦可包括允許藉由例如兩個半胱胺酸殘基之間的二硫鍵二聚之天然存在或引入的殘基。The pMHC binding domain may include at least one immunoglobulin constant region. For example, the constant regions in VC1 and VC2 may be immunoglobulin constant regions. The constant region may correspond to the constant region from the TCRα chain or the TCRβ chain (TRAC or TRBC, respectively). Alternatively, the constant region of the pMHC binding domain may be a constant region from the antibody light chain or heavy chain (CL, CH1, CH2, CH3 or CH4). The constant region may be full-length or may be truncated. The TCR constant region may be truncated to remove the transmembrane domain and the cytoplasmic tail. In the case where the constant region is truncated, it is preferred to remove only the membrane-associated and cytoplasmic parts from the C-terminus. In the case where the pMHC binding domain comprises a TCRα or TCRβ chain sequence, VC1 and VC2 may each comprise a TCR variable region and a TCR constant region. Preferably, VC1 and VC2 do not comprise a transmembrane or cytoplasmic domain, i.e., preferably, the pMHC binding domain is soluble. Additional mutations may be introduced into the amino acid sequence of the constant region relative to the native constant region. The constant region may also include naturally occurring or introduced residues that allow dimerization by, for example, a disulfide bond between two cysteine residues.

若存在，本發明之分子之TCR部分可為αβ異二聚體。本發明之分子的α-β異二聚TCR部分可包含α鏈TRAC恆定區序列及/或β鏈TRBC1或TRBC2恆定區序列。如上文所描述，恆定區可呈可溶形式（亦即，不具有跨膜域或細胞質域）。恆定區中之一或兩者可含有相對於原生TRAC及/或TRBC1/2序列之突變、取代或缺失。術語TRAC及TRBC1/2亦涵蓋天然多型變異體，例如TRAC之位置4處N變為K（Bragado等人，International immunology. 1994年2月;6(2):223-30）。If present, the TCR portion of the molecule of the present invention may be an αβ heterodimer. The α-β heterodimeric TCR portion of the molecule of the present invention may include an α-chain TRAC constant region sequence and/or a β-chain TRBC1 or TRBC2 constant region sequence. As described above, the constant region may be in a soluble form (i.e., without a transmembrane domain or a cytoplasmic domain). One or both of the constant regions may contain a mutation, substitution or deletion relative to the native TRAC and/or TRBC1/2 sequence. The terms TRAC and TRBC1/2 also encompass natural polymorphic variants, such as a change of N to K at position 4 of TRAC (Bragado et al., International immunology. February 1994; 6(2): 223-30).

α鏈及β鏈恆定區序列可藉由截短或取代修飾，以刪除TRAC之外顯子2之Cys4與TRBC1或TRBC2之外顯子2之Cys2之間的原生二硫鍵。α及/或β鏈恆定區序列在各別恆定域之殘基之間可具有引入之二硫鍵，如例如WO 2003/020763、WO 2004/033685及WO 2006/000830中所描述，及例如美國專利第7,329,731號、第7,569,664號及第8,361,794號中所描述，該等文獻中之各者的內容以引用之方式併入本文中。α及β恆定區可藉由取代在TRAC之位置Thr 48及TRBC1或TRBC2之位置Ser 57處之半胱胺酸殘基來修飾，該等半胱胺酸在TCR之α恆定區與β恆定區之間形成二硫鍵。TRBC1或TRBC2可另外包括在恆定域之位置75處之半胱胺酸至丙胺酸突變及在恆定域之位置89處之天冬醯胺酸至天冬胺酸突變。αβ異二聚體中存在之細胞外恆定區中之一者或兩者可在C端（C terminus/C termini）處截短，例如截短至多15個、或至多10個、或至多8個或更少個胺基酸。α鏈細胞外恆定區之C端可截短8個胺基酸。The alpha and beta chain homeostasis region sequences may be modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The alpha and/or beta chain homeostasis region sequences may have introduced disulfide bonds between the residues of the respective homeostasis domains, as described, for example, in WO 2003/020763, WO 2004/033685, and WO 2006/000830, and, for example, in U.S. Patent Nos. 7,329,731, 7,569,664, and 8,361,794, the contents of each of which are incorporated herein by reference. The α and β homeostasis regions may be modified by replacing cysteine residues at position Thr 48 of TRAC and position Ser 57 of TRBC1 or TRBC2, which form a disulfide bond between the α homeostasis region and the β homeostasis region of the TCR. TRBC1 or TRBC2 may additionally include a cysteine to alanine mutation at position 75 of the homeostasis domain and an asparagine to aspartate mutation at position 89 of the homeostasis domain. One or both of the extracellular homeostasis regions present in the αβ heterodimer may be truncated at the C-terminus/C-termini, for example, by up to 15, or up to 10, or up to 8 or fewer amino acids. The C-terminus of the α chain extracellular homeostasis region may be truncated by 8 amino acids.

VC1及VC2可變區及恆定區之胺基酸序列可對應於自然界中發現之胺基酸序列，或其可相對於天然蛋白質含有一或多個突變。可進行此類突變以增加pMHC結合域對給定抗原之親和力。另外或替代地，可併入突變以提高穩定性及可製造性。VC1及VC2序列可源自人類序列。The amino acid sequences of the VC1 and VC2 variable and constant regions may correspond to amino acid sequences found in nature, or they may contain one or more mutations relative to the native protein. Such mutations may be made to increase the affinity of the pMHC binding domain for a given antigen. Additionally or alternatively, mutations may be incorporated to improve stability and manufacturability. The VC1 and VC2 sequences may be derived from human sequences.

VC1及VC2序列可包含在恆定區中之一或多個經工程改造之半胱胺酸殘基，以在VC1與VC2之間形成非原生二硫鍵。用於在各別恆定區之殘基之間引入二硫鍵的適合位置描述於WO 2003/020763及WO 2004/033685中。單鏈TCR進一步描述於WO2004/033685；W098/39482；WO01/62908；Weidanz等人(1998) J Immunol Methods 221 (1 -2): 59-76；Hoo等人(1992) Proc Natl Acad Sci U S A 89(10): 4759-4763；Schodin (1996) Mol Immunol 33(9): 819-829）。The VC1 and VC2 sequences may comprise one or more engineered cysteine residues in the constant region to form a non-native disulfide bond between VC1 and VC2. Suitable positions for introducing disulfide bonds between residues in the respective constant regions are described in WO 2003/020763 and WO 2004/033685. Single-chain TCRs are further described in WO2004/033685; WO98/39482; WO01/62908; Weidanz et al. (1998) J Immunol Methods 221(1-2): 59-76; Hoo et al. (1992) Proc Natl Acad Sci USA 89(10): 4759-4763; Schodin (1996) Mol Immunol 33(9): 819-829).

VC1可包含TCRα或TCRβ可變區且VC2可包含TCRα及TCRβ可變區中之另一者。較佳地：（i） TCRα可變區包含SEQ ID NO: 3、4及5分別作為CDR1、CDR2及CDR3之CDR；及（ii） TCRβ可變區包含SEQ ID NO: 9、10及11分別作為CDR1、CDR2及CDR3之CDR。 VC1 may comprise a TCRα or TCRβ variable region and VC2 may comprise the other of the TCRα and TCRβ variable regions. Preferably: (i) The TCRα variable region comprises CDRs of SEQ ID NOs: 3, 4 and 5 as CDR1, CDR2 and CDR3, respectively; and (ii) The TCRβ variable region comprises CDRs of SEQ ID NOs: 9, 10 and 11 as CDR1, CDR2 and CDR3, respectively.

替代地，TCRα及TCRβ CDR序列可各自視情況相對於以上列舉之序列具有一個、兩個、三個或四個胺基酸取代。Alternatively, the TCRα and TCRβ CDR sequences may each have one, two, three or four amino acid substitutions relative to the sequences listed above, as appropriate.

分別地，TCRα可變區可包含作為CDR1、CDR2及CDR3，與SEQ ID NO: 3、4及5之序列至少90%、至少95%、至少98%或至少99%一致之CDR，及/或TCRβ可變區可包含作為CDR1、CDR2及CDR3，與SEQ ID NO: 9、10及11至少90%、至少95%、至少98%或至少99%一致之CDR。Respectively, the TCRα variable region may comprise CDRs that are at least 90%, at least 95%, at least 98% or at least 99% identical to the sequences of SEQ ID NOs: 3, 4 and 5 as CDR1, CDR2 and CDR3, and/or the TCRβ variable region may comprise CDRs that are at least 90%, at least 95%, at least 98% or at least 99% identical to the sequences of SEQ ID NOs: 9, 10 and 11 as CDR1, CDR2 and CDR3.

TCRα可變區可包含對應於SEQ ID NO: 3、4及5之序列之CDR，且包含與SEQ ID NO: 27、6、7及28之序列至少90%、至少95%、至少98%或至少99%一致之FR，及/或TCRβ可變區可包含對應於SEQ ID NO: 9、10及11之序列之CDR，且包含與SEQ ID NO: 29、12、13及30之序列至少90%、至少95%、至少98%或至少99%一致的FR。The TCRα variable region may comprise CDRs corresponding to the sequences of SEQ ID NOs: 3, 4 and 5, and comprise FRs that are at least 90%, at least 95%, at least 98% or at least 99% identical to the sequences of SEQ ID NOs: 27, 6, 7 and 28, and/or the TCRβ variable region may comprise CDRs corresponding to the sequences of SEQ ID NOs: 9, 10 and 11, and comprise FRs that are at least 90%, at least 95%, at least 98% or at least 99% identical to the sequences of SEQ ID NOs: 29, 12, 13 and 30.

TCRα可變區可與SEQ ID NO: 2之序列至少80%一致且TCRβ可變區可與SEQ ID NO: 8之序列至少80%一致。TCRα可變區可與SEQ ID NO: 2之序列至少90%、至少95%或至少98%一致且TCRβ可變區可與SEQ ID NO: 8之序列至少90%、至少95%或至少98%一致。較佳地，TCRα可變區具有提供於SEQ ID NO: 2中之序列且TCRβ可變區具有提供於SEQ ID NO: 8中之序列。The TCR alpha variable region may be at least 80% identical to the sequence of SEQ ID NO: 2 and the TCR beta variable region may be at least 80% identical to the sequence of SEQ ID NO: 8. The TCR alpha variable region may be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 2 and the TCR beta variable region may be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 8. Preferably, the TCR alpha variable region has the sequence provided in SEQ ID NO: 2 and the TCR beta variable region has the sequence provided in SEQ ID NO: 8.

VC1可包含TCRα或TCRβ恆定區且VC2可包含TCRα及TCRβ恆定區中之另一者。TCRα恆定區可與SEQ ID NO: 15之序列至少80%一致且TCRβ恆定區可與SEQ ID NO: 19之序列至少80%一致。TCRα恆定區可與SEQ ID NO: 15之序列至少90%、至少95%或至少98%一致且TCRβ恆定區可與SEQ ID NO: 19之序列至少90%、至少95%或至少98%一致。較佳地，TCRα恆定區具有提供於SEQ ID NO: 15中之序列且TCRβ恆定區具有提供於SEQ ID NO: 19中之序列。VC1 may include a TCRα or TCRβ constant region and VC2 may include the other of the TCRα and TCRβ constant regions. The TCRα constant region may be at least 80% identical to the sequence of SEQ ID NO: 15 and the TCRβ constant region may be at least 80% identical to the sequence of SEQ ID NO: 19. The TCRα constant region may be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 15 and the TCRβ constant region may be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 19. Preferably, the TCRα constant region has the sequence provided in SEQ ID NO: 15 and the TCRβ constant region has the sequence provided in SEQ ID NO: 19.

VC1可包含TCRα可變區及恆定區或TCRβ可變區及恆定區，且VC2可包含TCRα及TCRβ可變區及恆定區中之另一者。TCRα可變區及恆定區可包含與SEQ ID NO: 14之序列至少80%一致之胺基酸序列或由其組成，且TCRβ可變區及恆定區可包含與SEQ ID NO: 16之序列至少80%一致之胺基酸序列或由其組成。TCRα可變區及恆定區可包含與SEQ ID NO: 14之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成，且TCRβ可變區及恆定區可包含與SEQ ID NO: 16之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成。較佳地，TCRα可變區及恆定區包含提供於SEQ ID NO: 14中之胺基酸序列或由其組成，且TCRβ可變區及恆定區包含提供於SEQ ID NO: 16中之胺基酸序列或由其組成。VC1 may include a TCRα variable region and a constant region or a TCRβ variable region and a constant region, and VC2 may include the other of the TCRα and TCRβ variable regions and constant regions. The TCRα variable region and the constant region may include or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 14, and the TCRβ variable region and the constant region may include or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 16. The TCRα variable region and the constant region may include or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 14, and the TCRβ variable region and the constant region may include or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 16. Preferably, the TCRα variable region and the constant region comprise or consist of the amino acid sequence provided in SEQ ID NO: 14, and the TCRβ variable region and the constant region comprise or consist of the amino acid sequence provided in SEQ ID NO: 16.

所屬技術領域中具有通常知識者應瞭解，本發明之多域結合分子之形式可同樣適用除上文所述之TCR序列以外的TCR序列。舉例而言，其他適合之TCR鏈胺基酸序列提供於WO2011001152、WO2017109496、WO2017175006及WO2018234319中，及例如美國專利第8,519,100號、第11,639,374號、第11,505,590號及第11,427,624號中，該等文獻中之各者的內容以引用之方式併入本文中。Those skilled in the art will appreciate that the multi-domain binding molecule of the present invention may be similarly applicable to TCR sequences other than those described above. For example, other suitable TCR chain amino acid sequences are provided in WO2011001152, WO2017109496, WO2017175006, and WO2018234319, and, for example, in U.S. Patent Nos. 8,519,100, 11,639,374, 11,505,590, and 11,427,624, the contents of each of which are incorporated herein by reference.

如所屬技術領域中熟知，可以對蛋白質分子進行轉譯後修飾。醣基化係一種此類修飾，其包含寡醣部分與TCR或抗體鏈中之確定胺基酸之共價連接。舉例而言，天冬醯胺酸殘基或絲胺酸/蘇胺酸殘基為用於寡醣連接之熟知位置。特定蛋白質之醣基化狀態視多種因素而定，該等因素包括蛋白質序列、蛋白質構形及某些酶之可用性。此外，醣基化狀態（亦即寡醣類型、共價鍵聯及連接總數）可影響蛋白質功能。因此，當產生重組蛋白時，控制醣基化通常為合乎需要的。控制醣基化已用於改良基於抗體之治療劑。（Jefferis等人，(2009) Nat Rev Drug Discov 3月;8(3):226-34.）。醣基化可藉由使用例如特定細胞株（包括但不限於哺乳動物細胞株，諸如中國倉鼠卵巢（Chinese hamster ovary；CHO）細胞或人類胚胎腎（human embryonic kidney；HEK）細胞）或藉由化學修飾來控制。因為醣基化可改良藥物動力學、降低免疫原性且更接近地模擬原生人類蛋白質，此類修飾可為合乎需要的（Sinclair及Elliott, (2005) Pharm Sci.8月; 94(8):1626-35）。替代地，醣基化可導致製造缺乏一致性，其對於治療分子而言不合乎需要。醣基化風險較高的殘基，諸如天冬醯胺酸，可經諸如麩醯胺酸的替代胺基酸取代。As is well known in the art, protein molecules can be post-translationally modified. Glycosylation is one such modification that includes the covalent attachment of an oligosaccharide moiety to defined amino acids in a TCR or antibody chain. For example, aspartic acid residues or serine/threonine residues are well-known positions for oligosaccharide attachment. The glycosylation state of a particular protein depends on a variety of factors, including protein sequence, protein conformation, and the availability of certain enzymes. In addition, the glycosylation state (i.e., oligosaccharide type, covalent bonding, and total number of attachments) can affect protein function. Therefore, when producing recombinant proteins, it is generally desirable to control glycosylation. Controlling glycosylation has been used to improve antibody-based therapeutics. (Jefferis et al., (2009) Nat Rev Drug Discov Mar;8(3):226-34.). Glycosylation can be controlled by, for example, using specific cell lines (including but not limited to mammalian cell lines such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells) or by chemical modification. Such modifications may be desirable because glycosylation can improve pharmacokinetics, reduce immunogenicity, and more closely mimic native human proteins (Sinclair and Elliott, (2005) Pharm Sci. Aug;94(8):1626-35). Alternatively, glycosylation can result in a lack of manufacturing consistency, which is undesirable for therapeutic molecules. Residues with a higher risk of glycation, such as aspartic acid, can be replaced by alternative amino acids such as glutamine.

VC1及/或VC2可包含一或多個胺基酸取代，其移除一或多個醣基化位。在此情形下，取代係相對於原生（例如野生型）序列或未經修飾之序列。舉例而言：（i） VC1或VC2可包含TCRα可變區及恆定區，該TCRα可變區及恆定區包含如下位置處的一或多個胺基酸取代：選自由根據SEQ ID NO: 14編號之N24、N148、N182及N193組成之群；及/或（ii） VC1及VC2中之另一者可包含TCRβ可變區及恆定區，該TCRβ可變區及恆定區包含根據SEQ ID NO: 16編號之位置N184處之胺基酸取代。取代可為Asn至Gln（亦即N至Q）取代。較佳地，TCRα可變區及恆定區包含根據SEQ ID NO: 14編號之N24Q、N148Q、N182Q及N193Q取代，且TCRβ可變區及恆定區包含根據SEQ ID NO: 16編號之N184Q取代。 VC1 and/or VC2 may comprise one or more amino acid substitutions that remove one or more glycosylation sites. In this case, the substitutions are relative to the native (e.g., wild-type) sequence or the unmodified sequence. For example: (i) VC1 or VC2 may comprise a TCRα variable region and a constant region, the TCRα variable region and the constant region comprising one or more amino acid substitutions at the following positions: selected from the group consisting of N24, N148, N182, and N193 numbered according to SEQ ID NO: 14; and/or (ii) The other of VC1 and VC2 may comprise a TCRβ variable region and a constant region, the TCRβ variable region and the constant region comprising an amino acid substitution at position N184 numbered according to SEQ ID NO: 16. The substitution may be an Asn to Gln (i.e., N to Q) substitution. Preferably, the TCRα variable region and constant region comprise N24Q, N148Q, N182Q and N193Q substitutions numbered according to SEQ ID NO: 14, and the TCRβ variable region and constant region comprise N184Q substitution numbered according to SEQ ID NO: 16.

pMHC結合域可不完全去醣基化，亦即pMHC可保留一或多個來自其原生序列之醣基化位。舉例而言，pMHC結合域可在單一醣基化位處醣基化（亦即pMHC結合域可僅含有一個醣基化位）。單一醣基化位可在VC1或VC2之可變區中。單一醣基化位可位於根據SEQ ID NO: 16編號之TCRβ可變區之位置N18。有利地，本發明人已鑑別出，相較於其他醣基化及/或去醣基化變異體，除了保留對肽-MHC結合之親和力及目標細胞殺滅效能之外，具有此單一醣基化位之多域結合蛋白具有更佳之可製造性（例如蛋白質產率、對熱應力及聚集之抗性）。The pMHC binding domain may not be completely deglycosylated, i.e., the pMHC may retain one or more glycosylation sites from its native sequence. For example, the pMHC binding domain may be glycosylated at a single glycosylation site (i.e., the pMHC binding domain may contain only one glycosylation site). The single glycosylation site may be in the variable region of VC1 or VC2. The single glycosylation site may be located at position N18 of the TCRβ variable region numbered according to SEQ ID NO: 16. Advantageously, the inventors have identified that multi-domain binding proteins with this single glycosylation site have better manufacturability (e.g., protein yield, resistance to thermal stress and aggregation) compared to other glycosylation and/or deglycosylation variants, in addition to retaining affinity for peptide-MHC binding and target cell killing potency.

pMHC結合域結合與肽抗原複合之MHC。肽抗原可為疾病相關抗原。pMHC結合域可結合與MHC複合之腫瘤相關抗原肽。舉例而言，肽抗原可為源自如WO2011001152、WO2017109496、WO2017175006及WO2018234319中所描述之GP100、NYESO、MAGEA4或PRAME的肽。腫瘤相關抗原可為PIWIL1。舉例而言，pMHC結合域可結合SLSNRLYYL（SEQ ID NO: 56）HLA-A*02複合物。腫瘤相關抗原可為PRAME。較佳地，pMHC結合域結合SLLQHLIGL（SEQ ID NO: 1）HLA-A*02複合物。免疫細胞接合域 The pMHC binding domain binds to MHC complexed with a peptide antigen. The peptide antigen may be a disease-associated antigen. The pMHC binding domain may bind to a tumor-associated antigen peptide complexed with MHC. For example, the peptide antigen may be a peptide derived from GP100, NYESO, MAGEA4 or PRAME as described in WO2011001152, WO2017109496, WO2017175006 and WO2018234319. The tumor-associated antigen may be PIWIL1. For example, the pMHC binding domain may bind to SLSNRLYYL (SEQ ID NO: 56) HLA-A*02 complex. The tumor-associated antigen may be PRAME. Preferably, the pMHC binding domain binds to SLLQHLIGL (SEQ ID NO: 1) HLA-A*02 complex. Immune cell binding domain

如本文所用，「免疫細胞接合域（immune cell engaging domain）」為能夠結合免疫細胞上之目標及/或調節免疫反應，例如促進或遏制諸如T細胞活化之免疫反應的蛋白質域。免疫細胞接合域在本文中亦稱為「ICE」域。As used herein, an "immune cell engaging domain" is a protein domain that is capable of binding to a target on an immune cell and/or modulating an immune response, such as promoting or suppressing an immune response such as T cell activation. An immune cell engaging domain is also referred to herein as an "ICE" domain.

在一些具體實例中，免疫細胞接合域包含抗體輕鏈可變區（ICE-VL）及抗體重鏈可變區（ICE-VH）。如本文所用，「ICE-VL」及「ICE-VH」分別指免疫細胞接合域之輕鏈可變區及重鏈可變區。本文中亦可將「ICE-VL」及「ICE-VH」稱為「ICEVL」及「ICEVH」。因此，免疫細胞接合域可包含抗原結合位。抗體亦可為單域抗體（「ICE-SD」），諸如重鏈抗體之可變區（例如VHH）。In some specific examples, the immune cell engaging domain comprises an antibody light chain variable region (ICE-VL) and an antibody heavy chain variable region (ICE-VH). As used herein, "ICE-VL" and "ICE-VH" refer to the light chain variable region and the heavy chain variable region of the immune cell engaging domain, respectively. "ICE-VL" and "ICE-VH" may also be referred to herein as "ICEVL" and "ICEVH". Therefore, the immune cell engaging domain may comprise an antigen binding site. The antibody may also be a single domain antibody ("ICE-SD"), such as a variable region (e.g., VHH) of a heavy chain antibody.

免疫細胞接合域可為T細胞接合免疫效應子域。如本文所用，「T細胞接合免疫效應子域（T cell engaging immune effector domain）」為能夠結合T細胞上之目標以促進免疫反應的蛋白質域。T細胞接合免疫效應子域可包含抗體輕鏈可變區（TCE-VL）及抗體重鏈可變區（TCE-VH）。如本文所用，「TCE-VL」及「TCE-VH」係指T細胞接合免疫效應子域之輕鏈可變區及重鏈可變區。本文中「TCE-VL」及「TCE-VH」亦可稱為「TCEVL」及「TCEVH」。The immune cell engaging domain may be a T cell engaging immune effector domain. As used herein, a "T cell engaging immune effector domain" is a protein domain that can bind to a target on a T cell to promote an immune response. The T cell engaging immune effector domain may include an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH). As used herein, "TCE-VL" and "TCE-VH" refer to the light chain variable region and the heavy chain variable region of the T cell engaging immune effector domain. "TCE-VL" and "TCE-VH" herein may also be referred to as "TCEVL" and "TCEVH".

T細胞接合免疫效應子域可結合在T細胞之細胞表面上表現之蛋白質以促進T細胞之活化。舉例而言，T細胞接合免疫效應子域可為CD3效應子域。T細胞接合免疫效應子域可結合，例如，特異性結合CD3（亦即，T細胞接合免疫效應子域可為CD3結合蛋白）。T細胞接合免疫效應子可為抗體或其功能片段，例如單鏈可變片段（single-chain variable fragment；scFv）或類似大小的抗體樣骨架，或經由與CD3及/或TCR/CD3複合物交互作用活化T細胞之任何其他結合蛋白。抗體亦可為單域抗體，諸如重鏈抗體之可變區（例如VHH）。The T cell engaging immune effector domain can bind to a protein expressed on the cell surface of a T cell to promote the activation of the T cell. For example, the T cell engaging immune effector domain can be a CD3 effector domain. The T cell engaging immune effector domain can bind, for example, specifically bind to CD3 (i.e., the T cell engaging immune effector domain can be a CD3 binding protein). The T cell engaging immune effector can be an antibody or a functional fragment thereof, such as a single-chain variable fragment (scFv) or an antibody-like framework of similar size, or any other binding protein that activates T cells by interacting with CD3 and/or the TCR/CD3 complex. The antibody can also be a single domain antibody, such as a variable region (e.g., VHH) of a heavy chain antibody.

替代地，免疫細胞接合域可為免疫抑制子。如本文所用，術語「免疫抑制子（immune suppressor）」係指能夠抑制免疫反應，諸如抑制T細胞活化之任何分子，例如蛋白質。免疫抑制子可與目標（例如抗原）結合。舉例而言，免疫抑制子可為免疫檢查點促效子，亦即，誘導免疫檢查點信號傳導之分子。免疫抑制子可包含能夠結合抗原之抗原結合部分。免疫抑制子之抗原可位於免疫細胞，諸如T細胞上。結合分子可包含抗體或其抗原結合片段，例如抗體亦可為單域抗體，諸如重鏈抗體之可變區。替代地，抗體可為單鏈可變片段（scFv）或類似大小之抗體樣骨架，或經由誘導免疫檢查點信號傳導抑制T細胞之任何其他結合蛋白。此類免疫抑制子描述於下文中。Alternatively, the immune cell binding domain may be an immunosuppressor. As used herein, the term "immune suppressor" refers to any molecule, such as a protein, that is capable of suppressing an immune response, such as suppressing T cell activation. An immunosuppressor may bind to a target, such as an antigen. For example, an immunosuppressor may be an immune checkpoint agonist, that is, a molecule that induces immune checkpoint signaling. An immunosuppressor may comprise an antigen binding portion capable of binding to an antigen. The antigen of the immunosuppressor may be located on an immune cell, such as a T cell. The binding molecule may comprise an antibody or an antigen-binding fragment thereof, for example, the antibody may also be a single domain antibody, such as a variable region of a heavy chain antibody. Alternatively, the antibody may be a single chain variable fragment (scFv) or antibody-like scaffold of similar size, or any other binding protein that inhibits T cells by inducing immune checkpoint signaling. Such immunosuppressants are described below.

免疫細胞接合域可包含能夠結合抗原之抗原結合部分。免疫細胞接合域之抗原可位於免疫細胞，諸如T細胞上。結合分子可包含抗體或其抗原結合片段。如本文所用，術語「抗體（antibody）」意欲包括習知/原生抗體及經工程改造之抗體，尤其功能性抗體片段、單鏈抗體、單域抗體及雙特異性或多特異性抗體。在此上下文中，「原生（native）」或「習知（conventional）」係指具有與自然界中發現之抗體相同類型之域及域配置且包含抗體源性之CDR及FR序列的抗體。在原生/習知四鏈抗體，例如人類抗體中，兩條重鏈藉由雙硫鍵彼此連接且各重鏈藉由雙硫鍵連接至輕鏈。輕鏈（VL）及重鏈（VH）之可變域決定對抗原之結合識別及特異性。習知抗體結合位由主要來自「抗體互補決定區（antibody complementarity determining region）」（CDR）或高變區之殘基構成。偶爾，來自非高變或構架區（framework region；FR）之殘基影響總體域結構且因此影響結合位。CDR係指一起定義原生抗體結合位之結合親和力及特異性的胺基酸序列。習知四鏈抗體之輕鏈及重鏈各自具有三個CDR，分別稱為：CDR1-L、CDR2-L、CDR3-L及CDR1-H、CDR2-H、CDR3-H。因此，習知四鏈抗體抗原結合位包括六個CDR，其包含來自VH及VL中之各者的CDR組。The immune cell binding domain may comprise an antigen binding portion capable of binding to an antigen. The antigen of the immune cell binding domain may be located on an immune cell, such as a T cell. The binding molecule may comprise an antibody or an antigen binding fragment thereof. As used herein, the term "antibody" is intended to include conventional/native antibodies and engineered antibodies, particularly functional antibody fragments, single chain antibodies, single domain antibodies, and bispecific or multispecific antibodies. In this context, "native" or "conventional" refers to antibodies having domains and domain configurations of the same type as antibodies found in nature and comprising antibody-derived CDR and FR sequences. In native/conventional four-chain antibodies, such as human antibodies, the two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. The variable domains of the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity for the antigen. The conventional antibody binding site is composed of residues mainly from the "antibody complementarity determining region" (CDR) or hypervariable region. Occasionally, residues from non-hypervariable or framework regions (FR) affect the overall domain structure and therefore the binding site. CDR refers to the amino acid sequence that together defines the binding affinity and specificity of the native antibody binding site. It is known that the light chain and heavy chain of a tetraclonal antibody each have three CDRs, namely: CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H. Therefore, it is known that the antigen binding site of a tetraclonal antibody includes six CDRs, which include a set of CDRs from each of VH and VL.

「經工程改造（engineered）」之抗體形式包括功能性抗體片段、單鏈抗體、單域抗體及嵌合、人源化、雙特異性或多特異性抗體。經工程改造之抗體形式進一步包括其中TCR源性之CDR，可能包括額外3、2或1個N端及/或C端構架殘基，或整個TCR源性之可變域被移植至抗體重鏈或輕鏈上的構築體。「功能性抗體片段（functional antibody fragment）」係指保留結合其目標抗原的能力的全長抗體之一部分，或類似於全長抗體之一部分的蛋白質，尤其全長抗體之抗原結合區或可變區。功能性抗體「片段」之實例包括Fv、Fab、F(ab')2、Fab'、dsFv、(dsFv)2、scFv、sc(Fv)2及雙功能抗體。舉例而言，本發明之結合分子可包含scFv。"Engineered" antibody forms include functional antibody fragments, single-chain antibodies, single-domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies. Engineered antibody forms further include constructs in which TCR-derived CDRs, possibly including additional 3, 2 or 1 N-terminal and/or C-terminal framework residues, or the entire TCR-derived variable domain is grafted onto the antibody heavy or light chain. "Functional antibody fragment" refers to a portion of a full-length antibody that retains the ability to bind to its target antigen, or a protein similar to a portion of a full-length antibody, particularly the antigen binding region or variable region of a full-length antibody. Examples of functional antibody "fragments" include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and bifunctional antibodies. For example, the binding molecules of the present invention may comprise scFv.

抗體亦可為單域抗體，諸如重鏈抗體之可變區。就此而言，術語「單域抗體（single domain antibody）」係指由單一抗體可變域（例如重鏈可變域）組成之抗體。因此，免疫細胞接合域可包含例如VHH（亦即，重鏈抗體之可變域）。如所屬技術領域中已知，諸如VHH的單域抗體之抗原結合位可包含三個CDR（相對於習知四鏈抗體中之六個）。如本文所用，術語「抗體之抗原結合部分（antigen binding moiety of an antibody）」涵蓋此類結合位。替代地或另外，結合分子可包含Fab或Fv片段。術語「Fab」（「抗原結合片段（fragment antigen-binding）」）表示抗體之抗原結合片段，其包含抗體輕鏈（VL-CL）及抗體重鏈之可變域及CH1域（VH-CH1）。Fab片段之分子量通常為約50,000道爾頓。Fv片段為抗體Fab片段之N端部分且由一條輕鏈（VL）及一條重鏈（VH）之可變部分組成。The antibody may also be a single domain antibody, such as the variable region of a heavy chain antibody. In this regard, the term "single domain antibody" refers to an antibody composed of a single antibody variable domain (e.g., a heavy chain variable domain). Thus, the immunocell binding domain may comprise, for example, a VHH (i.e., a variable domain of a heavy chain antibody). As is known in the art, the antigen binding site of a single domain antibody such as a VHH may comprise three CDRs (as opposed to six in a known four-chain antibody). As used herein, the term "antigen binding moiety of an antibody" encompasses such binding sites. Alternatively or in addition, the binding molecule may comprise a Fab or Fv fragment. The term "Fab" ("fragment antigen-binding") refers to the antigen-binding fragment of an antibody, which comprises the antibody light chain (VL-CL) and the variable domain and CH1 domain (VH-CH1) of the antibody heavy chain. The molecular weight of the Fab fragment is usually about 50,000 Daltons. The Fv fragment is the N-terminal part of the antibody Fab fragment and consists of one light chain (VL) and one variable part of the heavy chain (VH).

免疫細胞接合域可包含抗體重鏈可變區（VH）及抗體輕鏈可變區（VL），該等可變區結合以形成能夠結合抗原之抗原結合部分。因此，抗原結合部分可包含VH及VL。舉例而言，免疫細胞接合域可包含含有VH及VL之scFv。The immunocell-binding domain may comprise an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), which combine to form an antigen-binding portion capable of binding to an antigen. Thus, the antigen-binding portion may comprise VH and VL. For example, the immunocell-binding domain may comprise a scFv comprising VH and VL.

其他適合的抗原結合部分為重鏈抗體（heavy chain antibody；hcAb）、單域抗體（single domain antibody；sdAb）、微型抗體（Tramontano等人(1994) J. Mol. Recognition 7, 9-24）、駱駝重鏈抗體之可變域（VHH）、新抗原受體之可變域（variable domain of the new antigen receptor；VNAR）、親和抗體（Nygren P.A. (2008) FEBS J. 275, 2668-2676）、阿爾法體（參見WO2010066740）、經設計錨蛋白重複域（DARPin）（Stumpp等人(2008) Drug Discovery Today 13, 695-701）、抗運載蛋白（anticalin）（Skerra等人(2008) FEBS J. 275, 2677-2683）、打結素（knottin）（Kolmar等人(2008) FEBS J. 275, 2684-2690）及經工程改造之CH2域（奈米抗體，參見Dimitrov DS (2009) mAbs 1, 26-28）。Other suitable antigen-binding moieties are heavy chain antibodies (hcAb), single domain antibodies (sdAb), minibodies (Tramontano et al. (1994) J. Mol. Recognition 7, 9-24), variable domains of camel heavy chain antibodies (VHH), variable domains of new antigen receptors (VNAR), affibodies (Nygren P.A. (2008) FEBS J. 275, 2668-2676), alphabodies (see WO2010066740), designed anchor protein repeat domains (DARPins) (Stumpp et al. (2008) Drug Discovery Today 13, 695-701), anticalins (Skerra et al. (2008) FEBS J. 275, 2668-2676), and alphabodies (see WO2010066740). 2677-2683), knottin (Kolmar et al. (2008) FEBS J. 275, 2684-2690) and engineered CH2 domains (nanoantibodies, see Dimitrov DS (2009) mAbs 1, 26-28).

抗原結合部分可為或包含：重鏈可變域，其包含以下、由以下組成或基本上由以下組成：四個構架區（分別FR1至FR4）及三個互補決定區（分別CDR1至CDR3）；或此類重鏈可變域之任何適合片段（其保留抗原結合位）。抗原結合部分可為重鏈抗體。抗原結合部分可為衍生自習知四鏈抗體之抗體之重鏈可變域序列，諸如但不限於衍生自人類抗體之VH序列。較佳地，抗原結合部分為或包含重鏈抗體（例如駱駝抗體）之可變域，諸如VHH（在本文中亦稱為「VHH域」）。較佳地，抗原結合部分為VHH。The antigen binding portion may be or comprise: a heavy chain variable domain comprising, consisting of, or consisting essentially of: four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively); or any suitable fragment of such a heavy chain variable domain (which retains the antigen binding site). The antigen binding portion may be a heavy chain antibody. The antigen binding portion may be a heavy chain variable domain sequence of an antibody derived from a known four-chain antibody, such as, but not limited to, a VH sequence derived from a human antibody. Preferably, the antigen binding portion is or comprises a variable domain of a heavy chain antibody (e.g., a camel antibody), such as a VHH (also referred to herein as a "VHH domain"). Preferably, the antigen binding portion is a VHH.

如本文中所描述，免疫細胞接合域可包含結合位於免疫細胞上之抗原的抗原結合部分（例如，抗體抗原結合部分）。在本發明之上下文中，「免疫細胞（immune cell）」可指例如T細胞或B細胞。特定言之，抗原結合部分之抗原可為T細胞表面抗原。As described herein, an immune cell binding domain may comprise an antigen binding portion (e.g., an antibody antigen binding portion) that binds to an antigen located on an immune cell. In the context of the present invention, an "immune cell" may refer to, for example, a T cell or a B cell. Specifically, the antigen of the antigen binding portion may be a T cell surface antigen.

免疫細胞接合域可為單鏈可變片段（scFv）。亦縮寫為「sFv」或「scFv」之「單鏈Fv」為包含連接至單一多肽鏈中之VH及VL抗體域的抗體片段。scFv多肽可進一步包含VH與VL域之間的多肽連接子，其使得scFv能夠形成用於抗原結合之所要結構。關於scFv之綜述，參見Pluckthun, The Pharmacology of Monoclonal Antibodies, 第113卷, Rosenburg及Moore編, Springer-Verlag, New York, 第269-315頁(1994)。The immune cell engaging domain may be a single chain variable fragment (scFv). A "single chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. The scFv polypeptide may further comprise 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 Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

免疫細胞接合域可為CD3效應子。CD3效應子包括但不限於抗CD3抗體或抗體片段，尤其抗CD3 scFv或抗體樣骨架。免疫細胞接合域可為可係抗CD3 scFv之T細胞接合免疫效應子域。其他免疫效應子包括但不限於結合T細胞上之抗原的抗體，包括其片段、衍生物及變異體。此類抗原包括CD28、4-1bb（CD137）或CD16或在免疫突觸發揮作用之任何分子。尤其較佳的免疫效應子係抗CD3抗體，或該抗CD3抗體的功能片段或變異體。如本文所用，術語「抗體（antibody）」涵蓋此類片段及變異體。抗CD3抗體之實例包括但不限於OKT3、UCHT-1、BMA-031及12F6。適用於本文所描述之組成物及方法的抗體片段及變異體/類似物包括微型抗體、Fab片段、F(ab') ₂片段、dsFv及scFv片段。 The immune cell engaging domain may be a CD3 effector. CD3 effectors include, but are not limited to, anti-CD3 antibodies or antibody fragments, particularly anti-CD3 scFv or antibody-like frameworks. The immune cell engaging domain may be a T cell engaging immune effector domain, which may be an anti-CD3 scFv. Other immune effectors include, but are not limited to, antibodies that bind to antigens on T cells, including fragments, derivatives and variants thereof. Such antigens include CD28, 4-1bb (CD137) or CD16 or any molecule that plays a role in immune synapses. Particularly preferred immune effectors are anti-CD3 antibodies, or functional fragments or variants of the anti-CD3 antibodies. As used herein, the term "antibody" encompasses such fragments and variants. Examples of anti-CD3 antibodies include, but are not limited to, OKT3, UCHT-1, BMA-031 and 12F6. Antibody fragments and variants/analogs suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab') ₂ fragments, dsFv and scFv fragments.

較佳地，免疫細胞接合域為包含以下之T細胞接合免疫效應子域：（i） VL區，其包含SEQ ID NO: 33、34及35分別作為CDR1、CDR2及CDR3之CDR；及（ii） VH區，其包含SEQ ID NO: 36、37及38分別作為CDR1、CDR2及CDR3之CDR。 Preferably, the immune cell engaging domain is a T cell engaging immune effector subdomain comprising: (i) VL region comprising CDRs of SEQ ID NO: 33, 34 and 35 as CDR1, CDR2 and CDR3, respectively; and (ii) VH region comprising CDRs of SEQ ID NO: 36, 37 and 38 as CDR1, CDR2 and CDR3, respectively.

替代地，免疫細胞接合域可為包含以下之T細胞接合免疫效應子域：（i） VL區，其包含SEQ ID NO: 33、34及35分別作為CDR1、CDR2及CDR3之CDR；及（ii） VH區，其包含SEQ ID NO: 48、37及38分別作為CDR1、CDR2及CDR3之CDR。 Alternatively, the immune cell engaging domain may be a T cell engaging immune effector subdomain comprising: (i) a VL region comprising CDRs of SEQ ID NOs: 33, 34 and 35 as CDR1, CDR2 and CDR3, respectively; and (ii) a VH region comprising CDRs of SEQ ID NOs: 48, 37 and 38 as CDR1, CDR2 and CDR3, respectively.

以上VL及VH CDR序列可相對於以上列舉之序列各自視情況具有一個、兩個、三個或四個胺基酸取代。The above VL and VH CDR sequences may each have one, two, three or four amino acid substitutions relative to the above listed sequences, as appropriate.

分別地，TCE-VL可包含作為CDR1、CDR2及CDR3，與SEQ ID NO: 33、34及35之序列至少90%、至少95%、至少98%或至少99%一致之CDR，及/或TCE-VH可包含作為CDR1、CDR2及CDR3，與SEQ ID NO: 36、37及38至少90%、至少95%、至少98%或至少99%一致之CDR。Respectively, TCE-VL may comprise CDRs that are at least 90%, at least 95%, at least 98% or at least 99% identical to the sequences of SEQ ID NOs: 33, 34 and 35 as CDR1, CDR2 and CDR3, and/or TCE-VH may comprise CDRs that are at least 90%, at least 95%, at least 98% or at least 99% identical to the sequences of SEQ ID NOs: 36, 37 and 38 as CDR1, CDR2 and CDR3.

替代地，分別地，TCE-VL可包含作為CDR1、CDR2及CDR3，與SEQ ID NO: 33、34及35之序列至少90%、至少95%、至少98%或至少99%一致之CDR，及/或TCE-VH可包含作為CDR1、CDR2及CDR3，與SEQ ID NO: 48、37及38至少90%、至少95%、至少98%或至少99%一致之CDR。Alternatively, the TCE-VL may comprise CDRs that are at least 90%, at least 95%, at least 98% or at least 99% identical to the sequences of SEQ ID NOs: 33, 34 and 35 as CDR1, CDR2 and CDR3, and/or the TCE-VH may comprise CDRs that are at least 90%, at least 95%, at least 98% or at least 99% identical to the sequences of SEQ ID NOs: 48, 37 and 38 as CDR1, CDR2 and CDR3, respectively.

TCE-VL可包含與SEQ ID NO: 31之序列至少80%一致之胺基酸序列或由其組成，且TCE-VH可包含與SEQ ID NO: 32之序列至少80%一致之胺基酸序列或由其組成。TCE-VL可包含與SEQ ID NO: 31之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成，且TCE-VH可包含與SEQ ID NO: 32之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成。較佳地，TCE-VL包含SEQ ID NO: 31中所提供之胺基酸序列或由其組成，且TCE-VH包含SEQ ID NO: 32中所提供之胺基酸序列或由其組成。TCE-VL may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 31, and TCE-VH may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 32. TCE-VL may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 31, and TCE-VH may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 32. Preferably, TCE-VL comprises or consists of the amino acid sequence provided in SEQ ID NO: 31, and TCE-VH comprises or consists of the amino acid sequence provided in SEQ ID NO: 32.

替代地，TCE-VL包含與SEQ ID NO: 31之序列至少80%一致之胺基酸序列或由其組成，且TCE-VH包含與SEQ ID NO: 41之序列至少80%一致之胺基酸序列或由其組成。TCE-VL可包含與SEQ ID NO: 31之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成，且TCE-VH可包含與SEQ ID NO: 41之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成。舉例而言，TCE-VL可包含SEQ ID NO: 31中所提供之胺基酸序列或由其組成，且TCE-VH可包含SEQ ID NO: 41中所提供之胺基酸序列或由其組成。Alternatively, TCE-VL comprises or consists of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 31, and TCE-VH comprises or consists of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 41. TCE-VL may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 31, and TCE-VH may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 41. For example, TCE-VL may comprise or consist of an amino acid sequence provided in SEQ ID NO: 31, and TCE-VH may comprise or consist of an amino acid sequence provided in SEQ ID NO: 41.

如上文所描述，免疫細胞接合域或T細胞接合免疫效應子域可為scFv。免疫細胞接合域或T細胞接合免疫效應子域可為包含與SEQ ID NO: 17或40之序列至少80%一致之胺基酸序列或由其組成之scFv。scFv可包含與SEQ ID NO: 17或40之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成。較佳地，scFv包含SEQ ID NO: 17中所提供之胺基酸序列或由其組成。替代地，scFv可包含SEQ ID NO: 40中提供之胺基酸序列或由其組成。As described above, the immune cell engaging domain or T cell engaging immune effector subdomain may be a scFv. The immune cell engaging domain or T cell engaging immune effector subdomain may be a scFv comprising or consisting of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 17 or 40. The scFv may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 17 or 40. Preferably, the scFv comprises or consists of the amino acid sequence provided in SEQ ID NO: 17. Alternatively, the scFv may comprise or consist of the amino acid sequence provided in SEQ ID NO: 40.

免疫細胞接合域可替代地為免疫抑制子。舉例而言，免疫抑制子之目標可為免疫檢查點分子，諸如PD-1（計劃性死亡1受體；Programmed Death 1 receptor）、A2AR（腺苷A2A受體；Adenosine A2A receptor）、A2BR（腺苷A2B受體；Adenosine A2B receptor）、B7-H3（B7同系物3；B7 Homolog 3，亦稱為CD276）、B7-H4（B7同系物4，亦稱為VTCN1）、BTLA（B及T淋巴球弱化子；B and T Lymphocyte Attenuator，亦稱為CD272）、CTLA-4（細胞毒性T淋巴球相關蛋白質4；Cytotoxic T-Lymphocyte-Associated protein 4，亦稱為CD152）、IDO（吲哚胺2,3-二氧酶）、CD200受體、KIR（殺手細胞免疫球蛋白樣受體；Killer-cell Immunoglobulin-like Receptor）、TIGIT（具有Ig及ITIM域之T細胞免疫受體）、LAG3（淋巴球活化基因-3；Lymphocyte Activation Gene-3）、NOX2（菸醯胺腺嘌呤二核苷酸磷酸NADPH氧化酶同功型物2）、TIM-3（T細胞免疫球蛋白域及黏蛋白域3；T-cell Immunoglobulin domain and Mucin domain 3）、VISTA（T細胞活化之V域Ig抑制子；V-domain Ig suppressor of T cell activation）、SIGLEC7（唾液酸結合免疫球蛋白型凝集素7；Sialic acid-binding immunoglobulin-type lectin 7，亦稱為CD328）及SIGLEC9（唾液酸結合免疫球蛋白型凝集素9，亦稱為CD329）。The immune cell engaging domain may alternatively be an immunosuppressor. For example, the target of the immunosuppressor may be an immune checkpoint molecule such as PD-1 (Programmed Death 1 receptor), A2AR (Adenosine A2A receptor), A2BR (Adenosine A2B receptor), B7-H3 (B7 Homolog 3, also known as CD276), B7-H4 (B7 Homolog 4, also known as VTCN1), BTLA (B and T Lymphocyte Attenuator, also known as CD272), CTLA-4 (Cytotoxic T-Lymphocyte-Associated protein 4), 4, also known as CD152), IDO (indoleamine 2,3-dioxygenase), CD200 receptor, KIR (Killer-cell Immunoglobulin-like Receptor), TIGIT (T-cell immunoreceptor with Ig and ITIM domains), LAG3 (Lymphocyte Activation Gene-3), NOX2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), VISTA (V-domain Ig suppressor of T cell activation), SIGLEC7 (Sialic acid-binding immunoglobulin-type lectin 7, also known as CD328) and SIGLEC9 (sialic acid-binding immunoglobulin-type lectin 9, also known as CD329).

就此而言，免疫抑制子可為以上免疫檢查點分子中之一或多者之促效子。因此，免疫抑制子可為免疫檢查點促效子（亦即用於抑制免疫活化）。適合之免疫檢查點促效子，包括原生配位體及抗體，綜述於例如Paluch等人Front Immunol, 2018, 9:2306。In this regard, an immunosuppressant may be an agonist of one or more of the above immune checkpoint molecules. Thus, an immunosuppressant may be an immune checkpoint agonist (i.e., one that inhibits immune activation). Suitable immune checkpoint agonists, including native ligands and antibodies, are summarized in, for example, Paluch et al. Front Immunol, 2018, 9:2306.

取代包含抗體之抗原結合部分，免疫抑制子可包含受體-配位體對中之一者，由此免疫抑制子能夠結合受體-配位體對中之另一者。目標配位體或受體可位於免疫細胞上。舉例而言，免疫抑制子可包含上文所描述之免疫檢查點分子之配位體。特定言之，免疫抑制子可包含能夠結合PD-1之PD-L1之一部分（例如可溶性細胞外區）。此類免疫抑制子可藉由與PD-1結合且刺激PD-1信號傳導而接合免疫細胞。Instead of comprising the antigen binding portion of an antibody, an immunosuppressant may comprise one of a receptor-ligand pair, whereby the immunosuppressant is able to bind to the other of the receptor-ligand pair. The target ligand or receptor may be located on an immune cell. For example, an immunosuppressant may comprise a ligand of an immune checkpoint molecule described above. Specifically, an immunosuppressant may comprise a portion of PD-L1 (e.g., a soluble extracellular domain) that is able to bind to PD-1. Such immunosuppressants may engage immune cells by binding to PD-1 and stimulating PD-1 signaling.

替代地，免疫抑制子可包含與免疫檢查點分子結合且可刺激其信號傳導的促效抗體。舉例而言，免疫抑制子可為或包含PD-1促效抗體（例如單域抗體）。此類PD-1促效子較佳地不與PD-L1競爭結合PD-1。PD-1促效子可為全長抗體或其片段，諸如scFv抗體或Fab片段，或單域抗體。此類抗體之實例提供於WO2011110621及WO2010029434及WO2018024237中。因此，免疫抑制子之抗原可為PD-1且免疫抑制子之抗原結合部分可為PD-1促效子。免疫抑制子之抗原結合部分可包含單域抗體，視情況VHH。舉例而言，免疫抑制子可為PD-1促效VHH。Alternatively, the immunosuppressant may comprise an agonist antibody that binds to an immune checkpoint molecule and can stimulate its signaling. For example, the immunosuppressant may be or comprise a PD-1 agonist antibody (e.g., a single domain antibody). Such PD-1 agonists preferably do not compete with PD-L1 for binding to PD-1. The PD-1 agonist may be a full-length antibody or a fragment thereof, such as an scFv antibody or a Fab fragment, or a single domain antibody. Examples of such antibodies are provided in WO2011110621 and WO2010029434 and WO2018024237. Therefore, the antigen of the immunosuppressant may be PD-1 and the antigen binding portion of the immunosuppressant may be a PD-1 agonist. The antigen binding portion of the immunosuppressant may comprise a single domain antibody, optionally a VHH. For example, the immunosuppressant may be a PD-1 agonist VHH.

如上文所描述，免疫抑制子可為PD-1促效子。如本文所用，術語「PD-1促效子（PD-1 agonist）」係指能夠結合PD-1及活化PD-1信號傳導之任何分子，包括例如PD-1配位體、PD-L1及PD-1促效抗體。PD-1路徑之活化下調免疫活性、促進周邊免疫耐受性及防止自體免疫性（Keir等人，Annu Rev Immunol, 26:677-704, 2008；Okazaki等人，Int Immunol 19:813-824, 2007）。半衰期延長域 As described above, the immunosuppressant may be a PD-1 agonist. As used herein, the term "PD-1 agonist" refers to any molecule that is capable of binding to PD-1 and activating PD-1 signaling, including, for example, PD-1 ligands, PD-L1, and PD-1 agonist antibodies. Activation of the PD-1 pathway downregulates immune activity, promotes peripheral immune tolerance, and prevents autoimmunity (Keir et al., Annu Rev Immunol, 26:677-704, 2008; Okazaki et al., Int Immunol 19:813-824, 2007). Half-life extension domain

如本文所用，「半衰期延長域（half-life extending domain）」係指用於相對於不具有半衰期延長域之多域結合蛋白，延長多域結合蛋白之半衰期的蛋白域。半衰期延長域包含第一IgG Fc區（FC1）及第二IgG Fc區（FC2），其中FC1區與FC2區二聚形成Fc域。如本文所用，術語「Fc區（Fc region）」用於指包含至少CH2域及CH3域序列之單一多肽鏈的區，而術語「Fc域（Fc domain）」係指兩個Fc區（亦即FC1及FC2）之二聚體。As used herein, a "half-life extending domain" refers to a protein domain that is used to extend the half-life of a multi-domain binding protein relative to a multi-domain binding protein without a half-life extending domain. The half-life extending domain comprises a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region and the FC2 region dimerize to form an Fc domain. As used herein, the term "Fc region" is used to refer to a region of a single polypeptide chain comprising at least a CH2 domain and a CH3 domain sequence, and the term "Fc domain" refers to a dimer of two Fc regions (i.e., FC1 and FC2).

如本文所用，術語「半衰期（half-life）」意謂結合分子之藥物動力學特性，其為結合分子在投予後之平均存留時間之度量。結合分子半衰期可表示為例如如在血清（亦即循環半衰期）或其他組織中量測，自患者身體（或其他哺乳動物）或其特定隔室消除50%的已知數量之結合分子所需之時間。As used herein, the term "half-life" means a pharmacokinetic property of a binding molecule that is a measure of the average residence time of a binding molecule after administration. Binding molecule half-life can be expressed as the time required to eliminate 50% of a known amount of the binding molecule from the body of a patient (or other mammal) or a specific compartment thereof, for example, as measured in serum (i.e., circulation half-life) or other tissues.

半衰期增加使得給與患者之藥物的量減少以及投予頻率降低。例如對於治療癌症、感染性疾病或自體免疫疾病或病狀，半衰期增加可為有益的。半衰期增加之結合分子亦可藉由修飾被鑑別為涉及Fc與FcRn受體之間交互作用的胺基酸殘基而產生。包含含有一或多個促進與FcRn之結合的修飾的Fc區之結合分子可具有相較於包含原生Fc區之結合分子，約5%、約10%、約15%、約20%、約25%、約30%、約35%、約40%、約45%、約50%、約60%、約65%、約70%、約80%、約85%、約90%、約95%、約100%、約125%、約150%或更多之增加之半衰期。包含含有一或多個促進與FcRn之結合的修飾的Fc區之結合分子可具有相較於包含原生Fc區之結合分子，約2倍、約3倍、約4倍、約5倍、約10倍、約20倍、約50倍或更多倍、或2倍至10倍、或5倍至25倍、或15倍至50倍之增加之半衰期。The increase in half-life reduces the amount of drug given to the patient and reduces the frequency of administration. For example, for the treatment of cancer, infectious diseases or autoimmune diseases or conditions, the increase in half-life can be beneficial. Binding molecules with increased half-life can also be produced by modifying amino acid residues identified as being involved in the interaction between Fc and FcRn receptors. Binding molecules comprising a modified Fc region containing one or more modifications that promote binding to FcRn can have an increased half-life of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150% or more compared to binding molecules comprising native Fc regions. A binding molecule comprising an Fc region comprising one or more modifications that promote binding to FcRn may have an increased half-life of about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold or more, or 2-fold to 10-fold, or 5-fold to 25-fold, or 15-fold to 50-fold, compared to a binding molecule comprising a native Fc region.

WO 2020/157211描述一種藉由使TCR-抗CD3融合蛋白融合至IgG Fc域而延長其半衰期之方法。本發明人意外地發現，本發明之多域結合分子保持由WO 2020/157211中所揭示之形式中的Fc域提供之半衰期延長，且另外，具有顯著更高效能。WO 2020/157211 describes a method for extending the half-life of a TCR-anti-CD3 fusion protein by fusing it to an IgG Fc domain. The inventors unexpectedly discovered that the multi-domain binding molecules of the present invention retain the half-life extension provided by the Fc domain in the form disclosed in WO 2020/157211, and in addition, have significantly higher potency.

免疫球蛋白Fc域可為任何抗體Fc域。Fc域為與細胞表面Fc受體及補體系統之一些蛋白質交互作用的抗體之尾區。Fc域包含皆具有兩個或三個重鏈恆定域（稱為CH2、CH3及CH4）之兩個多肽鏈（亦即兩個Fc「區」），及視情況，鉸鏈區。兩個Fc區鏈可藉由鉸鏈區內之一或多個二硫鍵連接。來自免疫球蛋白子類IgG1、IgG2及IgG4之Fc域結合FcRn且經歷FcRn介導之再循環，提供較長循環半衰期（3至4週），由此延長本發明之多域結合分子之半衰期。IgG與FcRn之交互作用位於Fc區中，涵蓋CH2及CH3域之部分。用於本發明之較佳免疫球蛋白Fc域包括但不限於來自IgG1或IgG4之Fc域。舉例而言，Fc域可為IgG1 Fc域，亦即FC1及FC2區可為IgG1 Fc區。Fc域可源自人類序列。The immunoglobulin Fc domain can be any antibody Fc domain. The Fc domain is the tail region of the antibody that interacts with cell surface Fc receptors and some proteins of the complement system. The Fc domain comprises two polypeptide chains (i.e., two Fc "regions"), each having two or three heavy chain constant domains (called CH2, CH3 and CH4), and, as the case may be, a hinge region. The two Fc region chains can be linked by one or more disulfide bonds in the hinge region. Fc domains from immunoglobulin subclasses IgG1, IgG2 and IgG4 bind to FcRn and undergo FcRn-mediated recycling, providing a longer circulation half-life (3 to 4 weeks), thereby extending the half-life of the multi-domain binding molecules of the present invention. The interaction between IgG and FcRn is located in the Fc region, covering parts of the CH2 and CH3 domains. Preferred immunoglobulin Fc domains for use in the present invention include, but are not limited to, Fc domains from IgG1 or IgG4. For example, the Fc domain may be an IgG1 Fc domain, i.e., the FC1 and FC2 regions may be IgG1 Fc regions. The Fc domain may be derived from a human sequence.

FC1區可包含與SEQ ID NO: 42之序列至少80%一致之胺基酸序列或由其組成，且FC2區可包含與SEQ ID NO: 43之序列至少80%一致之胺基酸序列或由其組成。FC1區可包含與SEQ ID NO: 42之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成，且FC2區可包含與SEQ ID NO: 43之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成。較佳地，FC1區包含SEQ ID NO: 42中所提供之胺基酸序列或由其組成，且FC2區包含SEQ ID NO: 43中所提供之胺基酸序列或由其組成。如所屬技術領域中具有通常知識者將瞭解，上文針對FC1及FC2提供之序列反之亦適合。舉例而言，FC1區可包含SEQ ID NO: 43中所提供之胺基酸序列或由其組成，且FC2區可包含SEQ ID NO: 42中所提供之胺基酸序列或由其組成。The FC1 region may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 42, and the FC2 region may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 43. The FC1 region may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 42, and the FC2 region may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 43. Preferably, the FC1 region comprises or consists of an amino acid sequence provided in SEQ ID NO: 42, and the FC2 region comprises or consists of an amino acid sequence provided in SEQ ID NO: 43. As will be appreciated by those of ordinary skill in the art, the sequences provided above for FC1 and FC2 are also suitable conversely. For example, the FC1 region may comprise or consist of the amino acid sequence provided in SEQ ID NO: 43, and the FC2 region may comprise or consist of the amino acid sequence provided in SEQ ID NO: 42.

Fc區可包含相對於野生型或未經修飾之Fc序列的突變。突變包括取代、***及缺失。此類突變可出於引入所需治療特性之目的進行。舉例而言，為了促進異二聚化，可將杵臼（knobs into holes；KiH）突變工程改造至CH3域中。因此，半衰期延長域可包含促進FC1區與FC2區之二聚之一或多個胺基酸取代。此類取代包括「杵臼」取代。在此情況下，一條鏈（亦即FC1或FC2區中之一者）經工程改造以含有大型突出殘基（亦即杵（knob）），諸如Y，且另一條鏈（亦即FC1及FC2區中之另一者）經工程改造以含有互補凹穴（亦即臼（hole））。舉例而言，杵可藉由用較大側鏈置換小胺基酸側鏈來構築。臼可藉由用較小側鏈置換大胺基酸側鏈來構築。不希望受理論束縛，認為此藉由相較於其他物種，例如FC1與FC2之均多聚體，優先形成異二聚體，而使FC1與FC2區之異二聚體穩定化，由此增強本發明之多域結合分子之穩定性及可製造性。The Fc region may comprise mutations relative to the wild-type or unmodified Fc sequence. Mutations include substitutions, insertions, and deletions. Such mutations may be made for the purpose of introducing desired therapeutic properties. For example, to promote heterodimerization, a knobs into holes (KiH) mutation may be engineered into the CH3 domain. Thus, the half-life extension domain may comprise one or more amino acid substitutions that promote dimerization of the FC1 region and the FC2 region. Such substitutions include "knobs into holes" substitutions. In this case, one chain (i.e., one of the FC1 or FC2 regions) is engineered to contain a large protruding residue (i.e., the knob), such as Y, and the other chain (i.e., the other of the FC1 and FC2 regions) is engineered to contain a complementary cavity (i.e., the hole). For example, a knob can be constructed by replacing a small amino acid side chain with a larger side chain. A hole can be constructed by replacing a large amino acid side chain with a smaller side chain. Without wishing to be bound by theory, it is believed that this stabilizes heterodimers of the FC1 and FC2 regions by preferentially forming heterodimers over other species, such as homomultimers of FC1 and FC2, thereby enhancing the stability and manufacturability of the multi-domain binding molecules of the present invention.

KiH突變之適合位置及取代，及促進Fc區之二聚的其他突變為所屬技術領域中已知的且包括以下中所描述者：Merchant等人，Nat Biotechnol 16:677 (1998)及Ridgway等人，Prot Engineering 9:617 (1996)及Atwell等人，J Mol Biol 270,1 (1997): 26-35。舉例而言，在兩個Fc區中形成對應杵及臼之取代可對應於下表中提供之一或多個對： FC1 及 FC2 區中之一者之 CH3 FC1 及 FC2 區中之另一者之 CH3 T366Y Y407T T366W Y407A T366W T366S:L368A:Y407V F405A T394W Y407T T366Y T366Y:F405A T394W:Y407T T366W:F405W T394S:Y407A F405W:Y407A T366W:T394S F405W T394S Suitable positions and substitutions for KiH mutations, and other mutations that promote dimerization of Fc regions are known in the art and include those described in Merchant et al., Nat Biotechnol 16:677 (1998) and Ridgway et al., Prot Engineering 9:617 (1996) and Atwell et al., J Mol Biol 270, 1 (1997): 26-35. For example, substitutions that form corresponding knobs and holes in the two Fc regions may correspond to one or more of the pairs provided in the following table: CH3 in one of the FC1 and FC2 regions CH3 of the other of FC1 and FC2 regions T366Y Y407T T366W Y407A T366W T366S:L368A:Y407V F405A T394W Y407T T366Y T366Y:F405A T394W:Y407T T366W:F405W T394S:Y407A F405W:Y407A T366W:T394S F405W T394S

上表中之取代如下表示：原始殘基，接著係使用EU編號系統之位置，且接著係導入之殘基（所有殘基以單字母胺基酸代碼給出）。多個取代由冒號隔開。Substitutions in the above table are indicated as follows: the original residue, followed by the position using the EU numbering system, and then the introduced residue (all residues are given as single-letter amino acid codes). Multiple substitutions are separated by colons.

FC1及FC2區可包含上表中之一或多個取代。舉例而言：（i）該FC1區及該FC2區中之一者可包含一或多個根據EU編號方案之胺基酸取代，該一或多個胺基酸取代選自由T366S、L368A、T394S、F405A、Y407A、Y407T及Y407V組成之群；及（ii）該FC1區及該FC2區中之另一者可包含一或多個根據EU編號方案之胺基酸取代，該一或多個胺基酸取代選自由T366W、T366Y、T366W、T394W及F405W組成之群。（i）及（ii）中之取代分別為形成臼及形成杵之取代。FC1區可包含（i）中之取代中之一或多者且FC2區可包含（ii）中之取代中之一或多者。 The FC1 and FC2 regions may comprise one or more substitutions in the table above. For example: (i) One of the FC1 and FC2 regions may comprise one or more amino acid substitutions according to the EU numbering scheme, the one or more amino acid substitutions selected from the group consisting of T366S, L368A, T394S, F405A, Y407A, Y407T and Y407V; and (ii) The other of the FC1 and FC2 regions may comprise one or more amino acid substitutions according to the EU numbering scheme, the one or more amino acid substitutions selected from the group consisting of T366W, T366Y, T366W, T394W and F405W. The substitutions in (i) and (ii) are pore-forming and knob-forming substitutions, respectively. The FC1 region may include one or more of the substitutions in (i) and the FC2 region may include one or more of the substitutions in (ii).

舉例而言：（i）該FC1區及該FC2區中之一者可包含一或多個根據EU編號方案之胺基酸取代，該一或多個胺基酸取代選自由T366S、L368A及Y407V組成之群；及（ii）該FC1區及該FC2區中之另一者可包含根據EU編號方案之T366W胺基酸取代。FC1區可包含（i）中之取代中之一或多者且FC2區可包含（ii）中之取代。 For example: (i) One of the FC1 region and the FC2 region may comprise one or more amino acid substitutions according to the EU numbering scheme, the one or more amino acid substitutions being selected from the group consisting of T366S, L368A and Y407V; and (ii) The other of the FC1 region and the FC2 region may comprise the T366W amino acid substitution according to the EU numbering scheme. The FC1 region may comprise one or more of the substitutions in (i) and the FC2 region may comprise the substitution in (ii).

較佳地，（i）FC1區及FC2區中之一者包含根據EU編號方案之T366S、L368A及Y407V胺基酸取代；且（ii）FC1區及FC2區中之另一者包含根據EU編號方案之T366W胺基酸取代。舉例而言，FC1區可包含根據EU編號方案之T366S、L368A及Y407V胺基酸取代；且FC2區可包含根據EU編號方案之T366W胺基酸取代。Preferably, (i) one of the FC1 region and the FC2 region comprises T366S, L368A and Y407V amino acid substitutions according to the EU numbering scheme; and (ii) the other of the FC1 region and the FC2 region comprises T366W amino acid substitution according to the EU numbering scheme. For example, the FC1 region may comprise T366S, L368A and Y407V amino acid substitutions according to the EU numbering scheme; and the FC2 region may comprise T366W amino acid substitution according to the EU numbering scheme.

Fc域亦可包含一或多個減弱Fc域之效應功能的突變。例示性效應功能包括但不限於補體依賴性細胞毒性（complement-dependent cytotoxicity；CDC）及/或抗體依賴性細胞毒性（antibody-dependent cellular cytotoxicity；ADCC）。減弱效應功能之修飾可為改變Fc域之醣基化模式的修飾，例如產生去醣基化Fc域之修飾。替代地，減弱效應功能之修飾可為不改變Fc域之醣基化模式的修飾。減弱效應功能之修飾可減少或消除與人類效應細胞之結合、與一或多種Fc受體之結合及/或與表現Fc受體之細胞的結合。舉例而言，該半衰期延長域可包含一或多個根據EU編號方案之胺基酸取代，該一或多個胺基酸取代選自由S228P、E233P、L234A、L235A、L235E、L235P、G236R、G237A、P238S、F241A、V264A D265A、H268A、D270A、N297A、N297G、N297Q、E318A、K322A、L328R、P329G、P329A、A330S、A330L、P331A及P331S組成之群。特定修飾包括位於人類IgG1之Fc區中的N297G或N297A取代（EU編號）。其他適合修飾包括人類IgG1之Fc區中使得效應功能減弱的L234A、L235A及P329G取代（EU編號）。本發明之多域結合分子中之Fc區可包含根據EU索引編號之殘基N297處之取代。舉例而言，取代可為N297G或N297A取代。其他適合突變（例如在殘基N297處）為所屬技術領域中具有通常知識者已知。The Fc domain may also comprise one or more mutations that reduce the effector function of the Fc domain. Exemplary effector functions include, but are not limited to, complement-dependent cytotoxicity (CDC) and/or antibody-dependent cellular cytotoxicity (ADCC). Modifications that reduce effector function may be modifications that alter the glycosylation pattern of the Fc domain, such as modifications that produce a deglycosylated Fc domain. Alternatively, modifications that reduce effector function may be modifications that do not alter the glycosylation pattern of the Fc domain. Modifications that reduce effector function may reduce or eliminate binding to human effector cells, binding to one or more Fc receptors, and/or binding to cells expressing Fc receptors. For example, the half-life extension domain may comprise one or more amino acid substitutions according to the EU numbering scheme, the one or more amino acid substitutions selected from the group consisting of S228P, E233P, L234A, L235A, L235E, L235P, G236R, G237A, P238S, F241A, V264A D265A, H268A, D270A, N297A, N297G, N297Q, E318A, K322A, L328R, P329G, P329A, A330S, A330L, P331A and P331S. Specific modifications include N297G or N297A substitutions in the Fc region of human IgG1 (EU numbering). Other suitable modifications include L234A, L235A and P329G substitutions (EU numbering) in the Fc region of human IgG1 that reduce effector function. The Fc region in the multi-domain binding molecule of the present invention may include a substitution at residue N297 according to the EU index numbering. For example, the substitution may be an N297G or N297A substitution. Other suitable mutations (e.g. at residue N297) are known to those of ordinary skill in the art.

具有降低的效應功能的Fc變異體係指相比於野生型Fc區（例如，不具有降低效應功能之突變的Fc區，但其可具有其他突變）所達成的效應功能，效應功能（例如CDC、ADCC及/或與FcR之結合等活性）降低至少10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%或更多的Fc變異體。具有降低之效應功能的Fc變異體可為相比於野生型Fc區消除所有可偵測效應功能的Fc變異體。量測效應功能之分析為所屬技術領域中已知的且在下文描述。An Fc variant with reduced effector function refers to an Fc variant whose effector function (e.g., CDC, ADCC, and/or binding to FcR, etc.) is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more compared to the effector function achieved by a wild-type Fc region (e.g., an Fc region that does not have a mutation that reduces effector function, but which may have other mutations). An Fc variant with reduced effector function may be an Fc variant that eliminates all detectable effector function compared to a wild-type Fc region. Assays for measuring effector function are known in the art and are described below.

可進行試管內及/或活體內細胞毒性分析以確認CDC及/或ADCC活性之降低/耗盡。舉例而言，可進行Fc受體（FcR）結合分析以確保Fc區或融合蛋白缺乏FcγR結合（因此可能缺乏ADCC活性），但保留FcRn結合能力。用於介導ADCC之初級細胞NK細胞僅表現FcγRIII，而單核球表現FcγRI、FcγRII及FcγRIII。FcR在造血細胞上之表現概述於Ravetch及Kinet, Annu. Rev. Immunol. 9:457-492 (1991)之第464頁之表3中。評估相關分子之ADCC活性之試管內分析的非限制性實例描述於以下中：美國專利第5,500,362號（參見例如Hellstrom, I.等人，Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)）及Hellstrom, I等人，Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985)；第5,821,337號（參見Bruggemann, M.等人，J. Exp. Med. 166:1351-1361 (1987)）。In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the Fc region or fusion protein lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells used to mediate ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of related molecules are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).

可將消除或減少與Fcy受體之結合及/或增加與FcRn之結合及/或防止Fab臂交換及/或移除蛋白酶位之取代引入至FC1及FC2區中。就此而言，半衰期延長域亦可包含一或多個防止或減少與活化受體之結合的胺基酸取代。半衰期延長域可包含一或多個防止或減少與FcγR之結合之胺基酸取代。舉例而言，FC1區及/或FC2區可包含根據EU編號方案之N297G胺基酸取代。FC1區及FC2區兩者均可包含N297G胺基酸取代。Substitutions that eliminate or reduce binding to Fcγ receptors and/or increase binding to FcRn and/or prevent Fab arm exchange and/or remove protease sites can be introduced into FC1 and FC2 regions. In this regard, the half-life extension domain can also include one or more amino acid substitutions that prevent or reduce binding to activation receptors. The half-life extension domain can include one or more amino acid substitutions that prevent or reduce binding to FcγR. For example, the FC1 region and/or the FC2 region can include an N297G amino acid substitution according to the EU numbering scheme. Both the FC1 region and the FC2 region can include an N297G amino acid substitution.

與未經修飾之半衰期延長域相比，半衰期延長域可包含一或多個胺基酸取代，其中一或多個胺基酸取代促進Fc域與FcRn之結合。量測與FcRn之結合的方法為已知的（參見例如Ghetie及Ward, Immunol. Today 18: (12): 592-8 (1997)；Ghetie等人，Nature Biotechnology 15 (7): 637-40 (1997)；Hinton等人，J. Biol. Chem. 279 (8): 6213-6 (2004)；WO 2004/92219（Hinton等人）。可例如在表現人類FcRn之轉殖基因小鼠或經轉染人類細胞株中或在投予具有變異Fc區之多肽的靈長類動物中分析人類FcRn高親和力結合多肽與FcRn的活體內結合及血清半衰期。WO 2004/42072（Presta）描述提高或降低與FcR之結合的抗體取代。亦參見例如Shields等人，J. Biol. Chem. 9(2): 6591-6604 (2001)。特定言之，Mackness等人，MAbs. 11:1276-1288 (2019)描述用於增強與FcRn之結合的抗體Fc區中適合之胺基酸取代。The half-life extension domain may comprise one or more amino acid substitutions compared to an unmodified half-life extension domain, wherein the one or more amino acid substitutions promote binding of the Fc domain to FcRn. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem. 279 (8): 6213-6 (2004); WO 2004/92219 (Hinton et al.). In vivo binding and serum half-life of a human FcRn high affinity binding polypeptide to FcRn can be analyzed, for example, in transgenic mice or transfected human cell lines expressing human FcRn or in primates to which a polypeptide with a variant Fc region is administered. WO 2004/42072 (Presta) describes antibody substitutions that increase or decrease binding to FcRs. See also, for example, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001). In particular, Mackness et al., MAbs. 11: 1276-1288 (2019) describes suitable amino acid substitutions in the Fc region of antibodies for enhancing binding to FcRn.

促進Fc域與FcRn之結合的Fc區中之修飾（例如，胺基酸取代、胺基酸***或胺基酸缺失）可處於一或多個選自由以下組成之群的如藉由Kabat中所闡述之EU索引編號的位置：234、235、236、237、238、239、240、241、243、244、245、247、251、252、254、255、256、262、263、264、265、266、267、268、269、279、280、284、292、296、297、298、299、305、313、316、325、326、327、328、329、330、331、332、333、334、339、341、343、370、373、378、392、416、419、421、440及443。視情況，Fc區可在所屬技術領域中已知之額外及/或替代位置處包含非天然存在之胺基酸殘基。Modifications in the Fc region that promote binding of the Fc domain to FcRn (e.g., amino acid substitutions, amino acid insertions or amino acid deletions) may be at one or more positions selected from the group consisting of 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323 3, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440, and 443. Optionally, the Fc region may comprise non-naturally occurring amino acid residues at additional and/or alternative positions known in the art.

更特定言之，Fc區可包含至少一個選自由以下組成之群之如藉由Kabat中所闡述之EU索引編號的取代：234D、234E、234N、234Q、234T、234H、234Y、234I、234V、234F、235A、235D、235R、235W、235P、235S、235N、235Q、235T、235H、235Y、235I、235V、235F、236E、239D、239E、239N、239Q、239F、239T、239H、239Y、240I、240A、240T、240M、241W、241 L、241Y、241E、241R、243W、243L、243Y、243R、243Q、244H、245A、247L、247V、247G、251F、252Y、254T、255L、256E、256M、262I、262A、262T、262E、263I、263A、263T、263M、264L、264I、264W、264T、264R、264F、264M、264Y、264E、265G、265N、265Q、265Y、265F、265V、265I、265L、265H、265T、266I、266A、266T、266M、267Q、267L、268E、269H、269Y、269F、269R、270E、280A、284M、292P、292L、296E、296Q、296D、296N、296S、296T、296L、2961、296H、269G、297S、297D、297E、298H、298I、298T、298F、299I、299L、299A、299S、299V、299H、299F、299E、305I、313F、316D、325Q、325L、325I、325D、325E、325A、325T、325V、325H、327G、327W、327N、327L、328S、328M、328D、328E、328N、328Q、328F、328I、328V、328T、328H、328A、329F、329H、329Q、330K、330G、330T、330C、330L、330Y、330V、330I、330F、330R、330H、331G、331A、331L、331M、331F、331W、331K、331Q、331E、331S、331V、331I、331C、331Y、331H、331R、331N、331D、331T、332D、332S、332W、332F、332E、332N、332Q、332T、332H、332Y、332A、339T、370E、370N、378D、392T、396L、416G、419H、421K、440Y及434W。視情況，Fc區可包含所屬技術領域中已知之額外及/或替代非天然存在之胺基酸殘基。More specifically, the Fc region may comprise at least one substitution selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 65I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H , 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 33 0R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W. Optionally, the Fc region may contain additional and/or alternative non-naturally occurring amino acid residues known in the art.

促進與FcRn之結合之Fc區中的修飾（例如，胺基酸取代、胺基酸***或胺基酸缺失）可處於一或多個選自由以下組成之群的如藉由Kabat中所闡述之EU索引編號的位置：234、235及331。舉例而言，Fc區可包含至少一個選自由以下組成之群之如藉由Kabat中所闡述之EU索引編號的取代：234F、235F、235Y及331S。Modifications in the Fc region that promote binding to FcRn (e.g., amino acid substitutions, amino acid insertions, or amino acid deletions) may be at one or more positions as numbered by the EU index as set forth in Kabat selected from the group consisting of: 234, 235, and 331. For example, the Fc region may comprise at least one substitution as numbered by the EU index as set forth in Kabat selected from the group consisting of: 234F, 235F, 235Y, and 331S.

促進與FcRn之結合之Fc區中的修飾（例如，胺基酸取代、胺基酸***或胺基酸缺失）可處於一或多個選自由以下組成之群的如藉由Kabat中所闡述之EU索引編號的位置：239、330及332。舉例而言，Fc區可包含至少一個選自由以下組成之群之如藉由Kabat中所闡述之EU索引編號的取代：239D、330L及332E。Modifications in the Fc region that promote binding to FcRn (e.g., amino acid substitutions, amino acid insertions, or amino acid deletions) may be at one or more positions as numbered by the EU index as specified in Kabat selected from the group consisting of: 239, 330, and 332. For example, the Fc region may comprise at least one substitution as numbered by the EU index as specified in Kabat selected from the group consisting of: 239D, 330L, and 332E.

促進與FcRn之結合之Fc區中的修飾（例如，胺基酸取代、胺基酸***或胺基酸缺失）可處於選自由以下組成之群的一或多個如藉由Kabat中所闡述之EU索引編號的位置：252、254及256。舉例而言，Fc區可包含至少一個選自由以下組成之群之如藉由Kabat中所闡述之EU索引編號的取代：252Y、254T及256E，如內容以全文引用的方式併入本文中之美國專利第7,083,784號中所描述。Fc區可包含所有以下如藉由Kabat中所闡述之EU索引編號的取代：252Y、254T及256E。Modifications in the Fc region that promote binding to FcRn (e.g., amino acid substitutions, amino acid insertions, or amino acid deletions) may be at one or more positions as numbered by the EU index as specified in Kabat selected from the group consisting of: 252, 254, and 256. For example, the Fc region may comprise at least one substitution as numbered by the EU index as specified in Kabat selected from the group consisting of: 252Y, 254T, and 256E, as described in U.S. Patent No. 7,083,784, the contents of which are incorporated herein by reference in their entirety. The Fc region may comprise all of the following substitutions as numbered by the EU index as specified in Kabat: 252Y, 254T, and 256E.

以上列出之促進與FcRn之結合的取代係相對於對應野生型Fc區（例如人類IgG1或IgG4 Fc區）且可存在於Fc域之FC1及FC2部分中之一者或較佳兩者中。換言之，取代係指通常不存在於對應野生型Fc區，例如人類IgG1或IgG4 Fc區中之胺基酸。就此而言，如本文所用，「取代（substitution）」係指多肽中所列胺基酸中之一者的存在且未必需要用另一胺基酸置換一個胺基酸。在一個具體實例中，FC1及/或FC2區包含根據EU編號方案編號之252Y、254T及256E胺基酸取代。The substitutions listed above that promote binding to FcRn are relative to the corresponding wild-type Fc region (e.g., human IgG1 or IgG4 Fc region) and may be present in one or preferably both of the FC1 and FC2 portions of the Fc domain. In other words, substitution refers to an amino acid that is not normally present in the corresponding wild-type Fc region, such as human IgG1 or IgG4 Fc region. In this regard, as used herein, "substitution" refers to the presence of one of the listed amino acids in the polypeptide and does not necessarily require replacement of one amino acid with another amino acid. In a specific example, the FC1 and/or FC2 regions include 252Y, 254T, and 256E amino acid substitutions numbered according to the EU numbering scheme.

另外或替代地，可出於製造原因進行突變，例如以移除或置換可經歷如本文所描述之轉譯後修飾，諸如醣基化的胺基酸。免疫球蛋白Fc可經由如本文所描述之連接子及/或鉸鏈序列與本發明分子中之其他域（亦即VC1或VC2）融合。替代地，可不使用連接子。Additionally or alternatively, mutations may be made for manufacturing reasons, for example to remove or replace amino acids that may undergo post-translational modifications, such as glycosylation, as described herein. The immunoglobulin Fc may be fused to other domains (i.e., VC1 or VC2) in the molecules of the invention via a linker and/or hinge sequence as described herein. Alternatively, no linker may be used.

FC1區可包含與SEQ ID NO: 57之序列至少80%一致之胺基酸序列或由其組成，且FC2區可包含與SEQ ID NO: 58之序列至少80%一致之胺基酸序列或由其組成。FC1區可包含與SEQ ID NO: 57之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成，且FC2區可包含與SEQ ID NO: 58之序列至少90%、至少95%或至少98%一致的胺基酸序列或由其組成。FC1區可包含SEQ ID NO: 57中所提供之胺基酸序列或由其組成，且FC2區可包含SEQ ID NO: 58中所提供之胺基酸序列或由其組成。如所屬技術領域中具有通常知識者將瞭解，上文針對FC1及FC2提供之序列反之亦適合。舉例而言，FC1區可包含SEQ ID NO: 57中所提供之胺基酸序列或由其組成，且FC2區可包含SEQ ID NO: 58中所提供之胺基酸序列或由其組成。The FC1 region may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 57, and the FC2 region may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 58. The FC1 region may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 57, and the FC2 region may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 58. The FC1 region may comprise or consist of an amino acid sequence provided in SEQ ID NO: 57, and the FC2 region may comprise or consist of an amino acid sequence provided in SEQ ID NO: 58. As will be appreciated by those of ordinary skill in the art, the sequences provided above for FC1 and FC2 are also applicable conversely. For example, the FC1 region may comprise or consist of the amino acid sequence provided in SEQ ID NO: 57, and the FC2 region may comprise or consist of the amino acid sequence provided in SEQ ID NO: 58.

本發明之分子中之兩個Fc區可包含CH2及CH3恆定域及鉸鏈序列之全部或一部分。鉸鏈序列可實質上或部分對應於來自IgG1、IgG2、IgG3或IgG4之鉸鏈區。鉸鏈序列可為IgG1鉸鏈序列，諸如SEQ ID NO: 44中提供之胺基酸序列。鉸鏈可包含核心鉸鏈域之全部或一部分及下鉸鏈區之全部或一部分。The two Fc regions in the molecules of the present invention may comprise all or a portion of the CH2 and CH3 homeostasis domains and a hinge sequence. The hinge sequence may correspond substantially or in part to a hinge region from IgG1, IgG2, IgG3, or IgG4. The hinge sequence may be an IgG1 hinge sequence, such as the amino acid sequence provided in SEQ ID NO: 44. The hinge may comprise all or a portion of the core hinge domain and all or a portion of the lower hinge region.

本發明之多域結合分子之適合半衰期延長形式亦描述於與此一起申請之申請案中，其名稱為「多域結合分子（Multi-Domain Binding Molecules）」，主張2022年8月18日申請之美國臨時申請案第63/371,863號之優先權，其內容以引用的方式併入本文中。形式及連接子 Suitable half-life extension forms of the multi-domain binding molecules of the present invention are also described in an application filed herewith, entitled "Multi-Domain Binding Molecules," claiming priority to U.S. Provisional Application No. 63/371,863 filed on August 18, 2022, the contents of which are incorporated herein by reference. Formats and Linkers

如本文所用，術語「形式（format）」係指本發明之多域結合分子中之各域（及各域中之各區）之位置及位向，及多肽鏈數目。例示性多域結合分子之形式的示意圖提供於圖1中。圖1中所示之例示性結合分子中之免疫細胞接合域為包含VH（TCE-VH）及VL（TCE-VL）之T細胞接合免疫效應子域。如上文所描述，其他類型之免疫細胞接合域（例如單域抗體、VHH等）亦為適合的。此類分子之pMHC結合域及免疫細胞接合域能夠分別地結合pMHC複合物及免疫細胞。就此而言，pMHC結合域及免疫細胞接合域可能夠分別同時結合pMHC複合物及免疫細胞。As used herein, the term "format" refers to the position and orientation of each domain (and each region in each domain) in the multi-domain binding molecule of the present invention, and the number of polypeptide chains. A schematic diagram of the format of an exemplary multi-domain binding molecule is provided in Figure 1. The immune cell binding domain in the exemplary binding molecule shown in Figure 1 is a T cell binding immune effector domain comprising VH (TCE-VH) and VL (TCE-VL). As described above, other types of immune cell binding domains (such as single domain antibodies, VHH, etc.) are also suitable. The pMHC binding domain and immune cell binding domain of such molecules are capable of binding to pMHC complexes and immune cells, respectively. In this regard, the pMHC binding domain and the immune cell binding domain may be capable of binding to pMHC complexes and immune cells, respectively, at the same time.

在本發明之多域結合分子之形式中，該免疫細胞接合域連接至VC1之N端，VC1經由其C端連接至該FC1區之N端，該FC1區經由其C端連接至VC2之N端，且VC2經由其C端連接至FC2之N端。各區在單一多肽鏈中共價連接。形式可表示為： N-ICE-VC1-FC1-VC2-FC2- C。本發明人已鑑別出，所測試之超過35種不同形式當中，此形式之分子具有最高活性（亦即效能及選擇性）及產率。 In the form of the multi-domain binding molecule of the present invention, the immune cell binding domain is linked to the N-terminus of VC1, VC1 is linked via its C-terminus to the N-terminus of the FC1 region, the FC1 region is linked via its C-terminus to the N-terminus of VC2, and VC2 is linked via its C-terminus to the N-terminus of FC2. The regions are covalently linked in a single polypeptide chain. The form can be represented as: N -ICE-VC1-FC1-VC2-FC2- C . The inventors have identified that this form of the molecule has the highest activity (i.e., potency and selectivity) and yield among more than 35 different forms tested.

在免疫細胞接合域為包含VH及VL之T細胞接合免疫效應子域的情況下，形式可表示為： N-(TCEVL-TCEVH或TCEVH-TCEVL)-VC1-FC1-VC2-FC2- C。本發明人已鑑別出，所測試之超過35種不同形式當中，此形式之分子具有最高活性（亦即效能及選擇性）及產率。 In the case where the immune cell engaging domain is a T cell engaging immune effector subdomain comprising VH and VL, the format can be represented as: N- (TCEVL-TCEVH or TCEVH-TCEVL)-VC1-FC1-VC2-FC2- C. The inventors have identified that this format of the molecule has the highest activity (i.e. potency and selectivity) and productivity among more than 35 different formats tested.

本發明之多域結合分子呈單鏈形式。在此上下文中，「單鏈（single-chain）」用以描述表現為單一多肽鏈之多域結合分子，其含有pMHC結合域、免疫細胞接合域及半衰期延長域。The multi-domain binding molecules of the present invention are in the form of a single chain. In this context, "single-chain" is used to describe a multi-domain binding molecule that is present as a single polypeptide chain, which contains a pMHC binding domain, an immune cell binding domain, and a half-life extension domain.

較佳地，VC1包含TCRβ可變區及恆定區，VC2包含TCRα可變區及恆定區，免疫細胞接合域為抗CD3 scFv，且Fc域為IgG1 Fc域。Preferably, VC1 comprises a TCRβ variable region and a constant region, VC2 comprises a TCRα variable region and a constant region, the immune cell engaging domain is an anti-CD3 scFv, and the Fc domain is an IgG1 Fc domain.

ICE、TCE-VH、TCE-VL、VC1、VC2、FC1及/或FC2區中之兩者或更多者可經由連接子及/或IgG鉸鏈序列彼此連接。連接子序列可為可撓性的，此在於其主要由諸如甘胺酸、丙胺酸及絲胺酸之胺基酸構成，該等胺基酸不具有可能限制可撓性之大型側鏈。此類連接子包括「甘胺酸-絲胺酸（glycine-serine）」連接子，其係指僅包含或主要包含甘胺酸及絲胺酸殘基之連接子，例如(GGGGS)n。替代地，具有較大剛性之連接子可為合乎需要的。更剛性連接子之實例包括具有(EAAAK)n之序列的形成α螺旋之連接子。可容易地確定連接子序列之可使用或最佳長度。通常，連接子序列之長度將小於約15個，諸如小於10個，或2至10個胺基酸。連接子之長度可為1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30個胺基酸。可用於多域結合分子中之適合連接子之實例為所屬技術領域中已知且包括WO2010/133828及Chen等人，Adv Drug Deliv Rev. 2013;65(10):1357-1369中所描述者。舉例而言，存在於本發明之多域結合蛋白中之一或多個連接子可具有選自以下之群的序列：GGGGS（SEQ ID NO: 18）、GGGSG（SEQ ID NO: 20）、GGSGG（SEQ ID NO: 21）、GSGGG（SEQ ID NO: 22）、GSGGGP（SEQ ID NO: 23）、GGEPS（SEQ ID NO: 24）、GGEGGGP（SEQ ID NO: 25）、GGEGGGSEGGGS（SEQ ID NO: 26）、GGGSGGGG（SEQ ID NO: 47）、GGGGSGGGGSGGGGSGGGGSGGGS（SEQ ID NO: 39）、GGGGSGGGGSGGGGS（SEQ ID NO: 49）、EAAAK（SEQ ID NO: 50）及EAAAKEAAAKEAAAK（SEQ ID NO: 51）。Two or more of the ICE, TCE-VH, TCE-VL, VC1, VC2, FC1 and/or FC2 regions may be linked to each other via a linker and/or IgG hinge sequence. The linker sequence may be flexible in that it is composed primarily of amino acids such as glycine, alanine and serine that do not have large side chains that may limit flexibility. Such linkers include "glycine-serine" linkers, which refer to linkers that contain only or primarily glycine and serine residues, such as (GGGGS)n. Alternatively, linkers with greater rigidity may be desirable. Examples of more rigid linkers include alpha-helix-forming linkers having a sequence of (EAAAK)n. The useful or optimal length of the linker sequence can be easily determined. Typically, the length of the linker sequence will be less than about 15, such as less than 10, or 2 to 10 amino acids. The length of the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. Examples of suitable linkers that can be used in multi-domain binding molecules are known in the art and include those described in WO2010/133828 and Chen et al., Adv Drug Deliv Rev. 2013; 65(10): 1357-1369. For example, one or more linkers present in the multi-domain binding protein of the present invention can have a sequence selected from the group consisting of GGGGS (SEQ ID NO: 18), GGGSG (SEQ ID NO: 20), GGSGG (SEQ ID NO: 21), GSGGG (SEQ ID NO: 22), GSGGGP (SEQ ID NO: 23), GGEPS (SEQ ID NO: 24), GGEGGGP (SEQ ID NO: 25), GGEGGGSEGGGS (SEQ ID NO: 26), GGGSGGGG (SEQ ID NO: 47), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 39), GGGGSGGGGSGGGGS (SEQ ID NO: 49), EAAAK (SEQ ID NO: 50), and EAAAKEAAAKEAAAK (SEQ ID NO: 51).

適合IgG鉸鏈序列為所屬技術領域中已知且包括SEQ ID NO: 44中提供之例示性IgG1鉸鏈序列。其他適合IgG鉸鏈序列包括SEQ ID NO: 52中所提供之截短IgG1鉸鏈序列及SEQ ID NO: 53中所提供之IgG4鉸鏈。Suitable IgG hinge sequences are known in the art and include the exemplary IgG1 hinge sequence provided in SEQ ID NO: 44. Other suitable IgG hinge sequences include the truncated IgG1 hinge sequence provided in SEQ ID NO: 52 and the IgG4 hinge provided in SEQ ID NO: 53.

ICE域可經由其C端連接至VC1之N端。就此而言，本發明之多域結合分子可具有以下形式： N-ICE-VC1-FC1-VC2-FC2- C。在免疫細胞接合域為包含VH及VL之T細胞接合免疫效應子域的情況下，TCE-VL區可經由其C端連接至TCE-VH區之N端且TCE-VH區可經由其C端連接至VC1之N端。就此而言，本發明之多域結合分子可具有以下形式： N-TCEVL-TCEVH-VC1-FC1-VC2-FC2- C。 The ICE domain may be linked to the N-terminus of VC1 via its C-terminus. In this regard, the multi-domain binding molecule of the present invention may have the following form: N -ICE-VC1-FC1-VC2-FC2- C . In the case where the immune cell engaging domain is a T cell engaging immune effector subdomain comprising VH and VL, the TCE-VL region may be linked to the N-terminus of the TCE-VH region via its C-terminus and the TCE-VH region may be linked to the N-terminus of VC1 via its C-terminus. In this regard, the multi-domain binding molecule of the present invention may have the following form: N -TCEVL-TCEVH-VC1-FC1-VC2-FC2- C .

VC1可包含TCRβ可變區及恆定區且VC2可包含TCRα可變區及恆定區。因此，VC1與VC2可二聚形成可溶性TCR。就此而言，較佳地，本發明之多域結合分子具有以下形式： N-ICE-TCRβ-FC1-TCRα-FC2- C或 N-TCEVL-TCEVH-TCRβ-FC1-TCRα-FC2- C（其中「TCRβ」係指TCRβ可變區及恆定區且「TCRα」係指TCRα可變區及恆定區）。 VC1 may include a TCRβ variable region and a constant region and VC2 may include a TCRα variable region and a constant region. Therefore, VC1 and VC2 can dimerize to form a soluble TCR. In this regard, preferably, the multi-domain binding molecule of the present invention has the following form: N -ICE-TCRβ-FC1-TCRα-FC2- C or N -TCEVL-TCEVH-TCRβ-FC1-TCRα-FC2 -C (wherein "TCRβ" refers to the TCRβ variable region and the constant region and "TCRα" refers to the TCRα variable region and the constant region).

若存在，TCE-VL區可經由包含甘胺酸-絲胺酸連接子之序列連接至TCE-VH區。較佳地，使TCE-VL區與TCE-VH區連接之序列為SEQ ID NO: 39中提供之胺基酸序列。If present, the TCE-VL region may be linked to the TCE-VH region via a sequence comprising a glycine-serine linker. Preferably, the sequence linking the TCE-VL region to the TCE-VH region is the amino acid sequence provided in SEQ ID NO: 39.

ICE域或TCE-VH區可經由包含甘胺酸-絲胺酸連接子或由其組成之序列連接至VC1。較佳地，使ICE域或TCE-VH區與VC1連接之序列為SEQ ID NO: 18中提供之胺基酸序列。The ICE domain or TCE-VH region can be linked to VC1 via a sequence comprising or consisting of a glycine-serine linker. Preferably, the sequence linking the ICE domain or TCE-VH region to VC1 is the amino acid sequence provided in SEQ ID NO: 18.

VC1可經由包含IgG鉸鏈序列之序列連接至FC1區且/或VC2可經由包含IgG鉸鏈序列之序列連接至FC2區。IgG鉸鏈序列可與SEQ ID NO: 44至少80%一致。較佳地，IgG鉸鏈序列與SEQ ID NO: 44至少90%、至少95%、至少98%或100%一致。VC1 may be linked to the FC1 region via a sequence comprising an IgG hinge sequence and/or VC2 may be linked to the FC2 region via a sequence comprising an IgG hinge sequence. The IgG hinge sequence may be at least 80% identical to SEQ ID NO: 44. Preferably, the IgG hinge sequence is at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 44.

將VC1連接至FC1區之序列可進一步包含甘胺酸-絲胺酸連接子及/或將VC2連接至FC2區之序列可進一步包含甘胺酸-絲胺酸連接子。較佳地，甘胺酸-絲胺酸連接子具有SEQ ID NO: 47中提供之序列。較佳地，此等序列呈以下形式，N端至C端：VC1-GS連接子-IgG鉸鏈-FC1及VC2-GS連接子-IgG鉸鏈-FC2。The sequence linking VC1 to the FC1 region may further comprise a glycine-serine linker and/or the sequence linking VC2 to the FC2 region may further comprise a glycine-serine linker. Preferably, the glycine-serine linker has the sequence provided in SEQ ID NO: 47. Preferably, these sequences are in the following format, N-terminus to C-terminus: VC1-GS linker-IgG hinge-FC1 and VC2-GS linker-IgG hinge-FC2.

FC1區可經由包含甘胺酸-絲胺酸連接子之序列連接至VC2。較佳地，連接FC1區VC2區之甘胺酸-絲胺酸連接子具有SEQ ID NO: 47中提供之序列。The FC1 region can be connected to VC2 via a sequence comprising a glycine-serine linker. Preferably, the glycine-serine linker connecting the FC1 region to the VC2 region has a sequence provided in SEQ ID NO: 47.

本發明之多域結合分子為單一多肽鏈（參見圖1）。多域結合分子可為可溶及/或重組及/或分離的。兩個例示性多域結合分子之全胺基酸序列提供於SEQ ID NO: 45及SEQ ID NO: 46中。The multi-domain binding molecules of the present invention are single polypeptide chains (see Figure 1). The multi-domain binding molecules can be soluble and/or recombinant and/or isolated. The full amino acid sequences of two exemplary multi-domain binding molecules are provided in SEQ ID NO: 45 and SEQ ID NO: 46.

多域結合分子可具有與SEQ ID NO: 45之序列至少80%一致的胺基酸序列。多域結合分子可具有與SEQ ID NO: 45之序列至少90%、至少95%或至少98%一致的胺基酸序列。較佳地，多域結合分子包含SEQ ID NO: 45中提供之胺基酸序列或由其組成。The multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 45. The multi-domain binding molecule may have an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 45. Preferably, the multi-domain binding molecule comprises or consists of the amino acid sequence provided in SEQ ID NO: 45.

多域結合分子可具有與SEQ ID NO: 46之序列至少80%一致的胺基酸序列。多域結合分子可具有與SEQ ID NO: 46之序列至少90%、至少95%或至少98%一致的胺基酸序列。多域結合分子可包含SEQ ID NO: 46中所提供之胺基酸序列或由其組成。The multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 46. The multi-domain binding molecule may have an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 46. The multi-domain binding molecule may comprise or consist of the amino acid sequence provided in SEQ ID NO: 46.

視情況，以上多域結合分子序列可進一步與一或多個其他多肽序列融合。Optionally, the above multi-domain binding molecule sequence may be further fused with one or more other polypeptide sequences.

以上序列係關於包含結合SLLQHLIGL（SEQ ID NO: 1）HLA-A*02複合物之TCR鏈的多域結合分子。所屬技術領域中具有通常知識者可藉由用不同相關TCR之序列置換SEQ ID NO: 45及SEQ ID NO: 46中之TCR鏈，而調整此等序列以使其針對另一目標。類似地，所屬技術領域中具有通常知識者可用另一T細胞接合免疫效應子域，例如，不同抗CD3 scFv序列置換SEQ ID NO: 45或SEQ ID NO: 46中之抗CD3 scFv序列（亦即，T細胞接合免疫效應子域）。The above sequences are for a multi-domain binding molecule comprising a TCR chain that binds the SLLQHLIGL (SEQ ID NO: 1) HLA-A*02 complex. One of ordinary skill in the art can adjust these sequences to target another target by replacing the TCR chain in SEQ ID NO: 45 and SEQ ID NO: 46 with the sequence of a different related TCR. Similarly, one of ordinary skill in the art can replace the anti-CD3 scFv sequence (i.e., T cell engaging immune effector domain) in SEQ ID NO: 45 or SEQ ID NO: 46 with another T cell engaging immune effector domain, for example, a different anti-CD3 scFv sequence.

較佳地： a） VC1包含TCRβ可變區及恆定區， b） VC2包含TCRα可變區及恆定區， c）免疫細胞接合域為T細胞接合免疫效應子域，該T細胞接合免疫效應子域為抗CD3 scFv， d） FC1具有SEQ ID NO: 42中所提供之胺基酸序列，或與其至少90%、或至少95%、或至少98%一致之胺基酸序列，及 e） FC2具有SEQ ID NO: 43中所提供之胺基酸序列，或與其至少90%、至少95%或至少98%一致之胺基酸序列。 Preferably: a) VC1 comprises a TCRβ variable region and a constant region, b) VC2 comprises a TCRα variable region and a constant region, c) the immune cell engagement domain is a T cell engagement immune effector subdomain, and the T cell engagement immune effector subdomain is an anti-CD3 scFv, d) FC1 has an amino acid sequence provided in SEQ ID NO: 42, or an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical thereto, and e) FC2 has an amino acid sequence provided in SEQ ID NO: 43, or an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical thereto.

多域結合分子較佳包含以下胺基酸序列，自N端至C端按以下次序： a）抗CD3 scFv（TCE-VL及TCE-VH）之胺基酸序列，視情況接著係提供於SEQ ID NO: 18中之連接子序列； b） TCRβ可變區及恆定區（VC1）之胺基酸序列； c）提供於SEQ ID NO: 47中之連接子序列，接著係提供於SEQ ID NO: 44中之IgG鉸鏈序列； d）具有提供於SEQ ID NO: 42中之序列的Fc區（FC1）； e）提供於SEQ ID NO: 47中之連接子序列； f） TCRα可變區及恆定區（VC2）之胺基酸序列； g）提供於SEQ ID NO: 47中之連接子序列，接著係提供於SEQ ID NO: 44中之IgG鉸鏈序列；及 h）具有提供於SEQ ID NO: 43中之序列的Fc區（FC2）。 The multi-domain binding molecule preferably comprises the following amino acid sequences, in the following order from N-terminus to C-terminus: a) the amino acid sequence of anti-CD3 scFv (TCE-VL and TCE-VH), optionally followed by the linker sequence provided in SEQ ID NO: 18; b) the amino acid sequence of TCRβ variable region and constant region (VC1); c) the linker sequence provided in SEQ ID NO: 47, followed by the IgG hinge sequence provided in SEQ ID NO: 44; d) an Fc region (FC1) having the sequence provided in SEQ ID NO: 42; e) the linker sequence provided in SEQ ID NO: 47; f) the amino acid sequence of TCRα variable region and constant region (VC2); g) provided in SEQ ID NO: 47, followed by the IgG hinge sequence provided in SEQ ID NO: 44; and h) an Fc region (FC2) having the sequence provided in SEQ ID NO: 43.

TCRβ恆定區可具有提供於SEQ ID NO: 19中之胺基酸序列及/或TCRα恆定區可具有提供於SEQ ID NO: 15中之胺基酸序列。多域結合分子可不包含除以上a）至h）中之序列以外的胺基酸序列。The TCRβ constant region may have the amino acid sequence provided in SEQ ID NO: 19 and/or the TCRα constant region may have the amino acid sequence provided in SEQ ID NO: 15. The multi-domain binding molecule may not comprise an amino acid sequence other than the sequences in a) to h) above.

抗CD3 scFv可以包含SEQ ID NO: 17中所提供之胺基酸序列或SEQ ID NO: 40中所提供之胺基酸序列或由其組成。胺基酸序列 The anti-CD3 scFv may comprise or consist of the amino acid sequence provided in SEQ ID NO: 17 or the amino acid sequence provided in SEQ ID NO: 40. Amino acid sequence

在本發明之範圍內的有本文所揭示之任何分子的表型靜默變異體。如本文所用，術語「表型靜默變異體（phenotypically silent variant）」應理解為指除上文所述者之外併入一或多種包括取代、***及缺失的其他胺基酸變化的變異體，且該變異體具有與不具有該（等）變化之對應分子類似的表型。出於本申請案之目的，表型包含結合親和力（K _D及/或結合半衰期）及特異性。可溶性多域結合分子之表型可包括免疫活化之效能及純化產率，以及結合親和力及特異性。 Within the scope of the present invention are phenotypically silent variants of any molecule disclosed herein. As used herein, the term "phenotypically silent variant" is understood to refer to a variant that incorporates one or more other amino acid changes, including substitutions, insertions, and deletions, in addition to those described above, and that has a phenotype similar to the corresponding molecule without the change(s). For the purposes of this application, phenotype includes binding affinity ( _KD and/or binding half-life) and specificity. The phenotype of a soluble multi-domain binding molecule may include potency of immune activation and purification yield, as well as binding affinity and specificity.

表型靜默變異體可含有一或多個保守取代及/或一或多個容許取代（tolerated substitution）。容許取代意謂不屬於如下文所提供之保守定義但仍然為表型靜默的彼等取代。所屬技術領域中具有通常知識者意識到，各種胺基酸具有類似特性且因此為『保守性（conservative）』的。蛋白質、多肽或肽之一或多個此類胺基酸通常可經一或多個其他此類胺基酸取代，而不消除該蛋白質、多肽或肽之所要活性。Phenotypically silent variants may contain one or more conservative substitutions and/or one or more tolerated substitutions. Tolerated substitutions are those substitutions that do not fall within the definition of conservative as provided below but are still phenotypically silent. One of ordinary skill in the art recognizes that various amino acids have similar properties and are therefore "conservative." One or more such amino acids in a protein, polypeptide, or peptide can often be substituted with one or more other such amino acids without eliminating the desired activity of the protein, polypeptide, or peptide.

因此，胺基酸甘胺酸、丙胺酸、纈胺酸、白胺酸及異白胺酸通常可彼此取代（具有脂族側鏈之胺基酸）。在此等可能取代中，較佳地，甘胺酸及丙胺酸用於彼此取代（因為其具有相對較短的側鏈），且纈胺酸、白胺酸及異白胺酸用於彼此取代（因為其具有疏水性的較大脂族側鏈）。通常可彼此取代之其他胺基酸包括：***酸、酪胺酸及色胺酸（具有芳族側鏈之胺基酸）；離胺酸、精胺酸及組胺酸（具有鹼性側鏈之胺基酸）；天冬胺酸及麩胺酸（具有酸性側鏈之胺基酸）；天冬醯胺酸及麩醯胺酸（具有醯胺側鏈之胺基酸）；以及半胱胺酸及甲硫胺酸（具有含硫側鏈之胺基酸）。應瞭解，本發明之範圍內的胺基酸取代可使用天然存在或非天然存在之胺基酸來進行。舉例而言，本文考慮丙胺酸上之甲基可經乙基置換，及/或可對肽主鏈進行輕微改變。無論是否使用天然胺基酸抑或使用合成胺基酸，較佳僅存在L-胺基酸。Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine are generally substituted for one another (amino acids with aliphatic side chains). Of these possible substitutions, preferably, glycine and alanine are substituted for one another (because they have relatively short side chains), and valine, leucine, and isoleucine are substituted for one another (because they have hydrophobic, larger aliphatic side chains). Other amino acids that are commonly substituted for one another include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamine (amino acids with acidic side chains); aspartic acid and glutamine (amino acids with amide side chains); and cysteine and methionine (amino acids with sulfur-containing side chains). It should be understood that amino acid substitutions within the scope of the present invention can be performed using naturally occurring or non-naturally occurring amino acids. For example, it is contemplated that the methyl group on alanine can be replaced by an ethyl group, and/or that the peptide backbone can be slightly altered. Regardless of whether natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.

此性質之取代通常稱為「保守性」或「半保守性（semi-conservative）」胺基酸取代。本發明因此擴展至使用包含上文所描述之胺基酸序列中之任一者，但在序列中具有一或多個保守取代及/或一或多個容許取代，使得分子或其任何域或區之胺基酸序列與本文所揭示之序列具有至少90%一致性，諸如90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%一致性之分子。Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions. The invention therefore extends to the use of molecules comprising any of the amino acid sequences described above, but having one or more conservative substitutions and/or one or more permissive substitutions in the sequence, such that the amino acid sequence of the molecule or any domain or region thereof has at least 90% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequences disclosed herein.

所屬技術領域中已知之「一致性（identity）」為兩個或更多個多肽序列或兩個或更多個聚核苷酸序列之間的關係，其係藉由將該等序列進行比較而確定。在所屬技術領域中，一致性亦意謂多肽或聚核苷酸序列之間的序列相關性程度，視具體情況可藉由此類序列串之間的匹配所確定。雖然存在多種用於量測兩個多肽或兩個聚核苷酸序列之間的一致性之方法，但常用於確定一致性之方法編碼於電腦程式中。確定兩個序列之間的一致性的較佳電腦程式包括但不限於GCG套裝程式（Devereux,等人，Nucleic Acids Research, 12, 387 (1984)、BLASTP、BLASTN及FASTA（Atschul等人，J. Molec. Biol. 215, 403 (1990)）。"Identity" is known in the art as a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Although there are a variety of methods for measuring the identity between two polypeptide or two polynucleotide sequences, methods commonly used to determine identity are encoded in computer programs. Preferred computer programs for determining the identity between two sequences include, but are not limited to, the GCG suite of programs (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

可使用諸如CLUSTAL程式之程式來比較胺基酸序列。此程式比較胺基酸序列且藉由視需要在任一序列中***空隙來找到最佳比對。有可能計算最佳比對之胺基酸一致性或類似性（一致性加胺基酸類型之保守性）。如BLASTx之程式將比對類似序列之最長片段且為該配合指派一個值。因此有可能獲得其中找到若干類似區，各自具有不同評分之比較。本發明中考慮兩種類型之一致性分析。Programs such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the best alignment by inserting gaps in either sequence as needed. It is possible to calculate the amino acid consistency or similarity (consistency plus conservation of amino acid type) of the best alignment. Programs such as BLASTx will align the longest fragments of similar sequences and assign a value to the match. It is therefore possible to obtain a comparison in which several similar regions are found, each with a different score. Two types of consistency analysis are considered in the present invention.

兩個胺基酸序列或兩個核酸序列之一致性百分比係藉由排比序列以達到最佳比較目的（例如，可將間隙引入第一序列中以實現最佳序列比對），且將對應位置處之胺基酸殘基或核苷酸進行比較來確定。「最佳比對（best alignment）」為產生最高一致性百分比的兩個序列之比對。一致性百分比係藉由所比較的序列中之一致胺基酸殘基或核苷酸之數目來確定（亦即，一致性%=一致位置數/總位置數×100）。The percent identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the first sequence to achieve optimal sequence alignment) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is the alignment of the two sequences that produces the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the compared sequences (i.e., % identity = number of identical positions/total number of positions × 100).

確定兩個序列之間的一致性百分比可使用所屬技術領域中具有通常知識者已知的數學演算法來實現。用於比較兩個序列之數學演算法的一實例為Karlin及Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268之演算法，如Karlin及Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877中所修改。Altschul等人(1990) J. Mol. Biol. 215:403-410之BLASTn及BLASTp程式併入了此類演算法。可使用BLASTn程式進行兩個核苷酸序列之間的一致性百分比的確定。可使用BLASTp程式進行兩個蛋白質序列之間的一致性百分比的確定。為了獲得出於比較目的之間隙比對，可如Altschul等人(1997) Nucleic Acids Res. 25:3389-3402中所描述使用間隙BLAST。替代地，PSI-Blast可用於執行偵測分子（相同）間的遠緣關係之迭代檢索。當利用BLAST、間隙BLAST及PSI-Blast程式時，可使用各別程式（例如，BLASTp及BLASTp）之預設參數。參見http://www.ncbi.nlm.nih.gov。預設通用參數可包括例如字長=3，預期臨限值=10。可選擇參數以針對短輸入序列而自動調整。用於比較序列之數學演算法的另一實例為Myers及Miller，CABIOS (1989)之演算法。作為CGC序列比對套裝軟體之一部分的ALIGN程式（2.0版）已併有此類演算法。所屬技術領域中已知的用於序列分析之其他演算法包括如Torellis及Robotti (1994) Comput. Appl. Biosci., 10 :3-5中所描述之ADVANCE及ADAM；以及Pearson及Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8中描述之FASTA。在FASTA內，ktup為設定檢索之靈敏度及速度的控制選項。出於評價本發明中之一致性百分比的目的，使用利用預設參數之BLASTp作為比較方法。此外，所述之一致性百分比對於胺基酸提供非整數值時（亦即，具有90%序列一致性之25個胺基酸之序列提供值「22.5」，將所得值向下捨入至下一整數，因此係「22」）。因此，在所提供之實例中，25個胺基酸中具有22個匹配之序列屬於90%序列一致性內。Determining the percent identity between two sequences can be accomplished using mathematical algorithms known to those of ordinary skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Altschul et al. (1990) J. Mol. Biol. 215:403-410 incorporates such algorithms in their BLASTn and BLASTp programs. Determination of the percent identity between two nucleotide sequences can be performed using the BLASTn program. Determination of the percent identity between two protein sequences can be performed using the BLASTp program. In order to obtain gap alignments for comparison purposes, gap BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform iterative searches to detect distant relationships between molecules (identical). When using BLAST, gap BLAST and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTp and BLASTp) can be used. See http://www.ncbi.nlm.nih.gov. Default universal parameters may include, for example, word length = 3, expected threshold = 10. Parameters can be selected to automatically adjust for short input sequences. Another example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such algorithms are incorporated into the ALIGN program (version 2.0) as part of the CGC sequence alignment software suite. Other algorithms known in the art for sequence analysis include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. In FASTA, ktup is a control option that sets the sensitivity and speed of the search. For the purpose of evaluating the percent identity in the present invention, BLASTp using default parameters was used as the comparison method. In addition, the percentage identity described provides non-integer values for amino acids (i.e., a sequence of 25 amino acids with 90% sequence identity provides a value of "22.5", which is rounded down to the next integer, thus "22"). Therefore, in the example provided, a sequence with 22 matches out of 25 amino acids is within 90% sequence identity.

如所屬技術領域中具有通常知識者將顯而易見，可將提供於其C端及/或N端處之序列截短或延長1、2、3、4、5或更多個殘基，而不實質上影響分子，例如TCR部分之功能特徵。提供於其C端及/或N端處之序列可截短或延長1、2、3、4或5個殘基。本發明涵蓋所有此類變異體。As will be apparent to one of ordinary skill in the art, the sequence provided at its C-terminus and/or N-terminus may be truncated or extended by 1, 2, 3, 4, 5 or more residues without substantially affecting the functional characteristics of the molecule, e.g., the TCR portion. The sequence provided at its C-terminus and/or N-terminus may be truncated or extended by 1, 2, 3, 4 or 5 residues. The present invention encompasses all such variants.

突變，包括保守及容許取代、***及缺失，可使用任何適當方法引入所提供之序列中，該方法包括但不限於基於聚合酶連鎖反應（polymerase chain reaction；PCR）、基於限制酶之選殖或非接合依賴性選殖（ligation independent cloning；LIC）程序的方法。此等方法在許多標準分子生物學教科書中詳述。關於聚合酶連鎖反應（PCR）及基於限制酶之選殖的其他細節，參見Sambrook及Russell, (2001) Molecular Cloning - A Laboratory Manual (第3版) CSHL Press。關於非接合依賴性選殖（LIC）程序之其他資訊可見於Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6中。本文提供之蛋白質序列可獲自重組表現、固態合成或所屬技術領域中已知之任何其他適當方法。評估多域結合分子之結合特徵及活性 Mutations, including conservative and permissive substitutions, insertions and deletions, can be introduced into a provided sequence using any suitable method, including but not limited to methods based on polymerase chain reaction (PCR), restriction enzyme-based cloning or ligation independent cloning (LIC) procedures. Such methods are described in detail in many standard molecular biology textbooks. For additional details on polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook and Russell, (2001) Molecular Cloning - A Laboratory Manual (3rd edition) CSHL Press. Additional information on LIC procedures can be found in Rashtchian, (1995) Curr Opin Biotechnol 6 (1): 30-6. The protein sequences provided herein can be obtained from recombinant expression, solid state synthesis, or any other appropriate method known in the art. Evaluation of Binding Characteristics and Activity of Multi-Domain Binding Molecules

確定結合親和力（與平衡常數K _D成反比）及結合半衰期（表示為T½）之方法為所屬技術領域中具有通常知識者已知。結合親和力及結合半衰期可分別使用表面電漿子共振（Surface Plasmon Resonance；SPR）或生物層干涉術（Bio-Layer Interferometry；BLI），例如使用BIAcore儀器或Octet儀器確定。應瞭解，使親和力加倍使K _D減半。T½經計算為ln2除以解離速率（k _off）。因此，使T½加倍使k _off減半。通常量測TCR之可溶性形式，亦即經截短以移除細胞質域及跨膜域殘基之彼等形式的TCR的K _D及k _off值。為考慮獨立量測之間的變化，且尤其對於解離時間超過20小時的交互作用，可使用相同分析方案量測給定蛋白質之結合親和力及/或結合半衰期若干次，例如3次或更多次，且取結果之平均值。為比較兩個樣品（亦即兩種不同蛋白質及/或相同蛋白質之兩種製劑）之間的結合數據，較佳使用相同分析條件（例如溫度）進行量測。關於TCR所述之量測方法亦可應用於本文所述之多域結合分子。 Methods for determining binding affinity (inversely proportional to the equilibrium constant _KD ) and binding half-life (expressed as T½) are known to those of ordinary skill in the art. Binding affinity and binding half-life can be determined using surface plasmon resonance (SPR) or bio-layer interferometry (BLI), for example using a BIAcore instrument or an Octet instrument, respectively. It will be appreciated that doubling the affinity halves the _KD . T½ is calculated as ln2 divided by the dissociation rate ( _koff ). Thus, doubling T½ halves the _koff . _KD and _koff values are typically measured for soluble forms of the TCR, i.e., forms of the TCR that have been truncated to remove residues of the cytoplasmic domain and transmembrane domain. To account for variations between independent measurements, and especially for interactions with dissociation times exceeding 20 hours, the binding affinity and/or binding half-life of a given protein can be measured several times, for example 3 or more times, using the same analytical protocol, and the results are averaged. To compare binding data between two samples (i.e., two different proteins and/or two preparations of the same protein), it is preferred to use the same analytical conditions (e.g., temperature) for measurement. The measurement methods described for TCRs can also be applied to the multi-domain binding molecules described herein.

本發明之某些多域結合分子能夠在試管內針對抗原陽性細胞，尤其呈現低水平的癌細胞之典型抗原（亦即，約5至100個，例如50個抗原/細胞（Bossi等人，(2013) Oncoimmunol. 1;2 (11) :e26840；Purbhoo等人，(2006). J Immunol 176(12): 7308-7316.）的細胞產生高效T細胞反應。此類TCR可以適合於併入本文所述之多域結合分子中。所量測之T細胞反應可為諸如干擾素γ或顆粒酶B之T細胞活化標記之釋放，或目標細胞殺滅，或諸如T細胞增殖之T細胞活化之其他度量。高效反應可為EC ₅₀值在nM-pM範圍內，例如500 nM或更低，較佳1 nM或更低或500 pM或更低之反應。 Certain multi-domain binding molecules of the present invention are capable of targeting antigen-positive cells in vitro, particularly cells that present low levels of typical antigens of cancer cells (i.e., about 5 to 100, for example 50 antigens/cell (Bossi et al., (2013) Oncoimmunol. 1;2(11):e26840; Purbhoo et al., (2006). J Immunol 176(12): 7308-7316.) produces an efficient T cell response. Such TCRs may be suitable for incorporation into the multi-domain binding molecules described herein. The T cell response measured may be the release of T cell activation markers such as interferon gamma or granzyme B, or target cell killing, or other measures of T cell activation such as T cell proliferation. An efficient response may be a response with an EC ₅₀ value in the nM-pM range, for example, 500 nM or less, preferably 1 nM or less or 500 pM or less.

替代地，本發明之某些結合分子可產生高效的消炎反應，諸如CD8+細胞殺滅及/或CD4+發炎抑制。此類結合分子可呈可溶形式且可包含係免疫抑制子，諸如PD-1促效子或介白素或細胞介素，諸如IL-2、IL-4、IL-10或IL-13之免疫細胞接合域。所量測之消炎反應可為CD8+細胞殺滅及/或CD4+發炎抑制及/或CD8+ T細胞信號傳導路徑抑制。用於評估消炎反應之適合方法將為所屬技術領域中已知且包括Jurkat NFAT細胞報告分析。較佳地，高效反應為IC ₅₀值在pM範圍內，亦即1000 pM或更低之反應。報告分析中獲得之最大抑制較佳大於50%，例如80%或更大。 Alternatively, certain binding molecules of the present invention may produce highly effective anti-inflammatory responses, such as CD8+ cell killing and/or CD4+ inflammation inhibition. Such binding molecules may be in soluble form and may include immune cell binding domains that are immunosuppressants, such as PD-1 agonists or interleukins or cytokines, such as IL-2, IL-4, IL-10 or IL-13. The anti-inflammatory response measured may be CD8+ cell killing and/or CD4+ inflammation inhibition and/or CD8+ T cell signaling pathway inhibition. Suitable methods for evaluating anti-inflammatory responses will be known in the art and include Jurkat NFAT cell reporter assays. Preferably, a highly effective response is a response with an IC ₅₀ value in the pM range, i.e., 1000 pM or less. The maximum inhibition obtained in the reported assay is preferably greater than 50%, such as 80% or greater.

本發明涵蓋之分子可具有改良之半衰期。用於判定蛋白質是否具有改良之半衰期的方法將為所屬技術領域中具有通常知識者顯而易見。舉例而言，評估蛋白質結合新生兒Fc受體（FcRn）之能力。就此而言，對於FcRn之結合親和力提高增加蛋白質之血清半衰期（參見例如Kim等人，Eur J Immunol., 24:2429, 1994）。The molecules encompassed by the present invention may have improved half-life. Methods for determining whether a protein has improved half-life will be apparent to one of ordinary skill in the art. For example, the ability of a protein to bind to the neonatal Fc receptor (FcRn) is assessed. In this regard, an increase in binding affinity for FcRn increases the serum half-life of a protein (see, e.g., Kim et al., Eur J Immunol., 24:2429, 1994).

本發明之蛋白質之半衰期亦可藉由藥物動力學研究，例如根據Kim等人，Eur J of Immunol 24: 542, 1994所述之方法量測。根據此方法，放射性標記之蛋白質經靜脈內注射至小鼠中且其血漿濃度隨時間推移，例如在注射之後3分鐘至72小時週期性地量測。替代地，可注射未標記之本發明蛋白質且使用ELISA週期性量測其血漿濃度。由此獲得之清除曲線應為雙相的，亦即α相及β相。為了確定蛋白質之活體內半衰期，計算β相之清除率且與野生型或未經修飾之蛋白質之清除率相比。核酸、載體及宿主細胞 The half-life of the protein of the invention can also be measured by pharmacokinetic studies, for example according to the method described by Kim et al., Eur J of Immunol 24: 542, 1994. According to this method, a radiolabeled protein is injected intravenously into mice and its plasma concentration is measured periodically over time, for example 3 minutes to 72 hours after injection. Alternatively, an unlabeled protein of the invention can be injected and its plasma concentration measured periodically using ELISA. The clearance curve thus obtained should be biphasic, i.e., an α phase and a β phase. To determine the in vivo half-life of the protein, the clearance rate of the β phase is calculated and compared with the clearance rate of the wild-type or unmodified protein. Nucleic Acids, Vectors and Host Cells

本發明提供一種編碼本發明之多域結合分子之核酸。核酸可為cDNA。核酸可為mRNA。核酸可為非天然存在及/或純化及/或工程改造的。核酸序列可根據利用之表現系統經密碼子最佳化。如所屬技術領域中具有通常知識者所知，表現系統可包括細菌細胞，諸如大腸桿菌（E. coli），或酵母細胞，或哺乳動物細胞，或昆蟲細胞，或其可為無細胞表現系統。The present invention provides a nucleic acid encoding a multi-domain binding molecule of the present invention. The nucleic acid may be cDNA. The nucleic acid may be mRNA. The nucleic acid may be non-naturally occurring and/or purified and/or engineered. The nucleic acid sequence may be codon optimized depending on the expression system utilized. As known to those of ordinary skill in the art, the expression system may include bacterial cells, such as E. coli, or yeast cells, or mammalian cells, or insect cells, or it may be a cell-free expression system.

本發明亦提供呈包含至少一種如上文所述之核酸的質體、載體、轉錄或表現卡匣形式之構築體。本發明亦提供重組宿主細胞，其包含一或多種上述構築體。如所提及，編碼本發明之特異性結合分子之核酸形成本發明之一態樣，包含自編碼本發明之特異性結合分子之核酸表現的產生特異性結合分子之方法亦如此。表現可能適宜藉由在適當條件下培養含有核酸之重組宿主細胞來達成。在藉由表現產生之後，可使用任何適合之技術分離及/或純化特異性結合分子，隨後視需要使用。The present invention also provides constructs in the form of plasmids, vectors, transcripts or expression cassettes comprising at least one nucleic acid as described above. The present invention also provides recombinant host cells comprising one or more of the above constructs. As mentioned, nucleic acids encoding specific binding molecules of the present invention form an aspect of the present invention, as do methods for producing specific binding molecules comprising expression of nucleic acids encoding specific binding molecules of the present invention. Expression may be suitably achieved by culturing recombinant host cells containing the nucleic acids under appropriate conditions. Following production by expression, the specific binding molecules may be isolated and/or purified using any suitable technique and then used as desired.

用於在多種不同宿主細胞中選殖及表現多肽之系統為熟知的。適合宿主細胞包括細菌、哺乳動物細胞、酵母及桿狀病毒系統。所屬技術領域中可用於表現異源多肽之哺乳動物細胞株包括中國倉鼠卵巢細胞、希拉細胞（HeLa cell）、幼倉鼠腎細胞、NSO小鼠黑色素瘤細胞及許多其他細胞。常見的較佳細菌宿主為大腸桿菌。抗體及抗體片段在諸如大腸桿菌之原核細胞中之表現在所屬技術領域中沿用已久。關於綜述，參見例如Plückthun, Bio/Technology 9:545-551 (1991)。所屬技術領域中具有通常知識者亦可用培養中之真核細胞中之表現作為產生特異性結合分子的選項，參見近期評述，例如Reff, Curr. Opinion Biotech. 4:573-576 (1993)；Trill等人，Curr. Opinion Biotech. 6:553-560 (1995)。Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and bacilli systems. Mammalian cell lines that are available in the art for expressing heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, hamster kidney cells, NSO mouse melanoma cells, and many others. A common and preferred bacterial host is E. coli. The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli has long been used in the art. For a general review, see, e.g., Plückthun, Bio/Technology 9:545-551 (1991). One of ordinary skill in the art can also use expression in eukaryotic cells in culture as an option for producing specific binding molecules; see, for recent reviews, e.g., Reff, Curr. Opinion Biotech. 4:573-576 (1993); Trill et al., Curr. Opinion Biotech. 6:553-560 (1995).

可選擇或構築含有適當調節序列，包括啟動子序列、終止子序列、聚腺苷酸化序列、強化子序列、標記基因及視需要之其他序列之適合的載體。載體可為所屬技術領域中已知之任何適合載體，包括視需要之質體或病毒載體（例如『噬菌體（phage）或噬菌粒（phagemid））。關於其他細節，參見例如Sambrook等人，Molecular Cloning: A Laboratory Manual: 第2版, Cold Spring Harbor Laboratory Press (1989)。用於操縱核酸，例如在核酸構築體製備、突變誘發、定序、將DNA引入細胞及基因表現，及蛋白質分析之許多已知技術及方案，詳細描述於Ausubel等人編，Short Protocols in Molecular Biology, 第2版, John Wiley & Sons (1992)中。Suitable vectors containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as needed can be selected or constructed. The vector can be any suitable vector known in the art, including plasmids or viral vectors (e.g., phages or phagemids) as needed. For further details, see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual: 2nd edition, Cold Spring Harbor Laboratory Press (1989). Many known techniques and protocols for manipulating nucleic acids, such as in nucleic acid construct preparation, mutation induction, sequencing, introduction of DNA into cells and gene expression, and protein analysis, are described in detail in Ausubel et al., ed., Short Protocols in Molecular Biology, 2nd edition, John Wiley & Sons (1992).

本發明亦提供一種含有如本文所揭示之核酸的宿主細胞。此外，本發明提供一種包含將此類核酸引入宿主細胞中之方法。引入可採用任何可用技術。對於真核細胞，適合技術可包括磷酸鈣轉染、DEAE-聚葡萄糖、電穿孔、脂質體介導之轉染及使用反轉錄病毒或其他病毒，例如牛痘或對於昆蟲細胞使用桿狀病毒進行之轉導。對於細菌細胞，適合技術可包括氯化鈣轉形、電穿孔及使用噬菌體轉染。引入之後可接著引起或允許自核酸表現，例如藉由在用於基因表現之條件下培養宿主細胞。The present invention also provides a host cell containing a nucleic acid as disclosed herein. In addition, the present invention provides a method comprising introducing such nucleic acid into a host cell. The introduction may be by any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-polydextrose, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia or bacilli for insect cells. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophages. The introduction may then be followed by causing or allowing expression of the nucleic acid, for example by culturing the host cell under conditions for gene expression.

適用於本發明之聚核苷酸及/或載體之選殖或表現的宿主細胞為所屬技術領域中已知的。適用於表現（醣基化）蛋白質之宿主細胞亦源自多細胞生物（無脊椎動物及脊椎動物）。無脊椎動物細胞之實例包括植物及昆蟲細胞。已鑑別出眾多可與昆蟲細胞聯合使用，尤其用於轉染草地黏蟲（Spodoptera frugiperda）細胞之桿狀病毒株系。植物細胞培養物亦可用作宿主。參見例如US 5,959,177、US 6,040,498、US 6,420,548、US 7,125,978及US 6,417,429（描述用於在轉殖基因植物中產生抗體之PLANTIBODIES ^TM技術）。脊椎動物細胞亦可用作宿主。舉例而言，適於在懸浮液中生長之哺乳動物細胞株可為有用的。有用的哺乳動物宿主細胞株之其他實例為由SV40轉形之猴腎CV1細胞株（COS-7）；人類胚腎細胞株（如例如在Graham, F.L.等人，J. Gen Virol. 36 (1977) 59-74中所述之293或293T細胞）；幼倉鼠腎細胞（baby hamster kidney；BHK）；小鼠塞特利氏細胞（mouse sertoli cell）（如例如在Mather, J.P., Biol. Reprod. 23 (1980) 243-252中所述之TM4細胞）；猴腎細胞（CV1）；非洲綠猴腎細胞（VERO-76）；人類子宮頸癌細胞（希拉）；犬腎細胞（MDCK；水牛鼠肝細胞（buffalo rat liver；BRL 3A）；人類肺細胞（W138）；人類肝細胞（Hep G2）；小鼠***腫瘤（mouse mammary tumor；MMT 060562）；TRI細胞（如例如在Mather, J.P.等人，Annals N.Y. Acad. Sci. 383 (1982) 44-68中所述）；MRC 5細胞；及FS4細胞。其他有用的哺乳動物宿主細胞株包括中國倉鼠卵巢（CHO）細胞，包括DHFR-CHO細胞（Urlaub, G.等人，Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220）；及骨髓瘤細胞株，諸如Y0、NS0及Sp2/0。關於適合於蛋白質生產之某些哺乳動物宿主細胞株之綜述，參見例如Yazaki, P.及Wu, A.M., Methods in Molecular Biology, 第248卷, Lo, B.K.C. (編), Humana Press, Totowa, NJ (2004), 第255-268頁。宿主細胞可為真核細胞，例如中國倉鼠卵巢（CHO）細胞或淋巴細胞（例如Y0、NS0、Sp20細胞）。 Host cells suitable for the propagation or expression of the polynucleotides and/or vectors of the present invention are known in the art. Suitable host cells for the expression of (glycosylated) proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous bacilliform virus strains have been identified that can be used in conjunction with insect cells, in particular for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describing PLANTIBODIES ^™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell strains adapted for growth in suspension can be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney cell lines (such as 293 or 293T cells described, for example, in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (such as, for example, in Mather, JP, Biol. Reprod. 23 (1980)). 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (Hela); canine kidney cells (MDCK; buffalo rat liver (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, for example, in Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (eds.), Humana Press, Totowa, NJ (2004), Pages 255-268. The host cell may be a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (eg, Y0, NS0, Sp20 cell).

本發明之核酸可整合至宿主細胞之基因體（例如染色體）中。根據標準技術，整合可藉由包括促進與基因體重組之序列來促進。製造多域結合分子之方法 The nucleic acids of the invention can be integrated into the genome (e.g., chromosome) of a host cell. Integration can be facilitated by including sequences that promote recombination with the genome, according to standard techniques. Methods of making multi-domain binding molecules

本文進一步提供製造本文所述之多域結合分子的方法。該等方法包含將本發明之宿主細胞維持於最佳條件下，該等最佳條件係用於表現本發明之核酸或表現載體之最佳條件，且分離該多域結合分子。Further provided herein are methods of making the multi-domain binding molecules described herein. The methods comprise maintaining a host cell of the invention under optimal conditions for expressing a nucleic acid or expression vector of the invention, and isolating the multi-domain binding molecule.

產生重組蛋白之方法為所屬技術領域中所熟知。編碼蛋白質之核酸可選殖至表現構築體或載體中，接著轉染至在其他情況下不會產生該蛋白質的宿主細胞，諸如大腸桿菌細胞、酵母細胞、昆蟲細胞或哺乳動物細胞，諸如猴COS細胞、中國倉鼠卵巢（CHO）細胞、人類胚胎腎（HEK）細胞或骨髓瘤細胞中。用於表現蛋白質之例示性哺乳動物細胞為CHO細胞、骨髓瘤細胞或HEK細胞。達成此等目的之分子選殖技術為所屬技術領域中已知的且描述於例如Ausubel等人(編者), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience（1988，包括迄今為止的所有更新）或Sambrook等人，Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)。廣泛多種選殖及試管內擴增方法適用於構築重組核酸。產生重組抗體之方法在所屬技術領域中亦已知，參見例如US4816567或US5530101。Methods for producing recombinant proteins are well known in the art. Nucleic acids encoding proteins can be cloned into expression constructs or vectors and then transfected into host cells that would not otherwise produce the protein, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells. Exemplary mammalian cells for expressing proteins are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques for achieving these purposes are known in the art and are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are applicable to the construction of recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art, see, for example, US4816567 or US5530101.

核酸可被***而可操作地連接於表現構築體或表現載體中之啟動子，以用於進一步選殖（DNA擴增）或用於在無細胞株統中或在細胞中表現。如本文所用，術語「啟動子（promoter）」應以最廣泛的情形解讀，且包括準確轉錄起始所需之基因體基因之轉錄調節序列，包括TATA盒或起始元件，具有或不具有例如回應於發育及/或外部刺激，或以組織特異性方式改變核酸表現之額外調節元件（例如上游活化序列、轉錄因子結合位、強化子及靜默子）。在本上下文中，術語「啟動子」亦用於描述賦予、活化或增強其可操作地連接之核酸之表現的重組、合成或融合核酸或衍生物。例示性啟動子可以含有一或多種特定調節元件的額外複本，以進一步增強該核酸的表現及/或改變其空間表現及/或時間表現。如本文所用，術語「可操作地連接於（operably linked to）」意謂啟動子相對於核酸定位，使得核酸之表現受啟動子控制。Nucleic acids can be inserted and operably linked to promoters in expression constructs or expression vectors for further cloning (DNA amplification) or for expression in cell-free strains or in cells. As used herein, the term "promoter" should be interpreted in the broadest sense and includes transcriptional regulatory sequences of genomic genes required for accurate transcription initiation, including TATA boxes or initiation elements, with or without additional regulatory elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers and silencers) that, for example, respond to development and/or external stimuli, or alter nucleic acid expression in a tissue-specific manner. In this context, the term "promoter" is also used to describe a recombinant, synthetic or fusion nucleic acid or derivative that confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance the expression of the nucleic acid and/or alter its spatial expression and/or temporal expression. As used herein, the term "operably linked to" means that the promoter is positioned relative to the nucleic acid so that the expression of the nucleic acid is controlled by the promoter.

用於在細胞中表現之多種載體為可商購的。載體組件一般包括但不限於以下中之一或多者：信號序列、編碼蛋白質之序列（例如，源自本文所提供之資訊）、強化子元件、啟動子及轉錄終止序列。所屬技術領域中具有通常知識者將瞭解用於表現蛋白質之適合序列。例示性信號序列包括原核分泌信號（例如pe1B、鹼性磷酸酶、青黴素酶、Ipp或熱穩定腸毒素II）、酵母分泌信號（例如轉化酶前導序列、因子前導序列或酸性磷酸酶前導序列）或哺乳動物分泌信號（例如單純疱疹病毒gD信號）。A variety of vectors for expression in cells are commercially available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a protein (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcriptional termination sequence. One of ordinary skill in the art will appreciate suitable sequences for expressing proteins. Exemplary signal sequences include prokaryotic secretion signals (e.g., pe1B, alkaline phosphatase, penicillinase, Ipp, or heat stable enterotoxin II), yeast secretion signals (e.g., invertase leader, factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex virus gD signal).

哺乳動物細胞中具有活性之例示性啟動子包括巨細胞病毒即刻早期（cytomegalovirus immediate early）啟動子（CMV-IE）、人類延長因子（elongation factor）1-a啟動子（EF1）、小核RNA啟動子（Ula及Ulb）、α-肌凝蛋白重鏈啟動子、猴病毒40啟動子（Simian virus 40；SV40）、勞氏肉瘤病毒啟動子（Rous sarcoma virus；RSV）、腺病毒主要晚期啟動子、β-肌動蛋白啟動子；包含CMV強化子/β-肌動蛋白啟動子或免疫球蛋白啟動子之雜交調節元件或其活性片段。有用哺乳動物宿主細胞株之實例為由SV40轉形之猴腎CV1細胞株（COS-7，ATCC CRL 1651）；人類胚腎細胞株（次選殖以在懸浮液培養物中生長之293或293細胞）；幼倉鼠腎細胞（BHK，ATCC CCL 10）；或中國倉鼠卵巢細胞（CHO）。Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-a promoter (EF1), small nuclear RNA promoters (Ula and Ulb), α-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), adenovirus major late promoter, β-actin promoter; hybrid regulatory elements comprising CMV enhancer/β-actin promoter or immunoglobulin promoter or active fragments thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

適合於在酵母細胞，諸如選自包含畢赤酵母（Pichia pastoris）、釀酒酵母（Saccharomyces cerevisiae）及粟酒裂殖酵母（S. pombe）之群的酵母細胞中表現之典型啟動子包括但不限於ADH1啟動子、GAL1啟動子、GALA啟動子、CUP1啟動子、PH05啟動子、nmt啟動子、RPR1啟動子或TEF1啟動子。Typical promoters suitable for expression in yeast cells, such as yeast cells selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, ADH1 promoter, GAL1 promoter, GALA promoter, CUP1 promoter, PH05 promoter, nmt promoter, RPR1 promoter or TEF1 promoter.

用於產生蛋白質之宿主細胞可視所用細胞類型而定，在多種培養基中培養。市售培養基，諸如Ham's F10（Sigma）、最低必需培養基（Minimal Essential Medium；MEM）（Sigma）、RPM1-1640（Sigma）及杜爾貝科氏改良伊格爾氏培養基（Dulbecco's Modified Eagle's Medium；DMEM）（Sigma），適用於培養哺乳動物細胞。用於培養本文所論述之其他細胞類型之培養基為所屬技術領域中已知的。Host cells used to produce proteins can be cultured in a variety of media, depending on the cell type used. Commercially available media, such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma), are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

分離蛋白質之方法為所屬技術領域中已知的。在蛋白質分泌於培養基中之情況下，可首先使用市售蛋白質濃縮過濾器，例如Amicon或Millipore Pellicon超過濾單元濃縮來自此類表現系統之上清液。在任何先前步驟中可包括諸如PMSF之蛋白酶抑制劑以抑制蛋白分解，且可包括抗生素以防止外來污染物之生長。替代地或另外，可例如使用連續離心自表現蛋白質之細胞過濾及/或分離上清液。Methods for isolating proteins are known in the art. Where the protein is secreted in the culture medium, the supernatant from such an expression system may first be concentrated using a commercially available protein concentrator filter, such as an Amicon or Millipore Pellicon superfilter unit. Protease inhibitors such as PMSF may be included in any prior step to inhibit protein degradation, and antibiotics may be included to prevent the growth of adventitious contaminants. Alternatively or in addition, the supernatant may be filtered and/or isolated from cells expressing the protein, for example using continuous centrifugation.

由細胞製備之蛋白質可使用例如離子交換、羥基磷灰石層析、疏水***互作用層析、凝膠電泳、透析、親和層析（例如，蛋白質A親和層析或蛋白質G層析）或前述之任何組合純化。Proteins produced by cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing.

此等方法為所屬技術領域中已知的且描述於例如WO99/57134或Ed Harlow及David Lane (編者) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)。Such methods are known in the art and are described, for example, in WO 99/57134 or in Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

所屬技術領域中具有通常知識者亦將意識到，蛋白質可經修飾以包括促進純化或偵測之標籤，例如聚組胺酸標籤、六組胺酸標籤、流感病毒血球凝集素（hemagglutinin；HA）標籤、猴病毒5（V5）標籤、LLAG標籤或麩胱甘肽S-轉移酶（glutathione S-transferase；GST）標籤。所得蛋白質隨後使用所屬技術領域中已知之方法，諸如親和純化來純化。舉例而言，包含六his標籤之蛋白質藉由以下來純化：使包含蛋白質之樣品與固定於固體或半固體支撐物上之特異性結合六his標籤的鎳-氮基三乙酸（nickel-nitrilotriacetic acid；Ni-NTA）接觸，洗滌樣品以移除未結合之蛋白質，且隨後洗提結合之蛋白質。替代地或另外，在親和純化方法中使用結合標籤之配位體或抗體。Those skilled in the art will also appreciate that the protein can be modified to include a tag that facilitates purification or detection, such as a polyhistidine tag, a hexahistidine tag, an influenza virus hemagglutinin (HA) tag, a simian virus 5 (V5) tag, a LLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as affinity purification. For example, a protein containing a hexa-his tag is purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to the hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and then eluting bound protein. Alternatively or additionally, a ligand or antibody that binds to the tag is used in an affinity purification method.

本發明之分子可適於高產率純化。產率可係基於在純化過程期間保留的材料之量（亦即相對於在再摺疊之前獲得的溶解材料之量，在純化過程結束時獲得的正確摺疊材料之量）來確定，及/或產率可係基於相對於原始培養物體積，在純化過程結束時獲得的正確摺疊材料之量。高產率意謂大於1%或大於5%或更高產率。高產率意謂大於1 mg/ml，或大於3 mg/ml，或大於5 mg/ml，或更高產率。醫藥組成物及醫學方法 The molecules of the invention may be suitable for high yield purification. The yield may be determined based on the amount of material retained during the purification process (i.e., the amount of properly folded material obtained at the end of the purification process relative to the amount of dissolved material obtained before refolding), and/or the yield may be based on the amount of properly folded material obtained at the end of the purification process relative to the original culture volume. High yield means greater than 1% or greater than 5% or higher yield. High yield means greater than 1 mg/ml, or greater than 3 mg/ml, or greater than 5 mg/ml, or higher yield. PHARMACEUTICAL COMPOSITIONS AND MEDICAL METHODS

對於向患者投予，本發明之分子、本發明之核酸、表現載體或細胞可作為醫藥組成物之一部分與一或多種醫藥學上可接受之載劑或賦形劑一起提供。此醫藥組成物可呈任何適合形式（例如，取決於向患者投予其所需之方法）。其可以單位劑型提供，且一般將提供於密封容器中且可作為套組之一部分提供。此類套組通常（儘管不一定）包括使用說明書。其可包括複數個該等單位劑型。For administration to a patient, a molecule of the invention, a nucleic acid of the invention, an expression vector or a cell of the invention may be provided as part of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or formulations. Such a pharmaceutical composition may be in any suitable form (e.g., depending on the method required for administering it to the patient). It may be provided in unit dosage form and will generally be provided in a sealed container and may be provided as part of a kit. Such kits typically (although not necessarily) include instructions for use. It may include a plurality of such unit dosage forms.

醫藥組成物可經調適用於藉由任何適當途徑，諸如非經腸（包括皮下、肌肉內、鞘內或靜脈內）、經腸（包括經口或經直腸）、吸入或鼻內途徑投予。此類組成物可藉由藥學技術中已知之任何方法製備，例如藉由在無菌條件下混合活性成分與載劑或賦形劑來製備。用於將蛋白質製備成適合形式（例如醫藥組成物）用於向個體投予之方法為所屬技術領域中已知的，且包括例如如以下中所述之方法：Remington's Pharmaceutical Sciences (第18版, Mack Publishing Co., Easton, Pa., 1990)及U.S. Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984)。The pharmaceutical composition can be adapted for administration by any appropriate route, such as parenteral (including subcutaneous, intramuscular, intrathecal or intravenous), enteral (including oral or rectal), inhalation or intranasal. Such compositions can be prepared by any method known in the pharmaceutical art, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions. Methods for preparing proteins into a suitable form (e.g., a pharmaceutical composition) for administration to an individual are known in the art and include, for example, methods described in Remington's Pharmaceutical Sciences (18th edition, Mack Publishing Co., Easton, Pa., 1990) and U.S. Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

醫藥組成物將通常包含溶解於醫藥學上可接受之載劑，例如水性載劑中的本發明之多域結合分子（或本發明之核酸、細胞或載體）之溶液。可使用多種水性載劑，例如緩衝生理食鹽水及其類似物。組成物可含有接近生理條件所需的醫藥學上可接受之輔助物質，諸如pH調節劑及緩衝劑、毒性調節劑及其類似物，例如乙酸鈉、氯化鈉、氯化鉀、氯化鈣、乳酸鈉及其類似物。在此等調配物中，本發明之分子之濃度可廣泛變化，且將主要基於流體體積、黏度、體重及其類似者，根據所選擇之特定投予模式及患者需求來選擇。例示性載劑包括水、生理食鹽水、林格氏溶液（Ringer's solution）、右旋糖溶液及5%人類血清白蛋白。亦可使用非水性媒劑，諸如混合油及油酸乙酯。脂質體亦可用作載劑。媒劑可含有少量增強等張性及化學穩定性之添加劑，例如緩衝劑及防腐劑。Pharmaceutical compositions will typically comprise a solution of the multi-domain binding molecules of the invention (or nucleic acids, cells or vectors of the invention) dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers may be used, such as buffered physiological saline and the like. The composition may contain pharmaceutically acceptable adjuvants required to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters and the like, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. In such formulations, the concentration of the molecules of the invention may vary widely and will be selected primarily based on fluid volume, viscosity, weight and the like, according to the particular mode of administration chosen and the needs of the patient. Exemplary carriers include water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be used as carriers. The vehicle may contain a small amount of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

本發明之分子可具有用作治療劑之理想安全輪廓。理想安全輪廓意謂除了展現良好特異性之外，本發明之分子可通過了進一步臨床前安全性測試。此類測試之實例包括：全血分析，其確認在全血存在下之極少細胞介素釋放，且因此在活體內引起可能的細胞介素釋放症候群之風險較低；及同種異體反應性測試，其確認識別替代HLA類型之潛能較低。The molecules of the invention may have an ideal safety profile for use as therapeutic agents. An ideal safety profile means that in addition to demonstrating good specificity, the molecules of the invention may have passed further preclinical safety testing. Examples of such testing include: whole blood analysis, which confirms minimal interleukin release in the presence of whole blood, and therefore a low risk of causing possible interleukin release syndrome in vivo; and alloreactivity testing, which confirms a low potential to recognize alternative HLA types.

本發明之分子之劑量可視待治療之疾病或病症、待治療之個體之年齡及狀況等而定，在寬廣限度之間變化。醫師將最終確定待使用之適當劑量。The dosage of the molecules of the invention can vary within wide limits depending on the disease or disorder to be treated, the age and condition of the individual to be treated, etc. The physician will ultimately determine the appropriate dosage to be used.

本發明之多域結合分子、醫藥組成物、載體、核酸及細胞可以實質上純形式，例如至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%或100%純提供。The multi-domain binding molecules, pharmaceutical compositions, vectors, nucleic acids and cells of the invention can be provided in a substantially pure form, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure.

本發明之多域結合分子可進一步與治療劑結合。可與本發明之分子結合之治療劑包括免疫調節劑及效應子、放射性化合物、酶（例如穿孔蛋白）或化學治療劑（例如順鉑）。為確保在所要位置發揮毒性功效，毒素可在連接至本文所述之多域結合分子之脂質體內部，使得化合物被緩慢釋放。此將防止在體內輸送期間之損害性作用，且確保毒素在本文所述之多域結合分子結合相關抗原呈現細胞之後具有最大功效。The multi-domain binding molecules of the present invention can be further combined with therapeutic agents. Therapeutic agents that can be combined with the molecules of the present invention include immunomodulators and effectors, radioactive compounds, enzymes (such as perforin) or chemotherapeutic agents (such as cisplatin). To ensure that the toxic effect is exerted at the desired location, the toxin can be inside the liposomes connected to the multi-domain binding molecules described herein so that the compound is released slowly. This will prevent damaging effects during in vivo transport and ensure that the toxin has maximum efficacy after the multi-domain binding molecules described herein bind to the relevant antigen presenting cells.

適合之治療劑之實例包括但不限於： ● 小分子細胞毒性劑，亦即分子量低於700道爾頓之能夠殺滅哺乳動物細胞之化合物。此類化合物亦可含有能夠具有細胞毒性功效之毒性金屬。此外，應瞭解此等小分子細胞毒性劑亦包括前藥，亦即在生理學條件下衰變或轉化以釋放細胞毒性劑之化合物。此類藥劑之實例包括順鉑、美登素（maytansine）衍生物、雷查黴素（rachelmycin）、卡奇黴素（calicheamicin）、多西他賽（docetaxel）、依託泊苷（etoposide）、吉西他濱（gemcitabine）、異環磷醯胺、伊立替康（irinotecan）、黴法蘭（melphalan）、米托蒽醌（mitoxantrone）、卟吩姆鈉光卟啉II（sorfimer sodiumphotofrin II）、替莫唑胺（temozolomide）、拓朴替康（topotecan）、葡萄糖醛酸曲美沙特（trimetreate glucuronate）、奧瑞斯他汀E（auristatin E）、長春新鹼及小紅莓； ● 肽細胞毒素，亦即能夠殺滅哺乳動物細胞之蛋白質或其片段。舉例而言，蓖麻毒素、白喉毒素、假單胞菌屬（pseudomonas）細菌外毒素A、DNA酶及RNA酶； ● 放射性核素，亦即衰變且同時發射α或β粒子或γ射線中之一或多者之元素之不穩定同位素。舉例而言，碘131、錸186、銦111、釔90、鉍210及213、錒225及砈213；螯合劑可用於促進此等放射性核素與多域結合分子之結合； ● 免疫刺激劑，亦即刺激免疫反應之免疫效應分子。舉例而言，細胞介素，諸如IL-2及IFN-γ， ● 超抗原及其突變體； ● TCR-HLA融合物，例如與肽-HLA複合物之融合物，其中該肽源自常見人類病原體，諸如埃-巴二氏病毒（Epstein Barr Virus；EBV）； ● 趨化因子，諸如IL-8、血小板因子4、黑色素瘤生長刺激性蛋白質等； ● 抗體或其片段，包括抗T細胞或NK細胞決定子抗體（例如抗CD3、抗CD28或抗CD16）； ● 抗體或其片段，其結合定位於免疫突觸之分子； ● 具有抗體樣結合特徵之替代蛋白質骨架； ● 補體活化劑； ● 異種蛋白質域、同種異體蛋白質域、病毒/細菌蛋白質域、病毒/細菌肽。 Examples of suitable therapeutic agents include, but are not limited to: ● Small molecule cytotoxic agents, i.e., compounds with a molecular weight less than 700 Daltons that are capable of killing mammalian cells. Such compounds may also contain toxic metals that can have cytotoxic effects. In addition, it should be understood that such small molecule cytotoxic agents also include prodrugs, i.e., compounds that decay or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, isocyclophosphamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine and cranberries; ● Peptide cytotoxins, i.e. proteins or fragments thereof that are capable of killing mammalian cells. Examples include ricin, diphtheria toxin, Pseudomonas exotoxin A, DNA enzymes and RNA enzymes; ● Radionuclides, i.e. unstable isotopes of elements that decay and simultaneously emit one or more of alpha or beta particles or gamma rays. Examples include iodine-131, rhodium-186, indium-111, yttrium-90, bismuth-210 and 213, ruthenium-225 and arsine-213; chelating agents may be used to promote the binding of these radionuclides to multidomain binding molecules; ● Immunostimulants, i.e. immune effector molecules that stimulate an immune response. For example, interleukins, such as IL-2 and IFN-γ, ● Superantigens and their mutants; ● TCR-HLA fusions, such as fusions with peptide-HLA complexes, wherein the peptide is derived from a common human pathogen, such as Epstein Barr Virus (EBV); ● Trending factors, such as IL-8, platelet factor 4, melanoma growth stimulating protein, etc.; ● Antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (such as anti-CD3, anti-CD28 or anti-CD16); ● Antibodies or fragments thereof, which bind to molecules localized to immune junctions; ● Alternative protein backbones with antibody-like binding characteristics; ● Complement activators; ● Heterologous protein domains, homologous protein domains, viral/bacterial protein domains, viral/bacterial peptides.

本發明之多域結合分子、核酸、載體、醫藥組成物及細胞可用於治療諸如癌症，尤其與腫瘤相關抗原之表現相關的癌症之疾病。舉例而言，癌症可與如WO2011001152、WO2017109496、WO2017175006及WO2018234319中，及例如對應美國專利第8,519,100號、第11,639,374號、第11,505,590號及第11,427,624號中所描述之GP100、NYESO、MAGEA4或PRAME之表現相關，該等文獻中之各者的內容以引用之方式併入本文中。The multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions and cells of the present invention can be used to treat diseases such as cancer, especially cancers associated with the expression of tumor-associated antigens. For example, cancer can be associated with the expression of GP100, NYESO, MAGEA4 or PRAME as described in WO2011001152, WO2017109496, WO2017175006 and WO2018234319, and for example in corresponding U.S. Patent Nos. 8,519,100, 11,639,374, 11,505,590 and 11,427,624, the contents of each of which are incorporated herein by reference.

待治療之癌症可為與PRAME表現相關之癌症。「與PRAME表現相關（associated with PRAME expression）」意謂該癌症包含表現PRAME之癌細胞。就此而言，癌症可為PRAME陽性癌症。癌症可已知與PRAME表現相關，且因此可不評估PRAME表現。替代地，可使用所屬技術領域中已知之任何方法，包括例如組織學方法評估PRAME表現。然而，本發明不意欲限於治療可藉由組織學方法偵測到PRAME表現之癌症。與PRAME表現相關之癌症包括但不限於黑色素瘤、肺癌、乳癌、卵巢癌、子宮內膜癌、食道癌、膀胱癌、頭頸癌、子宮癌、急性骨髓性白血病、慢性骨髓性白血病及霍奇金氏淋巴瘤（Hodgkin's lymphoma）。舉例而言，與PRAME表現相關之癌症可為黑色素瘤。黑色素瘤可為葡萄膜黑色素瘤或皮膚黑色素瘤。肺癌可為非小細胞肺癌（non-small cell lung carcinoma；NSCLC）或小細胞肺癌（small cell lung cancer；SCLC）。乳癌可為三陰性乳癌（triple-negative breast cancer；TNBC）。膀胱癌可為尿道上皮癌。食道癌可為胃食道接合部（gastroesophageal junction；GEJ）腺癌。卵巢癌可為上皮卵巢癌，諸如高惡性度漿液性卵巢癌。The cancer to be treated may be a cancer associated with PRAME expression. "Associated with PRAME expression" means that the cancer comprises cancer cells expressing PRAME. In this regard, the cancer may be a PRAME-positive cancer. The cancer may be known to be associated with PRAME expression, and therefore PRAME expression may not be assessed. Alternatively, PRAME expression may be assessed using any method known in the art, including, for example, histological methods. However, the present invention is not intended to be limited to the treatment of cancers in which PRAME expression can be detected by histological methods. Cancers associated with PRAME expression include, but are not limited to, melanoma, lung cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, bladder cancer, head and neck cancer, uterine cancer, acute myeloid leukemia, chronic myeloid leukemia, and Hodgkin's lymphoma. For example, a cancer associated with PRAME expression may be melanoma. The melanoma may be uveal melanoma or cutaneous melanoma. The lung cancer may be non-small cell lung carcinoma (NSCLC) or small cell lung cancer (SCLC). The breast cancer may be triple-negative breast cancer (TNBC). The bladder cancer may be urothelial carcinoma. The esophageal cancer may be adenocarcinoma of the gastroesophageal junction (GEJ). The ovarian cancer may be epithelial ovarian cancer, such as high-grade serous ovarian cancer.

本發明之多域結合分子、核酸、載體、醫藥組成物及細胞可用於治療感染性疾病。感染性疾病可由細菌、病毒、真菌或寄生蟲病原體引起。導致抗原呈現細胞呈現與來自病原體之肽結合的MHC之病原體之任何感染，可以適於用本發明之多域結合分子治療。本發明之多域結合分子尤其良好地適於其中抗原呈現細胞呈現低於天然免疫系統在無需額外治療的情況下清除感染的最佳水平之病原體肽的感染。感染性疾病可為慢性感染。例示性感染性疾病包括B型肝炎病毒（Hepatitis B virus；HBV）感染及人類免疫缺乏病毒（human immunodeficiency virus；HIV）感染。The multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions and cells of the present invention can be used to treat infectious diseases. Infectious diseases can be caused by bacterial, viral, fungal or parasitic pathogens. Any infection with a pathogen that causes an antigen presenting cell to present MHC bound to a peptide from the pathogen can be suitable for treatment with the multi-domain binding molecules of the present invention. The multi-domain binding molecules of the present invention are particularly well suited for infections in which antigen presenting cells present pathogen peptides at levels below the optimal level for the natural immune system to clear the infection without additional treatment. The infectious disease can be a chronic infection. Exemplary infectious diseases include hepatitis B virus (HBV) infection and human immunodeficiency virus (HIV) infection.

本發明之多域結合分子可用於治療自體免疫疾病，諸如第1型糖尿病之方法中。鑒於與全身性免疫抑制相關之潛在顯著不良事件，器官特異性免疫抑制而非全身性免疫抑制可為治療之有益途徑。在自體免疫性中，亦有越來越多的證據表明，PD-1路徑障礙在疾病發病機制中起重要作用。PD-1、PD-L1及PD-L2基因多形現象與若干自體免疫疾病相關。已在來自第1型糖尿病及克隆氏病（Crohn's disease）患者之樣品中觀測到異常低之PD-L1表現。因此，活化自體反應淋巴球上之PD-1可充當治療自體免疫疾病之機制。用於治療自體免疫疾病之有效治療劑包括具有有利風險輪廓（例如，高水平之目標及組織特異性）且能夠以較低頻率投予的彼等治療劑。The multi-domain binding molecules of the present invention can be used in methods for treating autoimmune diseases, such as type 1 diabetes. In view of the potentially significant adverse events associated with systemic immunosuppression, organ-specific immunosuppression rather than systemic immunosuppression may be a beneficial approach to treatment. In autoimmunity, there is also increasing evidence that PD-1 pathway disorders play an important role in disease pathogenesis. PD-1, PD-L1 and PD-L2 gene polymorphisms are associated with several autoimmune diseases. Abnormally low PD-L1 expression has been observed in samples from patients with type 1 diabetes and Crohn's disease. Therefore, activation of PD-1 on autoreactive lymphocytes may serve as a mechanism for treating autoimmune diseases. Effective therapeutics for the treatment of autoimmune diseases include those that have a favorable risk profile (e.g., high levels of target and tissue specificity) and that can be administered less frequently.

本發明亦提供： ● 本發明之多域結合分子、核酸、載體、醫藥組成物或細胞，其用於醫藥，較佳用於治療癌症或腫瘤或感染性疾病之方法中； ● 本發明之多域結合分子、核酸、載體、醫藥組成物或細胞，其用於醫藥，較佳用於治療癌症或腫瘤或自體免疫疾病或感染性疾病之方法中； ● 本發明之多域結合分子、核酸、載體、醫藥組成物或細胞在製造用於治療癌症或腫瘤或感染性疾病之藥劑中的用途； ● 本發明之多域結合分子、核酸、載體、醫藥組成物或細胞在製造用於治療癌症或腫瘤或自體免疫疾病或感染性疾病之藥劑中的用途； ● 一種治療患者之癌症或腫瘤或感染性疾病的方法，其包含向該患者投予本發明之多域結合分子、核酸、載體、醫藥組成物或細胞； ● 一種治療患者之癌症或腫瘤或自體免疫疾病或感染性疾病之方法，其包含向該患者投予本發明之多域結合分子、核酸、載體、醫藥組成物或細胞；及 ● 用於向人類個體投予的，包含本發明之多域結合分子、核酸、載體、醫藥組成物或細胞的可注射調配物。 The present invention also provides: ● The multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions or cells of the present invention are used in medicine, preferably in methods for treating cancer, tumors or infectious diseases; ● The multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions or cells of the present invention are used in medicine, preferably in methods for treating cancer, tumors or autoimmune diseases or infectious diseases; ● The use of the multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions or cells of the present invention in the manufacture of medicaments for treating cancer, tumors or infectious diseases; ● The use of the multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions or cells of the present invention in the manufacture of medicaments for treating cancer, tumors or autoimmune diseases or infectious diseases; ● A method for treating cancer or tumor or infectious disease in a patient, comprising administering to the patient a multi-domain binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the present invention; ● A method for treating cancer or tumor or autoimmune disease or infectious disease in a patient, comprising administering to the patient a multi-domain binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the present invention; and ● An injectable formulation comprising a multi-domain binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the present invention for administration to a human individual.

治療方法可進一步包括分開地、以組合形式或依序投予額外抗贅生劑。此類藥劑之實例為所屬技術領域中已知且可包括免疫活化劑及/或T細胞調節劑。套組及製品 The method of treatment may further include administering additional antimicrobial agents separately, in combination, or sequentially. Examples of such agents are known in the art and may include immune activators and/or T cell modulators. Kits and Preparations

在另一態樣中，提供一種套組或製品，其含有可用於治療及/或預防上文所描述之疾病的材料。In another aspect, a kit or article of manufacture is provided containing materials useful for treating and/or preventing the diseases described above.

套組可包含（a）容器，其包含視情況於醫藥學上可接受之載劑或稀釋劑中之本發明之分子、核酸、載體或細胞；及（b）藥品說明書，其具有用於治療個體之疾病（例如癌症、免疫疾病或自體免疫疾病）的說明書。套組可進一步包含（c）至少一種其他治療學上之活性化合物或藥物。The kit may include (a) a container comprising a molecule, nucleic acid, vector or cell of the invention in a pharmaceutically acceptable carrier or diluent as appropriate; and (b) a product package insert having instructions for use in treating a disease (e.g., cancer, immune disease or autoimmune disease) in an individual. The kit may further include (c) at least one other therapeutically active compound or drug.

藥品說明書可在容器上或與容器相關。適合的容器包括例如瓶子、小瓶、注射器等。容器可由各種材料，諸如玻璃或塑膠形成。容器保存或容納包含本發明之分子、核酸、載體或細胞之組成物，且可具有無菌接取口（例如，容器可為靜脈內溶液袋或具有可由皮下注射針刺穿之塞子的小瓶）。組成物中之至少一種活性劑為本發明之分子、核酸、載體或細胞。標籤或藥品說明書指示組成物用於治療符合治療條件之個體，例如患有或易患本文所述疾病之個體，其中關於所提供之組成物及任何其他藥劑之給藥量及時間間隔具有特定指導。套組可進一步包含額外容器，其包含醫藥學上可接受之稀釋劑緩衝液，諸如抑細菌注射用水（bacteriostatic water for injection；BWFI）、磷酸鹽緩衝生理食鹽水、林格氏溶液及/或右旋糖溶液。套組可進一步包括就商業及使用者角度而言所需之其他材料，包括其他緩衝劑、稀釋劑、過濾器、針及注射器。The instructions may be on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed of a variety of materials, such as glass or plastic. The container stores or contains a composition comprising a molecule, nucleic acid, vector, or cell of the invention and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a molecule, nucleic acid, vector, or cell of the invention. The label or instructions indicate that the composition is used to treat an individual who meets the treatment criteria, such as an individual suffering from or susceptible to a disease described herein, with specific instructions regarding the dosage and time intervals of the composition and any other agents provided. The kit may further include an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and/or dextrose solution. The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

套組視情況進一步包含容器，該容器包含第二藥劑，其中本發明之分子、核酸、載體或細胞為第一藥劑，且該套組進一步包含藥品說明書上關於以有效量用第二藥劑治療個體之說明書。The kit optionally further comprises a container comprising a second agent, wherein the molecule, nucleic acid, vector or cell of the present invention is the first agent, and the kit further comprises instructions on the drug package insert for treating a subject with the second agent in an effective amount.

本發明之除本文中所展示及描述之修改以外的各種修改將自前述說明而變得對所屬技術領域中具有通常知識者顯而易見，且屬於隨附申請專利範圍之範圍內。本發明之各態樣之較佳特徵如針對其他態樣中之各者，細節上作必要修改。本文所提及之文獻以引用之方式在法律所允許之最大範圍內併入。 序列之描述 Various modifications of the invention other than those shown and described herein will become apparent to those of ordinary skill in the art from the foregoing description and are within the scope of the appended patent applications. The preferred features of each aspect of the invention are as for each of the other aspects, mutatis mutandis. The documents mentioned herein are incorporated by reference to the maximum extent permitted by law. Description of Sequence

SEQ ID NO: 1HLA-A*02限制性肽：SLLQHLIGL SEQ ID NO: 1 HLA-A*02 restricted peptide: SLLQHLIGL

SEQ ID NO: 2例示性TCR之α鏈可變域之胺基酸序列。CDR（CDR1、CDR2及CDR3）加底線且分別指定為SEQ ID NO: 3、4及5，構架區（FR1、FR2、FR3及FR4）呈斜體且分別指定為SEQ ID NO: 27、6、7及28。此序列含有N24Q突變（加雙底線），其移除N連接型醣基化位。 GDAKTTQPNSMESNEEEPVHLPC Q HS TISGTDY IHWYRQLPSQGPEYVIH GLTSN VNNRMASLAIAEDRKSSTLILHRATLRDAAVYY CILILGHSRLGNYIATF GKGTKLSVIP SEQ ID NO: 2 Amino acid sequence of the alpha chain variable domain of an exemplary TCR. The CDRs (CDR1, CDR2, and CDR3) are underlined and designated as SEQ ID NOs: 3, 4, and 5, respectively, and the framework regions (FR1, FR2, FR3, and FR4) are italicized and designated as SEQ ID NOs: 27, 6, 7, and 28, respectively. This sequence contains the N24Q mutation (double underlined), which removes the N-linked glycosylation site. GDAKTTQPNSMESNEEEPVHLPC Q HS TISGTDY IHWYRQLPSQGPEYVIH GLTSN VNNRMASLAIAEDRKSSTLILHRATLRDAAVYY CILILGHSRLGNYIATF GKGTKLSVIP

SEQ ID NO: 8例示性TCR之TCRβ鏈可變域之胺基酸序列。CDR（CDR1、CDR2及CDR3）加底線且分別指定為SEQ ID NO: 9、10及11，構架區（FR1、FR2、FR3及FR4）呈斜體且分別指定為SEQ ID NO: 29、12、13及30。 DGGITQSPKYLFRKEGQNVTLSCEQN LNHDA MYWYRQDPGQGLRLIYY SQIMGDE QKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYL CASSWWTGGASPIRF GPGTRLTVT SEQ ID NO: 8 Amino acid sequence of the TCR beta chain variable domain of an exemplary TCR. The CDRs (CDR1, CDR2 and CDR3) are underlined and designated as SEQ ID NOs: 9, 10 and 11, respectively, and the framework regions (FR1, FR2, FR3 and FR4) are italicized and designated as SEQ ID NOs: 29, 12, 13 and 30, respectively. DGGITQSPKYLFRKEGQNVTLSCEQN LNHDA MYWYRQDPGQGLRLIYY SQIMGDE QKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYL CASSWWTGGASPIRF GPGTRLTVT

SEQ ID NO: 14例示性TCR之TCRα鏈之胺基酸序列。CDR（CDR1、CDR2及CDR3）加底線且分別指定為SEQ ID NO: 3、4及5，構架區（FR1、FR2、FR3及FR4）呈斜體且分別指定為SEQ ID NO: 27、6、7及28。恆定區以粗體展示且指定為SEQ ID NO: 15。在恆定區內，非原生半胱胺酸殘基加雙底線（在恆定區之位置48處），其經引入以產生鏈間二硫鍵。該序列亦含有N24Q、N148Q、N182Q及N193Q取代（加雙底線），其各自移除N連接型醣基化位。 GDAKTTQPNSMESNEEEPVHLPC Q HS TISGTDY IHWYRQLPSQGPEYVIH GLTSN VNNRMASLAIAEDRKSSTLILHRATLRDAAVYY CILILGHSRLGNYIATF GKGTKLSVIP NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT QVSQSKDSDVYITDK CVLDMRSMDFKSNSAVAWS QKSDFACANAF QNSIIPEDT SEQ ID NO: 14 Amino acid sequence of the TCRα chain of an exemplary TCR. The CDRs (CDR1, CDR2, and CDR3) are underlined and designated as SEQ ID NOs: 3, 4, and 5, respectively, and the framework regions (FR1, FR2, FR3, and FR4) are italicized and designated as SEQ ID NOs: 27, 6, 7, and 28, respectively. The constant region is shown in bold and designated as SEQ ID NO: 15. Within the constant region, a non-native cysteine residue is double-underlined (at position 48 of the constant region), which is introduced to generate an interchain disulfide bond. The sequence also contains N24Q, N148Q, N182Q, and N193Q substitutions (double-underlined), each of which removes an N-linked glycosylation site. GDAKTTQPNSMESNEEEPVHLPC Q HS TISGTDY IHWYRQLPSQGPEYVIH GLTSN VNNRMASLAIAEDRKSSTLILHRATLRDAAVYY CILILGHSRLGNYIATF GKGTKLSVIP NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRSMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDT

SEQ ID NO: 16例示性TCR之TCRβ鏈之胺基酸序列。CDR（CDR1、CDR2及CDR3）加底線且分別指定為SEQ ID NO: 9、10及11，構架區（FR1、FR2、FR3及FR4）呈斜體且分別指定為SEQ ID NO: 29、12、13及30。恆定區以粗體（無底線）展示且指定為SEQ ID NO: 19。在恆定區內，非原生半胱胺酸殘基加陰影（在恆定區之位置57處），其經引入以產生鏈間二硫鍵。該序列亦含有N184Q取代（加雙底線），其移除N連接型醣基化位。 DGGITQSPKYLFRKEGQNVTLSCEQN LNHDA MYWYRQDPGQGLRLIYY SQIMGDE QKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYL CASSWWTGGASPIRF GPGTRLTVT EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV CTDPQPLKEQPAL QDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD SEQ ID NO: 16 Amino acid sequence of the TCR beta chain of an exemplary TCR. The CDRs (CDR1, CDR2, and CDR3) are underlined and designated as SEQ ID NOs: 9, 10, and 11, respectively, and the framework regions (FR1, FR2, FR3, and FR4) are italicized and designated as SEQ ID NOs: 29, 12, 13, and 30, respectively. The constant region is shown in bold (no underline) and designated as SEQ ID NO: 19. Within the constant region, a non-native cysteine residue is shaded (at position 57 of the constant region), which is introduced to generate an interchain disulfide bond. The sequence also contains a N184Q substitution (double underlined), which removes the N-linked glycosylation site. DGGITQSPKYLFRKEGQNVTLSCEQN LNHDA MYWYRQDPGQGLRLIYY SQIMGDE QKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYL CASSWWTGGASPIRF GPGTRLTVT EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD

SEQ ID NO: 17例示性抗CD3 scFv（T細胞接合免疫效應子域），在本文中稱為「U0」。輕鏈可變域（VL）呈斜體且指定為SEQ ID NO: 31。輕鏈CDR（CDR1、CDR2及CDR3）加底線且指定為SEQ ID NO: 33、34及35。重鏈可變域（VH）以粗體展示且指定為SEQ ID NO: 32。重鏈CDR（CDR1、CDR2及CDR3）加底線且指定為SEQ ID NO: 36、37及38。連接VL及VH之甘胺酸-絲胺酸連接子以純文字展示且指定為SEQ ID NO: 39。 AIQMTQSPSSLSASVGDRVTITCRAS QDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIK GGGGSGGGGSGGGGSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAAS GYSFTGYTMNWVRQAPGKGLEWVAL INPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYC ARSGYYGDSDWYFDVWGQGTLVTVSS SEQ ID NO: 17 Exemplary anti-CD3 scFv (T cell engaging immune effector domain), referred to herein as "U0". The light chain variable domain (VL) is italicized and designated as SEQ ID NO: 31. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined and designated as SEQ ID NOs: 33, 34, and 35. The heavy chain variable domain (VH) is shown in bold and designated as SEQ ID NO: 32. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined and designated as SEQ ID NOs: 36, 37, and 38. The glycine-serine linker connecting the VL and VH is shown in plain text and designated as SEQ ID NO: 39. AIQMTQSPSSLSASVGDRVTITCRAS QDIRNY LNWYQQKPGKAPKLLIY YTS RLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPWT FGQGTKVEIK GGGGSGGGGSGGGGSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAAS GYSFTGYT MNWVRQAPGKGLEWVAL INPYKGVS TYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYC ARSGYYGDSDWYFDV WGQGTLVTVSS

SEQ ID NO: 40另一例示性抗CD3 scFv（T細胞接合免疫效應子域），在本文中稱為「U28」。此序列與以上SEQ ID NO: 17相同，除了其中加雙底線之兩個取代（T164A及I201F）。輕鏈可變域（VL）呈斜體且指定為SEQ ID NO: 31。輕鏈CDR（CDR1、CDR2及CDR3）加底線且指定為SEQ ID NO: 33、34及35。重鏈可變域（VH）以粗體展示且指定為SEQ ID NO: 41。重鏈CDR（CDR1、CDR2及CDR3）加底線且指定為SEQ ID NO: 48、37及38。連接VL及VH之甘胺酸-絲胺酸連接子以純文字展示且指定為SEQ ID NO: 39。 AIQMTQSPSSLSASVGDRVTITCRAS QDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIK GGGGSGGGGSGGGGSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAAS GYSFTGY AMNWVRQAPGKGLEWVAL INPYKGVSTYNQKFKDRFT FSVDKSKNTAYLQMNSLRAEDTAVYYC ARSGYYGDSDWYFDVWGQGTLVTVSS SEQ ID NO: 40 Another exemplary anti-CD3 scFv (T cell engaging immune effector domain), referred to herein as "U28". This sequence is identical to SEQ ID NO: 17 above, except for two substitutions (T164A and I201F) which are double underlined. The light chain variable domain (VL) is italicized and designated as SEQ ID NO: 31. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined and designated as SEQ ID NOs: 33, 34, and 35. The heavy chain variable domain (VH) is shown in bold and designated as SEQ ID NO: 41. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined and designated as SEQ ID NOs: 48, 37, and 38. The glycine-serine linker connecting the VL and VH is shown in plain text and designated as SEQ ID NO: 39. AIQMTQSPSSLSASVGDRVTITCRAS QDIRNY LNWYQQKPGKAPKLLIY YTS RLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPWT FGQGTKVEIK GGGGSGGGGSGGGGSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAAS GYSFTGY A MNWVRQAPGKGLEWVAL INPYKGVS TYNQKFKDRFT F SVDKSKNTAYLQMNSLRAEDTAVYYC ARSGYYGDSDWYFDV WGQGTLVTVSS

SEQ ID NO: 54未經修飾之人類IgG1 Fc區（CH2及CH3域） APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 54 Unmodified human IgG1 Fc region (CH2 and CH3 domains) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 42例示性IgG1 Fc區序列。此序列相對於上述未經修飾之IgG1 Fc序列（SEQ ID NO: 54）具有四個取代，該等取代加雙底線。此等取代為：用於抑制與FcγR之結合之N297G取代，以及用於增強與含有T366W取代（形成杵之取代）之另一Fc區（例如SEQ ID NO: 43）之二聚的T366S、L368A及Y407V取代（形成臼之取代）。此序列中之取代之編號係根據EU編號方案。 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 42 Exemplary IgG1 Fc region sequence. This sequence has four substitutions relative to the unmodified IgG1 Fc sequence above (SEQ ID NO: 54), which are double underlined. These substitutions are: N297G substitution for inhibiting binding to FcγRs, and T366S, L368A and Y407V substitutions (hole-forming substitutions) for enhancing dimerization with another Fc region (e.g., SEQ ID NO: 43) containing a T366W substitution (knob-forming substitution). The numbering of the substitutions in this sequence is according to the EU numbering scheme. APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 43另一例示性IgG1 Fc區序列。此序列相對於上述未經修飾之IgG1 Fc序列（SEQ ID NO: 54）具有兩個取代，該等取代加雙底線。此等取代為：用於抑制與FcγR之結合之N297G取代，以及用於增強與含有T366S、L368A及Y407V取代（形成臼之取代）之另一Fc區（例如SEQ ID NO: 42）之二聚的T366W取代（形成杵之取代）。此序列中之取代之編號係根據EU編號方案。 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 43 Another exemplary IgG1 Fc region sequence. This sequence has two substitutions relative to the unmodified IgG1 Fc sequence above (SEQ ID NO: 54), which are double underlined. These substitutions are: N297G substitution for inhibiting binding to FcγRs, and T366W substitution (knob-forming substitution) for enhancing dimerization with another Fc region (e.g., SEQ ID NO: 42) containing T366S, L368A, and Y407V substitutions (hole-forming substitutions). The numbering of the substitutions in this sequence is according to the EU numbering scheme. APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 57例示性IgG1 Fc區序列展示如下。此序列相對於以上未經修飾之IgG1 Fc序列（SEQ ID NO: 54）具有七個取代（粗體）。此等取代為：用於抑制與FcγR之結合之N297G取代，用於增強與含有T366W取代（形成杵之取代）之另一Fc區（例如SEQ ID NO: 58）之二聚的T366S、L368A及Y407V取代（形成臼之取代），及用於增加與FcRn之結合之取代M252Y、S254T及T256E。此序列中之取代之編號係根據EU編號方案。 APELLGGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 57 An exemplary IgG1 Fc region sequence is shown below. This sequence has seven substitutions (in bold) relative to the unmodified IgG1 Fc sequence above (SEQ ID NO: 54). These substitutions are: N297G substitution for inhibiting binding to FcγRs, T366S, L368A and Y407V substitutions (hole-forming substitutions) for enhancing dimerization with another Fc region (e.g., SEQ ID NO: 58) containing the T366W substitution (knob-forming substitution), and substitutions M252Y, S254T and T256E for increasing binding to FcRn. The numbering of the substitutions in this sequence is according to the EU numbering scheme. APELLGGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 58另一例示性IgG1 Fc區序列展示如下。此序列相對於以上未經修飾之IgG1 Fc序列（SEQ ID NO: 54）具有五個取代（粗體）。此等取代為：用於抑制與FcγR之結合之N297G取代，用於增強與含有T366S、L368A及Y407V取代（形成臼之取代）之另一Fc區（例如SEQ ID NO: 57）之二聚的T366W取代（形成杵之取代），及用於增加與FcRn之結合之取代M252Y、S254T及T256E。此序列中之取代之編號係根據EU編號方案。 APELLGGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 58 Another exemplary IgG1 Fc region sequence is shown below. This sequence has five substitutions (in bold) relative to the above unmodified IgG1 Fc sequence (SEQ ID NO: 54). These substitutions are: N297G substitution for inhibiting binding to FcγRs, T366W substitution (knob-forming substitution) for enhancing dimerization with another Fc region (e.g., SEQ ID NO: 57) containing T366S, L368A, and Y407V substitutions (hole-forming substitutions), and substitutions M252Y, S254T, and T256E for increasing binding to FcRn. The numbering of the substitutions in this sequence is according to the EU numbering scheme. APELLGGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 44例示性IgG1鉸鏈序列（相對於原生人類IgG1序列，含有位置5處之C至S取代，根據SEQ ID NO: 44編號）： EPKSSDKTHTCPPCP SEQ ID NO: 44 Exemplary IgG1 hinge sequence (relative to native human IgG1 sequence, containing a C to S substitution at position 5, numbered according to SEQ ID NO: 44): EPKSSDKTHTCPPCP

SEQ ID NO: 52截短IgG1鉸鏈序列： DKTHTCPPCP SEQ ID NO: 52 Truncated IgG1 hinge sequence: DKTHTCPPCP

SEQ ID NO: 53IgG4鉸鏈序列： ESKYGPPCPSCP SEQ ID NO: 53 IgG4 hinge sequence: ESKYGPPCPSCP

SEQ ID NO: 45命名為「mol093v11」之例示性多域單鏈結合分子之完整胺基酸序列。T細胞接合免疫效應子域（加底線）為SEQ ID NO: 17中所提供之抗CD3 scFv序列（「U0」）。pMHC結合域加雙底線且包含提供於SEQ ID NO: 16中之TCRβ鏈序列（在此情況下為「VC1」）（加雙底線，純文字），及提供於SEQ ID NO: 14中之TCRα鏈序列（在此情況下為「VC2」）（加雙底線，粗體文字）。半衰期延長域為Fc域，其係形成於SEQ ID NO: 42中提供的Fc區序列（斜體）（在此情況下為FC1區）與SEQ ID NO: 43中提供的Fc區序列（斜體及粗體）（在此情況下為FC2區）之間的二聚體。 AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGS DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGASPIRFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADGGGSGGGGEPKSSDKTHTCPPCP APELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGG GDAKTTQPNSMESNEEEPVHLPCQHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDT GGGSGGGGEPKSSDKTHTCPPCP APELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 45 is the complete amino acid sequence of an exemplary multi-domain single-chain binding molecule designated "mol093v11". The T cell engaging immune effector domain (underlined) is the anti-CD3 scFv sequence ("U0") provided in SEQ ID NO: 17. The pMHC binding domain is double-underlined and includes the TCRβ chain sequence (in this case "VC1") provided in SEQ ID NO: 16 (double-underlined, plain text), and the TCRα chain sequence (in this case "VC2") provided in SEQ ID NO: 14 (double-underlined, bold text). The half-life extension domain is an Fc domain that forms a dimer between the Fc region sequence provided in SEQ ID NO: 42 (italics), in this case the FC1 region, and the Fc region sequence provided in SEQ ID NO: 43 (italics and bold), in this case the FC2 region. AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS GGGGS DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGASPIRFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD GGGSGGGGEPKSSDKTHTCPPCP APELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGSGGGG GDAKTTQPNSMESNEEEPVHLPCQHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDT GGGSGGGGEPKSSDKTHTCPPCP APELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 46命名為「mol093v9」之例示性多域單鏈結合分子之完整胺基酸序列。T細胞接合免疫效應子域（加底線）為SEQ ID NO: 40中所提供之抗CD3 scFv序列（「U28」）。pMHC結合域加雙底線且包含提供於SEQ ID NO: 16中之TCRβ鏈序列（在此情況下為「VC1」）（加雙底線，純文字），及提供於SEQ ID NO: 14中之TCRα鏈序列（在此情況下為「VC2」）（加雙底線，粗體文字）。半衰期延長域為Fc域，其係形成於SEQ ID NO: 42中提供的Fc區序列（斜體）（在此情況下為FC1區）與SEQ ID NO: 43中提供的Fc區序列（斜體及粗體）（在此情況下為FC2區）之間的二聚體。 AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGS DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGASPIRFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADGGGSGGGGEPKSSDKTHTCPPCP APELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGG GDAKTTQPNSMESNEEEPVHLPCQHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDT GGGSGGGGEPKSSDKTHTCPPCP APELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 46 is the complete amino acid sequence of an exemplary multi-domain single-chain binding molecule designated "mol093v9". The T cell engaging immune effector domain (underlined) is the anti-CD3 scFv sequence ("U28") provided in SEQ ID NO: 40. The pMHC binding domain is double-underlined and comprises the TCRβ chain sequence (in this case "VC1") provided in SEQ ID NO: 16 (double-underlined, plain text), and the TCRα chain sequence (in this case "VC2") provided in SEQ ID NO: 14 (double-underlined, bold text). The half-life extension domain is an Fc domain that forms a dimer between the Fc region sequence provided in SEQ ID NO: 42 (italics), in this case the FC1 region, and the Fc region sequence provided in SEQ ID NO: 43 (italics and bold), in this case the FC2 region. AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS GGGGS DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGASPIRFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD GGGSGGGGEPKSSDKTHTCPPCP APELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGSGGGG GDAKTTQPNSMESNEEEPVHLPCQHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDT GGGSGGGGEPKSSDKTHTCPPCP APELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 55命名為「a40b23U28-mol93」之例示性多域單鏈結合分子之完整胺基酸序列。T細胞接合免疫效應子域（加底線）為SEQ ID NO: 40中所提供之抗CD3 scFv序列（「U28」）。此分子中之pMHC結合域結合人類PIWIL1（PIWI樣蛋白1）肽-MHC複合物。pMHC結合域加雙底線且包含TCRβ鏈序列（在此情況下為「VC1」）（加雙底線，純文字）及TCRα鏈序列（在此情況下為「VC2」）（加雙底線，粗體文字）。半衰期延長域為Fc域，其係形成於SEQ ID NO: 42中提供的Fc區序列（斜體）（在此情況下為FC1區）與SEQ ID NO: 43中提供的Fc區序列（斜體及粗體）（在此情況下為FC2區）之間的二聚體。 AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGS EAGVAQSPRYKIIEKRQSVAFWCNPISGHGTLYWYQQILGQGPKLLIQFHENGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSVDWVGDGERQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADGGGSGGGGEPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGG LAKTTQPISMDSYEGQEVNITCSHNYIAANDFITWYQQFPSQGPRFFIQGYKTNVQNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLAWGGTDLLPFGTGTRLQVFPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDT GGGSGGGGEPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 55 The complete amino acid sequence of an exemplary multi-domain single-chain binding molecule designated "a40b23U28-mol93". The T cell engaging immune effector domain (underlined) is the anti-CD3 scFv sequence ("U28") provided in SEQ ID NO: 40. The pMHC binding domain in this molecule binds to the human PIWIL1 (PIWI-like protein 1) peptide-MHC complex. The pMHC binding domain is double-underlined and includes the TCRβ chain sequence (in this case "VC1") (double-underlined, plain text) and the TCRα chain sequence (in this case "VC2") (double-underlined, bold text). The half-life extension domain is an Fc domain that forms a dimer between the Fc region sequence provided in SEQ ID NO: 42 (italics), in this case the FC1 region, and the Fc region sequence provided in SEQ ID NO: 43 (italics and bold), in this case the FC2 region. AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS GGGGS EAGVAQSPRYKIIEKRQSVAFWCNPISGHGTLYWYQQILGQGPKLLIQFHENGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSVDWVGDGERQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD GGGSGGGGEPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGSGGGG LAKTTQPISMDSYEGQEVNITCSHNYIAANDFITWYQQFPSQGPRFFIQGYKTNVQNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLAWGGTDLLPFGTGTRLQVFPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDT GGGSGGGGEPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 56由a40b23U28-mol93結合的PIWIL1肽（當呈與MHC之複合物時）之胺基酸序列。 SLSNRLYYL SEQ ID NO: 56 Amino acid sequence of the PIWIL1 peptide bound by a40b23U28-mol93 when in complex with MHC.

額外連接子序列： GGGGS（SEQ ID NO: 18）、GGGSG（SEQ ID NO: 20）、GGSGG（SEQ ID NO: 21）、GSGGG（SEQ ID NO: 22）、GSGGGP（SEQ ID NO: 23）、GGEPS（SEQ ID NO: 24）、GGEGGGP（SEQ ID NO: 25）、GGEGGGSEGGGS（SEQ ID NO: 26）、GGGSGGGG（SEQ ID NO: 47）、GGGGSGGGGSGGGGSGGGGSGGGS（SEQ ID NO: 39）、GGGGSGGGGSGGGGS（SEQ ID NO: 49）、EAAAK（SEQ ID NO: 50）及EAAAKEAAAKEAAAK（SEQ ID NO: 51）。 實施例 Additional linker sequences: GGGGS (SEQ ID NO: 18), GGGSG (SEQ ID NO: 20), GGSGG (SEQ ID NO: 21), GSGGG (SEQ ID NO: 22), GSGGGP (SEQ ID NO: 23), GGEPS (SEQ ID NO: 24), GGEGGGP (SEQ ID NO: 25), GGEGGGSEGGGS (SEQ ID NO: 26 ), GGGSGGGG (SEQ ID NO: 47), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 39), GGGGSGGGGSGGGGS (SEQ ID NO: 49), EAAAK (SEQ ID NO: 50) and EAAAKEAAAKEAAAK (SEQ ID NO: 51).

將參考以下實施例更充分地理解本發明。然而，其不應視為限制本發明之範圍。應理解，本文中所描述之實施例及具體實例僅為達成說明之目的，且所屬技術領域中具有通常知識者將想到之根據其之各種修改或變化應包括在本申請案之領域及隨附申請專利範圍之範圍內。實施例1-具有提高之效能的多域分子 The present invention will be more fully understood with reference to the following examples. However, they should not be considered as limiting the scope of the present invention. It should be understood that the embodiments and specific examples described herein are for illustrative purposes only, and that various modifications or variations therefrom that would occur to a person of ordinary skill in the art should be included within the scope of the present application and the scope of the accompanying patent applications. Example 1 - Multi-domain molecules with improved performance

包含TCR-抗CD3融合蛋白且合併有半衰期延長Fc域的多域分子先前已由WO 2020/157211描述且證實具有功能性。然而，隨後發現此類分子相對於分子之非Fc融合形式具有實質上降低之試管內活化T細胞之能力，且因此認為對於治療用途而言並非最佳的。進行進一步工程改造以鑑別具有改良之治療特性的新穎分子形式。Multi-domain molecules comprising a TCR-anti-CD3 fusion protein incorporating a half-life extending Fc domain have been previously described and demonstrated to be functional by WO 2020/157211. However, such molecules were subsequently found to have a substantially reduced ability to activate T cells in vitro relative to non-Fc fused forms of the molecule and were therefore considered suboptimal for therapeutic use. Further engineering was performed to identify novel molecular forms with improved therapeutic properties.

構築多域分子，其中各功能域配置在單一多肽鏈上。圖1a展示域配置之示意性圖示，且圖1b展示分子摺疊結構之假設圖示。A multidomain molecule is constructed in which each functional domain is arranged on a single polypeptide chain. Figure 1a shows a schematic diagram of the domain arrangement, and Figure 1b shows a hypothetical diagram of the folded structure of the molecule.

在第一實施例中，如先前所述（WO 2018/234319），多域單鏈分子之TCR域經設計以識別源自PRAME之HLA-A*02限制性肽SLLQHLIGL（SEQ ID NO: 1）。使用ELISpot分析，用IFNγ作為T細胞活化之讀取結果，研究此分子在抗原陽性癌細胞存在下驅動T細胞活化之能力。作為比較，使用WO 2020/157211中先前揭示之形式，用相同的TCR來構築多域分子，且與圖1中呈現之單鏈形式並行測試。In a first embodiment, the TCR domain of a multi-domain single-chain molecule was designed to recognize the HLA-A*02 restricted peptide SLLQHLIGL (SEQ ID NO: 1) derived from PRAME, as previously described (WO 2018/234319). The ability of this molecule to drive T cell activation in the presence of antigen-positive cancer cells was investigated using an ELISpot assay with IFNγ as a readout of T cell activation. As a comparison, a multi-domain molecule was constructed with the same TCR using the format previously disclosed in WO 2020/157211 and tested in parallel with the single-chain format presented in FIG1 .

圖2中所示之數據表明，與先前揭示之形式相比，單鏈分子能夠驅動實質上改良的針對抗原陽性癌細胞之T細胞反應。The data presented in Figure 2 demonstrate that the single-chain molecule is able to drive substantially improved T cell responses against antigen-positive cancer cells compared to previously disclosed formats.

製造具有替代域配置之其他37種分子形式，且測試試管內效能。此等形式中無一者比圖1中所描繪之分子表現更佳。在17種形式下觀測到很少或未觀測到反應；在15種形式下觀測到低水平反應且在2種形式下觀測到中等水平反應。其餘三種形式除針對抗原陽性細胞之低效能以外，亦展現增加的針對抗原陰性細胞株之交叉反應性。實施例2-製備靶向PRAME之單鏈多域分子 Thirty-seven other molecular formats with alternative domain configurations were made and tested for in vitro potency. None of these formats performed better than the molecule depicted in Figure 1. Little or no response was observed in 17 formats; low-level responses were observed in 15 formats and moderate-level responses were observed in 2 formats. The remaining three formats, in addition to low potency against antigen-positive cells, also exhibited increased cross-reactivity against antigen-negative cell lines. Example 2 - Preparation of single-chain multi-domain molecules targeting PRAME

使用圖1中所示之形式製備兩種多域分子（稱為mol093v9及mol093v11）。兩種分子之TCR區經設計以識別源自PRAME之HLA-A*02限制性肽SLLQHLIGL。兩種分子的不同之處在於抗CD3 scFv片段之胺基酸序列。mol093v9及mol093v11之完整胺基酸序列分別提供於SEQ ID NO: 46及45中。表現 Two multidomain molecules (referred to as mol093v9 and mol093v11) were prepared using the format shown in Figure 1. The TCR region of both molecules was designed to recognize the HLA-A*02 restricted peptide SLLQHLIGL derived from PRAME. The two molecules differed in the amino acid sequence of the anti-CD3 scFv fragment. The complete amino acid sequences of mol093v9 and mol093v11 are provided in SEQ ID NOs: 46 and 45, respectively .

使用Thermo ExpiCHOTM短暫表現方案，將mol093v9及mol093v11於Cho細胞中表現。簡言之，在轉染之前將培養細胞稀釋至6×10 ⁶之濃度。轉染後第14天收集細胞，其中在轉染後第1天溫度轉移至32℃。在轉染後第1天及第5天進行進料添加。藉由以300×g及17,500×g進行的兩個連續離心步驟來進行澄清。所得上清液通過0.45 μm及0.2 μm膜過濾器。純化 mol093v9 and mol093v11 were expressed in Cho cells using the Thermo ExpiCHOTM transient expression protocol. Briefly, cultured cells were diluted to a concentration of 6×10 ⁶ prior to transfection. Cells were harvested on day 14 post-transfection, with a temperature shift to 32°C on day 1 post-transfection. Feed additions were performed on days 1 and 5 post-transfection. Clarification was performed by two consecutive centrifugation steps at 300×g and 17,500×g. The resulting supernatant was passed through 0.45 μm and 0.2 μm membrane filters. Purification

藉由蛋白質A繼之以尺寸排阻層析步驟來純化經澄清上清液。準備15 cm床高MabSelect Extra蛋白質A樹脂管柱。向管柱中裝載50倍管柱體積之上清液且使用pH 3.0之檸檬酸鈉緩衝液洗提。在已收集三倍管柱體積之後，藉由添加2 M Tris將經洗提之產物中和，且經由0.2 µm膜過濾器過濾。使用切向流式過濾（利用Ultracel® 30 kDa膜之Pellicon® XL50）將蛋白質A洗提液濃縮至至少2 mg/mL，隨後裝載至HiLoad 26/600 Superdex SEC樹脂上。管柱以5%管柱體積裝載。將產物洗提至磷酸鹽檸檬酸鹽緩衝液中，且經由0.22 μm膜過濾器過濾相關洗提份。產率 The clarified supernatant was purified by Protein A followed by a size exclusion chromatography step. A 15 cm bed height MabSelect Extra Protein A resin column was prepared. 50 column volumes of supernatant were loaded onto the column and eluted with sodium citrate buffer, pH 3.0. After three column volumes had been collected, the eluted product was neutralized by the addition of 2 M Tris and filtered through a 0.2 µm membrane filter. The Protein A eluate was concentrated to at least 2 mg/mL using tangential flow filtration (Pellicon® XL50 with Ultracel® 30 kDa membrane) and subsequently loaded onto HiLoad 26/600 Superdex SEC resin. The column was loaded at 5% column volume. The product was eluted into phosphate-citrate buffer, and the relevant fractions were filtered through a 0.22 μm membrane filter.

使用Nanodrop分光光度計藉由280 nm下之吸光度量測純化材料之濃度。The concentration of the purified material was measured by absorbance at 280 nm using a Nanodrop spectrophotometer.

每公升上清液之所計算產率為mol093v9：9.6 mg/L，及mol093v11：17 mg/L。 穩定性 The calculated yield per liter of supernatant is mol093v9: 9.6 mg/L, and mol093v11: 17 mg/L. Stability

在冷凍/解凍循環及/或在i）熱應力及ii）攪拌之條件下14天之後，藉由SEC UPLC評估分子穩定性。結果提供於緊接在下面的表中，其展示在指定條件下之mol093v11單體純度。在各情況下，認為單體純度係可接受的。 樣品條件 Mol093 v11 （ U0 ） Mol093 v9 （ U28 ）總 HMW 總單體 總 LMW 總 HMW 總單體 總 LMW 對照（5℃） 3.3 96.7 ＜0.1 - - - 冷凍/解凍 3.5 96.5 ＜0.1 - - - 對照（5℃） 4.6 95.3 0.1 3.0 97.0 ＜0.1 攪拌 5.6 94.3 0.1 3.1 96.8 ＜0.1 30℃ 8.2 91.6 0.2 5.5 94.4 0.1 實施例3-靶向PRAME之多域分子的結合親和力及動力學 Molecular stability was assessed by SEC UPLC after freeze/thaw cycles and/or 14 days under conditions of i) thermal stress and ii) agitation. The results are provided in the table immediately below, which shows the monomer purity of mol093v11 under the specified conditions. In each case, the monomer purity was considered acceptable. Sample conditions Mol093 v11 ( U0 ) Mol093 v9 ( U28 ) Total HMW Total monomer Total LMW Total HMW Total monomer Total LMW Control (5℃) 3.3 96.7 <0.1 - - - Freeze/thaw 3.5 96.5 <0.1 - - - Control (5℃) 4.6 95.3 0.1 3.0 97.0 <0.1 Stir 5.6 94.3 0.1 3.1 96.8 <0.1 30℃ 8.2 91.6 0.2 5.5 94.4 0.1 Example 3 - Binding affinity and kinetics of multi-domain molecules targeting PRAME

為驗證分子之TCR及抗CD3部分結合對應的目標分子，藉由用T200 BIAcore上進行表面電漿子共振（SPR），隨後單次注射CD3（γε），來進行單循環動力學分析。方法 To verify that the TCR and anti-CD3 parts of the molecule bind to their target molecules, single-cycle kinetic analysis was performed by surface plasmon resonance (SPR) on a T200 BIAcore followed by a single injection of CD3 (γε).

所使用之晶片來自Serie-S生物素捕捉套組（Cytiva）。操作緩衝液為含有0.005% P20的pH 7.2之磷酸鹽緩衝生理食鹽水（phosphate buffer saline；PBS）。藉由三次連續注射比率為3+1之8 M鹽酸胍（GuHCl）及1 M氫氧化鈉（NaOH）之溶液使晶片再生。流動速率為20 μL/分鐘，接觸時間為120秒。用經PBS+P20以16:1稀釋的生物素捕捉試劑（CAPture reagent）活化晶片。流動速率為2 μL/分鐘持續300秒。所捕捉之量為1600反應單位（Response Unit；RU）。以10 μg/mL、以10 μL/分鐘之流動速率注射經生物素標記之pHLA持續120秒。The chip used was from the Serie-S Biotin Capture Kit (Cytiva). The operating buffer was phosphate buffer saline (PBS) at pH 7.2 containing 0.005% P20. The chip was regenerated by three consecutive injections of 8 M guanidine hydrochloride (GuHCl) and 1 M sodium hydroxide (NaOH) in a ratio of 3+1. The flow rate was 20 μL/min and the contact time was 120 sec. The chip was activated with biotin capture reagent (CAPture reagent) diluted 16:1 with PBS+P20. The flow rate was 2 μL/min for 300 sec. The amount captured was 1600 response units (RU). Biotinylated pHLA was injected at 10 μg/mL at a flow rate of 10 μL/min for 120 seconds.

對於單循環動力學分析，以60 μL/分鐘之流動速率注射mol093v9及mol093v11之連續稀釋液（最高濃度=15 nM），其中在各注射之間解離200秒，且對於第5次注射解離7200秒。For single-cycle kinetic analysis, serial dilutions of mol093v9 and mol093v11 (highest concentration = 15 nM) were injected at a flow rate of 60 μL/min with 200 sec dissociation between each injection and 7200 sec dissociation for the 5th injection.

隨後以300 nM的濃度及10 μL/分鐘的流動速率注射CD3（γε）持續60秒。CD3(γε) was then injected at a concentration of 300 nM and a flow rate of 10 μL/min for 60 seconds.

對於FcRn捕捉，最初向流量槽中添加PBS+P20 0.005% pH6.0。經生物素標記之FcRn之注射以5 μg/mL、以2 μL/分鐘之流動速率進行120秒。FcRn之注射後，在所有流量槽上以10 μL/分鐘注射5 μM生物素持續120秒。FcRn之量為450 RU。For FcRn capture, PBS + P20 0.005% pH 6.0 was initially added to the flow cell. Injection of biotinylated FcRn was performed at 5 μg/mL at a flow rate of 2 μL/min for 120 seconds. After the injection of FcRn, 5 μM biotin was injected at 10 μL/min for 120 seconds on all flow cells. The amount of FcRn was 450 RU.

mol093v9或mol093v11以15 nM、以10 μL/分鐘之流動速率注射300秒，且解離600秒。對於mol093v9及mol093v11，反應分別為152 RU及163 RU。mol093v9 or mol093v11 was injected at 15 nM, at a flow rate of 10 μL/min, for 300 sec, and dissociated for 600 sec. The responses were 152 RU and 163 RU for mol093v9 and mol093v11, respectively.

使用製造商軟體計算動力學參數。將解離相擬合至單一指數衰變方程式，使得能夠計算半衰期。由k _off/k _on計算平衡常數K _D。結果 The kinetic parameters were calculated using the manufacturer's software. The dissociation phase was fitted to a single exponential decay equation, allowing calculation of the half-life. The equilibrium constant K _D was calculated from k _off / _kon . Results

mol093v9及mol093v11展現對pHLA複合物的皮莫耳親和力以及高水平之CD3活性及與FcRn之結合。數據展示於圖3中且結合參數概述於緊接在下面之表中。 ka （ 1/Ms ） kd （ 1/s ） KD （ M ） KD （ pM ） t1/2 （ hr ） CD3 活性（ % ） FcRn 結合 mol93v9 3.55E+05 2.56E-05 7.20E-11 72.02 7.54 83.5% 是 mol93v11 2.86E+05 1.82E-05 6.38E-11 63.77 10.57 84.1% 是實施例4-藥物動力學 Mol093v9 and mol093v11 exhibited picomolar affinity for the pHLA complex as well as high levels of CD3 activity and binding to FcRn. The data are shown in Figure 3 and the binding parameters are summarized in the table immediately below. ka ( 1/Ms ) kd ( 1/s ) KD ( M ) KD ( pM ) t1/2 ( hr ) CD3 activity ( % ) FcRn binding mol93v9 3.55E+05 2.56E-05 7.20E-11 72.02 7.54 83.5% yes mol93v11 2.86E+05 1.82E-05 6.38E-11 63.77 10.57 84.1% yes Example 4 - Pharmacokinetics

在Tg32 SCID小鼠中評估藥物動力學特性。測試物品藉由IV推注以1 mg/Kg、每化合物4隻小鼠來給藥，且在21天時段內連續採集血液樣品。藉由電化學發光免疫分析以及用經生物素標記之PRAME肽-HLA進行捕捉且用經磺基標記（sulfo-tagged）之抗scFv抗體進行偵測，在血清中偵測樣品。圖4展示4隻個別小鼠之隨時間推移的血清濃度。藉由非隔室分析來提取PK參數。結果 Pharmacokinetic properties were evaluated in Tg32 SCID mice. Test articles were dosed by IV bolus at 1 mg/Kg, 4 mice per compound, and blood samples were collected serially over a 21-day period. Samples were detected in serum by electrochemical luminescence immunoassay with capture using biotinylated PRAME peptide-HLA and detection using sulfo-tagged anti-scFv antibodies. Figure 4 shows serum concentrations over time for 4 individual mice. PK parameters were extracted by non-compartmental analysis. Results

Mol93v9之終末t _1/2經計算為9天。非隔室分析之結果展示於緊接在下面之表中。分子 C _max （ ng/ml ） T _max （ min ） T _½ （ h ） T _1/2 之 SEM （ h ） AUC _inf （ h*ng/ml ） 清除率 （ ml/h/kg ） Mol093v9 21814 5 171 13.0 1515240 0.66 The terminal t1 _/2 of Mol93v9 was calculated to be 9 days. The results of the non-compartmental analysis are shown in the table immediately below. molecular _Cmax ( ng/ml ) _Tmax ( min ) T _½ ( h ) SEM of T _1/2 ( h ) AUC _inf ( h*ng/ml ) Clearance ( ml/h/kg ) Mol093v9 21814 5 171 13.0 1515240 0.66

使用NONMEM之2隔室模型化之結果展示於緊接在下面之表中。參數小鼠（ 25 g ） CL（mL/h） 0.0211 V _c（mL） 1.24 Q（mL/h） 0.0358 V _p（mL/kg） 3.60 比例誤差（%） 25.8 t _1/2 _α（hr） 13.2 t _1/2 _β（hr） 216 實施例5-試管內T細胞活化 The results of 2-compartment modeling using NONMEM are shown in the table immediately below. Parameters Mouse ( 25 g ) CL (mL/h) 0.0211 V _c (mL) 1.24 Q (mL/h) 0.0358 V _p (mL/kg) 3.60 Proportional error (%) 25.8 t _1/2 _α (hr) 13.2 t _1/2 _β (hr) 216 Example 5 - In vitro T cell activation

評估Mol093v9及Mol093v11針對呈現SLLQHLIGL-HLA-A*02複合物之細胞，介導CD3+ T細胞的強力及特異性活化的能力。干擾素-γ（IFN-γ）釋放用作T細胞活化讀取結果。方法 Mol093v9 and Mol093v11 were evaluated for their ability to mediate potent and specific activation of CD3+ T cells against cells presenting the SLLQHLIGL-HLA-A*02 complex. Interferon-γ (IFN-γ) release was used as a readout of T cell activation. Methods

根據製造商說明書使用人類IFN-γ ELISPOT套組（BD Biosciences）進行分析。簡言之，以1×10 ⁶/ml之密度在分析培養基（含有10%熱不活化FBS及1%青黴素-鏈黴素-L-麩醯胺酸之RPMI 1640）中製備目標細胞，且以50 μl之體積每孔50,000個細胞塗鋪。自新鮮供體血液分離之周邊血液單核細胞（peripheral blood mononuclear cell；PBMC）用作效應細胞，且以50 μl之體積以與目標細胞大致1:1之比率塗鋪（用於各實驗之PBMC的確切數目係供體依賴性的，且可調整以產生在適合分析範圍內的反應）。將融合分子自10 nM向下調定，以得到表示之最終濃度（跨越預期臨床上相關範圍）且以50 μl之體積添加至孔中。 The assay was performed using the human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, target cells were prepared at a density of 1×10 ⁶ /ml in assay medium (RPMI 1640 containing 10% heat-inactivated FBS and 1% penicillin-streptomycin-L-glutamine) and 50,000 cells were plated per well in a volume of 50 μl. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells and plated at an approximate 1:1 ratio to target cells in a volume of 50 μl (the exact number of PBMCs used for each experiment is donor dependent and can be adjusted to produce a response within the appropriate range for the assay). Fusion molecules were adjusted downward from 10 nM to obtain the final concentrations indicated (spanning the expected clinically relevant range) and added to the wells in a volume of 50 μl.

根據製造商說明書準備盤。將目標細胞、效應細胞及融合分子添加至相關孔中且用分析培養基補足至200 μl之最終體積。所有反應一式三份地進行。亦藉由省略融合分子製備對照孔。隨後培育盤隔夜（37℃/5% CO2）。次日，用洗滌緩衝液（1×PBS小袋，含有0.05% Tween-20，在去離子水中製得）洗滌盤三次。隨後，以50 μl之體積向各孔中添加初級偵測抗體。盤在室溫下培育2小時，隨後再次洗滌三次。藉由將50 μl經稀釋之鏈球菌親生物素蛋白-HRP添加至各孔中，且在室溫下培育1小時，且重複洗滌步驟來進行二級偵測。在使用之前不超過15分鐘，將一滴（20 μl）AEC色素原添加至各1 ml AEC受質中且混合，且將50 μl添加至各孔中。定期監測點之顯影，且在自來水中洗滌盤以終止顯影反應。使盤在室溫下乾燥至少2小時，隨後使用CTL分析儀與Immunospot軟體（Cellular Technology Limited）對點進行計數。Prepare the plates according to the manufacturer's instructions. Add target cells, effector cells and fusion molecules to the relevant wells and make up to a final volume of 200 μl with assay medium. All reactions were performed in triplicate. Control wells were also prepared by omitting the fusion molecule. The plates were then incubated overnight (37°C/5% CO2). The next day, the plates were washed three times with wash buffer (1× PBS sachet containing 0.05% Tween-20, prepared in deionized water). Primary detection antibodies were then added to each well in a volume of 50 μl. The plates were incubated for 2 hours at room temperature and then washed again three times. Secondary detection was performed by adding 50 μl of diluted streptavidin-HRP to each well and incubating for 1 hour at room temperature with repeated wash steps. No more than 15 minutes before use, one drop (20 μl) of AEC chromogen was added to each 1 ml AEC substrate and mixed, and 50 μl was added to each well. The development of the spots was monitored regularly and the plates were washed in tap water to stop the development reaction. The plates were allowed to dry at room temperature for at least 2 hours before the spots were counted using a CTL analyzer with Immunospot software (Cellular Technology Limited).

在此實施例中，以下細胞株用作目標細胞：抗原陽性： Mel624 - 人類黑色素瘤細胞株 NCI-H1755 - 非小細胞肺癌（NSCLC）細胞株 OV56 - 卵巢漿液癌細胞株 THP-1 - 急性單核球性白血病細胞株 NCI-H1703 - 肺鱗狀細胞癌細胞株 COV318 - 卵巢漿液癌細胞株抗原陰性： TY-KNU - 卵巢漿液性腺癌（HLA-A*02-ve；PRAME-ve） NCI-H1693 - 非小細胞肺癌（NSCLC）細胞株（HLA-A*02+ve；PRAME-ve）結果 In this example, the following cell lines were used as target cells: Antigen positive: Mel624 - human melanoma cell line NCI-H1755 - non-small cell lung cancer (NSCLC) cell line OV56 - ovarian plasma carcinoma cell line THP-1 - acute monocytic leukemia cell line NCI-H1703 - lung squamous cell carcinoma cell line COV318 - ovarian plasma carcinoma cell line Antigen negative: TY-KNU - ovarian plasma adenocarcinoma (HLA-A*02-ve; PRAME-ve) NCI-H1693 - non-small cell lung cancer (NSCLC) cell line (HLA-A*02+ve; PRAME-ve) Results

mol093v9及mol093v11在各種抗原陽性癌細胞存在下展現T細胞之強力活化。自數據計算且使用來自兩個個別供體之PBMC獲得EC ₅₀值。兩個供體之T細胞活化EC ₅₀值展示於緊接在下面之表中。圖5展示獲自供體1之數據。在抗原陰性細胞株中觀測到有限反應。 細胞株 Mol093v11 EC ₅₀ Mol093v9 EC ₅₀ 供體1 供體2 供體1 供體2 NCI-H1755 147.6 64.1 69.1 62.9 NCI-H1703 126.6 231.8 120.0 117.0 OV56 9.2 10.8 4.0 5.1 COV318 47.7 37.1 36.3 29.8 THP-1 27.0 19.7 18.0 14.7 比較數據 Mol093v9 and mol093v11 exhibited potent activation of T cells in the presence of various antigen-positive cancer cells. EC ₅₀ values were calculated from the data and obtained using PBMCs from two individual donors. The T cell activation EC ₅₀ values for the two donors are shown in the table immediately below. Figure 5 shows data obtained from donor 1. Limited responses were observed in antigen-negative cell lines. Cell lines Mol093v11 EC ₅₀ Mol093v9 EC ₅₀ Donor 1 Donor 2 Donor 1 Donor 2 NCI-H1755 147.6 64.1 69.1 62.9 NCI-H1703 126.6 231.8 120.0 117.0 OV56 9.2 10.8 4.0 5.1 COV318 47.7 37.1 36.3 29.8 THP-1 27.0 19.7 18.0 14.7 Comparison data

將由mol093v9驅動之T細胞活化與靶向相同PRAME肽但不包括半衰期延長Fc域之替代分子直接比較。此類分子描述於WO 2018/234319及美國專利第11,427,624號中，該等文獻中之各者的內容以引用之方式併入本文中。如上文所描述進行ELISPot分析。圖6展示兩種分子驅動類似強力的T細胞反應。實施例6-T細胞殺滅 T cell activation driven by mol093v9 was directly compared to an alternative molecule targeting the same PRAME peptide but not including a half-life extending Fc domain. Such molecules are described in WO 2018/234319 and U.S. Patent No. 11,427,624, each of which is incorporated herein by reference. ELISPot analysis was performed as described above. Figure 6 shows that both molecules drive similarly potent T cell responses. Example 6 - T Cell Killing

評估Mol093v9及Mol093v11介導抗原陽性癌細胞的強力及特異性殺滅之能力。方法 To evaluate the ability of Mol093v9 and Mol093v11 to mediate potent and specific killing of antigen-positive cancer cells.

分析使用以下進行：利用適用於阻抗讀取之96孔盤（xCELLigence E-盤96 PET部件號300600900）之xCELLigence平台，或利用CellPlayer 96孔凋亡蛋白酶-3/7細胞凋亡分析套組（Essen BioScience，目錄號4440）之Incucyte活細胞成像平台，且根據製造商說明書進行。目標細胞以各別最佳密度塗鋪（每孔添加之目標數目對於各細胞株不同，且先前已調定以確定最佳條件）且培育隔夜以允許其貼附。製備各種濃度之測試分子且將50 μl之各者添加至相關孔中以使得最終濃度在100 fM與10 nM之間。效應細胞以10:1之效應:目標細胞比率使用且以50 μl塗鋪。亦製備無融合物之對照樣品以及單獨含有效應細胞或單獨含有目標細胞之樣品。對於xCELLigence平台，使用分析培養基將盤中之最終體積調整至200 μl。使用標準化細胞指數（阻抗量測）確定細胞溶解百分比。對於Incucyte平台，以30 μM製得NucView分析試劑且將25 μl添加至每孔中，且使最終體積達到150 μl（得到5 μM最終濃度）。確定各影像中凋亡細胞之數目且以凋亡細胞數/mm2記錄。在所有情況下，分析一式三份地進行，在96小時內每2小時進行量測。結果 Assays were performed using either the xCELLigence platform using 96-well plates adapted for impedance reading (xCELLigence E-plate 96 PET part number 300600900) or the Incucyte live cell imaging platform using the CellPlayer 96-well caspase-3/7 apoptosis assay kit (Essen BioScience, catalog number 4440) and according to the manufacturer's instructions. Target cells were plated at the respective optimal density (the number of targets added per well was different for each cell line and was previously adjusted to determine the optimal conditions) and incubated overnight to allow for their attachment. Various concentrations of the test molecule were prepared and 50 μl of each was added to the relevant wells so that the final concentration was between 100 fM and 10 nM. Effector cells were used at a 10:1 effector:target cell ratio and plated at 50 μl. Control samples without fusions and samples containing effector cells alone or target cells alone were also prepared. For the xCELLigence platform, the final volume in the plate was adjusted to 200 μl using assay medium. Percent cell lysis was determined using normalized cell index (impedance measurement). For the Incucyte platform, NucView assay reagent was prepared at 30 μM and 25 μl was added to each well, bringing the final volume to 150 μl (giving a 5 μM final concentration). The number of apoptotic cells in each image was determined and recorded as apoptotic cells/mm2. In all cases, analyses were performed in triplicate, with measurements taken every 2 h for 96 h.

圖7中呈現之數據展示經使用xCELLigence平台所測定，在mol093v9及mol093v11存在下抗原陽性細胞之即時殺滅。EC ₅₀值展示於緊接在下面之表中且在低pM範圍內。偵測到之抗原陰性細胞株殺滅有限。 細胞株 Mol093v11 EC ₅₀ （ pM ） Mol093v9 EC ₅₀ （ pM ） NCI-H1755 6.2 4.1 NCI-H1703 16.7 12.0 OV56 22.3 14.2 COV318 460.2 111.8 比較數據 The data presented in Figure 7 demonstrates immediate killing of antigen-positive cells in the presence of mol093v9 and mol093v11 as determined using the xCELLigence platform. _EC50 values are shown in the table immediately below and are in the low pM range. Limited killing of antigen-negative cell lines was detected. Cell lines Mol093v11 EC ₅₀ ( pM ) Mol093v9 EC ₅₀ ( pM ) NCI-H1755 6.2 4.1 NCI-H1703 16.7 12.0 OV56 22.3 14.2 COV318 460.2 111.8 Comparison data

將由mol093v9驅動之T細胞活化與靶向相同PRAME肽但不包括半衰期延長Fc域之替代分子直接比較。此類分子描述於WO 2018/234319中。使用如上文所描述之Incucyte平台進行殺滅分析。圖8展示兩種分子驅動類似強力的殺滅反應。實施例7-針對高風險正常組織之極小反應性 T cell activation driven by mol093v9 was directly compared to an alternative molecule targeting the same PRAME peptide but without the half-life extending Fc domain. Such molecules are described in WO 2018/234319. Killing assays were performed using the Incucyte platform as described above. Figure 8 shows that both molecules drive similarly potent killing responses. Example 7 - Minimal Reactivity to High-Risk Normal Tissues

為了證明mol093v9及mol093v11之特異性，使用如上所述之相同ELISPOT方法及一組源自健康人類組織之正常細胞作為目標，進行進一步測試。正常組織包括心、肺、腎及皮膚。To demonstrate the specificity of mol093v9 and mol093v11, further testing was performed using the same ELISPOT method as described above and a panel of normal cells derived from healthy human tissues as targets. Normal tissues included heart, lung, kidney, and skin.

針對與來自健康供體之PBMC共培養的目標正常細胞批次，以介於50 pM至10 nM範圍內之六種不同濃度測試TCR-抗CD3融合分子。對照量測使用無融合分子之樣品及其中正常細胞換成NCI-H1755（抗原陽性）細胞之樣品進行。結果 The TCR-anti-CD3 fusion molecules were tested at six different concentrations ranging from 50 pM to 10 nM against batches of target normal cells co-cultured with PBMCs from healthy donors. Control measurements were performed using samples without fusion molecule and samples in which normal cells were replaced with NCI-H1755 (antigen-positive) cells.

圖9展示針對一個PBMC效應子供體用兩個正常細胞批次（心臟細胞（HCM27）及肺上皮細胞（HSAEpiC9））獲得之數據。對於至多並包括1.1 nM融合分子之mol093v9及mol093v11濃度，觀測到針對正常細胞之極小T細胞活化。 比較數據 Figure 9 shows data obtained with two normal cell batches (cardiac cells (HCM27) and lung epithelial cells (HSAEpiC9)) against one PBMC effector donor. Minimal T cell activation against normal cells was observed for mol093v9 and mol093v11 concentrations up to and including 1.1 nM fusion molecule. Comparison of Data

將mol093v9之正常細胞反應性與靶向相同PRAME肽但不包括半衰期延長Fc域之替代分子直接比較。此類分子描述於WO 2018/234319中。圖10展示兩種分子展示針對來自皮膚（黑色素細胞）及腎臟（腎近端小管）之正常細胞的類似反應性缺乏。實施例8-靶向PIWIL1肽-MHC複合物之多域單鏈結合分子展現強力及特異性T細胞活化 The normal cell reactivity of mol093v9 was directly compared to an alternative molecule targeting the same PRAME peptide but not including the half-life extending Fc domain. Such molecules are described in WO 2018/234319. Figure 10 shows that both molecules exhibit similar lack of reactivity against normal cells from the skin (melanocytes) and kidney (renal proximal tubules). Example 8 - Multi-domain single-chain binding molecules targeting the PIWIL1 peptide-MHC complex exhibit potent and specific T cell activation

設計另一多域單鏈結合分子，其包含靶向PIWIL1肽（SLSNRLYYL，SEQ ID NO: 56）-MHC複合物之pMHC結合域，而非結合PRAME肽-MHC複合物之上文所描述的mol093v9及mol093v11。稱為「a40b23U28-mol93」之所得分子之完整序列提供於SEQ ID NO: 55中。除了TCRα及TCRβ可變域之外，a40b23U28-mol93之序列與mol093v9一致。a40b23U28-mol93之TCRα可變域及TCRβ可變域分別對應於在GB申請案第2300226.4號中之SEQ ID NO: 28（「a40」）及36（「b23」）。Another multi-domain single-chain binding molecule was designed, which includes a pMHC binding domain that targets the PIWIL1 peptide (SLSNRLYYL, SEQ ID NO: 56)-MHC complex, rather than the mol093v9 and mol093v11 described above that bind the PRAME peptide-MHC complex. The complete sequence of the resulting molecule, referred to as "a40b23U28-mol93", is provided in SEQ ID NO: 55. The sequence of a40b23U28-mol93 is identical to mol093v9, except for the TCRα and TCRβ variable domains. The TCRα variable domain and TCRβ variable domain of a40b23U28-mol93 correspond to SEQ ID NOs: 28 ("a40") and 36 ("b23"), respectively, in GB application No. 2300226.4.

如以上實施例3中所描述使用SPR測定a40b23U28-mol93與SLSNRLYYL（SEQ ID NO: 56）-HLA-A*02複合物之結合。結合親和力（K _D）為50 pM且結合半衰期（t1/2）為11.8小時。此等結果與不具有半衰期延長（亦即Fc）域之等效TCR-抗CD3融合分子類似。 Binding of a40b23U28-mol93 to the SLSNRLYYL (SEQ ID NO: 56)-HLA-A*02 complex was determined using SPR as described above in Example 3. The binding affinity ( _KD ) was 50 pM and the binding half-life (t1/2) was 11.8 hours. These results are similar to equivalent TCR-anti-CD3 fusion molecules without a half-life extension (i.e., Fc) domain.

藉由使用ELISpot分析量測IFNγ分泌測定a40b23U28-mol93驅動T細胞活化之能力。根據製造商說明書使用人類IFN-γ ELISPOT套組（BD Biosciences）進行分析。自新鮮供體血液分離之周邊血液單核細胞（PBMC）用作效應細胞。在此分析中，使用KATOIII（胃癌）及CL11（結腸癌）作為抗原陽性目標細胞。NCI-H1755用作抗原陰性細胞。使用PRISM軟體來繪製數據且從曲線計算出EC ₅₀值。 The ability of a40b23U28-mol93 to drive T cell activation was determined by measuring IFNγ secretion using an ELISpot assay. The assay was performed using the human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells. KATOIII (gastric cancer) and CL11 (colon cancer) were used as antigen-positive target cells in this assay. NCI-H1755 was used as antigen-negative cells. Data were plotted using PRISM software and EC ₅₀ values were calculated from the curves.

圖11展示a40b23U28-mol93針對兩種抗原陽性細胞株產生在低pM範圍內的EC ₅₀值（KATO-III：42.1 pM，及CL11：164.0 pM），且在抗原陰性細胞存在下產生很少至無反應（在小於1 nM之a40b23U28-mol93濃度下）。不具有半衰期延長（亦即，Fc）域之等效TCR-抗CD3融合分子對於KATO-III及CL11細胞之EC ₅₀分別為12.9 pM及52.7 pM。 Figure 11 shows that a40b23U28-mol93 produced _EC50 values in the low pM range against two antigen-positive cell lines (KATO-III: 42.1 pM, and CL11: 164.0 pM), and produced little to no response in the presence of antigen-negative cells (at a40b23U28-mol93 concentrations less than 1 nM). An equivalent TCR-anti-CD3 fusion molecule without a half-life extension (i.e., Fc) domain had an _EC50 of 12.9 pM and 52.7 pM for KATO-III and CL11 cells, respectively.

此等數據表明包含靶向PIWIL1肽-MHC複合物之pMHC結合域的本發明之多域單鏈結合分子保持與不具有半衰期延長（亦即，Fc）域之等效TCR-抗CD3融合分子類似的高親和力及效能，且保持對抗原陽性細胞之特異性。These data indicate that the multi-domain single-chain binding molecules of the present invention comprising a pMHC binding domain that targets the PIWIL1 peptide-MHC complex retain high affinity and potency similar to equivalent TCR-anti-CD3 fusion molecules without a half-life extension (i.e., Fc) domain, and retain specificity for antigen-positive cells.

在進一步實驗中，將a40b23U28-mol93與稱為a40b23U28-mol14之替代多域分子形式進行比較。除了以如圖12中所描繪之雙鏈形式配置以外，a40b23U28-mol14具有與a40b23U28-mol93相同的個別域胺基酸序列。在此雙鏈形式中，第一鏈（圖12中之左側鏈）在N端至C端方向上包含TCR α鏈可變域、TCR α鏈恆定域及Fc區。第二鏈（圖12中之右側鏈）在N端至C端方向上包含抗CD3 scFv、TCR β鏈可變域、TCR β鏈恆定域及Fc區。在兩個分子之間比較針對KATOIII細胞株之T細胞活化。如圖12中所示，兩種分子均驅動T細胞活化；然而，Mol93比Mol14提供更強力反應。In further experiments, a40b23U28-mol93 was compared to an alternative multi-domain molecule format, called a40b23U28-mol14. A40b23U28-mol14 has the same individual domain amino acid sequences as a40b23U28-mol93, except that it is configured in a two-chain format as depicted in Figure 12. In this two-chain format, the first chain (the left chain in Figure 12) contains the TCR α chain variable domain, the TCR α chain constant domain, and the Fc region in the N-terminal to C-terminal direction. The second chain (the right chain in Figure 12) contains the anti-CD3 scFv, the TCR β chain variable domain, the TCR β chain constant domain, and the Fc region in the N-terminal to C-terminal direction. T cell activation against the KATOIII cell line was compared between the two molecules. As shown in Figure 12, both molecules drove T cell activation; however, Mol93 provided a more potent response than Mol14.

此實施例中所描述之結果展現，結合PIWIL1肽-MHC複合物之TCR可變域可併入本發明之多域單鏈結合分子中。換言之，無關於所用特定TCR序列（亦即靶向PRAME肽-MHC或PIWIL1肽-MHC），多域單鏈形式在延長半衰期方面有效（經由提供Fc域）而不顯著影響對目標之親和力或T細胞活化之效能。The results described in this example demonstrate that TCR variable domains that bind to the PIWIL1 peptide-MHC complex can be incorporated into the multi-domain single-chain binding molecules of the present invention. In other words, regardless of the specific TCR sequence used (i.e., targeting PRAME peptide-MHC or PIWIL1 peptide-MHC), the multi-domain single-chain format is effective in extending half-life (via provision of the Fc domain) without significantly affecting affinity for the target or potency of T cell activation.

無without

[圖1]為本發明之例示性多域單鏈結合分子之示意圖。圖1a展示自N端至C端的域配置之圖示，且圖1b展示分子摺疊結構之假設圖示。 [圖2]展示使用IFNγ作為T細胞活化之讀取結果的ELISpot分析之結果。作為比較，使用WO 2020/157211中先前揭示之形式，用相同的TCR來構築多域分子，且與圖1中呈現之單鏈形式並行測試。各形式之示意性圖示置於圖2中以指示對應數據點。 [圖3]展示表面電漿子共振實驗之圖式，該等實驗評估mol093v9及mol093v11與pHLA、CD3及FcRn中之各者的結合。 [圖4]展示在Tg32 SCID小鼠中評估之藥物動力學特性。小鼠藉由IV推注以1 mg/Kg給藥，且在21天時段內連續取樣血液。樣品在血清中藉由電化學發光免疫分析進行偵測。圖式展示4隻個別小鼠之隨時間推移的血清濃度。 [圖5]呈現展示ELISPot分析之結果的圖式，該等分析中試管內評估mol093v9及mol093v11之T細胞活化。 [圖6]呈現展示ELISPot分析之結果的圖式，該等分析中將mol093v9之T細胞活化與靶向相同PRAME肽但不包括半衰期延長Fc域之替代分子（WO 2018/234319）直接比較。圖6展示兩種分子驅動類似強力的T細胞反應。 [圖7]展示展現經使用xCELLigence平台所測定，在mol093v9及mol093v11存在下抗原陽性細胞之即時殺滅的圖式。 [圖8]呈現展示T細胞殺滅分析之結果的圖式，該等分析中將mol093v9與靶向相同PRAME肽但不包括半衰期延長Fc域之替代分子（WO 2018/234319）直接比較。圖8展示mol093v9展現與非HLE形式之分子（「Mol001」）類似的殺滅數據。 [圖9]展示針對一個PBMC效應子供體用兩個正常細胞批次（心臟細胞（HCM27）及肺上皮細胞（HSAEpiC9））獲得之ELISPOT T細胞活化分析之數據。對於至多並包括1.1 nM融合分子之mol093v9及mol093v11濃度，觀測到針對正常細胞之極小T細胞活化。 [圖10]呈現展示ELISPOT T細胞活化分析之結果的圖式，該等分析中將mol093v9之正常細胞反應性與靶向相同PRAME肽但不包括半衰期延長Fc域之替代分子（WO 2018/234319）直接比較。圖10展示兩種分子展示針對來自皮膚（黑色素細胞）及腎臟（腎近端小管）之正常細胞的類似反應性缺乏。 [圖11]為展示在抗原陽性及抗原陰性癌細胞株存在下，TCR抗CD3融合分子a40b23U28-mol93之藉由IFNγ釋放所量測之T細胞活化的圖式。 [圖12]為展示在抗原陽性細胞存在下，半衰期延長之TCR抗CD3融合分子a40b23U28-mol93及a40b23U28-mol14之藉由IFNγ釋放所量測之T細胞活化的圖式。 [FIG. 1] is a schematic diagram of an exemplary multi-domain single-chain binding molecule of the present invention. FIG. 1a shows a diagram of the domain configuration from the N-terminus to the C-terminus, and FIG. 1b shows a hypothetical diagram of the folded structure of the molecule. [FIG. 2] shows the results of an ELISpot analysis using IFNγ as a readout of T cell activation. For comparison, a multi-domain molecule was constructed using the same TCR using the format previously disclosed in WO 2020/157211 and tested in parallel with the single-chain format presented in FIG. 1. Schematic diagrams of each format are placed in FIG. 2 to indicate the corresponding data points. [FIG. 3] shows a diagram of surface plasmon resonance experiments evaluating the binding of mol093v9 and mol093v11 to each of pHLA, CD3 and FcRn. [Figure 4] Pharmacokinetic properties evaluated in Tg32 SCID mice are shown. Mice were dosed at 1 mg/Kg by IV bolus and blood was sampled continuously over a 21 day period. Samples were detected in serum by electrochemical luminescent immunoassay. Graphs show serum concentrations over time for 4 individual mice. [Figure 5] Graphs showing the results of ELISPot assays in which T cell activation of mol093v9 and mol093v11 was evaluated in vitro are presented. [Figure 6] Graphs showing the results of ELISPot assays in which T cell activation of mol093v9 was directly compared to an alternative molecule targeting the same PRAME peptide but not including a half-life extending Fc domain (WO 2018/234319) are presented. Figure 6 shows that both molecules drive similarly potent T cell responses. [Figure 7] Shows graphs showing immediate killing of antigen-positive cells in the presence of mol093v9 and mol093v11 as measured using the xCELLigence platform. [Figure 8] Presents graphs showing the results of T cell killing assays in which mol093v9 was directly compared to an alternative molecule targeting the same PRAME peptide but not including a half-life extending Fc domain (WO 2018/234319). Figure 8 shows that mol093v9 exhibits similar killing data to the non-HLE form of the molecule ("Mol001"). [Figure 9] shows data from an ELISPOT T cell activation assay obtained with two normal cell batches (cardiac cells (HCM27) and lung epithelial cells (HSAEpiC9)) against one PBMC effector donor. Minimal T cell activation against normal cells was observed for mol093v9 and mol093v11 concentrations up to and including 1.1 nM fusion molecule. [Figure 10] presents a graph showing the results of an ELISPOT T cell activation assay in which the normal cell reactivity of mol093v9 was directly compared to an alternative molecule targeting the same PRAME peptide but not including a half-life extending Fc domain (WO 2018/234319). Figure 10 shows that both molecules exhibit similar lack of reactivity against normal cells from the skin (melanocytes) and kidney (renal proximal tubules). [Figure 11] is a graph showing T cell activation measured by IFNγ release of the TCR anti-CD3 fusion molecule a40b23U28-mol93 in the presence of antigen-positive and antigen-negative cancer cell lines. [Figure 12] is a graph showing T cell activation measured by IFNγ release of the half-life extended TCR anti-CD3 fusion molecules a40b23U28-mol93 and a40b23U28-mol14 in the presence of antigen-positive cells.

TW202413410A_112131156_SEQL.xmlTW202413410A_112131156_SEQL.xml

Claims

一種多域單鏈結合分子，其包含： i）肽-主要組織相容性複合物（peptide-major histocompatibility complex；pMHC）結合域，其包含連接至恆定區之第一可變區（VC1）及連接至恆定區之第二可變區（VC2），其中VC1與VC2二聚形成該pMHC結合域； ii）免疫細胞接合（immune cell engaging；ICE）域；及 iii）半衰期延長域，其包含第一IgG Fc區（FC1）及第二IgG Fc區（FC2），其中該FC1區與該FC2區二聚形成Fc域；其中該ICE域連接至VC1之N端，VC1經由其C端連接至該FC1區之N端，該FC1區經由其C端連接至VC2之N端，且VC2經由其C端連接至該FC2區之N端；及其中該pMHC結合域及該T細胞接合免疫效應子域分別能夠結合pMHC複合物及免疫細胞。 A multi-domain single-chain binding molecule comprising: i) a peptide-major histocompatibility complex (pMHC) binding domain comprising a first variable region (VC1) connected to a constant region and a second variable region (VC2) connected to a constant region, wherein VC1 and VC2 dimerize to form the pMHC binding domain; ii) an immune cell engaging (ICE) domain; and iii) a half-life extension domain comprising a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region and the FC2 region dimerize to form an Fc domain; wherein the ICE domain is connected to the N-terminus of VC1, VC1 is connected to the N-terminus of the FC1 region via its C-terminus, the FC1 region is connected to the N-terminus of VC2 via its C-terminus, and VC2 is connected to the N-terminus of the FC2 region via its C-terminus; and The pMHC binding domain and the T cell-engaging immune effector domain are capable of binding to pMHC complexes and immune cells, respectively.

如請求項1之多域結合分子，其中該免疫細胞接合域包含： i）抗體輕鏈可變區（ICE-VL）及抗體重鏈可變區（ICE-VH）；或 ii）單域抗體（ICE-SD）。 A multi-domain binding molecule as claimed in claim 1, wherein the immune cell binding domain comprises: i) an antibody light chain variable region (ICE-VL) and an antibody heavy chain variable region (ICE-VH); or ii) a single domain antibody (ICE-SD).

如請求項1或請求項2之多域結合分子，其中該免疫細胞為T細胞且該免疫細胞接合域選自： i）T細胞接合免疫效應子域；或 ii）T細胞接合免疫抑制子。 A multi-domain binding molecule as claimed in claim 1 or claim 2, wherein the immune cell is a T cell and the immune cell binding domain is selected from: i) a T cell binding immune effector domain; or ii) a T cell binding immune suppressor.

一種多域單鏈結合分子，其包含： i）肽-主要組織相容性複合物（pMHC）結合域，其包含連接至恆定區之第一可變區（VC1）及連接至恆定區之第二可變區（VC2），其中VC1與VC2二聚形成該pMHC結合域； ii）T細胞接合（T cell engaging）免疫效應子域，其包含抗體輕鏈可變區（TCE-VL）及抗體重鏈可變區（TCE-VH）；及 iii）半衰期延長域，其包含第一IgG Fc區（FC1）及第二IgG Fc區（FC2），其中該FC1區與該FC2區二聚形成Fc域；其中該T細胞接合免疫效應子域連接至VC1之N端，VC1經由其C端連接至該FC1區之N端，該FC1區經由其C端連接至VC2之N端，且VC2經由其C端連接至該FC2區之N端；及其中該pMHC結合域及該T細胞接合免疫效應子域分別能夠結合pMHC複合物及T細胞。 A multi-domain single-chain binding molecule comprising: i) a peptide-major histocompatibility complex (pMHC) binding domain comprising a first variable region (VC1) connected to a constant region and a second variable region (VC2) connected to a constant region, wherein VC1 and VC2 dimerize to form the pMHC binding domain; ii) a T cell engaging (T cell engaging) immune effector subdomain comprising an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH); and iii) a half-life extension domain comprising a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region and the FC2 region dimerize to form an Fc domain; wherein the T cell-engaging immune effector domain is connected to the N-terminus of VC1, VC1 is connected to the N-terminus of the FC1 region via its C-terminus, the FC1 region is connected to the N-terminus of VC2 via its C-terminus, and VC2 is connected to the N-terminus of the FC2 region via its C-terminus; and wherein the pMHC binding domain and the T cell-engaging immune effector domain are capable of binding to a pMHC complex and a T cell, respectively.

如請求項1之多域結合分子，其中該T細胞接合免疫效應子為ScFv。The multi-domain binding molecule of claim 1, wherein the T cell engaging immune effector is ScFv.

如前述請求項中任一項之多域結合分子，其中（i）VC1包含（a）TCRα可變區及恆定區或（b）TCRβ可變區及恆定區，及（ii）VC2包含（a）及（b）中之另一者。 A multi-domain binding molecule as claimed in any of the preceding claims, wherein (i) VC1 comprises (a) a TCRα variable region and a constant region or (b) a TCRβ variable region and a constant region, and (ii) VC2 comprises the other of (a) and (b).

如請求項6之多域結合分子，其中VC1包含該TCRβ可變區及恆定區且VC2包含該TCRα可變區及恆定區。A multi-domain binding molecule as claimed in claim 6, wherein VC1 comprises the TCRβ variable region and the constant region and VC2 comprises the TCRα variable region and the constant region.

如請求項1至5中任一項之多域結合分子，其中（i）VC1包含（a）重鏈抗體可變區或（b）輕鏈抗體可變區，及（ii）VC2包含（a）及（b）中之另一者。 A multi-domain binding molecule as claimed in any one of claims 1 to 5, wherein (i) VC1 comprises (a) a heavy chain antibody variable region or (b) a light chain antibody variable region, and (ii) VC2 comprises the other of (a) and (b).

如請求項4之或附屬於請求項4時如請求項5至8中任一項之多域結合分子，其中該TCE-VL區經由其C端連接至該TCE-VH區之N端且該TCE-VH區經由其C端連接至VC1之N端。A multi-domain binding molecule as claimed in claim 4 or as any one of claims 5 to 8 when appended to claim 4, wherein the TCE-VL region is linked via its C-terminus to the N-terminus of the TCE-VH region and the TCE-VH region is linked via its C-terminus to the N-terminus of VC1.

如前述請求項中任一項之多域結合分子，其中該免疫細胞接合域或該T細胞接合免疫效應子域為經由與CD3及/或TCR/CD3複合物之交互作用活化T細胞之CD3效應子域。A multi-domain binding molecule as claimed in any of the preceding claims, wherein the immune cell engaging domain or the T cell engaging immune effector domain is a CD3 effector domain that activates T cells through interaction with CD3 and/or TCR/CD3 complex.

如前述請求項中任一項之多域結合分子，其中該免疫細胞接合域或該T細胞接合免疫效應子域為抗CD3 scFv。The multi-domain binding molecule of any of the preceding claims, wherein the immune cell engaging domain or the T cell engaging immune effector domain is an anti-CD3 scFv.

如前述請求項中任一項之多域結合分子，其中該pMHC結合域結合與MHC複合之腫瘤相關抗原肽。A multi-domain binding molecule as claimed in any preceding claim, wherein the pMHC binding domain binds a tumor associated antigenic peptide complexed with MHC.

如前述請求項中任一項之多域結合分子，其中該ICE、該TCE-VH、該TCE-VL、該VC1、該VC2、該FC1及該FC2區中之兩者或更多者經由連接子及/或IgG鉸鏈序列彼此連接。A multi-domain binding molecule as in any of the preceding claims, wherein two or more of the ICE, the TCE-VH, the TCE-VL, the VC1, the VC2, the FC1 and the FC2 regions are linked to each other via a linker and/or an IgG hinge sequence.

如請求項13之多域結合分子，其中該連接子或該等連接子具有選自以下之群之序列：GGGGS（SEQ ID NO: 18）、GGGSG（SEQ ID NO: 20）、GGSGG（SEQ ID NO: 21）、GSGGG（SEQ ID NO: 22）、GSGGGP（SEQ ID NO: 23）、GGEPS（SEQ ID NO: 24）、GGEGGGP（SEQ ID NO: 25）、GGEGGGSEGGGS（SEQ ID NO: 26）、GGGSGGGG（SEQ ID NO: 47）、GGGGSGGGGSGGGGSGGGGSGGGS（SEQ ID NO: 39）、GGGGSGGGGSGGGGS（SEQ ID NO: 49）、EAAAK（SEQ ID NO: 50）及EAAAKEAAAKEAAAK（SEQ ID NO: 51）。The multi-domain binding molecule of claim 13, wherein the linker or the linkers have a sequence selected from the following group: GGGGS (SEQ ID NO: 18), GGGSG (SEQ ID NO: 20), GGSGG (SEQ ID NO: 21), GSGGG (SEQ ID NO: 22), GSGGGP (SEQ ID NO: 23), GGEPS (SEQ ID NO: 24), GGEGGGP (SEQ ID NO: 25), GGEGGGSEGGGS (SEQ ID NO: 26), GGGSGGGG (SEQ ID NO: 47), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 39), GGGGSGGGGSGGGGS (SEQ ID NO: 49), EAAAK (SEQ ID NO: 50) and EAAAKEAAAKEAAAK (SEQ ID NO: 51).

如前述請求項中任一項之多域結合分子，其中VC1經由包含IgG鉸鏈序列之序列連接至該FC1區及/或VC2經由包含IgG鉸鏈序列之序列連接至該FC2區。A multi-domain binding molecule as claimed in any preceding claim, wherein VC1 is linked to the FC1 region via a sequence comprising an IgG hinge sequence and/or VC2 is linked to the FC2 region via a sequence comprising an IgG hinge sequence.

如請求項15之多域結合分子，其中該IgG鉸鏈序列與SEQ ID NO: 44至少80%一致。A multi-domain binding molecule as claimed in claim 15, wherein the IgG hinge sequence is at least 80% identical to SEQ ID NO: 44.

如請求項15或請求項16之多域結合分子，其中將VC1連接至該FC1區之序列進一步包含甘胺酸-絲胺酸連接子及/或將VC2連接至該FC2區之序列進一步包含甘胺酸-絲胺酸連接子。The multi-domain binding molecule of claim 15 or claim 16, wherein the sequence linking VC1 to the FC1 region further comprises a glycine-serine linker and/or the sequence linking VC2 to the FC2 region further comprises a glycine-serine linker.

如請求項17之多域結合分子，其中該甘胺酸-絲胺酸連接子具有SEQ ID NO: 47中提供之序列。A multi-domain binding molecule as claimed in claim 17, wherein the glycine-serine linker has the sequence provided in SEQ ID NO: 47.

如請求項4之或附屬於請求項4時如請求項5至18中任一項之多域結合分子，其中該TCE-VL區經由包含甘胺酸-絲胺酸連接子之序列連接至該TCE-VH區，視情況其中該序列提供於SEQ ID NO: 39中。A multi-domain binding molecule as claimed in claim 4 or as claimed in any one of claims 5 to 18 when appended to claim 4, wherein the TCE-VL region is linked to the TCE-VH region via a sequence comprising a glycine-serine linker, optionally wherein the sequence is provided in SEQ ID NO: 39.

如請求項4之或附屬於請求項4時如請求項5至19中任一項之多域結合分子，其中該TCE-VH區經由包含甘胺酸-絲胺酸連接子之序列連接至VC1，視情況其中該序列提供於SEQ ID NO: 18中。A multi-domain binding molecule as claimed in claim 4 or as claimed in any one of claims 5 to 19 when appended to claim 4, wherein the TCE-VH region is linked to VC1 via a sequence comprising a glycine-serine linker, optionally wherein the sequence is provided in SEQ ID NO: 18.

如前述請求項中任一項之多域結合分子，其中該FC1區經由包含甘胺酸-絲胺酸連接子之序列連接至VC2，視情況其中該序列提供於SEQ ID NO: 47中。A multi-domain binding molecule as claimed in any of the preceding claims, wherein the FC1 region is linked to VC2 via a sequence comprising a glycine-serine linker, optionally wherein the sequence is provided in SEQ ID NO: 47.

如前述請求項中任一項之多域結合分子，其中該半衰期延長域包含促進該FC1區與該FC2區之二聚之一或多個胺基酸取代。The multi-domain binding molecule of any of the preceding claims, wherein the half-life extension domain comprises one or more amino acid substitutions that promote dimerization of the FC1 region and the FC2 region.

如請求項22之多域結合分子，其中：（i）該FC1區及該FC2區中之一者包含一或多個根據EU編號方案之胺基酸取代，該一或多個胺基酸取代選自由T366S、L368A、T394S、F405A、Y407A、Y407T及Y407V組成之群；及（ii）該FC1區及該FC2區中之另一者包含一或多個根據EU編號方案之胺基酸取代，該一或多個胺基酸取代選自由T366W、T366Y、T366W、T394W及F405W組成之群。 A multi-domain binding molecule as claimed in claim 22, wherein: (i) one of the FC1 region and the FC2 region comprises one or more amino acid substitutions according to the EU numbering scheme, the one or more amino acid substitutions being selected from the group consisting of T366S, L368A, T394S, F405A, Y407A, Y407T and Y407V; and (ii) the other of the FC1 region and the FC2 region comprises one or more amino acid substitutions according to the EU numbering scheme, the one or more amino acid substitutions being selected from the group consisting of T366W, T366Y, T366W, T394W and F405W.

如請求項22之多域結合分子，其中：（i）該FC1區及該FC2區中之一者包含一或多個根據EU編號方案之胺基酸取代，該一或多個胺基酸取代選自由T366S、L368A及Y407V組成之群；及（i）該FC1區及該FC2區中之另一者包含根據EU編號方案之T366W胺基酸取代。 A multi-domain binding molecule as claimed in claim 22, wherein: (i) one of the FC1 region and the FC2 region comprises one or more amino acid substitutions according to the EU numbering scheme, the one or more amino acid substitutions being selected from the group consisting of T366S, L368A and Y407V; and (i) the other of the FC1 region and the FC2 region comprises a T366W amino acid substitution according to the EU numbering scheme.

如前述請求項中任一項之多域結合分子，其中該半衰期延長域包含減弱該Fc域之效應功能的一或多個胺基酸取代。A multi-domain binding molecule as claimed in any of the preceding claims, wherein the half-life extension domain comprises one or more amino acid substitutions that reduce the effector function of the Fc domain.

如請求項25之多域結合分子，其中該半衰期延長域包含一或多個根據EU編號方案之胺基酸取代，該一或多個胺基酸取代選自由S228P、E233P、L234A、L235A、L235E、L235P、G236R、G237A、P238S、F241A、V264A D265A、H268A、D270A、N297A、N297G、N297Q、E318A、K322A、L328R、P329G、P329A、A330S、A330L、P331A及P331S組成之群。A multi-domain binding molecule as claimed in claim 25, wherein the half-life extension domain comprises one or more amino acid substitutions according to the EU numbering scheme, and the one or more amino acid substitutions are selected from the group consisting of S228P, E233P, L234A, L235A, L235E, L235P, G236R, G237A, P238S, F241A, V264A D265A, H268A, D270A, N297A, N297G, N297Q, E318A, K322A, L328R, P329G, P329A, A330S, A330L, P331A and P331S.

如請求項25或請求項26之多域結合分子，其中該半衰期延長域包含阻止或減少與FcγR之結合之一或多個胺基酸取代。The multi-domain binding molecule of claim 25 or claim 26, wherein the half-life extension domain comprises one or more amino acid substitutions that prevent or reduce binding to FcγR.

如請求項27之多域結合分子，其中該FC1區及/或該FC2區包含根據EU編號方案之N297G胺基酸取代。A multi-domain binding molecule as claimed in claim 27, wherein the FC1 region and/or the FC2 region comprises an N297G amino acid substitution according to the EU numbering scheme.

如前述請求項中任一項之多域結合分子，其中該半衰期延長域包含促進與FcRn之結合之一或多個胺基酸取代。The multi-domain binding molecule of any of the preceding claims, wherein the half-life extension domain comprises one or more amino acid substitutions that promote binding to FcRn.

如請求項29之多域結合分子，其中該FC1區及/或該FC2區包含根據EU編號方案編號之M252Y、S254T及T256E胺基酸取代。A multi-domain binding molecule as claimed in claim 29, wherein the FC1 region and/or the FC2 region comprises M252Y, S254T and T256E amino acid substitutions numbered according to the EU numbering scheme.

如請求項4之或附屬於請求項4時如請求項5至30中任一項之多域結合分子，其中該T細胞接合免疫效應子域包含：（i） VL區，其包含SEQ ID NO: 33、34及35分別作為CDR1、CDR2及CDR3之CDR；及（ii） VH區，其包含SEQ ID NO: 36、37及38分別作為CDR1、CDR2及CDR3之CDR。 A multi-domain binding molecule as claimed in claim 4 or as claimed in any one of claims 5 to 30 when appended to claim 4, wherein the T cell engaging immune effector subdomain comprises: (i) a VL region comprising CDRs of SEQ ID NOs: 33, 34 and 35 as CDR1, CDR2 and CDR3, respectively; and (ii) a VH region comprising CDRs of SEQ ID NOs: 36, 37 and 38 as CDR1, CDR2 and CDR3, respectively.

如請求項4之或附屬於請求項4時如請求項5至31中任一項之多域結合分子，其中該T細胞接合免疫效應子域包含與SEQ ID NO: 31之序列至少80%一致之VL區及與SEQ ID NO: 32之序列至少80%一致之VH區。A multi-domain binding molecule as claimed in claim 4 or as claimed in any one of claims 5 to 31 when appended to claim 4, wherein the T cell engaging immune effector domain comprises a VL region that is at least 80% identical to the sequence of SEQ ID NO: 31 and a VH region that is at least 80% identical to the sequence of SEQ ID NO: 32.

如前述請求項中任一項之多域結合分子，其中該FC1區及該FC2區為IgG1 Fc區。The multi-domain binding molecule of any preceding claim, wherein the FC1 region and the FC2 region are IgG1 Fc regions.

如前述請求項中任一項之多域結合分子，其中： i）該FC1區包含與SEQ ID NO: 42之序列至少80%一致之胺基酸序列，且該FC2區包含與SEQ ID NO: 43之序列至少80%一致之胺基酸序列；或 ii）該FC1區包含與SEQ ID NO: 57之序列至少80%一致之胺基酸序列，且該FC2區包含與SEQ ID NO: 58之序列至少80%一致之胺基酸序列。 A multi-domain binding molecule as claimed in any of the preceding claims, wherein: i) the FC1 region comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 42, and the FC2 region comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 43; or ii) the FC1 region comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 57, and the FC2 region comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 58.

如前述請求項中任一項之多域結合分子，其自N端至C端按以下次序包含以下胺基酸序列： a）抗CD3 scFv之胺基酸序列，視情況接著係提供於SEQ ID NO: 18中之連接子序列； b） TCRβ可變區及恆定區之胺基酸序列； c）提供於SEQ ID NO: 47中之連接子序列，接著係提供於SEQ ID NO: 44中之IgG鉸鏈序列； d）具有提供於SEQ ID NO: 42中之序列的Fc區； e）提供於SEQ ID NO: 47中之連接子序列； f） TCRα可變區及恆定區之胺基酸序列； g）提供於SEQ ID NO: 47中之連接子序列，接著係提供於SEQ ID NO: 44中之IgG鉸鏈序列；及 h）具有提供於SEQ ID NO: 43中之序列的Fc區。 A multi-domain binding molecule as claimed in any of the preceding claims, comprising the following amino acid sequences in the following order from N-terminus to C-terminus: a) the amino acid sequence of an anti-CD3 scFv, optionally followed by a linker sequence provided in SEQ ID NO: 18; b) the amino acid sequences of a TCRβ variable region and a constant region; c) the linker sequence provided in SEQ ID NO: 47, followed by an IgG hinge sequence provided in SEQ ID NO: 44; d) an Fc region having a sequence provided in SEQ ID NO: 42; e) the linker sequence provided in SEQ ID NO: 47; f) the amino acid sequences of a TCRα variable region and a constant region; g) the linker sequence provided in SEQ ID NO: 47, followed by an IgG hinge sequence provided in SEQ ID NO: 44; and h) An Fc region having the sequence provided in SEQ ID NO: 43.

一種核酸，其編碼如前述請求項中任一項之多域結合分子。A nucleic acid encoding a multi-domain binding molecule as claimed in any one of the preceding claims.

一種表現載體，其包含如請求項36之核酸。An expression vector comprising the nucleic acid of claim 36.

一種宿主細胞，其包含如請求項36之核酸或如請求項37之表現載體。A host cell comprising the nucleic acid of claim 36 or the expression vector of claim 37.

一種製造如請求項1至35中任一項之多域結合分子的方法，其包含將如請求項38之宿主細胞維持於最佳條件下，該等最佳條件係用於表現如請求項36之核酸或如請求項37之表現載體的最佳條件，且分離該多域結合分子。A method for producing a multi-domain binding molecule as described in any one of claims 1 to 35, comprising maintaining a host cell as described in claim 38 under optimal conditions, which are optimal conditions for expressing a nucleic acid as described in claim 36 or an expression vector as described in claim 37, and isolating the multi-domain binding molecule.

一種醫藥組成物，其包含如請求項1至35中任一項之多域結合分子、如請求項36之核酸、如請求項37之表現載體或如請求項38之宿主細胞。A pharmaceutical composition comprising the multi-domain binding molecule of any one of claims 1 to 35, the nucleic acid of claim 36, the expression vector of claim 37, or the host cell of claim 38.

一種如請求項1至35中任一項之多域結合分子、如請求項36之核酸、如請求項37之表現載體、如請求項38之宿主細胞或如請求項40之醫藥組成物，其用作藥劑。A multi-domain binding molecule as claimed in any one of claims 1 to 35, a nucleic acid as claimed in claim 36, an expression vector as claimed in claim 37, a host cell as claimed in claim 38 or a pharmaceutical composition as claimed in claim 40, for use as a medicament.

一種如請求項1至35中任一項之多域結合分子、如請求項36之核酸、如請求項37之表現載體、如請求項38之宿主細胞或如請求項40之醫藥組成物，其用於治療癌症、自體免疫疾病或感染性疾病。A multi-domain binding molecule as described in any one of claims 1 to 35, a nucleic acid as described in claim 36, an expression vector as described in claim 37, a host cell as described in claim 38, or a pharmaceutical composition as described in claim 40, for use in treating cancer, autoimmune diseases, or infectious diseases.

如請求項43之多域結合分子、核酸、表現載體、宿主細胞或醫藥組成物，其中該癌症與PRAME表現相關。The multi-domain binding molecule, nucleic acid, expression vector, host cell or pharmaceutical composition of claim 43, wherein the cancer is associated with PRAME expression.