TW202402803A - Anti-ctla4 antibodies and methods of making and using the same - Google Patents

Anti-ctla4 antibodies and methods of making and using the same Download PDF

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TW202402803A
TW202402803A TW112135939A TW112135939A TW202402803A TW 202402803 A TW202402803 A TW 202402803A TW 112135939 A TW112135939 A TW 112135939A TW 112135939 A TW112135939 A TW 112135939A TW 202402803 A TW202402803 A TW 202402803A
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antibody
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amino acid
acid sequence
hvr
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培志 羅
杜方勇
忠宗 潘
劉桂中
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英屬開曼群島商天演藥業有限公司
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Abstract

Provided herein are cross-reactive antibodies (or antigen binding fragments thereof) that bind to human CTLA4, activatable antibodies that bind to human CTLA4, nucleic acid molecules encoding the same, pharmaceutical compositions thereof, and methods of their therapeutic use (e.g., for treatment of cancer).

Description

抗CTLA4抗體及其製備及使用方法Anti-CTLA4 antibodies and methods of preparation and use thereof

本發明係關於結合至人類細胞毒性T-淋巴細胞蛋白4 (CTLA4)之交叉反應性抗體、結合至人類CTLA4之精度/背景依賴性可活化抗體、編碼該等抗體之核酸、其醫藥組合物及其治療用途。The present invention relates to cross-reactive antibodies that bind to human cytotoxic T-lymphocyte protein 4 (CTLA4), precision/context-dependent activatable antibodies that bind to human CTLA4, nucleic acids encoding such antibodies, pharmaceutical compositions thereof, and Its therapeutic uses.

CTLA4為用於下調T-細胞活化且維持免疫原體內穩態之蛋白質之免疫球蛋白(Ig)超家族之成員。已顯示活體內抗體介導之CTLA4之阻斷增強同源鼠科前列腺癌模型中之抗癌免疫反應(Kwon等人(1997) Proc Natl Acad Sci USA, 94(15):8099-103)。此外,顯示CTLA4功能之阻斷在荷瘤小鼠之腫瘤生長之各階段增強抗腫瘤T細胞反應(Yang等人(1997) Cancer Res 57(18):4036-41;Hurwitz等人(1998) Proc Natl Acad Sci USA 95 (17):10067-7)。然而,適於人類使用之基於抗體之療法之開發仍困難,因為自臨床前動物模型至人類安全性之轉變經常係差的。因此,存在對在不同物種(諸如人類及實驗動物(例如,小鼠、猴、大鼠等))間具交叉反應性以使能同時進行動物模型研究且提供適宜人類治療候選之抗CTLA4抗體之需要。此外,存在對開發更安全抗CTLA4抗體之需要,該等抗體僅於某些情況下(諸如於蛋白酶濃化腫瘤微環境中)具活性。CTLA4 is a member of the immunoglobulin (Ig) superfamily of proteins used to downregulate T-cell activation and maintain immunogen homeostasis. Antibody-mediated blockade of CTLA4 has been shown to enhance anti-cancer immune responses in a syngeneic murine prostate cancer model in vivo (Kwon et al. (1997) Proc Natl Acad Sci USA, 94(15):8099-103). Furthermore, blockade of CTLA4 function was shown to enhance anti-tumor T cell responses at all stages of tumor growth in tumor-bearing mice (Yang et al. (1997) Cancer Res 57(18):4036-41; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-7). However, the development of antibody-based therapies suitable for human use remains difficult because translation of safety from preclinical animal models to humans is often poor. Therefore, there is a need for anti-CTLA4 antibodies that are cross-reactive between different species, such as humans and experimental animals (e.g., mice, monkeys, rats, etc.) to enable simultaneous animal model studies and provide suitable human therapeutic candidates. need. Furthermore, there is a need to develop safer anti-CTLA4 antibodies that are active only under certain circumstances, such as in the protease-concentrated tumor microenvironment.

本文中所引用之所有參考文獻(包括專利申請案、專利公開案、非專利文獻及UniProtKB/Swiss-Prot/GenBank寄存編號)之全文以引用的方式併入本文中,如同各個別參考文獻明確且個別地指示為以引用的方式併入般。The entire text of all references cited herein (including patent applications, patent publications, non-patent documents, and UniProtKB/Swiss-Prot/GenBank accession numbers) are incorporated by reference in their entirety as if each individual reference was expressly and Individually indicated as being incorporated by reference.

為滿足以上及其他需要,本文中揭示結合至人類CTLA4之抗體(例如,交叉反應性抗體)及其抗原結合片段。本發明之抗CTLA4抗體或其抗原結合片段具有下列功能性質中之至少一者(例如,一者、一些或所有):(a) 以500 nM或更低之K D結合至人類、食蟹獼猴、小鼠、大鼠及/或狗CTLA4,(b)具有對人類CTLA4之拮抗活性;(c)在多達100 nM之濃度下不結合至人類PD-1、PD-L1、PD-L2、LAG3、TIM3、B7-H3、CD95、CD120a、OX40、CD40、BTLA、VISTA、ICOS、及/或B7-H4;(d)與猴、小鼠、大鼠及/或狗CTLA4交叉反應;(e)誘導ADCC效應(例如,對Treg);(f)活化人類PBMC (例如,刺激IL-2及/或IFNγ之分泌);(g)能抑制腫瘤細胞生長及建立對腫瘤細胞之免疫記憶;(h)具有對癌症之治療效應;及(i)阻斷人類CTLA4結合至人類CD80及/或人類CD86 (參見以下實例1至5)。 To address these and other needs, disclosed herein are antibodies (eg, cross-reactive antibodies) and antigen-binding fragments thereof that bind to human CTLA4. The anti-CTLA4 antibody or antigen-binding fragment thereof of the invention has at least one (e.g., one, some or all) of the following functional properties: (a) Binds to humans, cynomolgus monkeys with a KD of 500 nM or less , mouse, rat and/or dog CTLA4, (b) has antagonistic activity against human CTLA4; (c) does not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS, and/or B7-H4; (d) Cross-reacts with monkey, mouse, rat, and/or dog CTLA4; (e) ) Induces ADCC effects (for example, on Tregs); (f) Activates human PBMC (for example, stimulates the secretion of IL-2 and/or IFNγ); (g) Can inhibit tumor cell growth and establish immune memory to tumor cells; ( h) have a therapeutic effect on cancer; and (i) block the binding of human CTLA4 to human CD80 and/or human CD86 (see Examples 1 to 5 below).

本文中揭示精度/背景依賴性可活化抗體,當以活性形式時,該等抗體結合至人類CTLA4,但是以減能形式時不結合,即,僅於裂解可裂解部分(CM)以移除掩蔽部分(MM)後,該等可活化抗體結合至CTLA4 (具活性)。於一些實施例中,本文中所述之發現之掩蔽部分(MM)能有效掩蔽抗體活性及/或減少或完全抑制抗原結合,而於一些實施例中,缺乏化學上不穩定殘基甲硫胺酸及/或色胺酸。此外,本文中所識別及所述之可活化抗體在治療多種癌症類型上與其親本抗體一樣有效,同時於易感動物(NOD小鼠)中具有顯著降低之細胞毒性。Disclosed herein are precision/context-dependent activatable antibodies that bind to human CTLA4 when in the active form, but do not bind when in the attenuated form, i.e., only cleave the cleavable moiety (CM) to remove the mask After moiety (MM), these activatable antibodies bind to CTLA4 (active). In some embodiments, the discovered masking moiety (MM) described herein is effective in masking antibody activity and/or reducing or completely inhibiting antigen binding, and in some embodiments, lacks the chemically labile residue methionine. acid and/or tryptophan. Furthermore, the activatable antibodies identified and described herein are as effective as their parent antibodies in treating multiple cancer types while having significantly reduced cytotoxicity in susceptible animals (NOD mice).

因此,於一態樣中,本文中提供抗CTLA4抗體(例如,人類抗體),該抗體結合人類CTLA4且與來自選自由食蟹獼猴、小鼠、大鼠及狗組成之群之至少一種非人類動物之CTLA4多肽交叉反應。於一些實施例中,該抗體結合至食蟹獼猴CTLA4及小鼠CTLA4。於可與上述實施例中之任一者組合之一些實施例中,該抗體以約350 nM或更低(例如,約300 nM或更低,約200 nM或更低,約100 nM或更低,約50 nM或更低,約10 nM或更低)之解離常數(K D) 結合至人類CTLA4、食蟹獼猴CTLA4、小鼠CTLA4、大鼠CTLA4及/或狗CTLA4。於一些實施例中,藉由表面電漿子共振(SPR)量測K D。於一些實施例中,抗體與CTLA4之結合誘導對表現CTLA4之細胞之抗體依賴性細胞毒性(ADCC)。於一些實施例中,抗體與CTLA4之結合誘導對Treg細胞之ADCC。於一些實施例中,本文中所述之抗CTLA4抗體之結合誘導對表現CTLA4之人類細胞或人類Treg細胞之抗體依賴性細胞毒性(ADCC),其中該抗CTLA4抗體之ADCC活性高於活體外伊匹單抗(ipilimumab)之ADCC活性,且其中兩種抗體包含野生型人類IgG1 Fc區。於一些實施例中,本文中所述之抗CTLA4抗體之結合誘導對表現CTLA4之人類細胞或人類Treg細胞之抗體依賴性細胞毒性(ADCC),其中該抗CTL4抗體之ADCC活性為活體外伊匹單抗之ADCC活性的兩倍或更高,且其中兩種抗體包含野生型人類IgG1 Fc區。於一些實施例中,該抗CTLA4抗體ADCC活性之EC50為活體外伊匹單抗ADCC活性之EC50之50%或更低。量測ADCC活性之檢定述於實例3及15中。於一些實施例中,該抗CTLA4抗體於腫瘤微環境中選擇性地耗盡Treg細胞(例如,降低腫瘤浸潤淋巴細胞中之Treg細胞之百分比),相較於小鼠癌症模型中之PBMC或脾。參見例如,實例18。 Accordingly, in one aspect, provided herein are anti-CTLA4 antibodies (e.g., human antibodies) that bind human CTLA4 and at least one non-human species selected from the group consisting of cynomolgus monkeys, mice, rats, and dogs. CTLA4 peptide cross-reactivity in animals. In some embodiments, the antibody binds to cynomolgus monkey CTLA4 and mouse CTLA4. In some embodiments that can be combined with any of the above embodiments, the antibody is present at about 350 nM or less (e.g., about 300 nM or less, about 200 nM or less, about 100 nM or less , about 50 nM or less, about 10 nM or less), binds to human CTLA4, cynomolgus monkey CTLA4, mouse CTLA4, rat CTLA4 and/or dog CTLA4. In some embodiments, K D is measured by surface plasmon resonance (SPR). In some embodiments, binding of an antibody to CTLA4 induces antibody-dependent cellular cytotoxicity (ADCC) on cells expressing CTLA4. In some embodiments, binding of the antibody to CTLA4 induces ADCC on Treg cells. In some embodiments, binding of an anti-CTLA4 antibody described herein induces antibody-dependent cellular cytotoxicity (ADCC) on human cells or human Treg cells expressing CTLA4, wherein the ADCC activity of the anti-CTLA4 antibody is higher than in vitro. ADCC activity of ipilimumab, and two of the antibodies contained wild-type human IgG1 Fc region. In some embodiments, binding of an anti-CTLA4 antibody described herein induces antibody-dependent cellular cytotoxicity (ADCC) on human cells or human Treg cells expressing CTLA4, wherein the ADCC activity of the anti-CTL4 antibody is i.p. The ADCC activity of the monoclonal antibodies was twice or greater, and two of the antibodies contained the wild-type human IgG1 Fc region. In some embodiments, the EC50 of the ADCC activity of the anti-CTLA4 antibody is 50% or less of the EC50 of the ADCC activity of ipilimumab in vitro. Assays to measure ADCC activity are described in Examples 3 and 15. In some embodiments, the anti-CTLA4 antibody selectively depletes Treg cells in the tumor microenvironment (e.g., reduces the percentage of Treg cells in tumor-infiltrating lymphocytes) compared to PBMC or spleen in a mouse cancer model. . See, e.g., Example 18.

於可與上述實施例中之任一者組合之一些實施例中,該抗體特異性結合至包含人類CTLA4之配位體結合位點(諸如人類CTLA4之CD80及/或CD86結合位點)處之胺基酸殘基之抗原決定基。於一些實施例中,該抗體特異性結合至類似於人類CTLA4之配位體結合位點(諸如人類CTLA4之CD80及/或CD86結合位點)之抗原決定基。於一些實施例中,該抗體特異性結合至包含人類CTLA4之胺基酸殘基Y105及L106之抗原決定基,其中該等胺基酸殘基之編號係根據SEQ ID NO: 207。於一些實施例中,該抗體不結合至人類CTLA4之殘基I108,其中該等胺基酸殘基之編號係根據SEQ ID NO: 207。於一些實施例中,該抗CTLA4抗體阻斷CD80及/或CD86結合至人類CTLA4。於一些實施例中,針對阻斷CD80及/或CD86結合至人類CTLA4而言,該抗CTLA4抗體具有高於伊匹單抗之IC50之IC50。於一些實施例中,在CD86或CD80經板結合且CTLA4係於溶液中或CTLA4展示在細胞表面之檢定中,針對阻斷CD80及/或CD86結合至人類CTLA4,該抗CTLA4抗體具有為伊匹單抗之IC50的3.5倍或更高(包括3.9倍或更高)之IC50。參見實例13、表23、圖57A至57D及58。用於測試抗體之阻斷活性(配位體競爭)及IC50之檢定述於實例3及13中。In some embodiments that can be combined with any of the above embodiments, the antibody specifically binds to a ligand binding site comprising human CTLA4, such as the CD80 and/or CD86 binding site of human CTLA4. The epitope of an amino acid residue. In some embodiments, the antibody specifically binds to an epitope similar to the ligand binding site of human CTLA4, such as the CD80 and/or CD86 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope comprising amino acid residues Y105 and L106 of human CTLA4, wherein the amino acid residues are numbered according to SEQ ID NO: 207. In some embodiments, the antibody does not bind to residue 1108 of human CTLA4, wherein the amino acid residues are numbered according to SEQ ID NO: 207. In some embodiments, the anti-CTLA4 antibody blocks CD80 and/or CD86 binding to human CTLA4. In some embodiments, the anti-CTLA4 antibody has an IC50 greater than the IC50 of ipilimumab for blocking CD80 and/or CD86 binding to human CTLA4. In some embodiments, for blocking CD80 and/or CD86 binding to human CTLA4 in an assay in which CD86 or CD80 is plate-bound and CTLA4 is in solution or CTLA4 is displayed on the cell surface, the anti-CTLA4 antibody has a The IC50 of the monoclonal antibody is 3.5 times or higher (including 3.9 times or higher). See Example 13, Table 23, Figures 57A-57D and 58. Assays used to test the blocking activity (ligand competition) and IC50 of antibodies are described in Examples 3 and 13.

於可與上述實施例中之任一者組合之一些實施例中,該抗體包含重鏈可變區及輕鏈可變區,a)其中該重鏈可變區包含HVR-H1、HVR-H2及HVR-H3,其中該HVR-H1包含如選自由以下組成之群之式之胺基酸序列:式(I):X1TFSX2YX3IHWV (SEQ ID NO: 1),其中X1為F或Y,X2為D或G,且X3為A、G或W;式(II):YSIX1SGX2X3WX4WI (SEQ ID NO: 2),其中X1為S或T,X2為H或Y,X3為H或Y,且X4為A、D或S;及式(III):FSLSTGGVAVX1WI (SEQ ID NO: 3),其中X1為G或S;其中該HVR-H2包含如選自由以下組成之群之式之胺基酸序列:式(IV):IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID NO: 4),其中X1為D或E,X2為S或Y,且X3為P或Q;式(V):IGX1ISPSX2GX3TX4YAQKFQGRV (SEQ ID NO: 5),其中X1為I或W,X2為G或S,X3為G或S,且X4為K或N;及式(VI):VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 6),其中X1為A、G或S,X2為S或Y,X3為G或S,且X4為S或T;且其中該HVR-H3包含如選自由以下組成之群之式之胺基酸序列:式(VII):ARX1X2X3X4FDX5 (SEQ ID NO: 7),其中X1為G、R或S,X2為A、I或Y,X3為D、V或Y,X4為A、E或Y,且X5為I或Y;式(VIII):ARX1GX2GYFDX3 (SEQ ID NO: 8),其中X1為D或L,X2為F或Y,且X3為V或Y;式(IX):ARX1X2X3X4AX5X6FDY (SEQ ID NO: 9),其中X1為L或R,X2為I或P,X3為A或Y,X4為S或T,X5為T或Y,且X6為A或Y;式(X):ARDX1X2X3GSSGYYX4GFDX5 (SEQ ID NO: 10),其中X1為I或V,X2為A或H,X3為P或S,X4為D或Y,且X5為F或V,及b)其中該輕鏈可變區包含HVR-L1、HVR-L2及HVR-L3,其中該HVR-L1包含如選自由以下組成之群之式之胺基酸序列:式(XI):RASQX1X2X3SX4LX5 (SEQ ID NO: 11),其中X1為G或S,X2為I或V,X3為G或S,X4為S或Y,且X5為A或N;式(XII):RASQX1VX2X3RX4LA (SEQ ID NO: 12),其中X1為S或T,X2為F、R或S,X3為G或S,且X4為F或Y;及(XIII):RASX1SVDFX2GX3SFLX4 (SEQ ID NO: 13),其中X1為E或Q,X2為D、F、H或Y,X3為F、I或K,且X4為A、D或H;其中該HVR-L2包含如式(XIV):X1ASX2X3X4X5GX6 (SEQ ID NO: 14)之胺基酸序列,其中X1為A或D,X2為N、S或T,X3為L或R,X4為A、E或Q,X5為S或T,且X6為I或V;且其中該HVR-L3包含如選自由以下組成之群之式之胺基酸序列:式(XV):YCX1X2X3X4X5X6PX7T (SEQ ID NO: 15),其中X1為E、Q或V,X2為H或Q,X3為A、G、H、R或S,X4為D、L、S或Y,X5為E、G、P、Q或S,X6為L、T、V或W,且X7為F、L、P、W或Y;式(XVI):YCQQX1X2X3WPPWT (SEQ ID NO: 16),其中X1為S或Y,X2為D或Y,且X3為Q或Y;及式(XVII):YCQX1YX2SSPPX3YT (SEQ ID NO: 17),其中X1為H或Q,X2為T或V,且X3為E或V。於一些實施例中,該HVR-H1包含選自由SEQ ID NO: 18至29組成之群之胺基酸序列,該HVR-H2包含選自由SEQ ID NO: 30至39組成之群之胺基酸序列,該HVR-H3包含選自由SEQ ID NO: 40至52組成之群之胺基酸序列,該HVR-L1包含選自由SEQ ID NO: 53至65組成之群之胺基酸序列,該HVR-L2包含選自由SEQ ID NO: 66至69組成之群之胺基酸序列,且該HVR-L3包含選自由SEQ ID NO: 70至81組成之群之胺基酸序列。於一些實施例中,該抗體包含:a)包含SEQ ID NO: 18之胺基酸序列之HVR-H1,包含SEQ ID NO: 30之胺基酸序列之HVR-H2,包含SEQ ID NO: 40之胺基酸序列之HVR-H3,包含SEQ ID NO: 53之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 70之胺基酸序列之HVR-L3;b)包含SEQ ID NO: 19之胺基酸序列之HVR-H1,包含SEQ ID NO: 31之胺基酸序列之HVR-H2,包含SEQ ID NO: 41之胺基酸序列之HVR-H3,包含SEQ ID NO: 54之胺基酸序列之HVR-L1,包含SEQ ID NO: 67之胺基酸序列之HVR-L2,及包含SEQ ID NO: 71之胺基酸序列之HVR-L3;c)包含SEQ ID NO: 20之胺基酸序列之HVR-H1,包含SEQ ID NO: 32之胺基酸序列之HVR-H2,包含SEQ ID NO: 42之胺基酸序列之HVR-H3,包含SEQ ID NO: 55之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 72之胺基酸序列之HVR-L3;d)包含SEQ ID NO: 21之胺基酸序列之HVR-H1,包含SEQ ID NO: 33之胺基酸序列之HVR-H2,包含SEQ ID NO: 43之胺基酸序列之HVR-H3,包含SEQ ID NO: 56之胺基酸序列之HVR-L1,包含SEQ ID NO: 68之胺基酸序列之HVR-L2,及包含SEQ ID NO: 73之胺基酸序列之HVR-L3;e)包含SEQ ID NO: 22之胺基酸序列之HVR-H1,包含SEQ ID NO: 34之胺基酸序列之HVR-H2,包含SEQ ID NO: 44之胺基酸序列之HVR-H3,包含SEQ ID NO: 57之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 74之胺基酸序列之HVR-L3;f)包含SEQ ID NO: 23之胺基酸序列之HVR-H1,包含SEQ ID NO: 35之胺基酸序列之HVR-H2,包含SEQ ID NO: 45之胺基酸序列之HVR-H3,包含SEQ ID NO: 58之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 75之胺基酸序列之HVR-L3;g)包含SEQ ID NO: 24之胺基酸序列之HVR-H1,包含SEQ ID NO: 32之胺基酸序列之HVR-H2,包含SEQ ID NO: 46之胺基酸序列之HVR-H3,包含SEQ ID NO: 59之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 76之胺基酸序列之HVR-L3;h)包含SEQ ID NO: 25之胺基酸序列之HVR-H1,包含SEQ ID NO: 36之胺基酸序列之HVR-H2,包含SEQ ID NO: 47之胺基酸序列之HVR-H3,包含SEQ ID NO: 60之胺基酸序列之HVR-L1,包含SEQ ID NO: 69之胺基酸序列之HVR-L2,及包含SEQ ID NO: 77之胺基酸序列之HVR-L3;i)包含SEQ ID NO: 26之胺基酸序列之HVR-H1,包含SEQ ID NO: 37之胺基酸序列之HVR-H2,包含SEQ ID NO: 48之胺基酸序列之HVR-H3,包含SEQ ID NO: 61之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 78之胺基酸序列之HVR-L3;j)包含SEQ ID NO: 27之胺基酸序列之HVR-H1,包含SEQ ID NO: 32之胺基酸序列之HVR-H2,包含SEQ ID NO: 49之胺基酸序列之HVR-H3,包含SEQ ID NO: 62之胺基酸序列之HVR-L1,包含SEQ ID NO: 67之胺基酸序列之HVR-L2,及包含SEQ ID NO: 79之胺基酸序列之HVR-L3;k)包含SEQ ID NO: 28之胺基酸序列之HVR-H1,包含SEQ ID NO: 37之胺基酸序列之HVR-H2,包含SEQ ID NO: 50之胺基酸序列之HVR-H3,包含SEQ ID NO: 63之胺基酸序列之HVR-L1,包含SEQ ID NO: 67之胺基酸序列之HVR-L2,及包含SEQ ID NO: 80之胺基酸序列之HVR-L3;l)包含SEQ ID NO: 18之胺基酸序列之HVR-H1,包含SEQ ID NO: 38之胺基酸序列之HVR-H2,包含SEQ ID NO: 51之胺基酸序列之HVR-H3,包含SEQ ID NO: 64之胺基酸序列之HVR-L1,包含SEQ ID NO: 67之胺基酸序列之HVR-L2,及包含SEQ ID NO: 81之胺基酸序列之HVR-L3;或m)包含SEQ ID NO: 29之胺基酸序列之HVR-H1,包含SEQ ID NO: 39之胺基酸序列之HVR-H2,包含SEQ ID NO: 52之胺基酸序列之HVR-H3,包含SEQ ID NO: 65之胺基酸序列之HVR-L1,包含SEQ ID NO: 68之胺基酸序列之HVR-L2,及包含SEQ ID NO: 77之胺基酸序列之HVR-L3。於可與上述實施例中之任一者組合之一些實施例中,該重鏈可變區包含選自由SEQ ID NO: 82至94組成之群之胺基酸序列,及/或該輕鏈可變區包含選自由SEQ ID NO: 95至107組成之群之胺基酸序列。於可與上述實施例中之任一者組合之一些實施例中,該抗體包含:a)包含SEQ ID NO: 82之胺基酸序列或具有與SEQ ID NO: 82之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 95之胺基酸序列或具有與SEQ ID NO: 95之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;b)包含SEQ ID NO: 83之胺基酸序列或具有與SEQ ID NO: 83之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 96之胺基酸序列或具有與SEQ ID NO: 96之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;c)包含SEQ ID NO: 84之胺基酸序列或具有與SEQ ID NO: 84之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 97之胺基酸序列或具有與SEQ ID NO: 97之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;d)包含SEQ ID NO: 85之胺基酸序列或具有與SEQ ID NO: 85之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 98之胺基酸序列或具有與SEQ ID NO: 98之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;e)包含SEQ ID NO: 86之胺基酸序列或具有與SEQ ID NO: 86之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 99之胺基酸序列或具有與SEQ ID NO: 99之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;f)包含SEQ ID NO: 87之胺基酸序列或具有與SEQ ID NO: 87之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 100之胺基酸序列或具有與SEQ ID NO: 100之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;g)包含SEQ ID NO: 88之胺基酸序列或具有與SEQ ID NO: 88之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 101之胺基酸序列或具有與SEQ ID NO: 101之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;h)包含SEQ ID NO: 89之胺基酸序列或具有與SEQ ID NO: 89之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 102之胺基酸序列或具有與SEQ ID NO: 102之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;i)包含SEQ ID NO: 90之胺基酸序列或具有與SEQ ID NO: 90之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 103之胺基酸序列或具有與SEQ ID NO: 103之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;j)包含SEQ ID NO: 91之胺基酸序列或具有與SEQ ID NO: 91之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 104之胺基酸序列或具有與SEQ ID NO: 104之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;k)包含SEQ ID NO: 92之胺基酸序列或具有與SEQ ID NO: 92之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 105之胺基酸序列或具有與SEQ ID NO: 105之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;l)包含SEQ ID NO: 93之胺基酸序列或具有與SEQ ID NO: 93之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 106之胺基酸序列或具有與SEQ ID NO: 106之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區;或m)包含SEQ ID NO: 94之胺基酸序列或具有與SEQ ID NO: 94之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之重鏈可變區,及包含SEQ ID NO: 107之胺基酸序列或具有與SEQ ID NO: 107之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體之輕鏈可變區。 In some embodiments that can be combined with any of the above embodiments, the antibody includes a heavy chain variable region and a light chain variable region, a) wherein the heavy chain variable region includes HVR-H1, HVR-H2 And HVR-H3, wherein the HVR-H1 includes an amino acid sequence of the formula selected from the group consisting of: Formula (I): X1TFSX2YX3IHWV (SEQ ID NO: 1), wherein X1 is F or Y, and X2 is D Or G, and X3 is A, G or W; Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO: 2), where X1 is S or T, X2 is H or Y, X3 is H or Y, and D or S; and Formula (III): FSLSTGGVAVX1WI (SEQ ID NO: 3), wherein X1 is G or S; wherein the HVR-H2 includes an amino acid sequence of a formula selected from the group consisting of: Formula (IV ): IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID NO: 4), where X1 is D or E, X2 is S or Y, and X3 is P or Q; Formula (V): IGX1ISPSX2GX3TX4YAQKFQGRV (SEQ ID NO: 5), where X1 is I or W, X2 is G or S, X3 is G or S, and X4 is K or N; and formula (VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 6), where X1 is A, G or S, and , X3 is G or S, and Where X1 is G, R or S, X2 is A, I or Y, X3 is D, V or Y, X4 is A, E or Y, and X5 is I or Y; Formula (VIII): ARX1GX2GYFDX3 (SEQ ID NO: 8), where X1 is D or L, X2 is F or Y, and X3 is V or Y; Formula (IX): ARX1X2X3X4AX5X6FDY (SEQ ID NO: 9), where X1 is L or R, is I or P, X3 is A or Y, X4 is S or T, X5 is T or Y, and X6 is A or Y; formula (X): ARDX1X2X3GSSGYYX4GFDX5 (SEQ ID NO: 10), where , X2 is A or H, X3 is P or S, X4 is D or Y, and X5 is F or V, and b) wherein the light chain variable region includes HVR-L1, HVR-L2 and HVR-L3, wherein The HVR-L1 includes an amino acid sequence of the formula selected from the group consisting of: Formula (XI): RASQX1X2X3SX4LX5 (SEQ ID NO: 11), where X1 is G or S, X2 is I or V, and X3 is G Or S, X4 is S or Y, and X5 is A or N; Formula (XII): RASQX1VX2X3RX4LA (SEQ ID NO: 12), where X1 is S or T, X2 is F, R or S, and X3 is G or S , and X4 is F or Y; and (XIII): RASX1SVDFX2GX3SFLX4 (SEQ ID NO: 13), where X1 is E or Q, X2 is D, F, H or Y, X3 is F, I or K, and A, D or H; wherein the HVR-L2 includes the amino acid sequence of formula (XIV): X1ASX2X3X4X5GX6 (SEQ ID NO: 14), where X1 is A or D, X2 is N, S or T, and or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V; and wherein the HVR-L3 includes an amino acid sequence of the formula selected from the group consisting of: Formula (XV) : YCX1X2X3X4X5X6PX7T (SEQ ID NO: 15), where X1 is E, Q or V, X2 is H or Q, X3 is A, G, H, R or S, X4 is D, L, S or Y, X5 is E , G, P, Q or S, X6 is L, T, V or W, and X7 is F, L, P, W or Y; Formula (XVI): YCQQX1X2X3WPPWT (SEQ ID NO: 16), where X1 is S Or Y, X2 is D or Y, and X3 is Q or Y; and formula (XVII): YCQX1YX2SSPPX3YT (SEQ ID NO: 17), where X1 is H or Q, X2 is T or V, and X3 is E or V . In some embodiments, the HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 18 to 29, and the HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 30 to 39 Sequence, the HVR-H3 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 40 to 52, the HVR-L1 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 53 to 65, the HVR -L2 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 66 to 69, and the HVR-L3 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 70 to 81. In some embodiments, the antibody comprises: a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, comprising SEQ ID NO: 40 HVR-H3 containing the amino acid sequence of SEQ ID NO: 53, HVR-L1 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L2 containing the amino acid sequence of SEQ ID NO: 70 HVR-L3 of the amino acid sequence; b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41 HVR-H3 of the amino acid sequence, HVR-L1 of the amino acid sequence of SEQ ID NO: 54, HVR-L2 of the amino acid sequence of SEQ ID NO: 67, and the amine of SEQ ID NO: 71 HVR-L3 of the amino acid sequence; c) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and the amine of SEQ ID NO: 42 HVR-H3 with the amino acid sequence, HVR-L1 with the amino acid sequence of SEQ ID NO: 55, HVR-L2 with the amino acid sequence of SEQ ID NO: 66, and the amino group of SEQ ID NO: 72 HVR-L3 of acid sequence; d) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, and the amino group of SEQ ID NO: 43 HVR-H3 with the acid sequence, HVR-L1 with the amino acid sequence of SEQ ID NO: 56, HVR-L2 with the amino acid sequence of SEQ ID NO: 68, and HVR-L2 with the amino acid sequence of SEQ ID NO: 73 HVR-L3 of the sequence; e) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44 HVR-H3 of the sequence, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L2 comprising the amino acid sequence of SEQ ID NO: 74 HVR-L3; f) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45 HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 58, HVR-L2 including the amino acid sequence of SEQ ID NO: 66, and HVR-L2 including the amino acid sequence of SEQ ID NO: 75 HVR-L3; g) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 24, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 46 HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 59, HVR-L2 including the amino acid sequence of SEQ ID NO: 66, and HVR including the amino acid sequence of SEQ ID NO: 76 -L3; h) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, HVR comprising the amino acid sequence of SEQ ID NO: 47 -H3, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69, and HVR- comprising the amino acid sequence of SEQ ID NO: 77 L3; i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR- comprising the amino acid sequence of SEQ ID NO: 48 H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 61, HVR-L2 including the amino acid sequence of SEQ ID NO: 66, and HVR-L3 including the amino acid sequence of SEQ ID NO: 78 ; j) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 49 , HVR-L1 comprising the amino acid sequence of SEQ ID NO: 62, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79; k) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 28, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 80; l ) HVR-H1 including the amino acid sequence of SEQ ID NO: 18, HVR-H2 including the amino acid sequence of SEQ ID NO: 38, HVR-H3 including the amino acid sequence of SEQ ID NO: 51, including HVR-L1 having the amino acid sequence of SEQ ID NO: 64, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81; or m ) HVR-H1 including the amino acid sequence of SEQ ID NO: 29, HVR-H2 including the amino acid sequence of SEQ ID NO: 39, HVR-H3 including the amino acid sequence of SEQ ID NO: 52, including HVR-L1 has the amino acid sequence of SEQ ID NO: 65, HVR-L2 has the amino acid sequence of SEQ ID NO: 68, and HVR-L3 has the amino acid sequence of SEQ ID NO: 77. In some embodiments that may be combined with any of the above embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 82 to 94, and/or the light chain may The variable region includes an amino acid sequence selected from the group consisting of SEQ ID NO: 95 to 107. In some embodiments that may be combined with any of the above embodiments, the antibody comprises: a) comprising the amino acid sequence of SEQ ID NO: 82 or having an amino acid sequence that is at least about A heavy chain variable region of a variant thereof that has 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity, and includes the amino acid sequence of SEQ ID NO: 95 or has The light chain variable region of a variant thereof that has at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO: 95; b ) comprises the amino acid sequence of SEQ ID NO: 83 or has a sequence that is at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) the same as the amino acid sequence of SEQ ID NO: 83 The heavy chain variable region of its variant is identical to, and comprises the amino acid sequence of SEQ ID NO: 96 or has at least about 90% (e.g., at least about 92%, The light chain variable region of a variant thereof with 95%, 98%, 99% or more sequence identity; c) comprising the amino acid sequence of SEQ ID NO: 84 or having an amino group with SEQ ID NO: 84 A heavy chain variable region of a variant thereof that has at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity of the acid sequence, and includes the amine group of SEQ ID NO: 97 The acid sequence or the light chain of a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 97 may Variable region; d) comprises the amino acid sequence of SEQ ID NO: 85 or has at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or The heavy chain variable region of a variant thereof has a sequence identity of at least about 90% (e.g., at least The light chain variable region of a variant thereof having about 92%, 95%, 98%, 99% or more sequence identity; e) comprising the amino acid sequence of SEQ ID NO: 86 or having the same amino acid sequence as SEQ ID NO: The heavy chain variable region of a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity to the amino acid sequence of 86, and comprising SEQ ID NO: The amino acid sequence of SEQ ID NO: 99 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO: 99 The light chain variable region; f) comprises the amino acid sequence of SEQ ID NO: 87 or has at least about 90% (e.g., at least about 92%, 95%, 98%) of the amino acid sequence of SEQ ID NO: 87 , 99% or more) sequence identity of the heavy chain variable region of its variant, and comprising the amino acid sequence of SEQ ID NO: 100 or having at least about 90% of the amino acid sequence of SEQ ID NO: 100 (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity to the light chain variable region of a variant thereof; g) comprising the amino acid sequence of SEQ ID NO: 88 or having the same The heavy chain variable region of a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 88, and comprising The amino acid sequence of SEQ ID NO: 101 may have at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO: 101 The light chain variable region of its variant; h) comprises the amino acid sequence of SEQ ID NO: 89 or has at least about 90% (e.g., at least about 92%, 95%) of the amino acid sequence of SEQ ID NO: 89 %, 98%, 99% or more) sequence identity of the heavy chain variable region of its variant, and comprising the amino acid sequence of SEQ ID NO: 102 or having the amino acid sequence of SEQ ID NO: 102 Light chain variable regions of variants thereof that have at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity; i) comprise the amino acid of SEQ ID NO: 90 The sequence or the heavy chain variable of a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 90 region, and comprise the amino acid sequence of SEQ ID NO: 103 or have at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) of the amino acid sequence of SEQ ID NO: 103 ) The light chain variable region of a variant thereof having sequence identity; j) comprising the amino acid sequence of SEQ ID NO: 91 or having at least about 90% (e.g., at least about 90%) the amino acid sequence of SEQ ID NO: 91 The heavy chain variable region of a variant thereof having 92%, 95%, 98%, 99% or more sequence identity, and comprising the amino acid sequence of SEQ ID NO: 104 or having the same amino acid sequence as SEQ ID NO: 104 A light chain variable region of a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) amino acid sequence identity; k) comprising SEQ ID NO: 92 The amino acid sequence of SEQ ID NO: 92 or a variant thereof that has at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO: 92 A heavy chain variable region, and comprising the amino acid sequence of SEQ ID NO: 105 or having at least about 90% (e.g., at least about 92%, 95%, 98%, 99) of the amino acid sequence of SEQ ID NO: 105 % or more) sequence identity of the light chain variable region of its variant; 1) comprising the amino acid sequence of SEQ ID NO: 93 or having at least about 90% ( For example, a heavy chain variable region of a variant thereof that has at least about 92%, 95%, 98%, 99% or more sequence identity, and includes the amino acid sequence of SEQ ID NO: 106 or has the same amino acid sequence as SEQ ID NO. The light chain variable region of a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity to the amino acid sequence of NO: 106; or m) comprising The amino acid sequence of SEQ ID NO: 94 may have at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO: 94 The heavy chain variable region of its variant, and comprising the amino acid sequence of SEQ ID NO: 107 or having at least about 90% (e.g., at least about 92%, 95%) the amino acid sequence of SEQ ID NO: 107 , 98%, 99% or more) sequence identity of the light chain variable region of its variant.

於一些實施例中,本文中所述之抗CTLA4抗體包含重鏈可變區及輕鏈可變區,其中該抗體之1、2、3、4、5或6個HVR包含表A中所示之HVR序列。於一些實施例中,該抗CTLA4抗體包含包含HVR-H1、HVR-H2及HVR-H3之重鏈可變區,其中該HVR-H1包含SEQ ID NO: 23之胺基酸序列,或該HVR-H2包含SEQ ID NO: 35之胺基酸序列,或該HVR-H3包含SEQ ID NO: 45之胺基酸序列。於一些實施例中,該抗CTLA4抗體包含包含HVR-L1、HVR-L2及HVR-L3之輕鏈可變區,其中該HVR-L1包含SEQ ID NO: 58之胺基酸序列,或該HVR-L2包含SEQ ID NO: 66之胺基酸序列,或該HVR-L3包含SEQ ID NO: 75之胺基酸序列。於一些實施例中,該抗體之HVR-H2包含SEQ ID NO: 35之胺基酸序列。於一些實施例中,該抗CTLA4抗體包含(a)包含包含SEQ ID NO: 23之胺基酸序列之HVR-H1,包含SEQ ID NO: 35之胺基酸序列之HVR-H2,及包含SEQ ID NO: 45之胺基酸序列之HVR-H3之重鏈可變區,及/或包含包含SEQ ID NO: 58之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 75之胺基酸序列之HVR-L3之輕鏈可變區。於一些實施例中,該抗體之HVR中之1、2、3、4、5或6者可包含1、2或3個保守胺基酸取代於該等HVR中。於一些實施例中,該抗CTLA4抗體包含(b)包含SEQ ID NO: 87之胺基酸序列或具有與SEQ ID NO: 87之胺基酸序列至少90% (例如,91%、92%、93%、95%、96%、97%、98%或99%)序列同一性之胺基酸序列之重鏈可變區,及/或包含SEQ ID NO: 100之胺基酸序列或具有與SEQ ID NO: 100之胺基酸序列至少90% (例如,91%、92%、93%、95%、96%、97%、98%或99%)序列同一性之胺基酸序列之輕鏈可變區。In some embodiments, the anti-CTLA4 antibodies described herein comprise a heavy chain variable region and a light chain variable region, wherein 1, 2, 3, 4, 5, or 6 HVRs of the antibody comprise those shown in Table A The HVR sequence. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region comprising HVR-H1, HVR-H2 and HVR-H3, wherein the HVR-H1 comprises the amino acid sequence of SEQ ID NO: 23, or the HVR -H2 comprises the amino acid sequence of SEQ ID NO: 35, or the HVR-H3 comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-CTLA4 antibody comprises a light chain variable region comprising HVR-L1, HVR-L2 and HVR-L3, wherein the HVR-L1 comprises the amino acid sequence of SEQ ID NO: 58, or the HVR -L2 comprises the amino acid sequence of SEQ ID NO: 66, or the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, HVR-H2 of the antibody comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CTLA4 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and SEQ. The heavy chain variable region of HVR-H3 having the amino acid sequence of ID NO: 45, and/or HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, and comprising the amino acid of SEQ ID NO: 66 The sequence of HVR-L2, and the light chain variable region of HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, 1, 2, 3, 4, 5 or 6 of the HVR of the antibody may comprise 1, 2 or 3 conservative amino acid substitutions in the HVR. In some embodiments, the anti-CTLA4 antibody comprises (b) comprising the amino acid sequence of SEQ ID NO: 87 or having at least 90% (e.g., 91%, 92%, The heavy chain variable region of an amino acid sequence with 93%, 95%, 96%, 97%, 98% or 99%) sequence identity, and/or comprising the amino acid sequence of SEQ ID NO: 100 or having an amino acid sequence with SEQ ID NO: 100 Amino acid sequences with at least 90% (e.g., 91%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity. chain variable region.

於可與上述實施例中之任一者組合之一些實施例中,該抗體為抗體片段。於一些實施例中,該片段為Fab、Fab’、Fab’-SH、F(ab’) 2、Fv或scFv片段。於可與上述實施例中之任一者組合之一些實施例中,該抗體包含IgG1、IgG2、IgG3或IgG4 Fc區(諸如人類IgG1、IgG2、IgG3或IgG4 Fc區)。於一些實施例中,包含人類IgG1或變異體之抗體具有增強之ADCC活性。於一些實施例中,該抗體包含人類IgG1,具有減少之岩藻糖基化(或未經岩藻糖基化)。於一些實施例中,該抗體為人類抗體。 In some embodiments that can be combined with any of the above embodiments, the antibody is an antibody fragment. In some embodiments, the fragment is a Fab, Fab', Fab'-SH, F(ab') 2 , Fv or scFv fragment. In some embodiments that can be combined with any of the above embodiments, the antibody comprises an IgG1, IgG2, IgG3 or IgG4 Fc region (such as a human IgG1, IgG2, IgG3 or IgG4 Fc region). In some embodiments, antibodies comprising human IgG1 or variants have enhanced ADCC activity. In some embodiments, the antibody comprises human IgG1 with reduced fucosylation (or no fucosylation). In some embodiments, the antibody is a human antibody.

本發明之其他態樣係關於與本文中所述抗體中之任一者競爭或交叉競爭結合至人類CTLA4之抗體。本文中亦提供結合至與本文中所述抗體中之任一者相同之抗原決定基及/或基本上相同之抗原決定基之抗體。Other aspects of the invention relate to antibodies that compete or cross-compete for binding to human CTLA4 with any of the antibodies described herein. Also provided herein are antibodies that bind to the same epitope and/or substantially the same epitope as any of the antibodies described herein.

本發明之其他態樣係關於一種可活化抗體,其包含:a)第一多肽,自N端至C端包含掩蔽部分(MM)、可裂解部分(CM)及靶結合部分(TBM),其中該MM包含如式(XVIII):X mCX nCZ o(SEQ ID NO: 134)之胺基酸序列,其中m為2至10,n為3至10,且o為1至10,其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸,且其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸;其中當該CM未裂解時,該MM抑制可活化抗體結合至人類CTLA4;其中該CM包含至少第一裂解位點;且其中該TBM包含抗體重鏈可變區(VH);及b)包含抗體輕鏈可變區(VL)之第二多肽;且其中當該CM被裂解時,該可活化抗體經由VH及VL結合至人類CTLA4。於一些實施例中,m為3至10。 Other aspects of the invention relate to an activatable antibody comprising: a) a first polypeptide comprising a masking moiety (MM), a cleavable moiety (CM) and a target binding moiety (TBM) from the N-terminus to the C-terminus, Wherein the MM includes an amino acid sequence such as formula ( XVIII) : Each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y Amino acid, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein when the CM When uncleaved, the MM inhibits binding of an activatable antibody to human CTLA4; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody heavy chain variable region (VH); and b) comprises an antibody light chain variable a second polypeptide of region (VL); and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 via VH and VL. In some embodiments, m is 3 to 10.

本發明之其他態樣係關於一種可活化抗體,其包含:a)第一多肽,自N端至C端包含掩蔽部分(MM)、可裂解部分(CM)及靶結合部分(TBM),其中該MM包含如式(XVIII):X mCX nCZ o(SEQ ID NO: 134)之胺基酸序列,其中m為2至10,n為3至10,且o為1至10,其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸,且其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸;其中當該CM未裂解時,該MM抑制可活化抗體結合至人類CTLA4;其中該CM包含至少第一裂解位點;且其中該TBM包含抗體輕鏈可變區(VL);及b)包含抗體重鏈可變區(VH)之第二多肽;且其中當該CM被裂解時,該可活化抗體經由VH及VL結合至人類CTLA4。於一些實施例中,m為3至10。 Other aspects of the invention relate to an activatable antibody comprising: a) a first polypeptide comprising a masking moiety (MM), a cleavable moiety (CM) and a target binding moiety (TBM) from the N-terminus to the C-terminus, Wherein the MM includes an amino acid sequence such as formula ( XVIII) : Each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y Amino acid, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein when the CM When uncleaved, the MM inhibits binding of an activatable antibody to human CTLA4; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region (VL); and b) comprises an antibody heavy chain variable a second polypeptide of region (VH); and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 via VH and VL. In some embodiments, m is 3 to 10.

本發明之其他態樣係關於一種可活化抗體,其包含:自N端至C端包含掩蔽部分(MM)、可裂解部分(CM)及靶結合部分(TBM)之多肽,其中該MM包含如式(XVIII):X mCX nCZ o(SEQ ID NO: 134)之胺基酸序列,其中m為2至10,n為3至10,且o為1至10,其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸,且其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸;其中當該CM未裂解時,該MM抑制可活化抗體結合至人類CTLA4;其中該CM包含至少第一裂解位點;其中該TBM自N端至C端包含抗體輕鏈可變區(VL)及抗體重鏈可變區(VH);且其中當該CM被裂解時,該可活化抗體經由VH及VL結合至人類CTLA4。於一些實施例中,m為3至10。 Other aspects of the invention relate to an activatable antibody comprising: a polypeptide comprising a masking moiety (MM), a cleavable moiety (CM) and a target binding moiety (TBM) from the N-terminus to the C-terminus, wherein the MM comprises as Formula (XVIII) : the amino acid sequence of An amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, And wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein when the CM is not cleaved, the MM inhibits binding of an activatable antibody to human CTLA4; wherein the CM includes at least a first cleavage site; wherein the TBM includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) from the N-terminus to the C-terminus ; and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 via VH and VL. In some embodiments, m is 3 to 10.

本發明之其他態樣係關於一種可活化抗體,其包含:自N端至C端包含掩蔽部分(MM)、可裂解部分(CM)及靶結合部分(TBM)之多肽,其中該MM包含如式(XVIII):X mCX nCZ o(SEQ ID NO: 134)之胺基酸序列,其中m為2至10,n為3至10,且o為1至10,其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸,且其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸;其中當該CM未裂解時,該MM抑制可活化抗體結合至人類CTLA4;其中該CM包含至少第一裂解位點;其中該TBM自N端至C端包含抗體重鏈可變區(VH)及抗體輕鏈可變區(VL);且其中當該CM被裂解時,該可活化抗體經由VH及VL結合至人類CTLA4。 Other aspects of the invention relate to an activatable antibody comprising: a polypeptide comprising a masking moiety (MM), a cleavable moiety (CM) and a target binding moiety (TBM) from the N-terminus to the C-terminus, wherein the MM comprises as Formula (XVIII) : the amino acid sequence of An amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, And wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein when the CM is not cleaved, the MM inhibits binding of an activatable antibody to human CTLA4; wherein the CM includes at least a first cleavage site; wherein the TBM includes an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) from N-terminus to C-terminus ; and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 via VH and VL.

於如上述可活化抗體中之任一者之一些實施例中,m為2、3、4、5或6。於一些實施例中,m為6。於一些實施例中,n為6至8。於一些實施例中,n為6。於一些實施例中,o為1至2。於一些實施例中,o為2。於可與上述實施例中之任一者組合之一些實施例中,各X非M、W或C。於可與上述實施例中之任一者組合之一些實施例中,式(XVIII)之X m中之各X獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。於可與上述實施例中之任一者組合之一些實施例中,式(XVIII)之X n中之各X獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。於一些實施例中,該MM包含選自由X mCPDHPYPCXX (SEQ ID NO:181)、X mCDAFYPYCXX (SEQ ID NO:182)、X mCDSHYPYCXX (SEQ ID NO:183)及X mCVPYYYACXX (SEQ ID NO:184)組成之群之胺基酸序列,其中m為2至10,且其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸。於一些實施例中,各X非M、W或C。於一些實施例中,各X獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。於可與上述實施例中之任一者組合之一些實施例中,該掩蔽部分(MM)包含選自SEQ ID NO: 141至147之胺基酸序列。於可與上述實施例中之任一者組合之一些實施例中,該MM在其N端處另包含另外胺基酸序列。於一些實施例中,該另外胺基酸序列包括SEQ ID NO: 148之胺基酸序列。 In some embodiments of any of the activatable antibodies described above, m is 2, 3, 4, 5, or 6. In some embodiments, m is 6. In some embodiments, n is 6-8. In some embodiments, n is 6. In some embodiments, o is 1 to 2. In some embodiments, o is 2. In some embodiments that can be combined with any of the above embodiments, each X is other than M, W, or C. In some embodiments that can be combined with any of the above embodiments, each X in X m of formula (XVIII) is independently selected from D, A, Y, S, T, N, I, L, Amino acids of the group consisting of F, V, H, and P. In some embodiments that can be combined with any of the above embodiments, each X in X n of formula (XVIII) is independently selected from D, A, Y, S, T, N, I, L, Amino acids of the group consisting of F, V, H, and P. In some embodiments, the MM includes Xm CPDHPYPCXX (SEQ ID NO : 181), Xm CDAFYPYCXX (SEQ ID NO:182), Xm CDSHYPYCXX (SEQ ID NO:183), and NO: 184) Amino acid sequence of the group consisting of, wherein m is 2 to 10, and each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M , N, P, Q, R, S, T, V, W, and Y. In some embodiments, each X is other than M, W, or C. In some embodiments, each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments that can be combined with any of the above embodiments, the masking moiety (MM) comprises an amino acid sequence selected from SEQ ID NO: 141 to 147. In some embodiments that can be combined with any of the above embodiments, the MM further comprises an additional amino acid sequence at its N-terminus. In some embodiments, the additional amino acid sequence includes the amino acid sequence of SEQ ID NO: 148.

於可與上述實施例中之任一者組合之一些實施例中,該第一裂解位點為選自由以下組成之群之蛋白酶之蛋白酶裂解位點:尿激酶型纖維蛋白溶酶原活化因子(uPA)、基質金屬蛋白酶-1 (MMP-1)、MMP-2、MMP-3、MMP-8、MMP-9、MMP-14、菸草蝕紋病毒(TEV)蛋白酶、胞漿素(plasmin)、凝血酶(Thrombin)、X因子、PSA、PSMA、組織蛋白酶(Cathepsin) D、組織蛋白酶K、組織蛋白酶S、ADAM10、ADAM12、ADAMTS、卡斯蛋白酶(Caspase)-1、卡斯蛋白酶-2、卡斯蛋白酶-3、卡斯蛋白酶-4、卡斯蛋白酶-5、卡斯蛋白酶-6、卡斯蛋白酶-7、卡斯蛋白酶-8、卡斯蛋白酶-9、卡斯蛋白酶-10、卡斯蛋白酶-11、卡斯蛋白酶-12、卡斯蛋白酶-13、卡斯蛋白酶-14、及TACE。於可與上述實施例中之任一者組合之一些實施例中,該CM另包含至第一裂解位點C端之第一連接子(L 1)。於一些實施例中,該L 1包含選自由SEQ ID NO: 156至163組成之群之胺基酸序列。於可與上述實施例中之任一者組合之一些實施例中,該CM另包含第二裂解位點。於一些實施例中,該第二裂解位點為L 1之C端。於一些實施例中,該第二裂解位點為選自由以下組成之群之蛋白酶之蛋白酶裂解位點:尿激酶型纖維蛋白溶酶原活化因子(uPA)、基質金屬蛋白酶-1 (MMP-1)、MMP-2、MMP-3、MMP-8、MMP-9、MMP-14、菸草蝕紋病毒(TEV)蛋白酶、胞漿素、凝血酶、X因子、PSA、PSMA、組織蛋白酶D、組織蛋白酶K、組織蛋白酶S、ADAM10、ADAM12、ADAMTS、卡斯蛋白酶-1、卡斯蛋白酶-2、卡斯蛋白酶-3、卡斯蛋白酶-4、卡斯蛋白酶-5、卡斯蛋白酶-6、卡斯蛋白酶-7、卡斯蛋白酶-8、卡斯蛋白酶-9、卡斯蛋白酶-10、卡斯蛋白酶-11、卡斯蛋白酶-12、卡斯蛋白酶-13、卡斯蛋白酶-14、及TACE。於一些實施例中,該第一及第二裂解位點係不同。於可與上述實施例中之任一者組合之一些實施例中,該CM另包含至第二裂解位點C端之第二連接子(L 2)。於一些實施例中,該L 2包含選自由SEQ ID NO: 156至163組成之群之胺基酸序列。於可與上述實施例中之任一者組合之一些實施例中,該CM另包含至第一裂解位點N端之第三連接子(L 3)。於可與上述實施例中之任一者組合之一些實施例中,該CM包含至少第一蛋白酶裂解位點且經選自由以下組成之群之一或多種蛋白酶裂解:尿激酶型纖維蛋白溶酶原活化因子(uPA)、基質金屬蛋白酶-1 (MMP-1)、MMP-2、MMP-3、MMP-8、MMP-9、MMP-14、菸草蝕紋病毒(TEV)蛋白酶、胞漿素、凝血酶、X因子、PSA、PSMA、組織蛋白酶D、組織蛋白酶K、組織蛋白酶S、ADAM10、ADAM12、ADAMTS、卡斯蛋白酶-1、卡斯蛋白酶-2、卡斯蛋白酶-3、卡斯蛋白酶-4、卡斯蛋白酶-5、卡斯蛋白酶-6、卡斯蛋白酶-7、卡斯蛋白酶-8、卡斯蛋白酶-9、卡斯蛋白酶-10、卡斯蛋白酶-11、卡斯蛋白酶-12、卡斯蛋白酶-13、卡斯蛋白酶-14、及TACE。 In some embodiments that can be combined with any of the above embodiments, the first cleavage site is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator ( uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, Thrombin (Thrombin), factor Caspsin-3, Caspsin-4, Caspsin-5, Caspsin-6, Caspsin-7, Caspsin-8, Caspsin-9, Caspsin-10, Caspsin-10 -11, Caspsin-12, Caspsin-13, Caspsin-14, and TACE. In some embodiments that can be combined with any of the above embodiments, the CM further includes a first linker (L 1 ) to the C-terminus of the first cleavage site. In some embodiments, L 1 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 156 to 163. In some embodiments, which can be combined with any of the above embodiments, the CM further comprises a second cleavage site. In some embodiments, the second cleavage site is the C-terminus of L 1 . In some embodiments, the second cleavage site is a protease cleavage site of a protease selected from the group consisting of urokinase plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1 ), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, cytoplasmin, thrombin, factor X, PSA, PSMA, cathepsin D, tissue Protease K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase Caspsin-7, Caspsin-8, Caspsin-9, Caspsin-10, Caspsin-11, Caspsin-12, Caspsin-13, Caspsin-14, and TACE. In some embodiments, the first and second cleavage sites are different. In some embodiments that can be combined with any of the above embodiments, the CM further includes a second linker ( L2 ) to the C-terminus of the second cleavage site. In some embodiments, the L 2 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 156 to 163. In some embodiments that can be combined with any of the above embodiments, the CM further includes a third linker ( L3 ) to the N-terminus of the first cleavage site. In some embodiments that may be combined with any of the above embodiments, the CM includes at least a first protease cleavage site and is cleaved by one or more proteases selected from the group consisting of: urokinase-type plasmin Protoactivator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, cytoplasmin , thrombin, factor -4. Caspsin-5, Caspsin-6, Caspsin-7, Caspsin-8, Caspsin-9, Caspsin-10, Caspsin-11, Caspsin-12 , caspase-13, caspase-14, and TACE.

於可與上述實施例中之任一者組合之一些實施例中,該可活化抗體包含掩蔽部分(MM)及可裂解部分(CM),包含如式(XXIX):EVGSYX1X2X3X4X5X6CX7X8X9X10X11X12CX13X14SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 164)之胺基酸序列,其中X1為A、D、I、N、P、或Y,X2為A、F、N、S、或V,X3為A、H、L、P、S、V、或Y,X4為A、H、S、或Y,X5為A、D、P、S、V、或Y,X6為A、D、L、S、或Y,X7為D、P、或V,X8為A、D、H、P、S、或T,X9為A、D、F、H、P、或Y,X10為L、P、或Y,X11為F、P、或Y,X12為A、P、S、或Y,X13為A、D、N、S、T、或Y,且X14為A、S、或Y。於可與上述實施例中之任一者組合之一些實施例中,該可活化抗體包含選自由SEQ ID NO: 165至179組成之群之胺基酸序列。In some embodiments that can be combined with any of the above embodiments, the activatable antibody includes a masking moiety (MM) and a cleavable moiety (CM), including formula (XXIX): EVGSYX1X2X3X4X5X6CX7X8X9X10X11X12CX13X14SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 164 ), where X1 is A, D, I, N, P, or Y, X2 is A, F, N, S, or V, and X3 is A, H, L, P, S, V, or Y, X4 is A, H, S, or Y, X5 is A, D, P, S, V, or Y, X6 is A, D, L, S, or Y, X7 is D, P, or V , X8 is A, D, H, P, S, or T, X9 is A, D, F, H, P, or Y, X10 is L, P, or Y, X11 is F, P, or Y, X12 is A, P, S, or Y, X13 is A, D, N, S, T, or Y, and X14 is A, S, or Y. In some embodiments that can be combined with any of the above embodiments, the activatable antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 165 to 179.

於可與上述實施例中之任一者組合之一些實施例中,該VL包含包含SEQ ID NO: 58之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 75之胺基酸序列之HVR-L3。於可與上述實施例中之任一者組合之一些實施例中,該VL包含SEQ ID NO: 100之胺基酸序列或具有與SEQ ID NO: 100之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體。於可與上述實施例中之任一者組合之一些實施例中,該VH包含包含SEQ ID NO: 23之胺基酸序列之HVR-H1,包含SEQ ID NO: 35之胺基酸序列之HVR-H2,及包含SEQ ID NO: 45之胺基酸序列之HVR-H3。於可與上述實施例中之任一者組合之一些實施例中,該VH包含SEQ ID NO: 87之胺基酸序列或具有與SEQ ID NO: 87之胺基酸序列至少約90% (例如,至少約92%、95%、98%、99%或更多)序列同一性之其變異體。In some embodiments that can be combined with any of the above embodiments, the VL includes HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, HVR comprising the amino acid sequence of SEQ ID NO: 66 -L2, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments that may be combined with any of the above embodiments, the VL comprises or has at least about 90% the amino acid sequence of SEQ ID NO: 100 (e.g., , variants thereof that have at least about 92%, 95%, 98%, 99% or more sequence identity. In some embodiments that may be combined with any of the above embodiments, the VH includes an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, an HVR comprising the amino acid sequence of SEQ ID NO: 35 -H2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments that may be combined with any of the above embodiments, the VH comprises or has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 87 (e.g., , variants thereof that have at least about 92%, 95%, 98%, 99% or more sequence identity.

本發明之其他態樣係關於醫藥組合物,該醫藥組合物包含本文中所述抗體及/或可活化抗體中之任一者及醫藥上可接受之載劑。Other aspects of the invention relate to pharmaceutical compositions comprising any of the antibodies and/or activatable antibodies described herein and a pharmaceutically acceptable carrier.

本發明之其他態樣係關於編碼本文中所述抗體及/或可活化抗體中之任一者之多核苷酸。於一些實施例中,該多核苷酸包含選自SEQ ID NO: 108至133之序列。Other aspects of the invention relate to polynucleotides encoding any of the antibodies and/or activatable antibodies described herein. In some embodiments, the polynucleotide comprises a sequence selected from SEQ ID NO: 108 to 133.

本發明之其他態樣係關於包含本文中所述多核苷酸中之任一者之載體。於一些實施例中,該載體為表現載體及/或顯示載體。Other aspects of the invention relate to vectors comprising any of the polynucleotides described herein. In some embodiments, the vector is an expression vector and/or a display vector.

本發明之其他態樣係關於包含本文中所述多核苷酸及/或載體中之任一者之宿主細胞。於一些實施例中,該宿主細胞為真核細胞。於一些實施例中,該宿主細胞為中國倉鼠卵巢(CHO)細胞。Other aspects of the invention relate to host cells comprising any of the polynucleotides and/or vectors described herein. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cells are Chinese hamster ovary (CHO) cells.

本發明之其他態樣係關於一種製備抗體或可活化抗體之方法,其包括在適於產生抗體或可活化抗體之條件下培養本文中所述宿主細胞中之任一者。於一些實施例中,該方法進一步包括回收由該細胞產生之抗體或可活化抗體。Other aspects of the invention relate to a method of producing an antibody or activatable antibody, which includes culturing any of the host cells described herein under conditions suitable for producing the antibody or activatable antibody. In some embodiments, the method further includes recovering the antibody or activatable antibody produced by the cell.

本發明之其他態樣係關於一種治療有需要受試者之癌症或延遲其癌症進展之方法,其包括對該受試者投與有效量之本文中所述抗體、可活化抗體及/或醫藥組合物中之任一者。於一些實施例中,該癌症為肝癌、消化系統癌(例如,結腸癌、結腸直腸癌)、肺癌、骨癌、心臟癌、腦癌、腎癌、膀胱癌、血液癌(例如,白血病)、皮膚癌、乳癌、甲狀腺癌、胰癌、頭頸癌、眼相關癌、男性生殖系統癌(例如,前列腺癌、睾丸癌)、或女性生殖系統癌(例如,子宮癌、子宮頸癌)。本發明之其他態樣係關於一種減少有需要受試者中之實體腫瘤大小之方法,其中該實體腫瘤具有約400至1000 mm 3之大小,該方法包括對該受試者投與有效量之本文中所述抗體、可活化抗體及/或醫藥組合物中之任一者。於一些實施例中,該實體腫瘤具有約400至800 mm 3之大小。於一些實施例中,該方法進一步包括對該受試者投與有效量之至少一種另外治療劑。於一些實施例中,該至少一種另外治療劑係選自由病毒基因療法、免疫檢查點抑制劑、靶療法、放射療法、疫苗接種療法及化療組成之群。於一些實施例中,該至少一種另外治療劑係選自由以下組成之群:鉑美特(pomalyst)、瑞復美(revlimid)、來那度胺(lenalidomide)、泊馬度胺(pomalidomide)、沙利度胺(thalidomide)、DNA烷基化含鉑衍生物、順鉑(cisplatin)、5-氟尿嘧啶、環磷醯胺(cyclophosphamide)、抗CD137抗體、抗PD-1抗體、抗PD-L1抗體、抗CD20抗體、抗CD40抗體、抗DR5抗體、抗CD1d抗體、抗TIM3抗體、抗SLAMF7抗體、抗KIR受體抗體、抗OX40抗體、抗HER2抗體、抗ErbB-2抗體、抗EGFR抗體、西妥昔單抗(cetuximab)、利妥昔單抗(rituximab)、曲妥珠單抗(trastuzumab)、派姆單抗(pembrolizumab)、放射療法、單劑量輻射、分次輻射、焦點輻射、全器官輻射、IL-12、IFNα、GM-CSF、嵌合抗原受體、授受性轉移之T細胞、抗癌疫苗、及溶瘤病毒。於一些實施例中,該方法包括在用於移除受試者中之腫瘤之手術之前或於該手術後對該受試者投與有效量之本文中所述之抗CTLA4抗體、可活化抗體或醫藥組合物。於一些實施例中,該抗CD137抗體包括包含胺基酸序列FSLSTGGVGVGWI (SEQ ID NO: 223)之HVR-H1,包含胺基酸序列LALIDWADDKYYSPSLKSRL (SEQ ID NO:224)之HVR-H2,及包含胺基酸序列ARGGSDTVIGDWFAY (SEQ ID NO: 225)之HVR-H3之抗體重鏈可變區,及包括包含胺基酸序列RASQSIGSYLA (SEQ ID NO: 226)之HVR-L1,包含胺基酸序列DASNLETGV (SEQ ID NO: 227)之HVR-L2,及包含胺基酸序列YCQQGYYLWT (SEQ ID NO: 228)之HVR-L3之抗體輕鏈可變區。於一些實施例中,該抗CD137抗體包括包含SEQ ID NO: 229之胺基酸序列或具有與SEQ ID NO: 229之序列至少90% (例如,91%、92%、93%、94%、95%、96%、97%、98%或99%)序列同一性之抗體重鏈可變區;及/或包含SEQ ID NO: 230之胺基酸序列或具有與SEQ ID NO: 230之序列至少90% (例如,91%、92%、93%、94%、95%、96%、97%、98%或99%)序列同一性之抗體輕鏈可變區。 EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSS (SEQ ID NO: 229) DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIK (SEQ ID NO: 230) Other aspects of the invention relate to a method of treating cancer or delaying the progression of cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody, activatable antibody and/or medicament described herein. any of the compositions. In some embodiments, the cancer is liver cancer, digestive system cancer (eg, colon cancer, colorectal cancer), lung cancer, bone cancer, heart cancer, brain cancer, kidney cancer, bladder cancer, blood cancer (eg, leukemia), Skin cancer, breast cancer, thyroid cancer, pancreatic cancer, head and neck cancer, eye-related cancer, male reproductive system cancer (eg, prostate cancer, testicular cancer), or female reproductive system cancer (eg, uterine cancer, cervical cancer). Other aspects of the invention relate to a method of reducing the size of a solid tumor in a subject in need thereof, wherein the solid tumor has a size of about 400 to 1000 mm, the method comprising administering to the subject an effective amount of Any of the antibodies, activatable antibodies, and/or pharmaceutical compositions described herein. In some embodiments, the solid tumor has a size of approximately 400 to 800 mm. In some embodiments, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, vaccination therapy, and chemotherapy. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of: pomalyst, revlimid, lenalidomide, pomalidomide, Thalidomide, DNA alkylated platinum-containing derivatives, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CD137 antibody, anti-PD-1 antibody, anti-PD-L1 antibody , anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, Western cetuximab, rituximab, trastuzumab, pembrolizumab, radiation therapy, single-dose radiation, fractionated radiation, focal radiation, whole organ Radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptors, receptive transferred T cells, anti-cancer vaccines, and oncolytic viruses. In some embodiments, the method includes administering to the subject an effective amount of an anti-CTLA4 antibody, activatable antibody described herein before surgery for removing a tumor in the subject or after the surgery. or pharmaceutical compositions. In some embodiments, the anti-CD137 antibody includes HVR-H1 comprising the amino acid sequence FSLSTGGVGVGWI (SEQ ID NO: 223), HVR-H2 comprising the amino acid sequence LALIDWADDKYYSPSLKSRL (SEQ ID NO: 224), and amine The antibody heavy chain variable region of HVR-H3 with the amino acid sequence ARGGSDTVIGDWFAY (SEQ ID NO: 225), and HVR-L1 including the amino acid sequence RASQSIGSYLA (SEQ ID NO: 226), including the amino acid sequence DASNLETGV ( HVR-L2 of SEQ ID NO: 227), and the antibody light chain variable region of HVR-L3 comprising the amino acid sequence YCQQGYYLWT (SEQ ID NO: 228). In some embodiments, the anti-CD137 antibody comprises the amino acid sequence of SEQ ID NO: 229 or has at least 90% (e.g., 91%, 92%, 93%, 94%, An antibody heavy chain variable region with 95%, 96%, 97%, 98% or 99%) sequence identity; and/or comprising the amino acid sequence of SEQ ID NO: 230 or having a sequence with SEQ ID NO: 230 Antibody light chain variable regions with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity. EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSS (SEQ ID NO: 229) DIQLTQSPSSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIK (SEQ ID NO: 230)

應瞭解,可組合以上及本文中所述之各種實施例之性質中之一者、一些或所有以形成本發明之其他實施例。本發明之此等及其他態樣將對熟習此項技術者變得顯而易見。本發明之此等及其他實施例進一步由隨後實施方式進行描述。It should be understood that one, some, or all of the properties of the various embodiments described above and herein may be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the invention are further described in the following description.

相關申請案之交互參照Cross-references to related applications

本申請案主張2019年2月2日申請之國際申請案第PCT/CN2019/074580號之優先權,其全文以引用的方式併入本文中。 以ASCII文本檔案之序列表之提交 This application claims priority over International Application No. PCT/CN2019/074580, filed on February 2, 2019, the full text of which is incorporated herein by reference. Submission of sequence listing as ASCII text file

以ASCII文本檔案之下列提交內容之全文以引用的方式併入本文中:序列表之電腦可讀形式(CRF) (檔案名稱:695402000542SEQLIST.TXT,記錄日期:2019年6月5日,大小:102 KB)。 I. 一般技術 The full text of the following submission in an ASCII text file is incorporated herein by reference: Computer Readable Form (CRF) of Sequence Listing (File Name: 695402000542SEQLIST.TXT, Record Date: June 5, 2019, Size: 102 KB). I. General technology

本文中所述或提及之技術及程序一般係熟知且通常由熟習此項技術者使用習知方法學採用,諸如例如,廣泛利用述於以下之方法學:Sambrook等人, Molecular Cloning: A Laboratory Manual第3版(2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology(F.M. Ausubel等人編輯,(2003)); Methods in Enzymology系列(Academic Press, Inc.): PCR 2: A Practical Approach(M.J. MacPherson、B.D. Hames及G.R. Taylor編輯(1995));Harlow及Lane編輯(1988) Antibodies, A Laboratory Manual、及 Animal Cell Culture(R.I. Freshney編輯(1987)); Oligonucleotide Synthesis(M.J. Gait編輯,1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J.E. Cellis編輯,1998) Academic Press; Animal Cell Culture(R.I. Freshney)編輯,1987); Introduction to Cell and Tissue Culture(J.P. Mather及P.E. Roberts,1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures(A. Doyle、J.B. Griffiths及D.G. Newell編輯,1993-8) J. Wiley and Sons; Handbook of Experimental Immunology(D.M. Weir及C.C. Blackwell編輯); Gene Transfer Vectors for Mammalian Cells(J.M. Miller及M.P. Calos編輯,1987); PCR: The Polymerase Chain Reaction, (Mullis等人編輯,1994); Current Protocols in Immunology(J.E. Coligan等人編輯,1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology(C.A. Janeway及P. Travers,1997); Antibodies(P. Finch,1997); Antibodies: A Practical Approach(D. Catty.編輯,IRL Press,1988至1989); Monoclonal Antibodies: A Practical Approach(P. Shepherd及C. Dean編輯,Oxford University Press, 2000); Using Antibodies: A Laboratory Manual(E. Harlow及D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies(M. Zanetti及J. D. Capra編輯,Harwood Academic Publishers, 1995);及 Cancer: Principles and Practice of Oncology(V.T. DeVita等人編輯,J.B. Lippincott Company, 1993)。 II. 定義 The techniques and procedures described or referred to herein are generally well known and commonly employed by those skilled in the art using conventional methodologies, such as, for example, the methodologies widely used in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (edited by FM Ausubel et al., (2003)); Methods in Enzymology Series (Academic Press, Inc.): PCR 2 : A Practical Approach (edited by MJ MacPherson, BD Hames and GR Taylor (1995)); edited by Harlow and Lane (1988) Antibodies, A Laboratory Manual , and Animal Cell Culture (edited by RI Freshney (1987)); Oligonucleotide Synthesis (edited by MJ Gait Editor, 1984); Methods in Molecular Biology , Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, 1998) Academic Press; Animal Cell Culture (RI Freshney), 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, JB Griffiths and DG Newell, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (edited by DM Weir and CC Blackwell) ; Gene Transfer Vectors for Mammalian Cells (edited by JM Miller and MP Calos, 1987); PCR: The Polymerase Chain Reaction , (edited by Mullis et al., 1994); Current Protocols in Immunology (edited by JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty., IRL Press, 1988 to 1989 ); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (edited by VT DeVita et al., JB Lippincott Company, 1993). II.Definition _

在詳細描述本發明之前,應瞭解,本發明揭示內容不限於特定組合物或生物系統,當然其可變化。亦應瞭解,本文中所用之術語係僅出於描述特定實施例之目的,且不旨在限制。Before the present invention is described in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

如本文中所用,除非內容另有明確指定,否則單數形式「一(a/an)」及「該」包括複數指示物。因此,例如,提及「一分子」視情況包括兩種或更多種此等分子之組合,及類似者。As used herein, the singular forms "a/an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and the like.

如本文中所用,術語「約」係指為此技術領域之熟習者容易知曉之各自值的通常誤差範圍。本文中提及「約」值或參數包括(且描述)指向該值或參數本身之實施例。As used herein, the term "about" refers to the usual error range for the respective value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that refer to the value or parameter itself.

應瞭解,本文中所述之本發明之態樣及實施例包括「包含」、「由…組成」及「基本上由…組成」態樣及實施例。It should be understood that aspects and embodiments of the invention described herein include "comprising", "consisting of" and "consisting essentially of" aspects and embodiments.

如本文中所用,術語「及/或」,諸如「A及/或B」之短語意欲包括A及B二者;A或B;A (單獨);及B (單獨)。同樣,如本文中所用,術語「及/或」,諸如「A、B及/或C」之短語意欲涵蓋下列實施例各者:A、B及C;A、B或C;A或C;A或B;B或C;A及C;A及B;B及C;A (單獨);B (單獨);及C (單獨)。As used herein, the term "and/or", phrases such as "A and/or B" are intended to include both A and B; A or B; A (alone); and B (alone). Likewise, as used herein, the term "and/or", phrases such as "A, B and/or C" are intended to cover each of the following embodiments: A, B and C; A, B or C; A or C ; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

術語「胺基酸」係指天然產生及合成胺基酸,以及胺基酸類似物及功能類似於天然產生胺基酸之胺基酸擬似物。天然產生胺基酸為藉由遺傳密碼編碼之彼等,以及後期經修飾之彼等胺基酸,例如,羥基脯胺酸、γ-羧基麩胺酸及O-磷酸絲胺酸。術語「胺基酸類似物」係指具有與天然產生胺基酸相同之基本化學結構但是C端羧基、N端胺基或側鏈官能基已經化學修飾成另一官能基。術語「胺基酸擬似物」係指具有不同於胺基酸之一般化學結構之結構但是功能類似於天然產生胺基酸之化學化合物。如本文中所用,二十種習知胺基酸及其縮略語遵循習知用法。參見例如, Immunology—A Synthesis(第2版,E. S. Golub及D. R. Gren編輯,Sinauer Associates, Sunderland, Mass. (1991))。 The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. The term "amino acid analogue" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid but that the C-terminal carboxyl group, N-terminal amine group, or side chain functional group has been chemically modified to another functional group. The term "amino acid mimetic" refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid but functions similarly to a naturally occurring amino acid. As used herein, the twenty common amino acids and their abbreviations follow common usage. See, for example, Immunology—A Synthesis (2nd ed., edited by ES Golub and DR Gren, Sinauer Associates, Sunderland, Mass. (1991)).

本文中術語「多肽」、「蛋白質」及「肽」可交換使用且可係指兩種或更多種胺基酸之聚合物。The terms "polypeptide", "protein" and "peptide" are used interchangeably herein and may refer to a polymer of two or more amino acids.

本文中可交換使用之「多核苷酸」或「核酸」係指任何長度之核苷酸之聚合物,且包括DNA及RNA。核苷酸可為去氧核糖核苷酸、核糖核苷酸、經修改之核苷酸或鹼及/或其類似物,或可藉由DNA或RNA聚合酶或藉由合成反應併入聚合物之任何受質。多核苷酸可包括經修飾之核苷酸(諸如甲基化核苷酸)及其類似物。若存在,則可在聚合物組裝之前或之後賦予核苷酸結構之修飾。核苷酸之序列可經非核苷酸組分中斷。多核苷酸可包含於合成後作出之修飾,諸如與標籤結合。其他類型之修改包括例如「帽」、天然產生核苷酸中之一或多者經類似物置換、內核苷酸修飾,諸如例如,具有不帶電鍵聯(例如,磷酸甲酯、磷酸三酯、磷醯胺、胺基甲酸酯等)及具有帶電鍵聯(例如,硫代磷酸酯、二硫代磷酸酯等)之彼等,含有側鏈部分(諸如例如,蛋白質(例如,核酸酶、毒素、抗體、信號肽、ply-L-離胺酸等))之彼等,具有嵌入劑(例如,吖啶、補骨脂素(psoralen)等)之彼等,含有螯合劑(例如,金屬、放射性金屬、硼、氧化金屬等)之彼等,含有烷基化劑之彼等,具有經修飾之鍵結(例如,α異頭物核酸等)之彼等,以及該(等)多核苷酸之未經修飾形式。此外,通常存在於糖中之羥基中之任一者可例如經膦酸酯基、磷酸酯基置換,經標準保護基保護,或經活化以製備另外核苷酸之另外鍵聯,或可與固體或半固體擔體結合。5’及3’端OH可經磷酸化或經胺或來自1至20個碳原子之有機封端基團部分取代。亦可將其他羥基衍生成標準保護基。多核苷酸亦可含有一般此項技術中已知之核糖或去氧核糖之類似形式,包括例如2’-O-甲基-、2’-O-烯丙基-、2’-氟-或2’-疊氮基-核糖、碳環糖類似物、α-異頭物糖、差向異構糖(諸如阿拉伯糖、木糖或來蘇糖)、吡喃糖、呋喃糖、景天庚糖、無環類似物及鹼性核苷類似物(諸如甲基核苷)。一或多個磷酸二酯鍵聯可經替代連接基團置換。此等替代連接基團包括(但不限於)其中磷酸酯經P(O)S (「硫代酯」)、P(S)S (「二硫代酯」)、(O)NR2 (「醯胺」)、P(O)R、P(O)OR’、CO或CH2 (「甲縮醛」)置換之實施例,其中各R或R’獨立地為H或經取代或未經取代之烷基(1至20個C),該烷基視情況含有醚(-O-)鍵聯、芳基、烯基、環烷基、環烯基或芳烷基。並非多核苷酸中之所有鍵聯必須相同。上述適用於本文中提及之所有多核苷酸,包括RNA及DNA。"Polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or analogs thereof, or may be incorporated into the polymer by DNA or RNA polymerase or by synthetic reactions any subject matter. Polynucleotides may include modified nucleotides (such as methylated nucleotides) and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may contain modifications made after synthesis, such as conjugation with a tag. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with analogues, internal nucleotide modifications such as, for example, having uncharged linkages (e.g., methyl phosphate, phosphotriester, phosphatides, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), containing side chain moieties such as, for example, proteins (e.g., nucleases, Toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those that contain chelating agents (e.g., metal , radioactive metals, boron, oxide metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and the polynucleosides(s) The unmodified form of acid. Furthermore, any of the hydroxyl groups typically present in sugars may be displaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to make additional linkages to additional nucleotides, or may be Solid or semi-solid support binding. The 5' and 3' terminal OH may be phosphorylated or partially substituted with amines or organic end-capping groups from 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized into standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro- or 2 '-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose), pyranose, furanose, sedumose , acyclic analogs and basic nucleoside analogs (such as methyl nucleosides). One or more phosphodiester linkages may be replaced by alternative linking groups. Such alternative linking groups include, but are not limited to, those in which the phosphate ester is replaced by P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR2 ("thioester") Examples of substitution of amine"), P(O)R, P(O)OR', CO or CH2 ("methylal"), where each R or R' is independently H or substituted or unsubstituted Alkyl (1 to 20 C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all linkages in a polynucleotide need to be identical. The above applies to all polynucleotides mentioned herein, including RNA and DNA.

術語「經單離核酸」係指基因組、cDNA或合成來源之核酸分子或其組合,其自存在於核酸之天然來源中之其他核酸分子分離。例如,關於基因組DNA,術語「經單離」包括自天然締合基因組DNA之染色體分離之核酸分子。較佳地,「經單離」核酸係無天然側接核酸之序列(即位於所關注核酸之5′及3′端之序列)。The term "isolated nucleic acid" refers to a nucleic acid molecule of genomic, cDNA or synthetic origin, or a combination thereof, that is isolated from other nucleic acid molecules present in the natural source of nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules isolated from chromosomes with which genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of natural flanking nucleic acid sequences (ie, sequences located at the 5' and 3' ends of the nucleic acid of interest).

術語「抗體」在本文中以廣義使用且具體而言涵蓋單株抗體(包括全長單株抗體)、多株抗體、多特異性抗體(例如,雙特異性抗體、三特異性抗體)及抗體片段(例如,Fab、Fab’、Fab’-SH、F(ab’) 2、Fv及/或單鏈可變片段或scFv),只要其展示所需生物活性。 The term "antibody" is used herein in a broad sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies) and antibody fragments (eg, Fab, Fab', Fab'-SH, F(ab') 2 , Fv and/or single chain variable fragment or scFv) as long as it exhibits the desired biological activity.

於一些實施例中,術語「抗體」係指具有由兩條相同重(H)鏈及兩條相同輕(L)鏈組成之基本四多肽鏈結構之抗原結合蛋白(即,免疫球蛋白)。各L鏈藉由一個共價二硫鍵連接至H鏈,而兩條H鏈藉由一或多個二硫鍵彼此連接,取決於H鏈同型。各重鏈在N端處具有可變區(本文中縮寫為V H),接著恆定區。該重鏈恆定區包含三個域C H1、C H2及C H3。各輕鏈在N端具有可變區(本文中縮寫為V L),接著恆定區在其另一端。該輕鏈恆定區包含一個域C L。將V L與V H對準及將C L與重鏈之第一恆定域(CH1)對準。V H及V L配對一起形成單抗原結合位點。IgM抗體由5個基本異四聚體單元連同稱作J鏈之另一多肽組成,因此含有10個抗原結合位點,而分泌之IgA抗體可聚合形成包含2至5個基本4鏈單元連同J鏈之多價組裝。 In some embodiments, the term "antibody" refers to an antigen-binding protein (i.e., immunoglobulin) having a basic four-polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. . Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds, depending on the H chain isotype. Each heavy chain has a variable region (abbreviated herein as VH ) at the N-terminus, followed by a constant region. The heavy chain constant region contains three domains CH1 , CH2 and CH3 . Each light chain has a variable region (abbreviated herein as VL ) at the N-terminus, followed by a constant region at the other end. The light chain constant region contains one domain CL . Align VL with VH and CL with the first constant domain (CH1) of the heavy chain. VH and VL pair together to form a single antigen binding site. IgM antibodies are composed of 5 basic heterotetrameric units together with another polypeptide called the J chain and therefore contain 10 antigen-binding sites, while secreted IgA antibodies can polymerize to form 2 to 5 basic 4-chain units together with Multivalent assembly of J chain.

可基於結構及序列分析將V H及V L區進一步細分成高可變性之區,稱作高可變區(HVR)。HVR散佈有更保守稱作框架區(FW)之區(參見例如,Chen等人(1999) J. Mol. Biol. (1999) 293, 865-881)。各V H及V L包含以下列順序自胺基端至羧基端排列之三個HVR及四個FW:FW-1_HVR-1_FW-2_HVR-2_FW-3_HVR-3_FW4。整篇本發明,將重鏈之三個HVR稱作HVR-H1、HVR-H2及HVR-H3。類似地,將輕鏈之三個HVR稱作HVR-L1、HVR-L2及HVR-L3。 The VH and VL regions can be further subdivided into regions of high variability, called hypervariable regions (HVR), based on structural and sequence analysis. HVR is interspersed with regions more conservatively termed framework regions (FW) (see, eg, Chen et al. (1999) J. Mol. Biol. (1999) 293, 865-881). Each VH and VL includes three HVRs and four FWs arranged in the following order from the amine terminus to the carboxyl terminus: FW-1_HVR-1_FW-2_HVR-2_FW-3_HVR-3_FW4. Throughout the present invention, the three HVRs of the heavy chain will be referred to as HVR-H1, HVR-H2 and HVR-H3. Similarly, the three HVRs of the light chain are referred to as HVR-L1, HVR-L2 and HVR-L3.

重鏈及輕鏈可變區含有與抗原相互作用之結合域。抗體之恆定區可介導免疫球蛋白結合至宿主組織或因子,包括免疫系統之各種細胞(例如,效應細胞)及經典補體系統之第一組分(Clq)。於輕鏈及重鏈內,可變區及恆定區藉由約12或更多個胺基酸之「J」區接合,其中該重鏈亦包含約10或更多個胺基酸之「D」區(參見例如,Fundamental Immunology第7章(Paul, W.編輯,第2版,Raven Press, N.Y). (1989))。The heavy and light chain variable regions contain binding domains that interact with antigens. The constant region of an antibody mediates immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 or more amino acids. ” area (see, e.g., Chapter 7 of Fundamental Immunology (Paul, W., ed., 2nd ed., Raven Press, N.Y.). (1989)).

可將來自任何脊椎動物物種之L鏈分配為兩種明確區別類型(稱作κ及λ)中之一者,基於其恆定域之胺基酸序列。取決於其重鏈之恆定域(CH)之胺基酸序列,可將抗體分配成不同類別或同型。存在五類抗體:IgA、IgD、IgE、IgG及gM,其各自具有指定為α、δ、ε、γ及μ之重鏈。可將抗體之IgG類別各自藉由γ重鏈(Y1至Y4)進一步分成四個子類別IgG1、IgG2、IgG3及IgG4。L chains from any vertebrate species can be assigned to one of two clearly distinguished types, termed kappa and lambda, based on the amino acid sequence of their constant domains. Antibodies can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain (CH) of their heavy chain. There are five classes of antibodies: IgA, IgD, IgE, IgG and gM, each with heavy chains designated alpha, delta, epsilon, gamma and mu. The IgG classes of antibodies can each be further divided into four subclasses, IgGl, IgG2, IgG3 and IgG4, by the gamma heavy chain (Y1 to Y4).

術語「抗體衍生物」或抗體之「衍生物」係指能結合至與抗體結合相同抗原(例如,CTLA4)且包含連接至另外分子實體之抗體之胺基酸序列的分子。含於抗體衍生物中之抗體之胺基酸序列可為全長重鏈、全長輕鏈、全長重鏈之任何部分、抗體之全長輕鏈之任何部分、抗體之任何其他片段、或完整抗體。另外分子實體可為化學或生物分子。另外分子實體之實例包括化學基團、胺基酸、肽、蛋白質(諸如酵素、抗體)及化學化合物。另外分子實體可具有任何效用,諸如用作檢測劑、標籤、標記、藥劑或治療劑。抗體之胺基酸序列可藉由化學偶合、基因融合、非共價締合或其他方式附接或連接至另外分子實體。術語「抗體衍生物」亦涵蓋嵌合抗體、人源化抗體及衍生自CTLA4抗體之胺基酸序列之修飾 (諸如保守胺基酸置換、新增及插入)之分子。The term "antibody derivative" or "derivative" of an antibody refers to a molecule that is capable of binding to the same antigen (eg, CTLA4) to which the antibody binds and that contains the amino acid sequence of the antibody linked to another molecular entity. The amino acid sequence of the antibody contained in the antibody derivative may be the full-length heavy chain, the full-length light chain, any portion of the full-length heavy chain, any portion of the full-length light chain of the antibody, any other fragment of the antibody, or the intact antibody. Additionally the molecular entities may be chemical or biological molecules. Examples of additional molecular entities include chemical groups, amino acids, peptides, proteins (such as enzymes, antibodies), and chemical compounds. Additionally the molecular entity may have any utility, such as being used as a detection agent, label, label, pharmaceutical or therapeutic agent. The amino acid sequence of an antibody can be attached or linked to another molecular entity by chemical coupling, genetic fusion, non-covalent association, or other means. The term "antibody derivatives" also encompasses chimeric antibodies, humanized antibodies, and molecules derived from modifications of the amino acid sequence of CTLA4 antibodies, such as conservative amino acid substitutions, additions, and insertions.

術語抗體之「抗原結合片段」或「抗原結合部分」係指保留結合至抗原之能力之抗體之一或多個部分,該抗體結合至該抗原(例如,CTLA4)。抗體之「抗原結合片段」之實例包括(i) Fab片段,由V L、V H、C L及C H1域組成之單價片段;(ii) F(ab′) 2片段,包含由鉸鏈區之二硫橋連接之兩個Fab片段之二價片段;(iii)由V H及C H1域組成之Fd片段;(iv)由抗體之單臂之V L及V H域組成之Fv片段,(v) dAb片段(Ward等人, Nature341:544-546 (1989)),其由V H域組成;及(vi)經單離之互補決定區(CDR)。 The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of an antibody that retains the ability to bind to the antigen to which the antibody binds (eg, CTLA4). Examples of "antigen-binding fragments" of antibodies include (i) Fab fragments, which are monovalent fragments consisting of the VL, VH , CL and CH1 domains; (ii) F(ab') 2 fragments, which consist of the hinge region A bivalent fragment of two Fab fragments connected by a disulfide bridge; (iii) an Fd fragment consisting of V H and CH1 domains; (iv) an Fv fragment consisting of the V L and V H domains of one arm of an antibody, ( v) dAb fragments (Ward et al., Nature 341:544-546 (1989)), consisting of VH domains; and (vi) isolated complementarity determining regions (CDRs).

術語「結合分子」涵蓋(1)抗體,(2)抗體之抗原結合片段,及(3)抗體之衍生物,各者如本文中所定義。The term "binding molecule" encompasses (1) antibodies, (2) antigen-binding fragments of antibodies, and (3) derivatives of antibodies, each as defined herein.

術語「CTLA4」在本申請案中使用且包含人類CTLA4 (例如,UniProt寄存編號P16410),以及其變異體、同工異型物及物種同源物(例如,小鼠CTLA4 (UniProt寄存編號P09793)、大鼠CTLA4 (UniProt寄存編號Q9Z1A7)、狗CTLA4 (UniProt寄存編號Q9XSI1)、食蟹獼猴CTLA4 (UniProt寄存編號G7PL88)等)。因此,如本文中所定義及揭示之結合分子(例如,抗體或可活化抗體)亦可結合來自除了人類外之物種之CTLA4。於其他情況下,結合分子可對人類CTLA4完全特異性且可不展示物種或其他類型之交叉反應性。The term "CTLA4" is used in this application and includes human CTLA4 (e.g., UniProt Registration No. P16410), as well as variants, isoforms, and species homologs thereof (e.g., mouse CTLA4 (UniProt Registration No. P09793), Rat CTLA4 (UniProt registration number Q9Z1A7), dog CTLA4 (UniProt registration number Q9XSI1), crab-eating macaque CTLA4 (UniProt registration number G7PL88), etc.). Thus, binding molecules (eg, antibodies or activatable antibodies) as defined and disclosed herein may also bind CTLA4 from species other than humans. In other cases, the binding molecule may be completely specific for human CTLA4 and may not exhibit species or other types of cross-reactivity.

術語「CTLA4抗體」係指如本文中所定義之能結合至人類CTLA4之抗體。The term "CTLA4 antibody" refers to an antibody capable of binding to human CTLA4 as defined herein.

術語「嵌合抗體」係指包含源自不同動物物種之胺基酸序列之抗體,諸如具有源自人類抗體之可變區及鼠科免疫球蛋白恆定區之彼等。The term "chimeric antibody" refers to antibodies that contain amino acid sequences derived from different animal species, such as those with variable regions derived from human antibodies and murine immunoglobulin constant regions.

術語「競爭結合」係指兩個抗體於其結合至結合靶中之相互作用。若在第二抗體之存在下第一抗體與其同源抗原決定基之結合相較於在第二抗體不存在下第一抗體之結合可檢測地減少,則第一抗體與第二抗體競爭結合。或者,在第一抗體之存在下第二抗體與其抗原決定基之結合亦可檢測地減少之情況可(但不必)為該案例。即,在第二抗體不抑制第一抗體結合至其各自抗原決定基下,第一抗體可抑制第二抗體結合至其抗原決定基。然而,在各抗體可檢測地抑制其他抗體與其同源抗原決定基之結合之情況下,無論是否至相同、更高或更低程度,將該等抗體稱作彼此「交叉競爭」以結合其各自抗原決定基。The term "competitive binding" refers to the interaction between two antibodies in their binding to a binding target. A first antibody competes with a second antibody for binding if the binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatively, this may (but need not) be the case where the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody. That is, the first antibody can inhibit the binding of the second antibody to its respective epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, to the extent that each antibody detectably inhibits the binding of the other antibody to its cognate epitope, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding to their respective Epitopes.

術語「抗原決定基」係指抗體(或其抗原結合片段)結合之抗原之部分。抗原決定基可自鄰接胺基酸或藉由蛋白質之三級折疊並置之非鄰接胺基酸二者形成。自鄰接胺基酸形成之抗原決定基通常保留暴露於變性劑,然而藉由三級折疊形成之抗原決定基通常在用變性溶劑處理中喪失。抗原決定基可包含各種數目之胺基酸於獨特空間構形中。測定抗原決定基之空間構形之方法包括例如x-射線結晶學、2維核磁共振、氘及氫交換與質譜法組合、或定點誘變、或與抗原及其複合結構與其結合抗體及其變異體之電腦建模組合使用之所有方法(參見例如,Epitope Mapping Protocols in Methods in Molecular Biology,第66卷,G. E. Morris編輯.(1996))。一旦確定抗原之所需抗原決定基,可例如使用本文中所述技術產生該抗原決定基之抗體。抗體之產生及表徵亦可闡明關於所需抗原決定基之資訊。自此資訊,然後可競爭篩選結合至相同抗原決定基之抗體。達成此之一種方法為進行交叉競爭研究以找到彼此競爭結合之抗體,即,競爭結合至抗原之抗體。基於其交叉競爭「分類」抗體之高通量方法述於PCT公開案第WO 03/48731號中。The term "epitope" refers to the portion of an antigen to which an antibody (or antigen-binding fragment thereof) binds. Epitopes can be formed from both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from adjacent amino acids generally remain on exposure to denaturing agents, whereas epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes may contain various numbers of amino acids in unique spatial configurations. Methods for determining the spatial configuration of epitopes include, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, deuterium and hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or binding to antigens and their complex structures, antibodies and their mutations All methods used in combination with computer modeling of organisms (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by G. E. Morris (1996)). Once the desired epitope of an antigen is determined, antibodies to that epitope can be generated, for example, using techniques described herein. The generation and characterization of antibodies can also elucidate information about the required epitopes. From this information, competitive screening of antibodies that bind to the same epitope can then be performed. One way to accomplish this is to perform cross-competition studies to find antibodies that compete for binding to each other, ie, antibodies that compete for binding to the antigen. A high-throughput method for "classifying" antibodies based on their cross-competition is described in PCT Publication No. WO 03/48731.

術語「生殖系」係指抗體基因及基因片段之核苷酸序列,因為其經由生殖細胞自親本傳給後代。生殖系序列不同於成熟B細胞中編碼抗體之核苷酸序列,該等核苷酸序列已在B細胞成熟過程期間藉由重組及高突變事件改變。The term "germline" refers to the nucleotide sequences of antibody genes and gene fragments as they are passed from parent to offspring via germ cells. Germline sequences differ from the nucleotide sequences encoding antibodies in mature B cells, which have been altered by recombination and hypermutation events during the B cell maturation process.

術語「糖基化位點」係指藉由真核細胞識別為糖殘基之連接位置之胺基酸殘基。在碳水化合物(諸如寡糖)之情況下連接之胺基酸通常為天冬醯胺(N-鍵聯)、絲胺酸(O-鍵聯)及蘇胺酸(O-鍵聯)殘基。特定連接位點通常藉由胺基酸序列(本文中稱作「糖基化位點序列」)信號傳導。用於N-連接之糖基化之糖基化位點序列為:-Asn-X-Ser-或-Asn-X-Thr-,其中X可為習知胺基酸中之任一者,除了脯胺酸。術語「N-連接」及「O-連接」係指用作糖分子與胺基酸殘基之間之連接位點之化學基團。N-連接之糖通過胺基連接;O-連接之糖通過羥基連接。術語「多醣佔用」係指連接至糖基化位點之碳水化合物部分之存在(即,佔用多醣位點)。在多肽上存在至少兩個潛在糖基化位點之情況下,無(0-多醣位點佔用)、一個(1-多醣位點佔用)或兩個(2-多醣位點佔用)位點可由碳水化合物部分佔用。The term "glycosylation site" refers to an amino acid residue recognized by eukaryotic cells as the site of attachment of a sugar residue. In the case of carbohydrates (such as oligosaccharides) the linked amino acids are usually asparagine (N-linked), serine (O-linked) and threonine (O-linked) residues . Specific attachment sites typically signal through amino acid sequences (referred to herein as "glycosylation site sequences"). The glycosylation site sequence used for N-linked glycosylation is: -Asn-X-Ser- or -Asn-X-Thr-, where X can be any of the conventional amino acids, except Proline. The terms "N-linked" and "O-linked" refer to chemical groups that serve as attachment sites between sugar molecules and amino acid residues. N-linked sugars are linked through amine groups; O-linked sugars are linked through hydroxyl groups. The term "polysaccharide occupancy" refers to the presence of a carbohydrate moiety linked to a glycosylation site (i.e., occupying a glycan site). In the case of at least two potential glycosylation sites on the polypeptide, none (0-glycan site occupied), one (1-glycan site occupied), or two (2-glycan site occupied) sites can be determined by The carbohydrate portion is occupied.

術語「宿主細胞」係指可工程改造以產生所關注蛋白質、蛋白質片段或肽之細胞系統。宿主細胞包括(不限於)經培養細胞,例如,源自囓齒動物(大鼠、小鼠、豚鼠或倉鼠)之哺乳動物培養細胞,諸如CHO、BHK、NSO、SP2/0、YB2/0;人類細胞(例如,HEK293F細胞、HEK293T細胞);或人類組織或雜交瘤細胞、酵母細胞、昆蟲細胞(例如,S2細胞)、細菌細胞(例如,大腸桿菌( E. coli)細胞)及包含於轉殖基因動物或經培養組織內之細胞。術語不僅涵蓋特定受試者細胞,而且涵蓋此細胞之後代。因為某些修飾可發生於後代中,由於突變或環境影響,此後代可不與親本細胞相同,但是仍包含於術語「宿主細胞」之範圍內。 The term "host cell" refers to a cellular system that can be engineered to produce a protein, protein fragment or peptide of interest. Host cells include, but are not limited to, cultured cells, for example, mammalian cultured cells derived from rodents (rat, mouse, guinea pig or hamster), such as CHO, BHK, NSO, SP2/0, YB2/0; human cells (e.g., HEK293F cells, HEK293T cells); or human tissue or hybridoma cells, yeast cells, insect cells (e.g., S2 cells), bacterial cells (e.g., Escherichia coli ( E. coli ) cells) and those included in the transformation Cells in genetic animals or cultured tissues. The term encompasses not only cells of a particular subject, but also descendants of such cells. Because certain modifications can occur in progeny, such progeny may not be identical to the parent cell due to mutations or environmental influences, but are still included within the scope of the term "host cell."

「人類抗體」為具有胺基酸序列者,該胺基酸序列對應於藉由人類或人類細胞產生或源自利用人類抗體庫或其他人類抗體編碼序列之非人類來源之抗體之胺基酸序列。人類抗體之此定義明確排除包含非人類抗原結合殘基之人源化抗體。A "human antibody" is one that has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by humans or human cells or derived from non-human sources utilizing human antibody libraries or other human antibody coding sequences. . This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues.

術語「人源化抗體」係指含有源自人類抗體序列之胺基酸殘基之嵌合抗體。人源化抗體可含有來自非人類動物或合成抗體之CDR或HVR中之一些或所有,而抗體之框架區及恆定區含有源自人類抗體序列之胺基酸殘基。The term "humanized antibody" refers to a chimeric antibody containing amino acid residues derived from human antibody sequences. Humanized antibodies may contain some or all of the CDRs or HVRs from non-human animals or synthetic antibodies, and the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

術語「說明性抗體」係指本發明中所述及如 A B中所列之彼等所指定之抗體中之任一者,及包含 A B中所列之抗體之6個HVR及/或VH及VL之任何抗體。此等抗體可為任何類別(例如,IgA、IgD、IgE、IgG及IgM)。因此,以上所識別之各抗體涵蓋具有V L及V H區之相同胺基酸序列之所有五類抗體。此外,IgG類別之抗體可為任何子類別(例如,IgG1、IgG2、IgG3及IgG4)。因此,以上於IgG子類別中所識別之各抗體涵蓋具有V L及V H區之相同胺基酸序列之所有四種子類別之抗體。五類以及四種IgG子類別之人類抗體之重鏈恆定區的胺基酸序列係此項技術中已知。 The term "illustrative antibody" refers to any of the designated antibodies described herein and as listed in Tables A and B , and the 6 HVRs comprising the antibodies listed in Tables A and B and/or any antibodies to VH and VL. These antibodies can be of any class (eg, IgA, IgD, IgE, IgG, and IgM). Therefore, each of the antibodies identified above covers all five types of antibodies having the same amino acid sequence of the VL and VH regions. Furthermore, antibodies of the IgG class can be of any subclass (eg, IgG1, IgG2, IgG3, and IgG4). Therefore, each of the antibodies identified above in the IgG subclass encompasses antibodies of all four subclasses having the same amino acid sequence of the VL and VH regions. The amino acid sequences of the heavy chain constant regions of human antibodies of five classes and four IgG subclasses are known in the art.

「經單離」抗體或結合分子(例如,可活化抗體)為已自其自然環境之組分分離者。於一些實施例中,抗體經純化為大於95%或99%純度,如藉由例如電泳(例如,SDS-PAGE、等電聚焦(IEF)、毛細管電泳)或層析(例如,離子交換或逆相HPLC)所測定。評論用於評估抗體純度之方法,參見例如,Flatman等人,J. Chromatogr. B 848:79-87 (2007)。An "isolated" antibody or binding molecule (eg, an activatable antibody) is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase electrophoresis). phase HPLC). For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

術語「K a」係指特定結合分子-抗原相互作用之締合速率常數,其中術語「k d」係指特定結合分子-抗原相互作用之解離速率常數。 The term " Ka " refers to the association rate constant of a specific binding molecule-antigen interaction, where the term " kd " refers to the dissociation rate constant of a specific binding molecule-antigen interaction.

術語「K D」係指特定抗體-抗原相互作用之平衡解離常數。其自k d與k a之比率(即,k d/k a)獲得且表示為莫耳濃度(M)。K D係用作抗體結合至其結合搭檔之親和力之量度。K D越小,抗體結合越緊密,或抗體與抗原之間之親和力越高。例如,具有奈莫耳(nM)解離常數之抗體較具有微莫耳(μM)解離常數之抗體與特定抗原結合更緊密。可使用此項技術中良好建立之方法測定抗體之K D值。一種測定抗體之K D之方法為藉由使用表面電漿子共振,通常使用生物感測器系統,諸如Biacore®系統。例如,使用BIACORE™系統(BIAcore檢定)之檢定程序述於本發明之至少實例3中。 The term " KD " refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. It is obtained from the ratio of k d to ka (ie, k d / ka ) and is expressed as molar concentration (M). KD is used as a measure of the affinity with which an antibody binds to its binding partner. The smaller the KD , the tighter the binding of the antibody, or the higher the affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant binds more tightly to a specific antigen than an antibody with a micromolar (μM) dissociation constant. The KD value of an antibody can be determined using methods well established in the art. One method of determining the K D of an antibody is by using surface plasmon resonance, typically using a biosensor system such as the Biacore® system. For example, an assay procedure using the BIACORE™ system (BIAcore assay) is described in at least Example 3 of the present invention.

術語「哺乳動物」係指哺乳綱類別之任何動物物種。哺乳動物之實例包括:人類;實驗動物,諸如大鼠、小鼠、倉鼠、兔、非人類靈長類動物及豚鼠;家養動物,諸如貓、狗、牛、綿羊、山羊、馬及豬;及被俘野生動物,諸如獅子、老虎、大象及類似者。The term "mammal" means any animal species within the class Mammalia. Examples of mammals include: humans; laboratory animals, such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; domestic animals, such as cats, dogs, cattle, sheep, goats, horses, and pigs; and Captured wild animals such as lions, tigers, elephants and the like.

關於哺乳動物之某種疾病病狀之術語「預防(prevent/preventing)」係指預防或延遲該疾病之發作,或預防其臨床或亞臨床症狀之表現。The term "prevent/preventing" regarding the symptoms of a certain disease in mammals refers to preventing or delaying the onset of the disease, or preventing the manifestation of its clinical or subclinical symptoms.

如本文中所用,兩個多肽序列之間之「序列同一性」指示該等序列之間相同之胺基酸之百分比。多肽之胺基酸序列同一性習知上可使用已知電腦程式,諸如Bestfit、FASTA或BLAST測定(參見例如,Pearson, Methods Enzymol.183:63-98 (1990);Pearson, Methods Mol. Biol.132:185-219 (2000);Altschul等人, J. Mol. Biol.215:403-410 (1990);Altschul等人, Nucelic Acids Res.25:3389-3402 (1997))。當使用Bestfit或任何其他序列比對程序測定特定序列是否與參考胺基酸序列為例如95%同一性時,設置參數使得在參考胺基酸序列之全長上計算同一性百分比且允許參考序列之胺基酸殘基之總數目之多達5%的同源性空隙。測定多肽之間之同一性百分比之此上述方法適用於本文中所揭示之所有蛋白質、片段或其變異體。 As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. The amino acid sequence identity of a polypeptide can conventionally be determined using known computer programs such as Bestfit, FASTA or BLAST (see, for example, Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucelic Acids Res. 25:3389-3402 (1997)). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, set the parameters so that the percent identity is calculated over the entire length of the reference amino acid sequence and allows for amines of the reference sequence. Homology gaps of up to 5% of the total number of amino acid residues. This above-described method of determining percent identity between polypeptides is applicable to all proteins, fragments, or variants thereof disclosed herein.

如本文中所用,術語「結合」、「結合至」、「特異性結合」、「特異性結合至」或「對…特異性」係指可量測且可再生相互作用,諸如靶與抗體之間之結合,其確定在分子(包括生物分子)異質群之存在下靶之存在。例如,結合至或特異性結合至靶(其可為抗原決定基)之抗體為結合此靶之抗體,結合此靶較其結合至其他靶具有更高親和力、抗體親抗原性(avidity)、更便利、及/或更多持續時間。於一實施例中,抗體與不相關靶之結合程度係小於抗體與該靶之結合之約10%,如例如藉由放射性免疫檢定(RIA)所量測。於某些實施例中,特異性結合至靶之抗體具有≤ 1μM、≤ 100 nM、≤ 10 nM、≤ 1 nM或≤ 0.1 nM之解離常數(Kd)。於某些實施例中,抗體特異性結合至蛋白質上之抗原決定基,該蛋白質在來自不同物種之蛋白質中係保守的。於另一實施例中,特異性結合可包括但不需要專一結合。As used herein, the terms "bind," "bind to," "specifically bind," "specifically bind to," or "specific for" refer to a measurable and reproducible interaction, such as between a target and an antibody. Binding between molecules that determines the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, and higher affinity than to other targets. Convenience, and/or more duration. In one embodiment, the degree of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to that target, as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding may include, but does not require, specific binding.

關於哺乳動物之某種疾病病狀之術語「治療(treat/treating/treatment)」係指造成患有該疾病病狀之哺乳動物之所需或有益效果。所需或有益效果可包括疾病之一或多種症狀之降低之頻率或嚴重度(即,腫瘤生長及/或轉移,或藉由免疫細胞之數目及/或活性介導之其他效果及類似者),或阻止或抑制疾病、病狀或病症之進一步發展。於治療哺乳動物之癌症之上下文中,所需或有益效果可包括抑制癌細胞之進一步生長或擴散、癌細胞之死亡、抑制癌症再發生、減少與癌症相關之疼痛、或提高哺乳動物之存活。該效果可係主觀或客觀。例如,若哺乳動物為人類,則人類可注意提高之精力或活力或減少之疼痛,因為主觀改善之症狀或對療法反應。或者,臨床醫師可通知腫瘤大小或腫瘤負擔之減少,基於身體檢查、實驗室參數、腫瘤標記物或射線照相發現。臨床醫師可觀察對治療反應之一些實驗室標誌包括測試之標準化,諸如白血球計數、紅血球計數、血小板計數、紅血球沉降率、及各種酵素水平。此外,臨床醫師可觀察可檢測腫瘤標記物之減少。或者,可使用其他測試評價客觀改善,諸如語圖、核磁共振測試及正電子放射測試。The term "treat/treating/treatment" with respect to a certain disease condition in mammals refers to causing a desired or beneficial effect on the mammal suffering from the disease condition. Desirable or beneficial effects may include reduced frequency or severity of one or more symptoms of disease (i.e., tumor growth and/or metastasis, or other effects mediated by the number and/or activity of immune cells, and the like) , or to prevent or inhibit the further development of a disease, condition or condition. In the context of treating cancer in a mammal, a desired or beneficial effect may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of recurrence of cancer, reduction of pain associated with cancer, or improved survival of the mammal. The effect can be subjective or objective. For example, if the mammal is a human, the human may note increased energy or vitality or reduced pain as a result of subjective improvement in symptoms or response to therapy. Alternatively, the clinician may be notified of a reduction in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers, or radiographic findings. Some laboratory markers that clinicians can observe for response to treatment include standardized tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. Additionally, clinicians can observe reductions in detectable tumor markers. Alternatively, other tests may be used to evaluate objective improvement, such as speech graphics, nuclear magnetic resonance testing, and positron emission testing.

術語「載體」係指能轉運外來核酸分子之核酸分子。外來核酸分子藉由重組技術(諸如連接或重組)連接至載體核酸分子。此允許外來核酸分子被操作、選擇、於宿主細胞或生物體中進一步操作或表現。載體可為質粒、噬菌體、轉位子、黏粒、染色體、病毒或病毒子。可在引入宿主細胞後將一種載體融合至宿主細胞之基因組,及從而連同宿主基因組(例如,非表小體哺乳動物載體)複製。另一種載體能於引入其之宿主細胞中自發複製(例如,具有複製之細菌來源之細菌載體及表小體哺乳動物載體)。能指導以可操作方式連接其之可表現外來核酸之表現之另一特定類型之載體通常被稱作「表現載體」。表現載體一般具有驅動可表現外來核酸之表現之控制序列。稱作「轉錄載體」之更簡單載體僅能被轉錄但是不能被轉譯:其可於靶細胞中複製但是不可表現。術語「載體」涵蓋所有類型之載體,不管其功能。能指導以可操作方式連接其之可表現核酸之表現之載體通常被稱作「表現載體」。「載體」之其他實例可包括顯示載體(例如,指導編碼多肽在病毒或細胞(諸如細菌細胞、酵母細胞、昆蟲細胞及/或哺乳動物細胞)表面上表現及顯示之載體)。The term "vector" refers to a nucleic acid molecule capable of transporting foreign nucleic acid molecules. Foreign nucleic acid molecules are linked to vector nucleic acid molecules by recombinant techniques such as ligation or recombination. This allows foreign nucleic acid molecules to be manipulated, selected, further manipulated or expressed in the host cell or organism. The vector can be a plasmid, phage, transposon, cosmid, chromosome, virus or virion. A vector can be fused to the genome of the host cell upon introduction into the host cell and thereby replicated together with the host genome (eg, a non-episomal mammalian vector). Another vector is capable of spontaneous replication in the host cell into which it is introduced (eg, bacterial vectors of bacterial origin and episome mammalian vectors with replicating bacteria). Another specific type of vector capable of directing the expression of an expressible foreign nucleic acid operably linked thereto is generally referred to as an "expression vector." Expression vectors typically have control sequences that drive the expression of foreign nucleic acids. Simpler vectors called "transcription vectors" can only be transcribed but not translated: they can be replicated in target cells but not expressed. The term "carrier" covers all types of carriers, regardless of their function. A vector capable of directing the expression of an expressible nucleic acid operably linked thereto is generally referred to as an "expression vector." Other examples of "vectors" may include display vectors (e.g., vectors that direct expression and display of the encoded polypeptide on the surface of a virus or cell, such as a bacterial cell, yeast cell, insect cell, and/or mammalian cell).

如本文中所用,「受試者」、「患者」或「個體」可係指人類或非人類動物。「非人類動物」可係指不歸類為人類之任何動物,諸如家養動物、農場動物或動物園動物、競技動物、寵物(諸如狗、馬、貓、牛等),以及用於研究之動物。研究動物可係指(不限於)線蟲類動物、節肢動物、脊椎動物、哺乳動物、青蛙、囓齒動物(例如,小鼠或大鼠)、魚(例如,斑馬魚或尖鼻魨)、禽(例如,雞)、狗、貓及非人類靈長類動物(例如,恆河猴、食蟹獼猴、黑猩猩等)。於一些實施例中,受試者、患者或個體為人類。As used herein, a "subject," "patient," or "individual" may refer to a human or non-human animal. "Non-human animal" may refer to any animal that is not classified as a human, such as domestic animals, farm or zoo animals, competitive animals, pets (such as dogs, horses, cats, cattle, etc.), and animals used for research. Research animals may refer to, but are not limited to, nematodes, arthropods, vertebrates, mammals, frogs, rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish), birds ( For example, chickens), dogs, cats, and non-human primates (for example, rhesus monkeys, crab-eating macaques, chimpanzees, etc.). In some embodiments, the subject, patient or individual is a human.

「有效量」係指至少劑量有效量且持續必要時間段以達成一或多種所需或指定效果,包括治療性或預防性結果。可於一或多種投與中提供有效量。出於本發明之目的,抗體、藥物、化合物或醫藥組合物之有效量為足以直接或間接達成預防性或治療性治療之量。如臨床背景中所瞭解,藥物、化合物或醫藥組合物之有效量可或可不結合另一種藥物、化合物或醫藥組合物達成(例如,如作為單藥療法或組合療法投與之有效量)。因此,可於投與一或多種治療劑之背景下考慮「有效量」,且若結合一或多種其他劑,則可考慮以有效量提供單藥劑,可為或達成所需結果。 III. 結合至人類 CTLA4 之結合分子 "Effective amount" means at least a dose effective for a period of time necessary to achieve one or more desired or specified effects, including therapeutic or prophylactic results. An effective amount can be provided in one or more administrations. For the purposes of the present invention, an effective amount of an antibody, drug, compound or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, preventive or therapeutic treatment. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition (eg, as an effective amount is administered as a monotherapy or in combination therapy). Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and, if combined with one or more other agents, may be considered to provide a single agent in an effective amount that achieves or achieves the desired result. III. Binding molecules that bind to human CTLA4

本發明部分係關於結合至人類CTLA4之經單離結合分子,其包括CTLA4抗體、CTLA4抗體之抗原結合片段及CTLA4抗體之衍生物。於一些實施例中,該等結合分子為本文中所述抗體中之任一者,包括關於HVR、可變區(VL、VH)及輕鏈及重鏈(例如,IgG1、IgG2、IgG4)之特定胺基酸序列所述之抗體。於一些實施例中,該等抗體為人類抗體。於一些實施例中,該等抗體為人源化抗體及/或嵌合抗體。於一些實施例中,本發明係關於結合至人類CTLA4且具有下列功能性質中之至少一者(例如,至少一者、至少兩者、至少三者、至少四者、至少五者、至少六者、至少七者、至少八者或所有九者)之結合分子:(a) 以500 nM或更低之K D結合至人類、食蟹獼猴、小鼠、大鼠及/或狗CTLA4;(b)具有對人類CTLA4之拮抗活性;(c) 在多達100 nM之濃度下不結合至人類PD-1、PD-L1、PD-L2、LAG3、TIM3、B7-H3、CD95、CD120a、OX40、CD40、BTLA、VISTA、ICOS及/或B7-H4;(d)與猴、小鼠、大鼠及/或狗CTLA4交叉反應;(e)誘導ADCC效應(例如,對Treg);(f)活化人類PBMC (例如,刺激IL-2及/或IFNγ之分泌);(g)能抑制腫瘤細胞生長;(h)具有對癌症之治療效應;及(i)阻斷人類CTLA4結合至人類CD80及/或人類CD86。於一些實施例中,本文中所述之抗CTLA4抗體相較於檢定中之伊匹單抗於阻斷CD80及/或CD86結合至人類CTLA4中具有更低活性,其中將人類CD80及/或CD86固定(或板結合)且人類CTLA4蛋白係於溶液中或在細胞表面上展示。參見圖:57C及57D及58。於一些實施例中,相較於PBMC或脾中之Treg耗盡,本文中所述之抗CTLA4抗體選擇性耗盡腫瘤微環境中之Treg細胞。於一些實施例中,本文中所述之抗CTLA4抗體相較於伊匹單抗於腫瘤微環境中具有更高Treg耗盡活性。參見圖:61A至B、62A至B及63。本文中亦提供與本文中所述抗體或抗原結合片段中之一或多者交叉競爭結合至人類CTLA4之一或多種抗CTLA4抗體或抗原結合片段。 The present invention relates in part to isolated binding molecules that bind to human CTLA4, including CTLA4 antibodies, antigen-binding fragments of CTLA4 antibodies, and derivatives of CTLA4 antibodies. In some embodiments, the binding molecules are any of the antibodies described herein, including those for HVR, variable regions (VL, VH), and light and heavy chains (e.g., IgG1, IgG2, IgG4) Antibodies described by specific amino acid sequences. In some embodiments, the antibodies are human antibodies. In some embodiments, the antibodies are humanized antibodies and/or chimeric antibodies. In some embodiments, the invention relates to binding to human CTLA4 and having at least one of the following functional properties (e.g., at least one, at least two, at least three, at least four, at least five, at least six , at least seven, at least eight, or all nine) binding molecules: (a) Bind to human, cynomolgus, mouse, rat and/or dog CTLA4 with a KD of 500 nM or less; (b) ) has antagonistic activity against human CTLA4; (c) does not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS and/or B7-H4; (d) Cross-reactivity with monkey, mouse, rat and/or dog CTLA4; (e) Induction of ADCC effects (e.g., on Tregs); (f) Activation Human PBMC (e.g., stimulate the secretion of IL-2 and/or IFNγ); (g) can inhibit tumor cell growth; (h) have therapeutic effects on cancer; and (i) block the binding of human CTLA4 to human CD80 and/or or human CD86. In some embodiments, anti-CTLA4 antibodies described herein have less activity in blocking CD80 and/or CD86 binding to human CTLA4 than ipilimumab in an assay, wherein human CD80 and/or CD86 The human CTLA4 protein is immobilized (or plate bound) in solution or displayed on the cell surface. See Figures: 57C and 57D and 58. In some embodiments, anti-CTLA4 antibodies described herein selectively deplete Treg cells in the tumor microenvironment compared to Treg depletion in PBMC or spleen. In some embodiments, anti-CTLA4 antibodies described herein have higher Treg depleting activity in the tumor microenvironment than ipilimumab. See Figures: 61A to B, 62A to B and 63. Also provided herein is one or more anti-CTLA4 antibodies or antigen-binding fragments that cross-compete for binding to human CTLA4 with one or more of the antibodies or antigen-binding fragments described herein.

於一些實施例中,抗體或抗原結合片段以約500 nM或更低(例如,約500 nM或更低,約450 nM或更低,約400 nM或更低,約350 nM或更低,約300 nM或更低,約250 nM或更低,約200 nM或更低,約150 nM或更低,約100 nM或更低,約90 nM或更低,約80 nM或更低,約70 nM或更低,約60 nM或更低,約50 nM或更低,約40 nM或更低,約30 nM或更低,約25 nM或更低,約20 nM或更低,約10 nM或更低,約1 nM或更低,約0.1 nM或更低等)之K D結合至人類、食蟹獼猴、小鼠、大鼠及/或狗CTLA4。於一些實施例中,抗體或抗原以約 350 nM或更低之K D結合片段結合至人類、食蟹獼猴、小鼠、大鼠及/或狗CTLA4。於一些實施例中,抗體或抗原結合片段以約100 nM或更低之K D結合至人類CTLA4。於一些實施例中,抗體或抗原結合片段以約50 nM或更低之K D結合至人類CTLA4。於一些實施例中,抗體或抗原結合片段以約10 nM或更低之K D結合至人類CTLA4。可使用此項技術中已知之任何方法(包括例如藉由表面電漿子共振、ELISA、等溫滴定量熱法、過濾結合檢定、EMSA等)進行量測抗體或抗原結合片段之K D之方法。於一些實施例中,藉由表面電漿子共振或ELISA量測K D(參見例如以下實例 3)。 In some embodiments, the antibody or antigen-binding fragment is present at about 500 nM or less (e.g., about 500 nM or less, about 450 nM or less, about 400 nM or less, about 350 nM or less, about 300 nM or less, about 250 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or lower, about 1 nM or lower, about 0.1 nM or lower, etc.) binds to human, cynomolgus monkey, mouse, rat and/or dog CTLA4. In some embodiments, the antibody or antigen binds to human, cynomolgus, mouse, rat and/or dog CTLA4 with a K binding fragment of about 350 nM or less. In some embodiments, the antibody or antigen-binding fragment binds to human CTLA4 with a KD of about 100 nM or less. In some embodiments, the antibody or antigen-binding fragment binds to human CTLA4 with a KD of about 50 nM or less. In some embodiments, the antibody or antigen-binding fragment binds to human CTLA4 with a KD of about 10 nM or less. Methods for measuring the KD of an antibody or antigen-binding fragment may be performed using any method known in the art (including, for example, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, etc.) . In some embodiments, KD is measured by surface plasmon resonance or ELISA (see, eg, Example 3 below).

於一些實施例中,本文中所述之抗體或抗原結合片段具有對人類CTLA4之拮抗活性。於一些實施例中,當表現人類CTLA4之細胞(例如,人類細胞)經抗體或抗原結合片段接觸時,該等抗體或抗原結合片段抑制人類CTLA4之一或多種活性(例如,CTLA4阻斷,如使用CLA4阻斷報告基因檢定藉由報告基因信號增加所量測)。 In some embodiments, the antibodies or antigen-binding fragments described herein have antagonistic activity against human CTLA4. In some embodiments, when cells expressing human CTLA4 (e.g., human cells) are contacted by antibodies or antigen-binding fragments, the antibodies or antigen-binding fragments inhibit one or more activities of human CTLA4 (e.g., CTLA4 blocking, such as Use CLA4 blocking reporter gene assay as measured by increase in reporter gene signal).

於一些實施例中,抗體或抗原結合片段與猴(例如,食蟹獼猴)、小鼠、大鼠及/或狗CTLA4交叉反應。於一些實施例中,抗體或抗原結合片段與猴CTLA4交叉反應。於一些實施例中,抗體或抗原結合片段與小鼠CTLA4交叉反應。於一些實施例中,抗體或抗原結合片段與大鼠CTLA4交叉反應。於一些實施例中,抗體或抗原結合片段與狗CTLA4交叉反應。於一些實施例中,抗體或抗原結合片段與猴及小鼠CTLA4;猴及大鼠CTLA4;猴及狗CTLA4;小鼠及大鼠CTLA4;小鼠及狗CTLA4;大鼠及狗CTLA4;猴、小鼠及大鼠CTLA4;猴、小鼠及狗CTLA4;猴、大鼠及狗CTLA4;小鼠、大鼠及狗CTLA4;或猴、小鼠、大鼠及狗CTLA4交叉反應。於一些實施例中,若抗體或抗原結合片段以低於約500 nM (例如,低於約1 nM、低於約10 nM、低於約25 nM、低於約50 nM、低於約75 nM、低於約100 nM、低於約150 nM、低於約200 nM、低於約250 nM、低於約300 nM、低於約350 nM等)之K D結合至非人類CTLA4分子,則抗體或抗原結合片段係交叉反應性。量測抗體交叉反應性之方法係此項技術中已知,包括(不限於)表面電漿子共振、ELISA、等溫滴定量熱法、過濾結合檢定、EMSA等。於一些實施例中,藉由ELISA量測交叉反應性(參見例如以下實例3)。 In some embodiments, the antibody or antigen-binding fragment cross-reacts with monkey (eg, cynomolgus monkey), mouse, rat, and/or dog CTLA4. In some embodiments, the antibody or antigen-binding fragment cross-reacts with monkey CTLA4. In some embodiments, the antibody or antigen-binding fragment cross-reacts with mouse CTLA4. In some embodiments, the antibody or antigen-binding fragment cross-reacts with rat CTLA4. In some embodiments, the antibody or antigen-binding fragment cross-reacts with dog CTLA4. In some embodiments, the antibody or antigen-binding fragment binds to monkey and mouse CTLA4; monkey and rat CTLA4; monkey and dog CTLA4; mouse and rat CTLA4; mouse and dog CTLA4; rat and dog CTLA4; monkey, Mouse and rat CTLA4; monkey, mouse, and dog CTLA4; monkey, rat, and dog CTLA4; mouse, rat, and dog CTLA4; or monkey, mouse, rat, and dog CTLA4 cross-reactive. In some embodiments, if the antibody or antigen-binding fragment is present at less than about 500 nM (e.g., less than about 1 nM, less than about 10 nM, less than about 25 nM, less than about 50 nM, less than about 75 nM , less than about 100 nM, less than about 150 nM, less than about 200 nM, less than about 250 nM, less than about 300 nM, less than about 350 nM, etc.), the antibody binds to a non-human CTLA4 molecule. Or the antigen-binding fragment is cross-reactive. Methods for measuring antibody cross-reactivity are known in the art and include, but are not limited to, surface plasmon resonance, ELISA, isothermal titration calorimetry, filtration binding assays, EMSA, etc. In some embodiments, cross-reactivity is measured by ELISA (see, eg, Example 3 below).

於一些實施例中,於抗體結合至表現CTLA4之細胞後,抗體誘導對表現CTLA4之細胞(例如,對表現CTLA4之人類細胞,諸如Treg)之ADCC效應。量測ADCC效應之方法(例如,活體外方法)係此項技術中已知,包括(不限於)經由以下實例3中所述之方法。於一些實施例中,抗體相對於對照(例如,同型對照或伊匹單抗)誘導ADCC效應超過約10% (例如,誘導ADCC超過約10%、超過約15%、超過約20%、超過約25%、超過約30%、超過約35%、超過約40%等)。 In some embodiments, upon binding of the antibody to the CTLA4-expressing cell, the antibody induces an ADCC effect on the CTLA4-expressing cell (eg, on CTLA4-expressing human cells such as Tregs). Methods for measuring ADCC effects (eg, in vitro methods) are known in the art, including, without limitation, via the method described in Example 3 below. In some embodiments, the antibody induces an ADCC effect of more than about 10% relative to a control (e.g., isotype control or ipilimumab) (e.g., induces ADCC by more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, etc.).

於一些實施例中,抗體或抗原結合片段能抑制腫瘤細胞生長及/或增殖。於一些實施例中,當與抗體或抗原結合片段接觸時,相對於不與抗體或抗原結合片段接觸之對應腫瘤細胞(或相對於與同型對照抗體接觸之對應腫瘤細胞)抑制腫瘤細胞生長及/或增殖至少約5% (例如,至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%或至少約99%)。於一些實施例中,當對受試者投與抗體或抗原結合片段時,抗體或抗原結合片段能減少受試者之腫瘤體積。於一些實施例中,抗體或抗原結合片段能減少受試者之腫瘤體積相對於受試者之初始腫瘤體積(例如,在投與抗體或抗原結合片段之前;如相較於投與同型對照抗體之受試者之對應腫瘤)至少約5% (例如,至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%或至少約99%)。監測腫瘤細胞生長/增殖、腫瘤體積及/或腫瘤抑制之方法係此項技術中已知,包括例如經由以下實例4中所述之方法。 In some embodiments, the antibody or antigen-binding fragment can inhibit tumor cell growth and/or proliferation. In some embodiments, when contacted with an antibody or antigen-binding fragment, inhibits tumor cell growth relative to corresponding tumor cells that are not contacted with the antibody or antigen-binding fragment (or relative to corresponding tumor cells that are contacted with an isotype control antibody) and/ or proliferate by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 99%). In some embodiments, the antibody or antigen-binding fragment reduces tumor volume in the subject when the antibody or antigen-binding fragment is administered to the subject. In some embodiments, the antibody or antigen-binding fragment is capable of reducing the subject's tumor volume relative to the subject's initial tumor volume (e.g., prior to administration of the antibody or antigen-binding fragment; such as compared to administration of an isotype control antibody of the subjects' corresponding tumors) at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%). Methods of monitoring tumor cell growth/proliferation, tumor volume, and/or tumor inhibition are known in the art, including, for example, via the methods described in Example 4 below.

於一些實施例中,抗體或抗原結合片段具有對癌症之治療效應。於一些實施例中,抗體或抗原結合片段減少癌症之一或多種徵兆或症狀。於一些實施例中,當投與抗體或抗原結合片段時,患有癌症之受試者變為部分或完全緩解。 In some embodiments, the antibody or antigen-binding fragment has a therapeutic effect on cancer. In some embodiments, the antibody or antigen-binding fragment reduces one or more signs or symptoms of cancer. In some embodiments, a subject with cancer goes into partial or complete remission when an antibody or antigen-binding fragment is administered.

於另一態樣中,本發明提供經單離抗體,其與本發明之說明性抗體(諸如TY21585、TY21586、TY21587、TY21588、TY21589、TY21580、TY21591、TY21686、TY21687、TY21689、TY21680、TY21691、及/或TY21692)中之任一者競爭或交叉競爭結合至人類CTLA4。於特定實施例中,本發明提供經單離抗體,其與本發明之說明性抗體中之任一者競爭或交叉競爭結合至人類 CTLA4上之相同抗原決定基。抗體與另一種抗體競爭或交叉競爭結合之能力可使用此項技術中已知之標準結合檢定(諸如BIAcore分析、ELISA檢定或流動式細胞測量術)測定。例如,吾人可允許本發明之說明性抗體在飽和條件下結合至人類CTLA4及然後量測測試抗體結合至CTLA4之能力。若測試抗體能與說明性抗體同時結合至CTLA4,則測試抗體結合至與說明性抗體不同之抗原決定基。然而,若測試抗體不能同時結合至CTLA4,則測試抗體結合至相同抗原決定基、重疊抗原決定基或緊鄰藉由說明性抗體結合之抗原決定基之抗原決定基。可使用各種方法(諸如ELISA、RIA、FACS或表面電漿子共振)進行此實驗。 In another aspect, the invention provides isolated antibodies that are identical to the illustrative antibodies of the invention (such as TY21585, TY21586, TY21587, TY21588, TY21589, TY21580, TY21591, TY21686, TY21687, TY21689, TY21680, TY21691, and or TY21692) competes or cross-competes for binding to human CTLA4. In specific embodiments, the invention provides isolated antibodies that compete or cross-compete with any of the illustrative antibodies of the invention for binding to the same epitope on human CTLA4. The ability of an antibody to compete or cross-compete for binding with another antibody can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, one can allow the illustrative antibodies of the invention to bind to human CTLA4 under saturating conditions and then measure the ability of the test antibody to bind to CTLA4. If the test antibody binds to CTLA4 at the same time as the illustrative antibody, then the test antibody binds to a different epitope than the illustrative antibody. However, if the test antibody cannot bind to CTLA4 simultaneously, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope immediately adjacent to the epitope bound by the illustrative antibody. This experiment can be performed using various methods such as ELISA, RIA, FACS or surface plasmon resonance.

於一些實施例中,抗體或抗原結合片段阻斷CTLA4與其結合搭檔中之一或多者之間(例如,人類CTLA4與人類CD80、人類CTLA4與人類CD86)之結合。於一些實施例中,抗體或抗原結合片段阻斷活體外CTLA4與其配位體之間之結合。於一些實施例中,抗體或抗原結合片段具有約500 nM或更低(例如,約500 nM或更低、約400 nM或更低、約300 nM或更低、約200 nM或更低、約100 nM或更低、約50 nM或更低、約25 nM或更低、約10 nM或更低、約1 nM或更低等)之半最大抑制濃度(IC 50)以阻斷CTLA4結合至CD80及/或CD86。於一些實施例中,抗體或抗原結合片段具有約100 nM或更低之半最大抑制濃度(IC 50)以阻斷CTLA4結合至CD80及/或CD86。於一些實施例中,當在約100 nM或更高(例如,約100 nM或更高、約500 nM或更高、約1 µM或更高、約10 µM或更高等)之濃度下提供時,抗體或抗原結合片段完全阻斷人類CTLA4結合至CD80及/或CD86。如本文中所用,術語「完全阻斷(complete blocking/completely blocks)」係指抗體或抗原結合片段減少第一蛋白與第二蛋白之間之結合至少約80% (例如,至少約80%、至少約85%、至少約90%、至少約95%、至少約99%等)的能力。量測抗體或抗原結合片段阻斷第一蛋白(例如,人類CTLA4)與第二蛋白(例如,人類CD80或人類CD86)之結合之能力的方法係此項技術中已知,包括(不限於)經由BIAcore分析、ELISA檢定及流動式細胞測量術(參見例如以下實例3)。於一些實施例中,本文中所述之抗CTLA4抗體具有較伊匹單抗更低阻斷配位體結合之活性。 CTLA4 抗體 In some embodiments, the antibody or antigen-binding fragment blocks binding between CTLA4 and one or more of its binding partners (eg, human CTLA4 and human CD80, human CTLA4 and human CD86). In some embodiments, the antibody or antigen-binding fragment blocks the binding between CTLA4 and its ligand in vitro. In some embodiments, the antibody or antigen-binding fragment has a concentration of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about Half-maximal inhibitory concentration (IC 50 ) of 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.) to block CTLA4 binding to CD80 and/or CD86. In some embodiments, the antibody or antigen-binding fragment has a half-maximal inhibitory concentration ( IC50 ) of about 100 nM or less to block CTLA4 binding to CD80 and/or CD86. In some embodiments, when provided at a concentration of about 100 nM or higher (e.g., about 100 nM or higher, about 500 nM or higher, about 1 µM or higher, about 10 µM or higher, etc.) , the antibody or antigen-binding fragment completely blocks the binding of human CTLA4 to CD80 and/or CD86. As used herein, the term "complete blocking/completely blocks" means that an antibody or antigen-binding fragment reduces the binding between a first protein and a second protein by at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.) capability. Methods for measuring the ability of an antibody or antigen-binding fragment to block the binding of a first protein (e.g., human CTLA4) to a second protein (e.g., human CD80 or human CD86) are known in the art, including (without limitation) Via BIAcore analysis, ELISA assays and flow cytometry (see, eg, Example 3 below). In some embodiments, anti-CTLA4 antibodies described herein have less activity than ipilimumab in blocking ligand binding. CTLA4 antibody

於一些態樣中,本發明提供結合至人類CTLA4之經單離抗體。於一些實施例中,抗體以1000 nM或更低(例如,50 nM或更低,10 nM或更低)之K D結合人類CTLA4,如藉由表面電漿子共振所量測。於一些實施例中,抗體與選自食蟹獼猴、小鼠、大鼠及狗之至少一種非人類物種交叉反應。 In some aspects, the invention provides isolated antibodies that bind to human CTLA4. In some embodiments, the antibody binds human CTLA4 with a K of 1000 nM or less (eg, 50 nM or less, 10 nM or less), as measured by surface plasmon resonance. In some embodiments, the antibody cross-reacts with at least one non-human species selected from the group consisting of cynomolgus monkey, mouse, rat, and dog.

於一些態樣中,本發明提供特異性結合至類似於人類CTLA4之配位體結合位點之抗原決定基之經單離抗體。於一些實施例中,抗體特異性結合至類似於人類CTLA4之CD80結合位點之抗原決定基。於一些實施例中,抗體特異性結合至類似於人類CTLA4之CD86結合位點之抗原決定基。於一些實施例中,抗體特異性結合至包含人類CTLA4之配位體結合位點(例如,CD80及/或CD86結合位點)中之一或多個胺基酸殘基之抗原決定基。於一些實施例中,抗體特異性結合至不同於伊匹單抗之抗原決定基之人類CTLA4之抗原決定基。於一些實施例中,抗原決定基不包含人類CTLA4之CC’環路基序中之胺基酸殘基。於一些實施例中,抗原決定基不包含人類CTLA4之胺基酸殘基L106或I108。於一些實施例中,抗體特異性結合至包含人類CTLA4之胺基酸殘基Y105及L106,但是非I108之抗原決定基,其中該等胺基酸殘基之編號係根據SEQ ID NO: 207。 KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPE (SEQ ID NO: 207) In some aspects, the invention provides isolated antibodies that specifically bind to an epitope similar to the ligand binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope similar to the CD80 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope similar to the CD86 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope comprising one or more amino acid residues in the ligand binding site of human CTLA4 (eg, the CD80 and/or CD86 binding site). In some embodiments, the antibody specifically binds to an epitope of human CTLA4 that is different from an epitope of ipilimumab. In some embodiments, the epitope does not comprise amino acid residues in the CC' loop motif of human CTLA4. In some embodiments, the epitope does not comprise amino acid residues L106 or I108 of human CTLA4. In some embodiments, the antibody specifically binds to an epitope comprising amino acid residues Y105 and L106 of human CTLA4, but not I108, wherein the amino acid residues are numbered according to SEQ ID NO: 207. KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSITGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPE (SEQ ID NO: 207)

於一態樣中,本發明提供包含重鏈可變區及輕鏈可變區之經單離抗體,a)其中該重鏈可變區包含HVR-H1、HVR-H2及HVR-H3,其中該HVR-H1包含如選自以下之式之胺基酸序列:式(I):X1TFSX2YX3IHWV (SEQ ID NO: 1),其中X1為F或Y,X2為D或G,且X3為A、G或W;式(II):YSIX1SGX2X3WX4WI (SEQ ID NO: 2),其中X1為S或T,X2為H或Y,X3為H或Y,且X4為A、D或S;及式(III):FSLSTGGVAVX1WI (SEQ ID NO: 3),其中X1為G或S;該HVR-H2包含如選自以下之式之胺基酸序列:式(IV):IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID NO: 4),其中X1為D或E,X2為S或Y,且X3為P或Q;式(V):IGX1ISPSX2GX3TX4YAQKFQGRV (SEQ ID NO: 5),其中X1為I或W,X2為G或S,X3為G或S,且X4為K或N;及式(VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 6),其中X1為A、G或S,X2 為S或Y,X3為G或S,且X4為S或T;且該HVR-H3包含如選自以下之式之胺基酸序列:式(VII):ARX1X2X3X4FDX5 (SEQ ID NO: 7),其中X1為G、R或S,X2為A、I或Y,X3為D、V或Y,X4為A、E或Y,且X5為I或Y;式(VIII):ARX1GX2GYFDX3 (SEQ ID NO: 8),其中X1為D或L,X2為F或Y,且X3為V或Y;式(IX):ARX1X2X3X4AX5X6FDY (SEQ ID NO: 9),其中X1為L或R,X2為 I或P,X3為A或Y,X4為S或T,X5為T或Y,且X6為A或Y;及式(X):ARDX1X2X3GSSGYYX4GFDX5 (SEQ ID NO: 10),其中X1為I或V,X2為A或H,X3為P或S,X4為D或Y,且X5為F或V;及/或b)其中該輕鏈可變區包含HVR-L1、HVR-L2及HVR-L3,其中該HVR-L1包含如選自以下之式之胺基酸序列:式(XI):RASQX1X2X3SX4LX5 (SEQ ID NO: 11),其中X1為G或S,X2為I或V,X3為G或S,X4為S或Y,且X5為A或N;式(XII):RASQX1VX2X3RX4LA (SEQ ID NO: 12),其中X1為S或T,X2為F、R或S,X3為G或S,且X4為F或Y;及式(XIII):RASX1SVDFX2GX3SFLX4 (SEQ ID NO: 13),其中X1為E或Q,X2為D、F、H或Y,X3為F、I或K,且X4為A、D或H;該HVR-L2包含如式(XIV):X1ASX2X3X4X5GX6 (SEQ ID NO: 14)之胺基酸序列,其中X1為A或D,X2為N、S或T,X3為L或R,X4為A、E或Q,X5為S或T,且X6為I或V;且該HVR-L3包含如選自以下之式之胺基酸序列:式(XV):YCX1X2X3X4X5X6PX7T (SEQ ID NO: 15),其中X1為E、Q或V,X2為H或Q,X3為A、G、H、R或S,X4為D、L、S或Y,X5為E、G、P、Q或S,X6為L、T、V或W,且X7為F、L、P、W或Y;式(XVI):YCQQX1X2X3WPPWT (SEQ ID NO: 16),其中X1為S或Y,X2為D或Y,且X3為Q或Y;及式(XVII):YCQX1YX2SSPPX3YT (SEQ ID NO: 17),其中X1為H或Q,X2為T或V,且X3為E或V。In one aspect, the invention provides an isolated antibody comprising a heavy chain variable region and a light chain variable region, a) wherein the heavy chain variable region comprises HVR-H1, HVR-H2 and HVR-H3, wherein The HVR-H1 includes an amino acid sequence selected from the following formula: Formula (I): X1TFSX2YX3IHWV (SEQ ID NO: 1), wherein X1 is F or Y, X2 is D or G, and X3 is A, G Or W; Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO: 2), where X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D or S; and Formula (III) : FSLSTGGVAVX1WI (SEQ ID NO: 3), where X1 is G or S; the HVR-H2 includes an amino acid sequence selected from the following formula: Formula (IV): IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID NO: 4), where X1 is D or E, X2 is S or Y, and X3 is P or Q; Formula (V): IGX1ISPSX2GX3TX4YAQKFQGRV (SEQ ID NO: 5), where X1 is I or W, X2 is G or S, and , and X4 is K or N; and formula (VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 6), where X1 is A, G or S, X2 is S or Y, X3 is G or S, and X4 is S or T ; And the HVR-H3 includes an amino acid sequence selected from the following formula: Formula (VII): ARX1X2X3X4FDX5 (SEQ ID NO: 7), where X1 is G, R or S, X2 is A, I or Y, X3 is D, V or Y, X4 is A, E or Y, and X5 is I or Y; Formula (VIII): ARX1GX2GYFDX3 (SEQ ID NO: 8), where X1 is D or L, X2 is F or Y, And X3 is V or Y; Formula (IX): ARX1X2X3X4AX5X6FDY (SEQ ID NO: 9), where X1 is L or R, X2 is I or P, X3 is A or Y, X4 is S or T, X5 is T or Y, and X6 is A or Y; and formula (X): ARDX1X2X3GSSGYYX4GFDX5 (SEQ ID NO: 10), where X1 is I or V, X2 is A or H, X5 is F or V; and/or b) wherein the light chain variable region comprises HVR-L1, HVR-L2 and HVR-L3, wherein the HVR-L1 comprises an amino acid sequence selected from the following formula: Formula (XI): RASQX1X2X3SX4LX5 (SEQ ID NO: 11), where X1 is G or S, X2 is I or V, X3 is G or S, X4 is S or Y, and X5 is A or N; Formula (XII): RASQX1VX2X3RX4LA (SEQ ID NO: 12), wherein X1 is S or T, X2 is F, R or S, X3 is G or S, and ), where X1 is E or Q, X2 is D, F, H or Y, X3 is F, I or K, and X4 is A, D or H; the HVR-L2 contains the following formula (XIV): ID NO: 14) Amino acid sequence, wherein X1 is A or D, X2 is N, S or T, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V; and the HVR-L3 includes an amino acid sequence selected from the following formula: Formula (XV): YCX1X2X3X4X5X6PX7T (SEQ ID NO: 15), where X1 is E, Q or V, and X2 is H or Q, X3 is A, G, H, R or S, X4 is D, L, S or Y, X5 is E, G, P, Q or S, X6 is L, T, V or W, and X7 is F, L , P, W or Y; Formula (XVI): YCQQX1X2X3WPPWT (SEQ ID NO: 16), where X1 is S or Y, X2 is D or Y, and X3 is Q or Y; and Formula (XVII): YCQX1YX2SSPPX3YT (SEQ ID NO: 17), where X1 is H or Q, X2 is T or V, and X3 is E or V.

於一些實施例中,抗體包含:a)包含選自SEQ ID NO: 18至29之胺基酸序列之HVR-H1;包含選自SEQ ID NO: 30至39之胺基酸序列之HVR-H2;及包含選自SEQ ID NO: 40至52之胺基酸序列之HVR-H3;及/或b)包含選自SEQ ID NO: 53至65之胺基酸序列之HVR-L1;包含選自SEQ ID NO: 66至69之胺基酸序列之HVR-L2;及包含選自SEQ ID NOS: 70至81之胺基酸序列之HVR-L3。於一些實施例中,抗體包含針對下 A中所述之示例性抗體中之任一者所示之HVR中之一者、兩者、三者、四者、五者或所有六者。 A :抗 CTLA4 HVR 序列 In some embodiments, the antibody comprises: a) HVR-H1 comprising an amino acid sequence selected from SEQ ID NO: 18 to 29; HVR-H2 comprising an amino acid sequence selected from SEQ ID NO: 30 to 39 ; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 40 to 52; and/or b) HVR-L1 comprising an amino acid sequence selected from SEQ ID NO: 53 to 65; comprising HVR-L2 having the amino acid sequence of SEQ ID NOS: 66 to 69; and HVR-L3 comprising the amino acid sequence selected from SEQ ID NOS: 70 to 81. In some embodiments, the antibody comprises one, two, three, four, five, or all six of the HVRs shown for any of the exemplary antibodies described in Table A below. Table A : Anti -CTLA4 HVR sequences

於一些實施例中,抗體包含包含SEQ ID NO: 18之胺基酸序列之HVR-H1,包含SEQ ID NO: 30之胺基酸序列之HVR-H2,包含SEQ ID NO: 40之胺基酸序列之HVR-H3,包含SEQ ID NO: 53之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 70之胺基酸序列之HVR-L3。於一些實施例中,抗體包含包含SEQ ID NO: 19之胺基酸序列之HVR-H1,包含SEQ ID NO: 31之胺基酸序列之HVR-H2,包含SEQ ID NO: 41之胺基酸序列之HVR-H3,包含SEQ ID NO: 54之胺基酸序列之HVR-L1,包含SEQ ID NO: 67之胺基酸序列之HVR-L2,及包含SEQ ID NO: 71之胺基酸序列之HVR-L3。於一些實施例中,抗體包含包含SEQ ID NO: 20之胺基酸序列之HVR-H1,包含SEQ ID NO: 32之胺基酸序列之HVR-H2,包含SEQ ID NO: 42之胺基酸序列之HVR-H3,包含SEQ ID NO: 55之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 72之胺基酸序列之HVR-L3。於一些實施例中,抗體包含包含SEQ ID NO: 21之胺基酸序列之HVR-H1,包含SEQ ID NO: 33之胺基酸序列之HVR-H2,包含SEQ ID NO: 43之胺基酸序列之HVR-H3,包含SEQ ID NO: 56之胺基酸序列之HVR-L1,包含SEQ ID NO: 68之胺基酸序列之HVR-L2,及包含SEQ ID NO: 73之胺基酸序列之HVR-L3。於一些實施例中,抗體包含包含SEQ ID NO: 22之胺基酸序列之HVR-H1,包含SEQ ID NO: 34之胺基酸序列之HVR-H2,包含SEQ ID NO: 44之胺基酸序列之HVR-H3,包含SEQ ID NO: 57之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 74之胺基酸序列之HVR-L3。於一些實施例中,抗體包括包含SEQ ID NO: 23之胺基酸序列之HVR-H1,包含SEQ ID NO: 35之胺基酸序列之HVR-H2,包含SEQ ID NO: 45之胺基酸序列之HVR-H3,包含SEQ ID NO: 58之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 75之胺基酸序列之HVR-L3。於一些實施例中,抗體包括包含SEQ ID NO: 24之胺基酸序列之HVR-H1,包含SEQ ID NO: 32之胺基酸序列之HVR-H2,包含SEQ ID NO: 46之胺基酸序列之HVR-H3,包含SEQ ID NO: 59之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 76之胺基酸序列之HVR-L3。於一些實施例中,抗體包括包含SEQ ID NO: 25之胺基酸序列之HVR-H1,包含SEQ ID NO: 36之胺基酸序列之HVR-H2,包含SEQ ID NO: 47之胺基酸序列之HVR-H3,包含SEQ ID NO: 60之胺基酸序列之HVR-L1,包含SEQ ID NO: 69之胺基酸序列之HVR-L2,及包含SEQ ID NO: 77之胺基酸序列之HVR-L3。於一些實施例中,抗體包括包含SEQ ID NO: 26之胺基酸序列之HVR-H1,包含SEQ ID NO: 37之胺基酸序列之HVR-H2,包含SEQ ID NO: 48之胺基酸序列之HVR-H3,包含SEQ ID NO: 61之胺基酸序列之HVR-L1,包含SEQ ID NO: 66之胺基酸序列之HVR-L2,及包含SEQ ID NO: 78之胺基酸序列之HVR-L3。於一些實施例中,抗體包括包含SEQ ID NO: 27之胺基酸序列之HVR-H1,包含SEQ ID NO: 32之胺基酸序列之HVR-H2,包含SEQ ID NO: 49之胺基酸序列之HVR-H3,包含SEQ ID NO: 62之胺基酸序列之HVR-L1,包含SEQ ID NO: 67之胺基酸序列之HVR-L2,及包含SEQ ID NO: 79之胺基酸序列之HVR-L3。於一些實施例中,抗體包括包含SEQ ID NO: 28之胺基酸序列之HVR-H1,包含SEQ ID NO: 37之胺基酸序列之HVR-H2,包含SEQ ID NO: 50之胺基酸序列之HVR-H3,包含SEQ ID NO: 63之胺基酸序列之HVR-L1,包含SEQ ID NO: 67之胺基酸序列之HVR-L2,及包含SEQ ID NO: 80之胺基酸序列之HVR-L3。於一些實施例中,抗體包含包含SEQ ID NO: 18之胺基酸序列之HVR-H1,包含SEQ ID NO: 38之胺基酸序列之HVR-H2,包含SEQ ID NO: 51之胺基酸序列之HVR-H3,包含SEQ ID NO: 64之胺基酸序列之HVR-L1,包含SEQ ID NO: 67之胺基酸序列之HVR-L2,及包含SEQ ID NO: 81之胺基酸序列之HVR-L3。於一些實施例中,抗體包含包含SEQ ID NO: 29之胺基酸序列之HVR-H1,包含SEQ ID NO: 39之胺基酸序列之HVR-H2,包含SEQ ID NO: 52之胺基酸序列之HVR-H3,包含SEQ ID NO: 65之胺基酸序列之HVR-L1,包含SEQ ID NO: 68之胺基酸序列之HVR-L2,及包含SEQ ID NO: 77之胺基酸序列之HVR-L3。In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 40 HVR-H3 of the sequence, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 53, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L2 comprising the amino acid sequence of SEQ ID NO: 70 HVR-L3. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41 Sequence HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 54, HVR-L2 including the amino acid sequence of SEQ ID NO: 67, and HVR-L2 including the amino acid sequence of SEQ ID NO: 71 HVR-L3. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 42 HVR-H3 of the sequence, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 55, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72 HVR-L3. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 43 Sequence HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 56, HVR-L2 including the amino acid sequence of SEQ ID NO: 68, and HVR-L2 including the amino acid sequence of SEQ ID NO: 73 HVR-L3. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44 HVR-H3 of the sequence, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L2 comprising the amino acid sequence of SEQ ID NO: 74 HVR-L3. In some embodiments, the antibody includes HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45 Sequence HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 58, HVR-L2 including the amino acid sequence of SEQ ID NO: 66, and HVR-L2 including the amino acid sequence of SEQ ID NO: 75 HVR-L3. In some embodiments, the antibody includes HVR-H1 comprising the amino acid sequence of SEQ ID NO: 24, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 46 HVR-H3 of the sequence, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L2 comprising the amino acid sequence of SEQ ID NO: 76 HVR-L3. In some embodiments, the antibody includes HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 47 Sequence HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 60, HVR-L2 including the amino acid sequence of SEQ ID NO: 69, and HVR-L2 including the amino acid sequence of SEQ ID NO: 77 HVR-L3. In some embodiments, the antibody includes HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 48 Sequence HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 61, HVR-L2 including the amino acid sequence of SEQ ID NO: 66, and HVR-L2 including the amino acid sequence of SEQ ID NO: 78 HVR-L3. In some embodiments, the antibody includes HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49 Sequence HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 62, HVR-L2 including the amino acid sequence of SEQ ID NO: 67, and HVR-L2 including the amino acid sequence of SEQ ID NO: 79 HVR-L3. In some embodiments, the antibody includes HVR-H1 comprising the amino acid sequence of SEQ ID NO: 28, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50 Sequence HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 63, HVR-L2 including the amino acid sequence of SEQ ID NO: 67, and HVR-L2 including the amino acid sequence of SEQ ID NO: 80 HVR-L3. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 51 Sequence HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 64, HVR-L2 including the amino acid sequence of SEQ ID NO: 67, and HVR-L2 including the amino acid sequence of SEQ ID NO: 81 HVR-L3. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 52 Sequence HVR-H3, HVR-L1 including the amino acid sequence of SEQ ID NO: 65, HVR-L2 including the amino acid sequence of SEQ ID NO: 68, and HVR-L2 including the amino acid sequence of SEQ ID NO: 77 HVR-L3.

於一些實施例中,抗體包含:a)包含選自SEQ ID NO: 82至94之胺基酸序列之重鏈可變區;及/或b)包含選自SEQ ID NO: 95至107之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含具有與選自SEQ ID NO: 82至94之序列至少90% (例如,至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%)序列同一性之胺基酸序列之重鏈可變區,及/或包含具有與選自SEQ ID NO: 95至107之序列至少90% (例如,至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%)序列同一性之胺基酸序列之輕鏈可變區。 於一些實施例中,抗體包含下 B中所述之示例性抗體中之任一者之重鏈可變區及輕鏈可變區。於一些實施例中,抗體包含針對下 B中所述之示例性抗體中之任一者所示之重鏈可變區之1、2或所有3個HVR,及/或輕鏈可變區之1、2或所有3個HVR。 B :抗 CTLA4 可變區胺基酸序列 抗體名稱: VH VL TY21585 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAIHWVRQAPGKGLEWIGIISPSSGSTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDIHSGSSGYYYGFDVWGQGTLVTVSS (SEQ ID NO: 82) DIQLTQSPSSLSASVGDRVTITCRASESVDFFGISFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYTSSPPVYTFGQGTKVEIKR (SEQ ID NO: 95) TY21586 EVQLVESGGGLVQPGGSLRLSCAASGYSITSGYYWAWIRQAPGKGLEWVSSISGSGSTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARDGFGYFDYWGQGTLVTVSS (SEQ ID NO: 83) DIQLTQSPSSLSASVGDRVTITCSASSSVSYVYWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQGLQTPWTFGQGTKVEIKR (SEQ ID NO: 96) TY21587 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDVAPGSSGYYDGFDFWGQGTLVTVSS (SEQ ID NO: 84) DIQLTQSPSSLSASVGDRVTITCRASQGIGSSLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDQWPPWTFGQGTKVEIKR (SEQ ID NO: 97) TY21588 EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWDWIRQAPGKGLEWVSGISGYGGSTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARHSYYGSGNFDYWGQGTLVTVSS (SEQ ID NO: 85) DIQLTQSPSSLSASVGDRVTITCRASESVDFFGKSFLHWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSWPPTFGQGTKVEIKR (SEQ ID NO: 98) TY21589 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWIHWVRQAPGKGLEWIGWISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGAYEFDYWGQGTLVTVSS (SEQ ID NO: 86) DIQLTQSPSSLSASVGDRVTITCRASQSVSSRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYPTPLTFGQGTKVEIKR (SEQ ID NO: 99) TY21580 EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS (SEQ ID NO: 87) DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR (SEQ ID NO: 100) TY21591 EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVAVSWIRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARRIATATYFDYWGQGTLVTVSS (SEQ ID NO: 88) DIQLTQSPSSLSASVGDRVTITCRASQTVFSRYLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYYWPPWTFGQGTKVEIKR (SEQ ID NO: 101) TY21686 EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVAVGWIRQAPGKGLEWVSAISGYGSTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARLPYSAYAFDYWGQGTLVTVSS (SEQ ID NO: 89) DIQLTQSPSSLSASVGDRVTITCRASQGVSSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKVEIKR (SEQ ID NO: 102) TY21687 EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAIHWVRQAPGKGLEWIGIISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARHPFAYWGQGTLVTVSS (SEQ ID NO: 90) DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGISFLDWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYVSSPPEYTFGQGTKVEIKR (SEQ ID NO: 103) TY21689 EVQLVESGGGLVQPGGSLRLSCAASGYTFSGYGIHWVRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARRIDAFDIWGQGTLVTVSS (SEQ ID NO: 91) DIQLTQSPSSLSASVGDRVTITCRASQSVDFDGFSFLHWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRDSWPYTFGQGTKVEIKR (SEQ ID NO: 104) TY21680 EVQLVESGGGLVQPGGSLRLSCAASGYTFSGYAIHWVRQAPGKGLEWIGIISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARLYDVAYWGQGTLVTVSS (SEQ ID NO: 92) DIQLTQSPSSLSASVGDRVTITCRASQSVDFHGKSFLHWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEQSLEVPFTFGQGTKVEIKR (SEQ ID NO: 105) TY21691 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAIHWVRQAPGKGLEWIGIISPSGGSTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARLGYGYFDVWGQGTLVTVSS (SEQ ID NO: 93) DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGISFLHWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQALQLPLTFGQGTKVEIKR (SEQ ID NO: 106) TY21692 EVQLVESGGGLVQPGGSLRLSCAASGYSITSGHYWSWIRQAPGKGLEWIGDISHSGSTYYSQSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGSRTGYFDYWGQGTLVTVSS (SEQ ID NO: 94) DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKVEIKR (SEQ ID NO: 107) In some embodiments, the antibody comprises: a) a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 82 to 94; and/or b) an amine selected from SEQ ID NO: 95 to 107 The amino acid sequence of the light chain variable region. In some embodiments, the antibody comprises a sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, A heavy chain variable region having an amino acid sequence with 97%, 98% or 99% sequence identity, and/or comprising at least 90% (e.g., at least 90%) with a sequence selected from SEQ ID NO: 95 to 107 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity of the light chain variable region of the amino acid sequence. In some embodiments, the antibody comprises the heavy chain variable region and the light chain variable region of any of the exemplary antibodies described in Table B below. In some embodiments, the antibody comprises 1, 2, or all 3 HVRs of the heavy chain variable region shown in any of the exemplary antibodies described in Table B below, and/or the light chain variable region 1, 2 or all 3 HVRs. Table B : Anti -CTLA4 variable region amino acid sequence Antibody name: VH : VL : TY21585 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAIHWVRQAPGKGLEWIGIISPSSGSTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDIHSGSSGYYYGFDVWGQGTLVTVSS (SEQ ID NO: 82) DIQLTQSPSSSLSASVGDRVTITCRASESVDFFGISFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYTSSPPVYTFGQGTKVEIKR (SEQ ID NO: 95) TY21586 EVQLVESGGGLVQPGGSLRLSCAASGYSITSGYYWAWIRQAPGKGLEWVSSISGSGSTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARDGFGYFDYWGQGTLVTVSS (SEQ ID NO: 83) DIQLTQSPSSSLSASVGDRVTITCSASSSVSYVYWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQGLQTPWTFGQGTKVEIKR (SEQ ID NO: 96) TY21587 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGIHWVRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDVAPGSSGYYDGFDFWGQGTLVTVSS (SEQ ID NO: 84) DIQLTQSPSSSLSASVGDRVTITCRASQGIGSSLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDQWPPWTFGQGTKVEIKR (SEQ ID NO: 97) TY21588 EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWDWIRQAPGKGLEWVSGISGYGGSTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARHSYYGSGNFDYWGQGTLVTVSS (SEQ ID NO: 85) DIQLTQSPSSSLSASVGDRVTITCRASESVDFFGKSFLHWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSWPPTFGQGTKVEIKR (SEQ ID NO: 98) TY21589 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWIHWVRQAPGKGLEWIGWISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGAYEFDYWGQGTLVTVSS (SEQ ID NO: 86) DIQLTQSPSSSLSASVGDRVTITCRASQSVSSRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYPTPLTFGQGTKVEIKR (SEQ ID NO: 99) TY21580 EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS (SEQ ID NO: 87) DIQLTQSPSSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR (SEQ ID NO: 100) TY21591 EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVAVSWIRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARRIATATYFDYWGQGTLVTVSS (SEQ ID NO: 88) DIQLTQSPSSSLSASVGDRVTITCRASQTVFSRYLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYYWPPWTFGQGTKVEIKR (SEQ ID NO: 101) TY21686 EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVAVGWIRQAPGKGLEWVSAISGYGSTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARLPYSAYAFDYWGQGTLVTVSS (SEQ ID NO: 89) DIQLTQSPSSSLSASVGDRVTITCRASQGVSSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKVEIKR (SEQ ID NO: 102) TY21687 EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAIHWVRQAPGKGLEWIGIISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARHPFAYWGQGTLVTVSS (SEQ ID NO: 90) DIQLTQSPSSSLSASVGDRVTITCRASQSVDFYGISFLDWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYVSSPPEYTFGQGTKVEIKR (SEQ ID NO: 103) TY21689 EVQLVESGGGLVQPGGSLRLSCAASGYTFSGYGIHWVRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARRIDAFDIWGQGTLVTVSS (SEQ ID NO: 91) DIQLTQSPSSSLSASVGDRVTITCRASQSVDFDGFSFLHWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRDSWPYTFGQGTKVEIKR (SEQ ID NO: 104) TY21680 EVQLVESGGGLVQPGGSLRLSCAASGYTFSGYAIHWVRQAPGKGLEWIGIISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARLYDVAYWGQGTLVTVSS (SEQ ID NO: 92) DIQLTQSPSSSLSASVGDRVTITCRASQSVDFHGKSFLHWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEQSLEVPFTFGQGTKVEIKR (SEQ ID NO: 105) TY21691 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAIHWVRQAPGKGLEWIGIISPSGGSTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARLGYGYFDVWGQGTLVTVSS (SEQ ID NO: 93) DIQLTQSPSSSLSASVGDRVTITCRASQSVDFYGISFLHWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQALQLPLTFGQGTKVEIKR (SEQ ID NO: 106) TY21692 EVQLVESGGGLVQPGGSLRLSCAASGYSITSGHYWSWIRQAPGKGLEWIGDISHSGSTYYSQSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGSRTGYFDYWGQGTLVTVSS (SEQ ID NO: 94) DIQLTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKVEIKR (SEQ ID NO: 107)

於一些實施例中,抗體包含包含SEQ ID NO: 82之胺基酸序列之重鏈可變區及包含SEQ ID NO: 95之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 83之胺基酸序列之重鏈可變區及包含SEQ ID NO: 96之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 84之胺基酸序列之重鏈可變區及包含SEQ ID NO: 97之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 85之胺基酸序列之重鏈可變區及包含SEQ ID NO: 98之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 86之胺基酸序列之重鏈可變區及包含SEQ ID NO: 99之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 87之胺基酸序列之重鏈可變區及包含SEQ ID NO: 100之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 88之胺基酸序列之重鏈可變區及包含SEQ ID NO: 101之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 89之胺基酸序列之重鏈可變區及包含SEQ ID NO: 102之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 90之胺基酸序列之重鏈可變區及包含SEQ ID NO: 103之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 91之胺基酸序列之重鏈可變區及包含SEQ ID NO: 104之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 92之胺基酸序列之重鏈可變區及包含SEQ ID NO: 105之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 93之胺基酸序列之重鏈可變區及包含SEQ ID NO: 106之胺基酸序列之輕鏈可變區。於一些實施例中,抗體包含包含SEQ ID NO: 94之胺基酸序列之重鏈可變區及包含SEQ ID NO: 107之胺基酸序列之輕鏈可變區。In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 82 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 83 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 84 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 85 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 86 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 88 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 101. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 89 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 102. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 90 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 103. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 91 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 92 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 106. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 107.

於一些實施例中,本發明之抗體與包含以下之抗體交叉競爭結合至人類CTLA4:a)包含選自SEQ ID NO: 18至29之胺基酸序列之HVR-H1;包含選自SEQ ID NO: 30至39之胺基酸序列之HVR-H2;及包含選自SEQ ID NO: 40至52之胺基酸序列之HVR-H3;及/或b)包含選自SEQ ID NO: 53至65之胺基酸序列之HVR-L1;包含選自SEQ ID NO: 66至69之胺基酸序列之HVR-L2;及包含選自SEQ ID NO: 70至81之胺基酸序列之HVR-L3。於一些實施例中,本發明之抗體與包含針對 A中所述之示例性抗體中之任一者所示之HVR中之一者、兩者、三者、四者、五者或所有六者的抗體交叉競爭結合至人類CTLA4。於一些實施例中,本發明之抗體與包含以下之抗體交叉競爭結合至人類CTLA4:a)包含選自SEQ ID NO: 82至94之胺基酸序列之重鏈可變區;及/或b)包含選自SEQ ID NO: 95至107之胺基酸序列之輕鏈可變區。於一些實施例中,本發明之抗體與包含針對 B中所述之示例性抗體中之任一者所示之VH及/或VL的抗體交叉競爭結合至人類CTLA4。 In some embodiments, the antibodies of the invention cross-compete for binding to human CTLA4 with an antibody comprising: a) HVR-H1 comprising an amino acid sequence selected from SEQ ID NO: 18 to 29; comprising an amino acid sequence selected from SEQ ID NO: 18 to 29; : HVR-H2 having an amino acid sequence of 30 to 39; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 40 to 52; and/or b) comprising an amino acid sequence selected from SEQ ID NO: 53 to 65 HVR-L1 comprising an amino acid sequence selected from SEQ ID NO: 66 to 69; and HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 70 to 81 . In some embodiments, the antibodies of the invention are associated with one, two, three, four, five, or all six of the HVRs shown for any of the exemplary antibodies described in Table A. Their antibodies cross-compete for binding to human CTLA4. In some embodiments, an antibody of the invention cross-competes for binding to human CTLA4 with an antibody comprising: a) a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 82 to 94; and/or b ) comprises a light chain variable region selected from the amino acid sequence of SEQ ID NO: 95 to 107. In some embodiments, the antibodies of the invention cross-compete for binding to human CTLA4 with an antibody comprising a VH and/or VL directed to any of the exemplary antibodies described in Table B.

本文中所述之CTLA4抗體可為任何類別,諸如IgG、IgM、IgE、IgA或IgD。於一些實施例中,該等CTLA4抗體為IgG類別,諸如IgG1、IgG2、IgG3或IgG4子類別。可使用此項技術中已知之方法將CTLA4抗體自一種類別或子類別轉變成另一種類別或子類別。用於產生所需類別或子類別之抗體之示例性方法包括以下步驟:單離編碼CTLA4抗體之重鏈之核酸及編碼CTLA4抗體之輕鏈之核酸,單離編碼V H區之序列,將該V H序列連接至編碼所需類別或子類別之重鏈恆定區之序列,表現細胞中之輕鏈基因及重鏈構築體,及收集CTLA4抗體。本發明之抗體可為單株抗體或多株抗體。本發明之抗體可為單特異性抗體或多特異性(例如,雙特異性抗體、三特異性抗體等)抗體。於一些實施例中,本文中所述之CTLA4抗體可包含一或多個Fc突變(例如,調節(增加或減少) ADCC或CDC活性之突變)。本發明之CTLA4抗體中可使用此項技術中已知之任何適宜Fc突變。 CTLA4 antibodies described herein can be of any class, such as IgG, IgM, IgE, IgA or IgD. In some embodiments, the CTLA4 antibodies are of the IgG class, such as IgGl, IgG2, IgG3, or IgG4 subclasses. CTLA4 antibodies can be converted from one class or subclass to another using methods known in the art. Exemplary methods for generating antibodies of a desired class or subclass include the steps of isolating a nucleic acid encoding the heavy chain of a CTLA4 antibody and a nucleic acid encoding a light chain of a CTLA4 antibody, isolating the sequence encoding the VH region, and isolating the sequence. VH sequences are linked to sequences encoding the heavy chain constant region of the desired class or subclass, the light chain genes and heavy chain constructs in the cell are represented, and CTLA4 antibodies are collected. The antibodies of the invention can be monoclonal antibodies or polyclonal antibodies. The antibodies of the invention can be monospecific antibodies or multispecific (eg, bispecific antibodies, trispecific antibodies, etc.) antibodies. In some embodiments, CTLA4 antibodies described herein may comprise one or more Fc mutations (eg, mutations that modulate (increase or decrease) ADCC or CDC activity). Any suitable Fc mutation known in the art may be used in the CTLA4 antibodies of the invention.

於一些實施例中,本發明之抗體為結合至第一及第二靶之雙特異性抗體,其中該第一靶為人類CTLA4。於一些實施例中,該雙特異性抗體結合至第一及第二靶,其中該第一靶為人類CTLA4,且其中該雙特異性抗體包含a)包含選自SEQ ID NO: 18至29之胺基酸序列之HVR-H1;包含選自SEQ ID NO: 30至39之胺基酸序列之HVR-H2;及包含選自SEQ ID NO: 40至52之胺基酸序列之HVR-H3;及/或b)包含選自SEQ ID NO: 53至65之胺基酸序列之HVR-L1;包含選自SEQ ID NO: 66至69之胺基酸序列之HVR-L2;及包含選自SEQ ID NO: 70至81之胺基酸序列之HVR-L3。於一些實施例中,該雙特異性抗體結合至第一及第二靶,其中該第一靶為人類CTLA4,且其中該雙特異性抗體包含針對 A中所述之示例性抗體中之任一者所示之HVR中之一者、兩者、三者、四者、五者或所有六者。於一些實施例中,該雙特異性抗體結合至第一及第二靶,其中該第一靶為人類CTLA4,且其中該雙特異性抗體包含:a)包含選自SEQ ID NO: 82至94之胺基酸序列之重鏈可變區;及/或b)包含選自SEQ ID NO: 95至107之胺基酸序列之輕鏈可變區。於一些實施例中,該雙特異性抗體結合至第一及第二靶,其中該第一靶為人類CTLA4,且其中該雙特異性抗體包含針對 B中所述之示例性抗體中之任一者所示之VH及/或VL。於一些實施例中,該第二靶為PD-1、PD-L1、PD-L2、LAG3、TIM3、B7-H3、CD95、CD120a、OX40、CD40、BTLA、VISTA、ICOS、Her1、Her2、Her3或B7-H4。 In some embodiments, the antibodies of the invention are bispecific antibodies that bind to first and second targets, wherein the first target is human CTLA4. In some embodiments, the bispecific antibody binds to first and second targets, wherein the first target is human CTLA4, and wherein the bispecific antibody comprises a) a compound selected from the group consisting of SEQ ID NOs: 18 to 29 HVR-H1 having an amino acid sequence; HVR-H2 comprising an amino acid sequence selected from SEQ ID NO: 30 to 39; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 40 to 52; and/or b) HVR-L1 comprising an amino acid sequence selected from SEQ ID NO: 53 to 65; HVR-L2 comprising an amino acid sequence selected from SEQ ID NO: 66 to 69; and comprising an amino acid sequence selected from SEQ ID NO: 66 to 69 ID NO: HVR-L3 of amino acid sequence 70 to 81. In some embodiments, the bispecific antibody binds to first and second targets, wherein the first target is human CTLA4, and wherein the bispecific antibody includes antibodies directed against any of the exemplary antibodies described in Table A. One, two, three, four, five or all six of the HVRs shown in one. In some embodiments, the bispecific antibody binds to first and second targets, wherein the first target is human CTLA4, and wherein the bispecific antibody comprises: a) a compound selected from the group consisting of SEQ ID NOs: 82 to 94 a heavy chain variable region having an amino acid sequence; and/or b) a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 95 to 107. In some embodiments, the bispecific antibody binds to first and second targets, wherein the first target is human CTLA4, and wherein the bispecific antibody includes antibodies directed against any of the exemplary antibodies described in Table B VH and/or VL indicated by one. In some embodiments, the second target is PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS, Her1, Her2, Her3 Or B7-H4.

本發明之抗體可藉由此項技術中已知之任何技術產生,包括習知單株抗體方法學,例如,標準體細胞雜交技術(參見例如,Kohler及Milstein, Nature256:495 (1975))、B淋巴細胞之病毒或致癌轉形、或如本文中詳細所述之重組抗體技術(參見例如,實例1及2)。於一些實施例中,本發明之抗體使用PCT申請案號PCT/CN2017/098333 (其全文以引用的方式併入本文中)及/或PCT申請案號PCT/CN2017/098299 (其全文以引用的方式併入本文中)中所述之庫及/或方法中之任一者產生。 Antibodies of the invention may be produced by any technique known in the art, including conventional monoclonal antibody methodologies, e.g., standard somatic cell hybridization techniques (see, e.g., Kohler and Milstein, Nature 256:495 (1975)), Viral or oncogenic transformation of B lymphocytes, or recombinant antibody technology as described in detail herein (see, eg, Examples 1 and 2). In some embodiments, the antibodies of the invention use PCT Application No. PCT/CN2017/098333 (the entire text of which is incorporated herein by reference) and/or PCT Application No. PCT/CN2017/098299 (the entire text of which is incorporated herein by reference). any of the libraries and/or methods described in (incorporated herein).

雜交瘤產生為極完善建立之程序。用於製備雜交瘤之常見動物系統為鼠科系統。用於單離經免疫脾細胞用於融合之免疫方案及技術係此項技術中已知。融合搭檔(例如,鼠科骨髓瘤細胞)及融合程序亦已知。可用於製備本發明提供之人類CTLA4抗體之一種熟知方法涉及使用XenoMouse™動物系統。XenoMouse™小鼠經小鼠株工程改造,該等小鼠株包含人類免疫球蛋白重鏈及輕鏈基因座之大片段且於小鼠抗體產生中有缺陷(參見例如,Green等人,(1994) Nature Genetics 7:13-21;WO2003/040170)。將動物用CTLA4抗原免疫。該CTLA4抗原為經單離及/或經純化之CTLA4。其可為CTLA4之片段,諸如CTLA4之細胞外域。動物之免疫可藉由此項技術中已知之任何方法進行(參見例如,Harlow及Lane,Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990)。用於將非人類動物(諸如小鼠、大鼠、綿羊、山羊、豬、牛及馬)免疫之方法係此項技術中熟知(參見例如,Harlow及Lane,見上;及美國專利案第5,994,619號)。可投與CTLA4抗原與佐劑以刺激免疫反應。示例性佐劑包括完全或不完全弗氏(Freund's)佐劑、RIBI (胞壁醯基二肽)或ISCOM (免疫刺激複合物)。於將動物用CTLA4抗原免疫後,自經免疫動物單離之細胞製備產生抗體之永生化細胞系。於免疫後,將小動物處死及將淋巴結及/或脾B細胞永生。將細胞永生化之方法包括(但不限於)用致癌基因轉移細胞、用致癌病毒感染細胞、將其在永生化細胞選擇之條件下培養、使其遭受致癌或突變化合物、使其與永生化細胞(例如骨髓瘤細胞)融合及使腫瘤抑制基因失活(參見例如Harlow及Lane,見上)。若使用與骨髓瘤細胞融合,則該等骨髓瘤細胞較佳地不分泌免疫球蛋白多肽(非分泌細胞系)。使用CTLA4、其部分或表現CTLA4之細胞篩選永生化細胞。針對所需性質(包括穩健生長、高抗體產生及所需抗體性質)選擇、選殖及進一步篩選產生CTLA4抗體之細胞(例如,雜交瘤),如下述所討論。雜交瘤可於活體內於同源動物中、於缺少免疫系統之動物(例如,裸鼠)中、或於細胞培養物中於活體外擴增。選擇、選殖及擴增雜交瘤之方法為一般技術者熟知。Hybridoma generation is a well-established procedure. A common animal system used to prepare hybridomas is the murine system. Immunization protocols and techniques for isolating isolated immunized splenocytes for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion procedures are also known. One well-known method that can be used to prepare human CTLA4 antibodies provided herein involves the use of the XenoMouse™ animal system. XenoMouse™ mice are engineered from mouse strains that contain large fragments of the human immunoglobulin heavy and light chain loci and are defective in mouse antibody production (see, e.g., Green et al., (1994) ) Nature Genetics 7:13-21; WO2003/040170). Animals were immunized with CTLA4 antigen. The CTLA4 antigen is isolated and/or purified CTLA4. It may be a fragment of CTLA4, such as the extracellular domain of CTLA4. Immunization of animals can be performed by any method known in the art (see, eg, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990). Methods for immunizing non-human animals, such as mice, rats, sheep, goats, pigs, cattle, and horses, are well known in the art (see, e.g., Harlow and Lane, supra; and U.S. Patent No. 5,994,619 No.). CTLA4 antigen can be administered with an adjuvant to stimulate an immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramidyl dipeptide), or ISCOM (immunostimulatory complex). After the animal is immunized with the CTLA4 antigen, an immortalized cell line producing the antibody is prepared from cells isolated from the immunized animal. After immunization, animals were sacrificed and lymph node and/or spleen B cells were immortalized. Methods of immortalizing cells include (but are not limited to) transferring cells with oncogenes, infecting cells with oncogenic viruses, culturing them under conditions for immortalized cell selection, exposing them to carcinogenic or mutagenic compounds, and exposing them to immortalized cells. (eg, myeloma cells) and inactivation of tumor suppressor genes (see, eg, Harlow and Lane, supra). If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Immortalized cells are screened using CTLA4, portions thereof, or cells expressing CTLA4. CTLA4 antibody-producing cells (eg, hybridomas) are selected, colonized, and further screened for desired properties, including robust growth, high antibody production, and desired antibody properties, as discussed below. Hybridomas can be expanded in vivo in syngeneic animals, in animals lacking an immune system (eg, nude mice), or in vitro in cell culture. Methods of selecting, breeding and amplifying hybridomas are well known to those of ordinary skill.

本發明之抗體亦可使用噬菌體展示或酵母展示方法製備。用於單離人類抗體之此等展示方法建立於此項技術中(參見例如,Knappik等人(2000) J. Mol. Biol. 296, 57-86;Feldhaus等人(2003) Nat Biotechnol 21:163-170;亦參見以下實例1及2之方法)。 抗原結合片段 Antibodies of the present invention can also be prepared using phage display or yeast display methods. Such display methods for isolated human antibodies are established in the art (see, eg, Knappik et al. (2000) J. Mol. Biol. 296, 57-86; Feldhaus et al. (2003) Nat Biotechnol 21:163 -170; see also the methods of Examples 1 and 2 below). antigen binding fragment

於一些其他態樣中,本發明提供本文中所述之CTLA4抗體中之任一者之抗原結合片段。In some other aspects, the invention provides antigen-binding fragments of any of the CTLA4 antibodies described herein.

抗原結合片段可包含本文中所述抗體中之任一者之任何序列。於一些實施例中,該抗原結合片段包含以下之胺基酸序列:(1) CTLA4抗體之輕鏈;(2) CTLA4抗體之重鏈;(3)來自CTLA4抗體之輕鏈可變區;(4)來自CTLA4抗體之重鏈可變區;(5) CTLA4抗體之一或多個HVR (例如,1、2、3、4、5或6個HVR);或(6)來自CTLA4抗體之輕鏈之三個HVR及來自CTLA4抗體之重鏈之三個HVR。Antigen-binding fragments may comprise any sequence of any of the antibodies described herein. In some embodiments, the antigen-binding fragment includes the following amino acid sequence: (1) the light chain of a CTLA4 antibody; (2) the heavy chain of a CTLA4 antibody; (3) the light chain variable region from a CTLA4 antibody; ( 4) heavy chain variable region from a CTLA4 antibody; (5) one or more HVRs (e.g., 1, 2, 3, 4, 5, or 6 HVRs) of a CTLA4 antibody; or (6) light chain variable region from a CTLA4 antibody three HVRs of the chain and three HVRs of the heavy chain from the CTLA4 antibody.

於一些實施例中,本發明提供選自 A B中所列之彼等之抗體之抗原結合片段。 In some embodiments, the invention provides antigen-binding fragments selected from the antibodies listed in Tables A and B.

於一些實施例中,CTLA4抗體之抗原結合片段包括:(i) Fab片段,其為由V L、V H、C L及C H1域組成之單價片段;(ii) F(ab′) 2片段,其為包含由鉸鏈區處之二硫橋連接之兩個Fab片段之二價片段;(iii)由V H及C H1域組成之Fd片段;(iv)由抗體之單臂之V L及V H域組成之Fv片段;(v) dAb片段(Ward等人,(1989) Nature 341:544-546),其由V H域組成;(vi)經單離CDR,及(vii)單鏈抗體(scFv),其為包含連接至抗體之V H區之抗體之V L區的多肽(參見例如,Bird等人(1988) Science 242:423-426;Huston等人(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883)。 抗體衍生物 In some embodiments, antigen-binding fragments of CTLA4 antibodies include: (i) Fab fragments, which are monovalent fragments consisting of V L , V H , CL and CH 1 domains; (ii) F(ab′) 2 Fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of V H and CH 1 domains; (iv) a V fragment consisting of a single arm of the antibody Fv fragment consisting of L and VH domains; (v) dAb fragment (Ward et al. (1989) Nature 341:544-546) consisting of VH domains; (vi) isolated CDRs, and (vii) Single chain antibodies (scFv), which are polypeptides comprising the V L region of an antibody linked to the V H region of the antibody (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Antibody derivatives

於一些其他態樣中,本發明提供本文中所述CTLA4抗體中之任一者之衍生物。In some other aspects, the invention provides derivatives of any of the CTLA4 antibodies described herein.

於一些實施例中,抗體衍生物係源自本發明之說明性抗體(例如,「親本抗體」)之胺基酸序列之修飾,同時保存親本抗體胺基酸序列之整體分子結構。可修飾親本抗體鏈之任何區域之胺基酸序列,諸如框架區、HVR區或恆定區。修飾之類型包括親本抗體之一或多個胺基酸之置換、插入、缺失或其組合。In some embodiments, antibody derivatives are derived from modifications of the amino acid sequence of an illustrative antibody of the invention (eg, a "parent antibody") while preserving the overall molecular structure of the amino acid sequence of the parent antibody. The amino acid sequence of any region of the parent antibody chain can be modified, such as the framework region, HVR region, or constant region. Types of modifications include substitution, insertion, deletion, or combinations thereof of one or more amino acids of the parent antibody.

於一些實施例中,該抗體衍生物包含與如SEQ ID NO: 82至107中之任一者中所述之胺基酸序列至少65%、至少75%、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%、或至少99%同一性之V L或V H區。於一些實施例中,該抗體衍生物包含與如SEQ ID NO: 18至29中之任一者中所述之胺基酸序列至少65%、至少75%、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%、或至少99%同一性之HVR-H1胺基酸序列區。於一些實施例中,該抗體衍生物包含與如SEQ ID NO: 30至39中之任一者中所述之胺基酸序列至少65%、至少75%、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%、或至少99%同一性之HVR-H2胺基酸序列區。於一些實施例中,該抗體衍生物包含與如SEQ ID NO: 40至52中之任一者中所述之胺基酸序列至少65%、至少75%、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%、或至少99%同一性之HVR-H3胺基酸序列區。於一些實施例中,該抗體衍生物包含與如SEQ ID NO: 53至65中之任一者中所述之胺基酸序列至少65%、至少75%、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%、或至少99%同一性之HVR-L1胺基酸序列區。於一些實施例中,該抗體衍生物包含與如SEQ ID NO: 66至69中之任一者中所述之胺基酸序列至少65%、至少75%、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%、或至少99%同一性之HVR-L2胺基酸序列區。於一些實施例中,該抗體衍生物包含與如SEQ ID NO: 70至81中之任一者中所述之胺基酸序列至少65%、至少75%、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%、或至少99%同一性之HVR-L3胺基酸序列區。 In some embodiments, the antibody derivative comprises at least 65%, at least 75%, at least 85%, at least 90%, at least A V L or V H region that is 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative comprises at least 65%, at least 75%, at least 85%, at least 90%, at least An HVR-H1 amino acid sequence region that is 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative comprises at least 65%, at least 75%, at least 85%, at least 90%, at least An HVR-H2 amino acid sequence region that is 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative comprises at least 65%, at least 75%, at least 85%, at least 90%, at least An HVR-H3 amino acid sequence region that is 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative comprises at least 65%, at least 75%, at least 85%, at least 90%, at least An HVR-L1 amino acid sequence region that is 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative comprises at least 65%, at least 75%, at least 85%, at least 90%, at least An HVR-L2 amino acid sequence region that is 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative comprises at least 65%, at least 75%, at least 85%, at least 90%, at least An HVR-L3 amino acid sequence region that is 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.

於一些特定實施例中,該衍生物包含如SEQ ID NO: 18至107中之任一者中所述之胺基酸序列之1、2、3、4、5、6、7、8、9、10、11、12、13、14、或15個保守或非保守置換,及/或1、2、3、4、5、6、7、8、9、10、11、12、13、14、或15個新增及/或缺失。In some specific embodiments, the derivative includes amino acid sequences 1, 2, 3, 4, 5, 6, 7, 8, 9 as described in any one of SEQ ID NOs: 18 to 107 , 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , or 15 new and/or missing.

胺基酸置換涵蓋保守置換及非保守置換二者。術語「保守胺基酸置換」意指一種胺基酸經另一種胺基酸置換,其中該等兩種胺基酸具有某些物理化學性質之相似性,諸如涉及之殘基之極性、電荷、溶解度、疏水性、親水性、及/或兩親性。例如,通常可於各下列基團內作出置換:(a)非極性(疏水性)胺基酸,諸如丙胺酸、白胺酸、異白胺酸、纈胺酸、脯胺酸、苯丙胺酸、色胺酸及甲硫胺酸;(b)極性中性胺基酸,諸如甘胺酸、絲胺酸、蘇胺酸、半胱胺酸、酪胺酸、天冬醯胺及麩胺醯胺;(c)帶正電荷(鹼性)胺基酸,諸如精胺酸、離胺酸及組胺酸;及(d)帶負電荷(酸性)胺基酸,諸如天冬胺酸及麩胺酸。Amino acid substitutions include both conservative substitutions and non-conservative substitutions. The term "conservative amino acid substitution" means the substitution of one amino acid by another amino acid, wherein the two amino acids share certain physicochemical properties of similarity, such as the polarity, charge, etc. of the residues involved. Solubility, hydrophobicity, hydrophilicity, and/or amphiphilicity. For example, substitutions may generally be made within each of the following groups: (a) non-polar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, Tryptophan and methionine; (b) polar neutral amino acids, such as glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine ; (c) Positively charged (basic) amino acids, such as arginine, lysine, and histidine; and (d) Negatively charged (acidic) amino acids, such as aspartic acid and glutamine acid.

可於抗體之胺基酸序列之任何位置中作出修改,包括HVR、框架區或恆定區。於一實施例中,本發明提供一種抗體衍生物,其含有本發明之說明性抗體之V H及V LHVR序列,又含有不同於該說明性抗體之彼等之框架序列。此等框架序列可獲自包含生殖系抗體基因序列之公共DNA資料庫或發表之參考文獻。例如,人類重鏈及輕鏈可變區基因之生殖系DNA序列可見於Genbank資料庫或「VBase」人類生殖系序列資料庫(Kaba等人,Sequences of Proteins of Immunological Interest,第5版,U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991);Tomlinson等人, J. Mol. Biol.227:776-798 (1992);及Cox等人, Eur. J. Immunol.24:827-836 (1994))。可用於構築抗體衍生物之框架序列包括結構上類似於本發明之說明性抗體所用之框架序列者。例如,可將說明性抗體之HVR-H1、HVR-H2及HVR-H3序列,及HVR-L1、HVR-L2及HVR-L3序列接枝至框架區,該等框架區具有與衍生框架序列之生殖系免疫球蛋白基因中發現之序列相同的序列,或可將HVR序列接枝至相較於該生殖系序列含有一或多個突變之框架區。 Modifications can be made anywhere in the amino acid sequence of the antibody, including the HVR, framework or constant regions. In one embodiment, the invention provides an antibody derivative that contains the VH and VL HVR sequences of the illustrative antibody of the invention, and contains framework sequences that are different from the illustrative antibody. Such framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database or the "VBase" human germline sequence database (Kaba et al., Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242 (1991); Tomlinson et al., J. Mol. Biol. 227:776-798 (1992); and Cox et al., Eur. J. Immunol. 24:827 -836 (1994)). Framework sequences useful in the construction of antibody derivatives include those that are structurally similar to those used in the illustrative antibodies of the invention. For example, the HVR-H1, HVR-H2, and HVR-H3 sequences, and the HVR-L1, HVR-L2, and HVR-L3 sequences of the illustrative antibodies can be grafted to framework regions having the same structure as the derived framework sequences. Sequences identical to those found in germline immunoglobulin genes, or HVR sequences may be grafted to framework regions containing one or more mutations compared to the germline sequence.

於一些實施例中,該抗體衍生物為包含本發明之說明性抗體之胺基酸序列之嵌合抗體。於一實例中,將來自一或多種說明性抗體之一或多個HVR與來自非人類動物(諸如小鼠或大鼠)之抗體之HVR組合。於另一實例中,嵌合抗體之所有HVR源自一或多種說明性抗體。於一些特定實施例中,嵌合抗體包含來自說明性抗體之重鏈可變區之1、2或3個HVR及/或來自說明性抗體之輕鏈可變區之1、2或3個HVR。可使用此項技術中已知之習知方法產生嵌合抗體。In some embodiments, the antibody derivative is a chimeric antibody comprising the amino acid sequence of an illustrative antibody of the invention. In one example, one or more HVRs from one or more illustrative antibodies are combined with HVRs from an antibody from a non-human animal, such as a mouse or rat. In another example, all HVRs of a chimeric antibody are derived from one or more illustrative antibodies. In some specific embodiments, the chimeric antibody comprises 1, 2, or 3 HVRs from the heavy chain variable region of the illustrative antibody and/or 1, 2, or 3 HVRs from the light chain variable region of the illustrative antibody. . Chimeric antibodies can be produced using conventional methods known in the art.

另一種類型修改為使V H及/或V L鏈之HVR區內之胺基酸殘基突變。可進行定點誘變或PCR介導之誘變以引入該(該等)突變及對抗體結合有影響,或可於此項技術中已知之活體外或活體內檢定中評價所關注之其他功能性質。通常,引入保守置換。突變可為胺基酸新增及/或缺失。此外,通常改變VHR區內之至多1、2、3、4或5個殘基。於一些實施例中,該抗體衍生物包含重鏈HVR及/或輕鏈HVR中之1、2、3或4個胺基酸置換。於另一實施例中,該胺基酸置換為將抗體中之一或多個半胱胺酸變成另一種殘基,諸如(不限於)丙胺酸或絲胺酸。該半胱胺酸可為標準或非標準半胱胺酸。於一實施例中,該抗體衍生物相對於說明性抗體之胺基酸序列具有重鏈HVR區中之1、2、3或4個保守胺基酸置換。 Another type of modification is to mutate amino acid residues within the HVR region of the VH and/or VL chain. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and their effect on antibody binding, or other functional properties of interest can be evaluated in in vitro or in vivo assays known in the art. . Typically, conservative substitutions are introduced. Mutations can be the addition and/or deletion of amino acids. In addition, typically up to 1, 2, 3, 4 or 5 residues within the VHR region are altered. In some embodiments, the antibody derivative comprises 1, 2, 3 or 4 amino acid substitutions in the heavy chain HVR and/or light chain HVR. In another embodiment, the amino acid substitution changes one or more cysteine residues in the antibody to another residue, such as (without limitation) alanine or serine. The cysteine can be standard or non-standard cysteine. In one embodiment, the antibody derivative has 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain HVR region relative to the amino acid sequence of the illustrative antibody.

亦可對V H及/或V L區內之框架殘基作出修改。通常,製備此等框架變異體以減少抗體之免疫原性。一種方法為使一或多個框架殘基「反突變」至對應生殖系序列。已經歷體細胞突變之抗體可含有不同於衍生抗體之生殖系序列之框架殘基。此等殘基可藉由比較抗體框架序列與衍生抗體之生殖系序列來識別。為使框架區序列返回其生殖系組態,可藉由例如定點誘變或PCR介導之誘變將體細胞突變「反突變」至生殖系序列。 Modifications can also be made to the framework residues within the VH and/or VL regions. Typically, such framework variants are prepared to reduce the immunogenicity of the antibody. One approach is to "backmute" one or more framework residues to the corresponding germline sequence. Antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence of the antibody from which they were derived. These residues can be identified by comparing the antibody framework sequence to the germline sequence of the derived antibody. To return the framework sequence to its germline configuration, somatic mutations can be "reverse mutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

此外,亦可於說明性抗體之Fc區內做出修改,通常以改變抗體之一或多種功能性質,諸如血清半衰期、補體固著、Fc受體結合及/或抗原依賴性細胞毒性。於一實例中,修改CH1之鉸鏈區使得該鉸鏈區中之半胱胺酸殘基之數目改變,例如,增加或減少。此方法進一步述於美國專利案第5,677,425號中。改變CH1之鉸鏈區中之半胱胺酸殘基之數目以例如促進輕鏈及重鏈之組裝或增加或減少抗體之穩定性。於另一種情況下,使抗體之Fc鉸鏈區突變以減少抗體之生物半衰期。Additionally, modifications may be made within the Fc region of an illustrative antibody, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In one example, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is changed, eg, increased or decreased. This method is further described in US Patent No. 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered to, for example, promote the assembly of light and heavy chains or increase or decrease the stability of the antibody. In another instance, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody.

此外,可根據此項技術中已知之常規實驗修飾本發明之抗體以改變其潛在糖基化位點或模式。於另一態樣中,本發明提供含有輕鏈或重鏈可變區中之至少一個突變之CTLA4抗體之衍生物,該至少一個突變改變可變區中之糖基化模式。此抗體衍生物可具有對結合抗原之增加之親和力及/或經修改之特異性。該等突變可將新穎糖基化位點添加於V區中,改變一或多個V區糖基化位點之位置或移除先已存在之V區糖基化位點。於一實施例中,本發明提供具有重鏈可變區中之天冬醯胺處之潛在N-連接之糖基化位點之CTLA4抗體的衍生物,其中一重鏈可變區中之潛在N-連接之糖基化位點經移除。於另一實施例中,本發明提供具有重鏈可變區中之天冬醯胺處之潛在N-連接之糖基化位點之CTLA4抗體的衍生物,其中兩個重鏈可變區中之潛在N-連接之糖基化位點經移除。改變抗體之糖基化模式之方法係此項技術中已知,諸如述於美國專利案第6,933,368號中之彼等,其揭示內容以引用的方式併入本文中。In addition, the antibodies of the invention can be modified to alter their potential glycosylation sites or patterns according to routine experimentation known in the art. In another aspect, the invention provides derivatives of CTLA4 antibodies containing at least one mutation in the light or heavy chain variable region that alters the glycosylation pattern in the variable region. Such antibody derivatives may have increased affinity for binding antigen and/or modified specificity. These mutations can add novel glycosylation sites to the V region, change the position of one or more V region glycosylation sites, or remove pre-existing V region glycosylation sites. In one embodiment, the invention provides derivatives of CTLA4 antibodies having a potential N-linked glycosylation site at asparagine in the heavy chain variable region, wherein a potential N-linked glycosylation site in the heavy chain variable region -The linked glycosylation sites are removed. In another embodiment, the invention provides derivatives of CTLA4 antibodies having a potential N-linked glycosylation site at asparagine in the heavy chain variable region, wherein in both heavy chain variable regions Potential N-linked glycosylation sites are removed. Methods of altering the glycosylation pattern of antibodies are known in the art, such as those described in U.S. Patent No. 6,933,368, the disclosure of which is incorporated herein by reference.

於另一態樣中,本發明提供一種抗體衍生物,其包括連接至另外分子實體之如本文中所述之CTLA4抗體或其抗原結合片段。另外分子實體之實例包括醫藥劑、肽或蛋白質、檢測劑或標籤、及抗體。In another aspect, the invention provides an antibody derivative comprising a CTLA4 antibody or antigen-binding fragment thereof as described herein linked to another molecular entity. Examples of additional molecular entities include pharmaceutical agents, peptides or proteins, detection agents or tags, and antibodies.

於一些實施例中,該抗體衍生物包括連接至醫藥劑之本發明之抗體。醫藥劑之實例包括細胞毒性劑或其他癌症治療劑,及放射性同位素。細胞毒性劑之特定實例包括紫杉醇(taxol)、細胞鬆弛素(cytochalasin) B、短桿菌肽(gramicidin) D、溴化乙錠(ethidium bromide)、依米丁(emetine)、絲裂黴素(mitomycin)、依託泊苷(etoposide)、替尼泊苷(tenoposide)、長春新鹼(vincristine)、長春鹼(vinblastine)、秋水仙素(colchicin)、多柔比星(doxorubicin)、柔紅黴素(daunorubicin)、二羥基蒽醌二酮、米托蒽醌(mitoxantrone)、光神黴素(mithramycin)、放線菌素(actinomycin) D、1-去氫睾酮、糖皮質激素(glucocorticoid)、普魯卡因(procaine)、丁卡因(tetracaine)、利多卡因(lidocaine)、普萘洛爾(propranolol)、及嘌呤黴素(puromycin)及其類似物或同系物。治療劑亦包括例如抗代謝劑(例如,胺甲喋呤(methotrexate)、6-巰基嘌呤、6-硫鳥嘌呤、阿糖胞苷(cytarabine)、5-氟尿嘧啶、達卡巴嗪(decarbazine))、烷基化劑(例如,二氯甲基二乙胺(mechlorethamine)、噻替派(thioepa)、苯丁酸氮芥(chlorambucil)、美法侖(melphalan)、卡莫司汀(carmustine) (BSNU)及洛莫司汀(lomustine) (CCNU)、環磷醯胺(cyclothosphamide)、白消安(busulfan)、二溴甘露醇、鏈脲佐菌素(streptozotocin)、絲裂黴素C及順-二氯二胺鉑(II) (DDP)順鉑(cisplatin))、蒽環黴素(例如,柔紅黴素(先前稱作道諾黴素(daunomycin))及多柔比星)、抗生素(例如,更生黴素(dactinomycin) (先前稱作放線菌素)、博來黴素(bleomycin)、光神黴素及安麯黴素(anthramycin) (AMC))、及抗有絲分裂劑(例如,長春新鹼及長春鹼)。可與抗體共軛用於診斷或治療使用之放射性同位素之實例包括(但不限於)碘 131、銦 111、釔 90及鑥 177。用於將抗體連接至醫藥劑之方法係此項技術中已知,諸如使用各種連接子技術。連接子類型之實例包括腙、硫醚、酯、二硫醚及含肽連接子。用於將治療劑連接至抗體之連接子及方法之進一步討論參見例如Saito等人, Adv. Drug Deliv. Rev.55:199-215 (2003);Trail等人, Cancer Immunol. Immunother.52:328-337 (2003);Payne, Cancer Cell3:207-212 (2003);Allen, Nat. Rev. Cancer2:750-763 (2002);Pastan及Kreitman, Curr. Opin. Investig. Drugs3:1089-1091 (2002);Senter及Springer (2001) Adv. Drug Deliv. Rev.53:247-264。 In some embodiments, the antibody derivative includes an antibody of the invention linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents, and radioactive isotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin ), etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin ( daunorubicin), dihydroxyanthraquinonedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, pluca procaine, tetracaine, lidocaine, propranolol, puromycin and their analogs or homologs. Therapeutic agents also include, for example, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), Alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU ) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis- Dichlordiamine platinum(II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics ( For example, dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vinblastine alkali and vinblastine). Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine -131 , indium -111 , yttrium -90 , and gallium -177 . Methods for linking antibodies to pharmaceutical agents are known in the art, such as the use of various linker technologies. Examples of linker types include hydrazone, thioether, ester, disulfide, and peptide-containing linkers. For further discussion of linkers and methods for linking therapeutic agents to antibodies, see, for example, Saito et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail et al., Cancer Immunol. Immunother. 52:328 -337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089- 1091 (2002); Senter and Springer (2001) Adv. Drug Deliv. Rev. 53:247-264.

於一些實施例中,該抗體衍生物為CTLA4抗體多聚體,其為CTLA4抗體之多聚形式,諸如抗體二聚體、三聚體或單聚抗體之更高階寡聚體。抗體多聚體內之個別單體可係相同或不同。此外,多聚體內之個別抗體可具有相同或不同結合特異性。抗體之多聚化可通過抗體之天然聚集達成。例如,一些百分比之經純化抗體製劑(例如,經純化IgG4分子)自發形成含有抗體均二聚體及其他更高階抗體多聚體之蛋白質聚集體。或者,抗體均二聚體可通過此項技術中已知之化學鍵聯技術(諸如通過使用交聯劑)形成。適宜交聯劑包括具有由適宜間隔子分開之兩個不同反應性基團之異雙官能性(諸如間-馬來醯亞胺苯甲醯基-N-羥基琥珀醯亞胺酯、4-(馬來醯亞胺甲基)環己烷-1-甲酸琥珀醯酯、及S-乙醯基硫代-乙酸N-琥珀醯酯)或同雙官能性(諸如辛二酸二琥珀醯酯)之彼等。此等連接子係自例如Pierce Chemical Company, Rockford, IL市面上可購得。亦可通過此項技術中已知之重組DNA技術將抗體多聚化。In some embodiments, the antibody derivative is a CTLA4 antibody multimer, which is a multimeric form of a CTLA4 antibody, such as an antibody dimer, a trimer, or a higher order oligomer of a monomeric antibody. The individual monomers within the antibody multimer may be the same or different. Furthermore, individual antibodies within a multimer may have the same or different binding specificities. Multimerization of antibodies can be achieved through natural aggregation of antibodies. For example, some percentages of purified antibody preparations (eg, purified IgG4 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher order antibody multimers. Alternatively, antibody homodimers can be formed by chemical linkage techniques known in the art, such as through the use of cross-linking agents. Suitable cross-linking agents include heterobifunctional ones having two different reactive groups separated by a suitable spacer (such as m-maleimide benzyl-N-hydroxysuccinimide ester, 4-( Maleimide methyl) cyclohexane-1-carboxylic acid succinyl ester, and S-acetylthio-acetic acid N-succinyl ester) or homobifunctional (such as disuccinyl suberate) The others. Such linkers are commercially available, for example, from Pierce Chemical Company, Rockford, IL. Antibodies can also be multimerized by recombinant DNA techniques known in the art.

由本發明提供之其他抗體衍生物之實例包括單鏈抗體、雙抗體、域抗體、奈米抗體及單抗體。「單鏈抗體」 (scFv)由包含連接至V H域之V L域之單多肽鏈組成,其中V L域及V H域配對以形成單價分子。單鏈抗體可根據此項技術中已知之方法製備(參見例如,Bird等人,(1988) Science 242:423-426及Huston等人,(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883)。「雙抗體」由兩條鏈組成,各鏈包含連接至藉由短肽連接子連接之相同多肽鏈上之輕鏈可變區之重鏈可變區,其中相同鏈上之兩個區彼此不配對,但是與另一條鏈上之互補域形成雙特異性分子。製備雙抗體之方法係此項技術中已知(參見例如,Holliger P.等人,(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448,及Poljak R. J.等人,(1994) Structure 2:1121-1123)。域抗體(dAb)為抗體之小功能結合單元,其對應於抗體之重鏈或輕鏈任一者之可變區。域抗體於細菌、酵母及哺乳動物細胞系統中良好表現。域抗體之進一步細節及其產生方法係此項技術中已知(參見例如,美國專利案第6,291,158號、第6,582,915號、第6,593,081號、第6,172,197號、第6,696,245號;歐洲專利案0368684及0616640;WO05/035572、WO04/101790、WO04/081026、WO04/058821、WO04/003019及WO03/002609)。奈米抗體源自抗體之重鏈。奈米抗體通常包含單個可變域及兩個恆定域(CH2及CH3)且保留原始抗體之抗原結合能力。奈米抗體可藉由此項技術中已知之方法製備(參見例如,美國專利案第6,765,087號、美國專利案第6,838,254號、WO 06/079372)。單抗體由IgG4抗體之一條輕鏈及一條重鏈組成。單抗體可藉由移除IgG4抗體之鉸鏈區來製備。單抗體之進一步細節及製備其之方法可見於WO2007/059782中。 IV. 靶向 CTLA4 之可活化結合多肽 Examples of other antibody derivatives provided by the present invention include single chain antibodies, diabodies, domain antibodies, nanobodies and single antibodies. A "single chain antibody" (scFv) consists of a single polypeptide chain comprising a VL domain linked to a VH domain , where the VL and VH domains pair to form a monovalent molecule. Single chain antibodies can be prepared according to methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879 -5883). A "diabody" consists of two chains, each chain comprising a heavy chain variable region linked to a light chain variable region on the same polypeptide chain linked by a short peptide linker, wherein the two regions on the same chain are mutually exclusive. Pairs, but with a complementary domain on the other chain, to form a bispecific molecule. Methods for preparing diabodies are known in the art (see, e.g., Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448, and Poljak RJ et al., (1994) Structure 2:1121-1123). Domain antibodies (dAbs) are small functional binding units of antibodies that correspond to the variable regions of either the heavy or light chain of the antibody. Domain antibodies perform well in bacterial, yeast and mammalian cell systems. Further details of domain antibodies and methods of producing them are known in the art (see, e.g., U.S. Patent Nos. 6,291,158, 6,582,915, 6,593,081, 6,172,197, 6,696,245; European Patent Nos. 0368684 and 0616640; WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 and WO03/002609). Nanobodies are derived from the heavy chains of antibodies. Nanobodies usually contain a single variable domain and two constant domains (CH2 and CH3) and retain the antigen-binding ability of the original antibody. Nanobodies can be prepared by methods known in the art (see, for example, US Patent No. 6,765,087, US Patent No. 6,838,254, WO 06/079372). A single antibody consists of one light chain and one heavy chain of an IgG4 antibody. Single antibodies can be prepared by removing the hinge region of IgG4 antibodies. Further details of single antibodies and methods of making them can be found in WO2007/059782. IV. Activatable binding peptides targeting CTLA4

本發明亦部分關於結合至人類CTLA4之精度/背景依賴性可活化結合多肽(即,可活化抗體),其包括包含本文中所述抗CTLA4抗體(例如,抗CTLA4抗體、抗CTLA4抗體結合片段及/或抗CTLA4抗體衍生物)中之任一者之可活化抗體、可活化抗CTLA4抗體之抗原結合片段、及/或可活化抗CTLA4抗體之衍生物。於一些實施例中,本文中所述之可活化抗CTLA4抗體可具有改善之安全特性。例如,本文中所述之抗CTLA4抗體可具有更好安全邊限,如由脾重量變化所評估。將脾大小隨著所投與藥物劑量之增加之變化用作基準以評估所用藥物候選之安全邊限。如圖48A至B中所示,本文中所述之可活化抗CTLA4抗體相對於親本抗體(無掩蔽部分之抗體)具有更好安全邊限。The present invention also relates in part to accuracy/context-dependent activatable binding polypeptides (i.e., activatable antibodies) that bind to human CTLA4, including anti-CTLA4 antibodies (e.g., anti-CTLA4 antibodies, anti-CTLA4 antibody binding fragments, and /or anti-CTLA4 antibody derivatives), an activatable antibody, an antigen-binding fragment that can activate an anti-CTLA4 antibody, and/or an activatable derivative of an anti-CTLA4 antibody. In some embodiments, the activatable anti-CTLA4 antibodies described herein may have improved safety profiles. For example, anti-CTLA4 antibodies described herein may have better safety margins, as assessed by changes in spleen weight. The change in spleen size with increasing doses of drug administered is used as a benchmark to evaluate the safety margin of the drug candidates used. As shown in Figures 48A-B, the activatable anti-CTLA4 antibodies described herein have a better safety margin relative to the parent antibody (the antibody without the masking moiety).

於一些實施例中,本發明之可活化抗體包含:(a)掩蔽部分(MM);(b)可裂解部分(CM);及(c)靶結合部分(TBM)。於一些實施例中,該MM為本文中所述之掩蔽部分中之任一者。於一些實施例中,該CM為本文中所述之可裂解部分中之任一者。於一些實施例中,該TBM為本文中所述之靶結合部分中之任一者(例如,包含抗體輕鏈可變區及/或抗體重鏈可變區(諸如本文中所述之抗CTLA4抗體中之任一者之VH及/或VL)之靶結合部分(TBM))。於一些實施例中,當該CM未經裂解時,該MM干預及/或抑制可活化抗體結合至其靶(例如,人類CTLA4或人類CD137)。於一些實施例中,當該CM經裂解時,該可活化抗體能結合至其靶(例如,人類CTLA4或人類CD137)。In some embodiments, activatable antibodies of the invention comprise: (a) a masking moiety (MM); (b) a cleavable moiety (CM); and (c) a target binding moiety (TBM). In some embodiments, the MM is any of the masked portions described herein. In some embodiments, the CM is any of the cleavable moieties described herein. In some embodiments, the TBM is any of the target binding moieties described herein (e.g., comprising an antibody light chain variable region and/or an antibody heavy chain variable region (such as an anti-CTLA4 antibody described herein) The target binding moiety (TBM) of any one of the VH and/or VL of the antibody. In some embodiments, when the CM is not cleaved, the MM interferes with and/or inhibits the binding of an activated antibody to its target (eg, human CTLA4 or human CD137). In some embodiments, the activatable antibody is capable of binding to its target (eg, human CTLA4 or human CD137) when the CM is cleaved.

於一些實施例中,該可活化抗體包含:(a)自N端至C端包含掩蔽部分(MM)、可裂解部分(CM)及靶結合部分(TBM)之多肽,其中該MM為本文中所述之掩蔽部分中之任一者,該CM為本文中所述之可裂解部分中之任一者,且其中該TBM包含抗體輕鏈可變區(VL);及(b)抗體重鏈可變區(VH)。In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising a masking moiety (MM), a cleavable moiety (CM) and a target binding moiety (TBM) from the N-terminus to the C-terminus, wherein the MM is as defined herein any of the masking moieties, the CM is any of the cleavable moieties described herein, and wherein the TBM comprises an antibody light chain variable region (VL); and (b) an antibody heavy chain Variable region (VH).

於一些實施例中,該可活化抗體包含:(a)自N端至C端包含掩蔽部分(MM)、可裂解部分(CM)及靶結合部分(TBM)之多肽,其中該MM為本文中所述之掩蔽部分中之任一者,該CM為本文中所述之可裂解部分中之任一者,且其中該TBM包含抗體重鏈可變區(VH);及(b)抗體輕鏈可變區(VL)。In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising a masking moiety (MM), a cleavable moiety (CM) and a target binding moiety (TBM) from the N-terminus to the C-terminus, wherein the MM is as defined herein any of the masking moieties, the CM is any of the cleavable moieties described herein, and wherein the TBM comprises an antibody heavy chain variable region (VH); and (b) an antibody light chain Variable region (VL).

於一些實施例中,該可活化抗體包含:自N端至C端包含掩蔽部分(MM)、可裂解部分(CM)及靶結合部分(TBM)之多肽,其中該MM為本文中所述之掩蔽部分中之任一者,該CM為本文中所述之可裂解部分中之任一者,且其中該TBM包含抗體重鏈可變區(VH)及抗體輕鏈可變區(VL)。In some embodiments, the activatable antibody comprises: a polypeptide comprising a masking moiety (MM), a cleavable moiety (CM) and a target binding moiety (TBM) from the N-terminus to the C-terminus, wherein the MM is as described herein Any of the masking moieties, the CM is any of the cleavable moieties described herein, and wherein the TBM comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL).

術語「可活化結合多肽」、「ABP」或「可活化抗體」包含包含靶結合部分(TBM)、可裂解部分(CM)及掩蔽部分(MM)之多肽。於一些實施例中,該TBM包含結合至靶之胺基酸序列。於一些實施例中,該TBM包含抗體之抗原結合域(ABD)或其抗體片段(例如,本文中所述之抗體或抗原結合片段中之任一者)。於一些實施例中,該抗原結合域包含包含本文中所述之重鏈可變區HVR中之一者、兩者或三者之重鏈可變區,及包含本文中所述之輕鏈可變區HVR中之一者、兩者或三者之輕鏈可變區(例如,如 A中所示之重鏈可變區HVR序列中之一者、兩者或三者,及/或輕鏈可變區HVR序列中之一者、兩者或三者,包括如 A中所示之示例性抗體中之任一者之所有六個HVR)。於一些實施例中,該抗原結合域包含包含本文中所述之重鏈可變區序列中之任一者之重鏈可變區,及包含本文中所述之輕鏈可變區序列中之任一者之輕鏈可變區(例如,如 B中所示之重鏈可變區序列及輕鏈可變區序列)。於一些實施例中,該TBM (例如,包含ABD)包含抗體輕鏈可變區(VL)及抗體重鏈可變區(VH),其中該VH及VL在MM不存在下形成結合至靶之結合域。於一些實施例中,該VH及VL例如於scFv中共價連接。於一些實施例中,該VH及VL不共價連接。於一些實施例中,該VH及VL形成Fab片段。於一些實施例中,該VH連接至抗體重鏈恆定區,及該VL連接至抗體輕鏈恆定區。 The term "activatable binding polypeptide", "ABP" or "activatable antibody" includes polypeptides comprising a target binding moiety (TBM), a cleavable moiety (CM) and a masking moiety (MM). In some embodiments, the TBM includes an amino acid sequence that binds to a target. In some embodiments, the TBM comprises an antigen-binding domain (ABD) of an antibody or an antibody fragment thereof (eg, any of the antibodies or antigen-binding fragments described herein). In some embodiments, the antigen-binding domain comprises a heavy chain variable region comprising one, two, or three of the heavy chain variable regions HVR described herein, and a light chain variable region comprising a HVR described herein. The light chain variable regions of one, two, or three of the variable region HVRs (e.g., one, two, or three of the heavy chain variable region HVR sequences as shown in Table A , and/or One, two, or three of the light chain variable region HVR sequences, including all six HVRs of any of the exemplary antibodies shown in Table A ). In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising any of the heavy chain variable region sequences described herein, and a heavy chain variable region comprising any of the light chain variable region sequences described herein. The light chain variable region of either (eg, the heavy chain variable region sequence and the light chain variable region sequence as shown in Table B ). In some embodiments, the TBM (e.g., comprising an ABD) includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein the VH and VL form binding to the target in the absence of MM. Combined domain. In some embodiments, the VH and VL are covalently linked, for example, in scFv. In some embodiments, the VH and VL are not covalently linked. In some embodiments, the VH and VL form Fab fragments. In some embodiments, the VH is linked to an antibody heavy chain constant region, and the VL is linked to an antibody light chain constant region.

於一些實施例中,該可活化抗體包含自N端至C端包含掩蔽部分(MM)-可裂解部分(CM)-VL之結構之多肽,且該可活化抗體另包含包含VH (例如,Fab片段)之第二多肽。於一些實施例中,該可活化抗體包含自N端至C端包含掩蔽部分(MM)-可裂解部分(CM)-VL-VH (例如,scFv)之結構之多肽。於一些實施例中,該可活化抗體包含自N端至C端包含掩蔽部分(MM)-可裂解部分(CM)-VH之結構之多肽,且該可活化抗體另包含包含VL (例如,Fab片段)之第二多肽。於一些實施例中,該可活化抗體包含自N端至C端包含掩蔽部分(MM)-可裂解部分(CM)-VH-VL (例如,scFv)之結構之多肽。In some embodiments, the activatable antibody comprises a polypeptide comprising a structure of masking moiety (MM)-cleavable moiety (CM)-VL from N-terminus to C-terminus, and the activatable antibody further comprises a VH (e.g., Fab fragment) of the second polypeptide. In some embodiments, the activatable antibody comprises a polypeptide comprising a structure of masking moiety (MM)-cleavable moiety (CM)-VL-VH (eg, scFv) from N-terminus to C-terminus. In some embodiments, the activatable antibody comprises a polypeptide comprising a structure of masking moiety (MM)-cleavable moiety (CM)-VH from N-terminus to C-terminus, and the activatable antibody further comprises a VL (e.g., Fab fragment) of the second polypeptide. In some embodiments, the activatable antibody comprises a polypeptide comprising a structure of masking moiety (MM)-cleavable moiety (CM)-VH-VL (eg, scFv) from N-terminus to C-terminus.

該CM一般包含可裂解之胺基酸序列,例如,用作能形成可還原二硫鍵之酵素及/或半胱胺酸-半胱胺酸對之受質。因而,當術語「裂解」、「可裂解」、「經裂解」及類似者結合CM使用時,該等術語涵蓋例如藉由蛋白酶之酶促裂解,以及半胱胺酸-半胱胺酸對之間之二硫鍵經由可由暴露於還原劑產生之二硫鍵之還原之中斷。The CM typically contains cleavable amino acid sequences, for example, serving as substrates for enzymes capable of forming reducible disulfide bonds and/or cysteine-cysteine pairs. Thus, when the terms "cleaved", "cleavable", "cleaved" and the like are used in connection with CM, these terms encompass enzymatic cleavage, for example by proteases, as well as cysteine-cysteine pairs. The disulfide bonds between the

該MM係指胺基酸序列,當可活化抗體之CM係完整時(例如,未藉由對應酵素裂解,及/或含有未經還原之半胱胺酸-半胱胺酸二硫鍵),該MM干預或抑制TBM結合至其靶。於一些實施例中,該MM干預或抑制TBM結合至其靶如此有效使得TBM與其靶之結合極其低及/或在檢測限以下(例如,於ELISA或流動式細胞測量術檢定中不可檢測結合)。該CM之胺基酸序列可與該MM重疊或包含於該MM內。應注意,為了方便,本文中使用「ABP」或「可活化抗體」係指呈其未經裂解(或「初始」)狀態以及其經裂解狀態二者之ABP或可活化抗體。對一般技術者將顯而易見於一些實施例中,經裂解之ABP可缺少MM,由於CM例如藉由蛋白酶之裂解導致至少MM之釋放(例如,在MM不藉由共價鍵(例如,半胱胺酸殘基之間之二硫鍵)接合至ABP之情況下)。以下更詳細描述示例性ABP。The MM refers to the amino acid sequence, when the CM of the activatable antibody is intact (for example, not cleaved by the corresponding enzyme, and/or contains unreduced cysteine-cysteine disulfide bonds), The MM interferes with or inhibits TBM binding to its target. In some embodiments, the MM interferes with or inhibits TBM binding to its target so effectively that the binding of TBM to its target is extremely low and/or below the detection limit (e.g., undetectable binding in an ELISA or flow cytometry assay) . The amino acid sequence of the CM may overlap with or be included within the MM. It should be noted that, for convenience, the use of "ABP" or "activatable antibody" herein refers to the ABP or activatable antibody in both its uncleaved (or "original") state and its cleaved state. It will be apparent to one of ordinary skill that in some embodiments, a cleaved ABP may lack a MM, since cleavage of the CM, e.g., by a protease, results in the release of at least the MM (e.g., where the MM is not bound by a covalent bond (e.g., cysteamine disulfide bonds between acid residues) when linked to ABP). Exemplary ABPs are described in more detail below.

於一些實施例中,該掩蔽部分(MM)干預、阻止、降低(能力)、防止、抑制靶結合部分結合至其靶,或與靶結合部分競爭結合至其靶(例如「非活性」可活化抗體)。於一些實施例中,僅當多肽尚未被活化(例如,藉由pH變化(增加或減少)活化,藉由溫度變化(增加或減少)活化,與第二分子(諸如小分子或蛋白質配位體)接觸後活化等)時,該掩蔽部分(MM)干預、阻止、降低、防止、抑制靶結合部分結合至其靶或與靶結合部分競爭結合至其靶。於一些實施例中,活化誘導裂解部分內之多肽之裂解。於一些實施例中,活化誘導多肽之構形變化(例如,掩蔽部分(MM)之置換),其導致掩蔽部分不再防止可活化抗體結合至其靶。於一些實施例中,僅當可裂解部分(CM)尚未藉由於該可裂解部分(CM)內裂解之一或多種蛋白酶裂解時,該掩蔽部分(MM)干預、阻止、降低靶結合部分結合至其靶之能力、防止、抑制靶結合部分結合至其靶或與靶結合部分競爭結合至其靶。於一些實施例中,在活化之前該掩蔽部分(MM)具有至少約2.0 (例如,至少約2.0、至少約3.0、至少約4.0、至少約5.0、至少約6.0、至少約7.0、至少約8.0、至少約9.0、至少約10、至少約25、至少約50、至少約75、至少約100、至少約150、至少約200、至少約300、至少約400、至少約500等)之掩蔽效率。於一些實施例中,量測掩蔽效率作為包含掩蔽部分(MM)之可活化抗體結合其靶(在活化之前)之親和力相對於缺少掩蔽部分之多肽結合其靶之親和力的差異(例如,對包含掩蔽部分(MM)之可活化抗體(在活化之前)之靶抗原(諸如CTLA4)之親和力相對於缺少掩蔽部分(MM)之親本抗體之親和力的差異,或對包含掩蔽部分(MM)之可活化抗體(在活化之前)之靶抗原(諸如CTLA4)之親和力相對於對活化後可活化抗體之靶抗原之親和力的差異)。於一些實施例中,掩蔽效率藉由包含掩蔽部分(MM)之可活化抗體(在活化之前)之結合之EC 50除以親本抗體之EC 50量測(例如,藉由ELISA藉由量測EC 50;參見例如實例8之方法)。於一些實施例中,量測掩蔽效率作為包含掩蔽部分(MM)之可活化抗體在活化之前結合其靶之親和力相對於包含掩蔽部分(MM)之可活化抗體於活化後結合其靶之親和力的差異(例如,在活化之前對可活化抗體之靶抗原(諸如CTLA4)之親和力相對於活化後可活化抗體之親和力的差異)。於一些實施例中,該掩蔽部分(MM)結合至靶結合部分(TBM),且防止可活化抗體結合至其靶(例如,「減能」可活化抗體)。於一些實施例中,該掩蔽部分(MM)具有結合至靶結合部分(TBM)之解離常數,其高於靶結合部分(TBM)對其靶之解離常數。 In some embodiments, the masking moiety (MM) interferes with, prevents, reduces (the ability of), prevents, inhibits, or competes with the target binding moiety for binding to its target (e.g., "inactive" activatable antibody). In some embodiments, only when the polypeptide has not been activated (e.g., activated by a change in pH (increase or decrease), activated by a change in temperature (increase or decrease), with a second molecule (such as a small molecule or protein ligand) ) upon post-contact activation, etc.), the masking moiety (MM) intervenes, prevents, reduces, prevents, inhibits, or competes with the target binding moiety from binding to its target. In some embodiments, activation induces cleavage of the polypeptide within the cleavage moiety. In some embodiments, activation induces a conformational change in the polypeptide (eg, displacement of the masking moiety (MM)) that results in the masking moiety no longer preventing the activatable antibody from binding to its target. In some embodiments, the masking moiety (MM) intervenes, prevents, or reduces the binding of the target binding moiety to the cleavable moiety (CM) only when the cleavable moiety (CM) has not been cleaved by one or more proteases that cleave within the cleavable moiety (CM). The ability of its target to prevent, inhibit, or compete with the target binding moiety from binding to its target. In some embodiments, the masked portion (MM) prior to activation has a value of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, A masking efficiency of at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, etc.). In some embodiments, masking efficiency is measured as the difference in the affinity with which an activatable antibody containing a masking moiety (MM) binds its target (prior to activation) relative to the affinity with which a polypeptide lacking the masking moiety binds its target (e.g., for Differences in the affinity of an activatable antibody (prior to activation) with a masking moiety (MM) for a target antigen (such as CTLA4) relative to the affinity of the parent antibody lacking the masking moiety (MM), or to the affinity of an activatable antibody containing the masking moiety (MM) The difference in affinity of an activating antibody (before activation) for its target antigen (such as CTLA4) relative to its affinity for the target antigen of the activatable antibody after activation). In some embodiments, masking efficiency is measured by dividing the EC 50 of binding of an activatable antibody (prior to activation) containing the masking moiety (MM) by the EC 50 of the parent antibody (e.g., by measuring EC 50 ; see e.g. the methods of Example 8). In some embodiments, masking efficiency is measured as the affinity with which an activatable antibody comprising a masking moiety (MM) binds its target prior to activation relative to the affinity with which an activatable antibody comprising a masking moiety (MM) binds its target after activation. Differences (eg, differences in affinity for the target antigen (such as CTLA4) of the activatable antibody before activation relative to affinity of the activatable antibody after activation). In some embodiments, the masking moiety (MM) binds to the target binding moiety (TBM) and prevents the activatable antibody from binding to its target (eg, "deenergizes" the activatable antibody). In some embodiments, the masking moiety (MM) has a dissociation constant for binding to the target binding moiety (TBM) that is higher than the dissociation constant of the target binding moiety (TBM) for its target.

於一些實施例中,於可活化抗體已經活化(例如,藉由於可裂解部分(CM)內裂解之一或多種蛋白酶處理活化,藉由pH變化(增加或減少)活化,藉由溫度變化(增加或減少)活化,與第二分子(諸如酵素或蛋白質配位體)接觸後活化等)後,該掩蔽部分(MM)不干預、阻止靶結合部分(TBM)結合至其靶、降低其結合之能力,不防止、抑制靶結合部分結合至其靶,或不與靶結合部分競爭結合至其靶。於一些實施例中,於可裂解部分(CM)已藉由於可裂解部分(CM)內裂解之一或多種蛋白酶裂解後,該掩蔽部分(MM)不干預、阻止靶結合部分(TBM)結合其靶、降低其結合之能力,不防止、抑制靶結合部分結合其靶,或不與靶結合部分競爭結合其靶。於一些實施例中,於活化後該掩蔽部分(MM)具有至多約1.75 (例如,至多約1.75、至多約1.5、至多約1.4、至多約1.3、至多約1.2、至多約1.1、至多約1.0、至多約0.9、至多約0.8、至多約0.7、至多約0.6、或至多約0.5等)之掩蔽效率(例如,可活化抗體於活化後相較於親本抗體之親和力之相對親和力)。In some embodiments, the activatable antibody has been activated (e.g., activated by cleavage of one or more proteases within the cleavable moiety (CM), activated by a change in pH (increased or decreased), activated by a change in temperature (increased or reduce) activation, activation after contact with a second molecule (such as an enzyme or protein ligand, etc.), the masking moiety (MM) does not interfere, preventing the target binding moiety (TBM) from binding to its target, reducing its binding The ability not to prevent, inhibit, or compete with the target binding moiety from binding to its target. In some embodiments, the masking moiety (MM) does not interfere with, preventing the target binding moiety (TBM) from binding to the cleavable moiety (CM) after it has been cleaved by one or more proteases that cleave within the cleavable moiety (CM). Target, reduce its ability to bind, do not prevent, inhibit the target binding moiety from binding to its target, or do not compete with the target binding moiety for binding to its target. In some embodiments, the masked portion (MM) after activation has at most about 1.75 (e.g., at most about 1.75, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1, at most about 1.0, The masking efficiency (e.g., the relative affinity of the activatable antibody after activation compared to the affinity of the parent antibody) is up to about 0.9, up to about 0.8, up to about 0.7, up to about 0.6, or up to about 0.5, etc.).

於一些實施例中,本發明之可活化抗體:含有包含固定位置處之一對半胱胺酸以確保可活化抗體具有受限構形之掩蔽部分(MM),及/或含有少量或無化學上不穩定殘基(諸如甲硫胺酸或色胺酸)。有利地,固定位置處之一對半胱胺酸殘基之納入確保可活化抗體具有受限構形,該等構形傾向於展示增加之結合親和力及/或特異性。此外,本發明之可活化抗體包含具有很少至無製造過程之不利殘基(諸如甲硫胺酸或色胺酸)之掩蔽部分。In some embodiments, the activatable antibodies of the invention: contain a masking moiety (MM) that includes a pair of cysteines at a fixed position to ensure that the activatable antibody has a restricted configuration, and/or contain little or no chemical unstable residues (such as methionine or tryptophan). Advantageously, the inclusion of a pair of cysteine residues at a fixed position ensures that the activatable antibody has a restricted configuration that tends to exhibit increased binding affinity and/or specificity. In addition, activatable antibodies of the invention comprise masking moieties with unfavorable residues such as methionine or tryptophan with little to no manufacturing process.

於一些實施例中,本發明之可活化抗體係背景依賴性(例如,於某些背景下(諸如於蛋白酶濃化腫瘤微環境中)經活化(僅能結合其靶))。於一些實施例中,本發明之可活化抗體提供超過更多傳統非可活化抗體之提高之安全性(例如,顯示降低之毒性,不誘導許多器官之重量之顯著改變,不改變肝臟組織病理學、血液學及/或血液生物化學等)。於一些實施例中,本發明之可活化抗體相較於更多傳統非可活化抗體具有改善之藥物動力學性質(例如,具有更長活體內半衰期)。 CTLA4 可活化抗體活性 In some embodiments, the activatable antibodies of the invention are context-dependent (eg, activated (only able to bind their target) in certain contexts, such as in a protease-concentrated tumor microenvironment). In some embodiments, activatable antibodies of the invention provide improved safety over more traditional non-activatable antibodies (e.g., exhibit reduced toxicity, do not induce significant changes in the weight of many organs, do not alter liver histopathology , hematology and/or blood biochemistry, etc.). In some embodiments, the activatable antibodies of the invention have improved pharmacokinetic properties (eg, have a longer in vivo half-life) compared to more traditional non-activatable antibodies. Anti- CTLA4 activates antibody activity

於一些實施例中,本發明係關於當呈活化形式時(例如,可活化抗體於可裂解部分中裂解(例如,用一或多種蛋白酶)後具活性,但是在於可裂解部分中裂解(例如,用一或多種蛋白酶)之前減能)結合至人類CTLA4之可活化抗體。於一些實施例中,當以活性形式時可活化抗體具有下列功能性質中之至少一者(例如,至少一者、至少兩者、至少三者、至少四者、至少五者、至少六者、至少七者、至少八者、或所有九者):(a)以500 nM或更低(例如,約10 nM或更低)之K D結合至人類、食蟹獼猴、小鼠、大鼠及/或狗CTLA4;(b)具有對人類CTLA4之拮抗活性;(c)在多達100 nM之濃度下不結合至人類PD-1、PD-L1、PD-L2、LAG3、TIM3、B7-H3、CD95、CD120a、OX40、CD40、BTLA、VISTA、ICOS、及/或B7-H4;(d)與猴、小鼠、大鼠及/或狗CTLA4交叉反應;(e)誘導ADCC效應(例如,對Treg);(f)活化人類PBMC (例如,刺激IL-2及/或IFNγ之分泌);(g)能抑制腫瘤細胞生長;(h)具有對癌症之治療效應;及(i)抑制人類CTLA4結合至人類CD80及/或人類CD86。本文中亦提供一或多種可活化抗體,其與靶向CTLA4之可活化抗體及/或本文中所述之抗CTLA4抗體中之一或多者交叉競爭結合至人類CTLA4。 In some embodiments, the invention relates to an activatable antibody that is active when in an activated form (e.g., an activatable antibody is cleaved in a cleavable moiety (e.g., with one or more proteases)), but is cleaved in a cleavable moiety (e.g., Activatable antibodies that bind to human CTLA4 are previously inactivated using one or more proteases. In some embodiments, the activatable antibody has at least one of the following functional properties when in an active form (e.g., at least one, at least two, at least three, at least four, at least five, at least six, At least seven, at least eight, or all nine): (a) Bind to humans, cynomolgus monkeys, mice, rats, and /or dog CTLA4; (b) has antagonistic activity against human CTLA4; (c) does not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3 at concentrations up to 100 nM , CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS, and/or B7-H4; (d) cross-reacts with monkey, mouse, rat, and/or dog CTLA4; (e) induces ADCC effects (e.g., for Tregs); (f) activate human PBMC (e.g., stimulate the secretion of IL-2 and/or IFNγ); (g) inhibit tumor cell growth; (h) have a therapeutic effect on cancer; and (i) inhibit human CTLA4 binds to human CD80 and/or human CD86. Also provided herein are one or more activatable antibodies that cross-compete for binding to human CTLA4 with an activatable antibody targeting CTLA4 and/or one or more of the anti-CTLA4 antibodies described herein.

於一些實施例中,當呈減能形式時,可活化抗體以約500 nM或更多之K D結合至人類、食蟹獼猴、小鼠、大鼠及/或狗CTLA4。於一些實施例中,當以活性形式時,可活化抗體以約500 nM或更低(例如,約500 nM或更低、約450 nM或更低、約400 nM或更低、約350 nM或更低、約300 nM或更低、約250 nM或更低、約200 nM或更低、約150 nM或更低、約100 nM或更低、約90 nM或更低、約80 nM或更低、約70 nM或更低、約60 nM或更低、約50 nM或更低、約40 nM或更低、約30 nM或更低、約25 nM或更低、約20 nM或更低、約10 nM或更低、約1 nM或更低、約0.1 nM或更低等)之K D結合至人類、食蟹獼猴、小鼠、大鼠及/或狗CTLA4。於一些實施例中,當以活性形式時,可活化抗體以約350 nM或更低之K D結合至人類、食蟹獼猴、小鼠、大鼠及/或狗CTLA4。於一些實施例中,當以活性形式時,可活化抗體以約100 nM或更低之K D結合至人類CTLA4。於一些實施例中,當以活性形式時,可活化抗體以約50 nM或更低之K D結合至人類CTLA4。於一些實施例中,當以活性形式時,可活化抗體以約10 nM或更低之K D結合至人類CTLA4。量測可活化抗體之K D之方法可使用此項技術中已知之任何方法進行,包括例如,藉由表面電漿子共振、ELISA、等溫滴定量熱法、過濾結合檢定、EMSA等。於一些實施例中,藉由ELISA量測K D(參見例如,以下實例)。 In some embodiments, when in an attenuated form, the activatable antibody binds to human, cynomolgus, mouse, rat and/or dog CTLA4 with a KD of about 500 nM or more. In some embodiments, when in an active form, the antibody can be activated at about 500 nM or less (e.g., about 500 nM or less, about 450 nM or less, about 400 nM or less, about 350 nM or less). Lower, about 300 nM or lower, about 250 nM or lower, about 200 nM or lower, about 150 nM or lower, about 100 nM or lower, about 90 nM or lower, about 80 nM or lower Low, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less , about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.) binds to human, cynomolgus monkey, mouse, rat and/or dog CTLA4. In some embodiments, the activatable antibody binds to human, cynomolgus, mouse, rat and/or dog CTLA4 with a KD of about 350 nM or less when in an active form. In some embodiments, the activatable antibody binds to human CTLA4 with a KD of about 100 nM or less when in an active form. In some embodiments, the activatable antibody binds to human CTLA4 with a KD of about 50 nM or less when in an active form. In some embodiments, the activatable antibody binds to human CTLA4 with a KD of about 10 nM or less when in an active form. Methods of measuring the KD of an activatable antibody can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filtration binding assays, EMSA, and the like. In some embodiments, KD is measured by ELISA (see, eg, Examples below).

於一些實施例中,當呈減能形式時,可活化抗體不具有對人類CTLA4之拮抗活性。於一些實施例中,當以活性形式(例如,誘導ADCC效應(諸如對Treg)、活化PBMC (諸如藉由活化、誘導及/或刺激IL-2及/或IFNγ分泌)、阻斷人類CTLA4結合至人類CD80及/或人類CD86等)時,可活化抗體具有對人類CTLA4之拮抗活性。於一些實施例中,當以活性形式時,可活化抗體抑制人類CTLA4之一或多種活性(例如,當表現人類CTLA4之細胞(諸如人類細胞)藉由可活化抗體接觸時,抑制人類CTLA4之一或多種活性)。In some embodiments, the activatable antibody does not have antagonistic activity against human CTLA4 when in an attenuated form. In some embodiments, when in an active form (e.g., inducing ADCC effects (such as on Tregs), activating PBMC (such as by activating, inducing and/or stimulating IL-2 and/or IFNγ secretion), blocking human CTLA4 binding to human CD80 and/or human CD86, etc.), the activatable antibody has antagonistic activity against human CTLA4. In some embodiments, the activatable antibody inhibits one or more activities of human CTLA4 when in an active form (e.g., inhibits one or more activities of human CTLA4 when a cell expressing human CTLA4, such as a human cell) is contacted by the activatable antibody. or multiple activities).

於一些實施例中,當呈減能形式時,可活化抗體不與猴(例如,食蟹獼猴)、小鼠、大鼠及/或狗CTLA4交叉反應。於一些實施例中,當以活性形式時,可活化抗體與猴(例如,食蟹獼猴)、小鼠、大鼠及/或狗CTLA4交叉反應。於一些實施例中,當以活性形式時,可活化抗體與猴CTLA4交叉反應。於一些實施例中,當以活性形式時,可活化抗體與小鼠CTLA4交叉反應。於一些實施例中,當以活性形式時,可活化抗體與大鼠CTLA4交叉反應。於一些實施例中,當以活性形式時,可活化抗體與狗CTLA4交叉反應。於一些實施例中,當以活性形式時,可活化抗體與以下交叉反應:猴及小鼠CTLA4;猴及大鼠CTLA4;猴及狗CTLA4;小鼠及大鼠CTLA4;小鼠及狗CTLA4;大鼠及狗CTLA4;猴、小鼠及大鼠CTLA4;猴、小鼠及狗CTLA4;猴、大鼠及狗CTLA4;小鼠、大鼠及狗CTLA4;或猴、小鼠、大鼠及狗CTLA4。於一些實施例中,當以活性形式時,可活化結合多肽在約350 nM下(例如,在約1nM下、在約10nM下、在約25nM下、在約50nM下、在約75nM下、在約100nM下、在約150 nM下、在約200 nM下、在約250 nM下、在約300 nM下、在約350 nM下)係交叉反應性。量測交叉反應性之方法係此項技術中已知,包括(不限於)表面電漿子共振、ELISA、等位滴定量熱法、過濾結合檢定、EMSA等。 In some embodiments, when in an attenuated form, the activatable antibody does not cross-react with monkey (eg, cynomolgus monkey), mouse, rat and/or dog CTLA4. In some embodiments, the activated antibody cross-reacts with monkey (eg, cynomolgus monkey), mouse, rat and/or dog CTLA4 when in active form. In some embodiments, the activated antibody cross-reacts with monkey CTLA4 when in an active form. In some embodiments, the activated antibody cross-reacts with mouse CTLA4 when in an active form. In some embodiments, the activated antibody cross-reacts with rat CTLA4 when in an active form. In some embodiments, the activated antibody cross-reacts with canine CTLA4 when in an active form. In some embodiments, when in active form, the activated antibodies cross-react with: monkey and mouse CTLA4; monkey and rat CTLA4; monkey and dog CTLA4; mouse and rat CTLA4; mouse and dog CTLA4; Rat and dog CTLA4; monkey, mouse, and rat CTLA4; monkey, mouse, and dog CTLA4; monkey, rat, and dog CTLA4; mouse, rat, and dog CTLA4; or monkey, mouse, rat, and dog CTLA4. In some embodiments, when in active form, the activatable binding polypeptide is at about 350 nM (e.g., at about 1 nM, at about 10 nM, at about 25 nM, at about 50 nM, at about 75 nM, at (at about 100 nM, at about 150 nM, at about 200 nM, at about 250 nM, at about 300 nM, at about 350 nM) is cross-reactive. Methods for measuring cross-reactivity are known in the art and include, but are not limited to, surface plasmon resonance, ELISA, isometric titration calorimetry, filtration binding assay, EMSA, etc.

於一些實施例中,當呈減能形式時,可活化抗體不誘導ADCC效應(例如,對表現CTLA4之人類細胞,諸如Treg)。於一些實施例中,當呈減能形式時,可活化抗體相較於對照結合多肽(例如,親本抗體)具有降低之ADCC效應(例如,對表現CTLA4之人類細胞,諸如Treg)。於一些實施例中,當以活性形式時,可活化抗體誘導ADCC效應(例如對表現CTLA4之人類細胞諸如Treg)。量測ADCC效應之方法(例如,活體外方法)係此項技術中已知,包括(不限於)經由以下實例中所述之方法。於一些實施例中,當呈減能形式時,可活化抗體誘導ADCC效應相對於對照(例如,親本抗體)少於約10% (例如,誘導ADCC少於約10%、少於約5%、少於約1%等)。於一些實施例中,當以活性形式時,可活化抗體誘導ADCC效應相對於對照(例如,同型對照)超過約10% (例如,誘導ADCC超過約10%、超過約15%、超過約20%、超過約25%、超過約30%、超過約35%、超過約40%等)。In some embodiments, the activatable antibody does not induce an ADCC effect when in an attenuated form (eg, on human cells expressing CTLA4, such as Tregs). In some embodiments, when in an attenuated form, the activatable antibody has a reduced ADCC effect (eg, on human cells expressing CTLA4, such as Tregs) compared to a control binding polypeptide (eg, the parent antibody). In some embodiments, when in active form, the activating antibody induces an ADCC effect (eg, on human cells expressing CTLA4 such as Tregs). Methods for measuring ADCC effects (eg, in vitro methods) are known in the art, including, without limitation, via the methods described in the Examples below. In some embodiments, when in an attenuated form, the activatable antibody induces an ADCC effect of less than about 10% (e.g., inducing less than about 10%, less than about 5%) relative to a control (e.g., parent antibody) , less than about 1%, etc.). In some embodiments, the activatable antibody induces an ADCC effect of more than about 10% relative to a control (e.g., an isotype control) when in an active form (e.g., inducing ADCC by more than about 10%, more than about 15%, more than about 20% , more than about 25%, more than about 30%, more than about 35%, more than about 40%, etc.).

於一些實施例中,可活化抗體能抑制腫瘤細胞生長及/或增殖。於一些實施例中,當與可活化抗體接觸時,相對於不與可活化抗體接觸之對應腫瘤細胞(或相對於與同型對照抗體接觸之對應腫瘤細胞)抑制腫瘤細胞生長及/或增殖至少約5% (例如,至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、或至少約99%)。於一些實施例中,當對受試者投與可活化抗體時,該等可活化抗體能減少受試者之腫瘤體積。於一些實施例中,相對於受試者之初始腫瘤體積(例如,在投與可活化抗體之前;相較於投與同型對照抗體之受試者之對應腫瘤),可活化抗體能減少受試者之腫瘤體積至少約5% (例如,至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、或至少約99%)。監測腫瘤細胞生長/增殖、腫瘤體積及/或腫瘤抑制之方法係此項技術中已知,包括例如經由以下實例中所述之方法。In some embodiments, the activatable antibody can inhibit tumor cell growth and/or proliferation. In some embodiments, when contacted with the activatable antibody, tumor cell growth and/or proliferation is inhibited by at least about 10% relative to corresponding tumor cells not contacted with the activatable antibody (or relative to corresponding tumor cells contacted with an isotype control antibody) 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%). In some embodiments, activatable antibodies can reduce tumor volume in a subject when the activatable antibodies are administered to the subject. In some embodiments, the activatable antibody can reduce the subject's tumor volume relative to the subject's initial tumor volume (e.g., prior to administration of the activatable antibody; compared to the corresponding tumor in a subject administered an isotype control antibody). The tumor volume is at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% , at least about 80%, at least about 90%, or at least about 99%). Methods of monitoring tumor cell growth/proliferation, tumor volume, and/or tumor inhibition are known in the art, including, for example, via the methods described in the Examples below.

於一些實施例中,可活化抗體具有對癌症之治療效應。於一些實施例中,可活化抗體減少癌症之一或多種徵兆或症狀。於一些實施例中,當投與可活化抗體時,患有癌症之受試者進入部分或完全緩解。In some embodiments, the activatable antibody has a therapeutic effect on cancer. In some embodiments, the activating antibody reduces one or more signs or symptoms of cancer. In some embodiments, subjects with cancer enter partial or complete remission when an activatable antibody is administered.

於一些實施例中,本發明提供經單離可活化抗體,當以活性形式時,其與包含以下之抗體競爭或交叉競爭結合至人類CTLA4:a)包含SEQ ID NO: 23之胺基酸序列之HVR-H1;包含SEQ ID NO: 35之胺基酸序列之HVR-H2;及包含SEQ ID NO: 45之胺基酸序列之HVR-H3;及/或b)包含SEQ ID NO: 58之胺基酸序列之HVR-L1;包含SEQ ID NO: 66之胺基酸序列之HVR-L2;及包含SEQ ID NO: 75之胺基酸序列之HVR-L3。於一些實施例中,本發明提供經單離可活化抗體,當以活性形式時,其與包含以下之抗體競爭或交叉競爭結合至人類CTLA4:a)包含SEQ ID NO: 87之胺基酸序列之重鏈可變區;及/或b)包含SEQ ID NO: 100之胺基酸序列之輕鏈可變區。可活化抗體與抗體競爭或交叉競爭結合之能力可使用此項技術中已知之標準結合檢定測定,諸如BIAcore分析、ELISA檢定或流動式細胞測量術。例如,吾人可允許抗體(例如,如上所述)在飽和條件下結合至人類CTLA4及然後量測測試可活化抗體(當以活性形式時)結合至CTLA4之能力。若測試可活化抗體能與抗體同時結合至CTLA4,則測試可活化抗體結合至與抗體不同的抗原決定基。然而,若測試可活化抗體不能同時結合至CTLA4,則測試可活化抗體結合至相同抗原決定基、重疊抗原決定基、或緊鄰藉由抗體結合之抗原決定基之抗原決定基。此實驗可使用各種方法(諸如ELISA、RIA、FACS或表面電漿子共振)進行。In some embodiments, the invention provides an isolated, activatable antibody that, when in an active form, competes or cross-competes for binding to human CTLA4 with an antibody comprising: a) an amino acid sequence comprising SEQ ID NO: 23 HVR-H1; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35; and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45; and/or b) comprising SEQ ID NO: 58 HVR-L1 with the amino acid sequence; HVR-L2 with the amino acid sequence of SEQ ID NO: 66; and HVR-L3 with the amino acid sequence of SEQ ID NO: 75. In some embodiments, the invention provides an isolated, activatable antibody that, when in an active form, competes or cross-competes for binding to human CTLA4 with an antibody comprising: a) an amino acid sequence comprising SEQ ID NO: 87 The heavy chain variable region; and/or b) the light chain variable region comprising the amino acid sequence of SEQ ID NO: 100. The ability of an activatable antibody to compete or cross-compete with the antibody for binding can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, one can allow an antibody (e.g., as described above) to bind to human CTLA4 under saturating conditions and then measure the ability of the test activatable antibody (when in active form) to bind to CTLA4. If the test-activatable antibody binds to CTLA4 at the same time as the antibody, then the test-activatable antibody binds to a different epitope than the antibody. However, if the test activatable antibody cannot simultaneously bind to CTLA4, the test activatable antibody binds to the same epitope, an overlapping epitope, or an epitope immediately adjacent to the epitope bound by the antibody. This experiment can be performed using various methods such as ELISA, RIA, FACS or surface plasmon resonance.

於一些實施例中,可活化抗體(當呈減能形式時)不抑制CTLA4與其結合搭檔中之一或多者之間(例如,人類CTLA4與人類CD80、人類CTLA4與人類CD86)之結合。於一些實施例中,可活化抗體(當以活性形式時)抑制CTLA4與其結合搭檔中之一或多者之間(例如,人類CTLA4與人類CD80、人類CTLA4與人類CD86)之結合。於一些實施例中,可活化抗體抑制活體外CTLA4與其配位體之間之結合。於一些實施例中,可活化抗體具有約500 nM或更低(例如,約500 nM或更低、約400 nM或更低、約300 nM或更低、約200 nM或更低、約100 nM或更低、約50 nM或更低、約25 nM或更低、約10 nM或更低、約1 nM或更低等)之半最大抑制濃度(IC 50)以抑制CTLA4結合至CD80及/或CD86。於一些實施例中,可活化抗體具有約100 nM或更低之半最大抑制濃度(IC 50)以抑制CTLA4結合至CD80及/或CD86。於一些實施例中,當在約100 nM或更高(例如,約100 nM或更高、約500 nM或更高、約1 µM或更高、約10 µM或更高等)之濃度下提供時,可活化抗體完全抑制人類CTLA4結合至CD80及/或CD86。如本文中所用,術語「完全抑制(complete inhibiting/completely inhibits)」係指可活化抗體減少第一蛋白與第二蛋白之間之結合至少約80% (例如,至少約80%、至少約85%、至少約90%、至少約95%、至少約99%等)的能力。量測多肽抑制第一蛋白(例如,人類CTLA4)與第二蛋白(例如,人類CD80或人類CD86)之結合之能力的方法係此項技術中已知,包括(不限於)經由BIAcore分析、ELISA檢定及流動式細胞測量術。 掩蔽部分 (MM) In some embodiments, the activatable antibody (when in an attenuated form) does not inhibit binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD86). In some embodiments, the activatable antibody (when in active form) inhibits binding between CTLA4 and one or more of its binding partners (eg, human CTLA4 and human CD80, human CTLA4 and human CD86). In some embodiments, the activatable antibody inhibits binding between CTLA4 and its ligand in vitro. In some embodiments, the activatable antibody has a concentration of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.) at half-maximal inhibitory concentration (IC 50 ) to inhibit CTLA4 binding to CD80 and/or Or CD86. In some embodiments, the activatable antibody has a half-maximal inhibitory concentration ( IC50 ) of about 100 nM or less to inhibit CTLA4 binding to CD80 and/or CD86. In some embodiments, when provided at a concentration of about 100 nM or higher (e.g., about 100 nM or higher, about 500 nM or higher, about 1 µM or higher, about 10 µM or higher, etc.) , activating antibodies completely inhibit the binding of human CTLA4 to CD80 and/or CD86. As used herein, the term "complete inhibiting/completely inhibits" refers to an activatable antibody that reduces the binding between a first protein and a second protein by at least about 80% (e.g., at least about 80%, at least about 85% , at least about 90%, at least about 95%, at least about 99%, etc.) capability. Methods for measuring the ability of a polypeptide to inhibit the binding of a first protein (e.g., human CTLA4) to a second protein (e.g., human CD80 or human CD86) are known in the art, including, without limitation, via BIAcore analysis, ELISA Assays and flow cytometry. Masked part (MM)

於一些實施例中,本發明係關於包含掩蔽部分(MM)之可活化抗體。於一些實施例中,該掩蔽部分(MM)包含如式(XVIII):X mCX nCZ o(SEQ ID NO: 134)之胺基酸序列,其中m為2至10,n為3至10,且o為1至10,其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸,且其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。於一些實施例中,X非W、M、及/或C。於一些實施例中,式(XVIII)之X m中之各X獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸及/或式(XVIII)之X n中之各X獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。於一些實施例中,該MM包含藉由如式(XX):(NNK) mTGY(NNK) nTGY(NHC) o(SEQ ID NO: 136)之多核苷酸序列編碼之多肽,其中各N獨立地為A、G、T、或C,其中各K獨立地為T或G,其中各Y獨立地為T或C,且其中各H獨立地為A、T、或C。 In some embodiments, the invention relates to activatable antibodies comprising a masking moiety (MM). In some embodiments, the masking moiety (MM) includes an amino acid sequence such as formula (XVIII): X m CX n CZ o (SEQ ID NO: 134), wherein m is 2 to 10 and n is 3 to 10 , and o is 1 to 10, where each X is independently selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Amino acids from the group consisting of V, W, and Y, and each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P of amino acids. In some embodiments, X is other than W, M, and/or C. In some embodiments, each X in X m of Formula (XVIII) is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. Each X in X n of amino acids and/or formula (XVIII) is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P Amino acids. In some embodiments, the MM includes a polypeptide encoded by a polynucleotide sequence of formula (XX): (NNK) m TGY(NNK) n TGY(NHC) o (SEQ ID NO: 136), wherein each N is independently A, G, T, or C, where each K is independently T or G, where each Y is independently T or C, and where each H is independently A, T, or C.

於一些實施例中,該掩蔽部分(MM)包含如式(XIX): Z mCZ nCZ o(SEQ ID NO: 135)之胺基酸序列,其中m為2至10,n為3至10,且o為1至10,且各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。 In some embodiments, the masking moiety (MM) includes an amino acid sequence of formula (XIX): Z m CZ n CZ o (SEQ ID NO: 135), wherein m is 2 to 10 and n is 3 to 10 , and o is 1 to 10, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

於一些實施例中,m為2至10、2至9、2至8、2至7、2至6、2至5、2至4、2至3、3至10、3至9、3至8、3至7、3至6、3至5、3至4、4至10、4至9、4至8、4至7、4至6、4至5、5至10、5至9、5至8、5至7、5至6、6至10、6至9、6至8、6至7、7至10、7至9、7至8、8至10、8至9、或9至10。於一些實施例中,m為6至8。於一些實施例中,m為3、4、5、6、7、8、9、或10。於一些實施例中,m為6。In some embodiments, m is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8. 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10. In some embodiments, m is 6 to 8. In some embodiments, m is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 6.

於一些實施例中,n為3至10、3至9、3至8、3至7、3至6、3至5、3至4、4至10、4至9、4至8、4至7、4至6、4至5、5至10、5至9、5至8、5至7、5至6、6至10、6至9、6至8、6至7、7至10、7至9、7至8、8至10、8至9、或9至10。於一些實施例中,n為6至8。於一些實施例中,n為3、4、5、6、7、8、9、或10。於一些實施例中,n為6。於一些實施例中,n為8。In some embodiments, n is 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10. In some embodiments, n is 6-8. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 6. In some embodiments, n is 8.

於一些實施例中,o為1至10、1至9、1至8、1至7、1至6、1至5、1至4、1至3、1至2、2至10、2至9、2至8、2至7、2至6、2至5、2至4、2至3、3至10、3至9、3至8、3至7、3至6、3至5、3至4、4至10、4至9、4至8、4至7、4至6、4至5、5至10、5至9、5至8、5至7、5至6、6至10、6至9、6至8、6至7、7至10、7至9、7至8、8至10、8至9、或9至10。於一些實施例中,o為1至2。於一些實施例中,o為1、2、3、4、5、6、7、8、9、或10。於一些實施例中,o為2。In some embodiments, o is 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9. 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10. In some embodiments, o is 1 to 2. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, o is 2.

於一些實施例中,該掩蔽部分(MM)包含如式(XXI):Z 6CX 6CZ 2(SEQ ID NO: 137)之胺基酸序列,其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸,且其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。 In some embodiments, the masking moiety (MM) includes an amino acid sequence of formula (XXI): Z 6 CX 6 CZ 2 (SEQ ID NO: 137), wherein each X is independently selected from the group consisting of A, C, Amino acids of the group consisting of D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and each Z is independently It is an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

於一些實施例中,該掩蔽部分(MM)包含如式(XXII):Z 6CX 8CZ 2(SEQ ID NO: 138)之胺基酸序列,其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸,且其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。 In some embodiments, the masking moiety (MM) includes an amino acid sequence of formula (XXII): Z 6 CX 8 CZ 2 (SEQ ID NO: 138), wherein each X is independently selected from the group consisting of A, C, Amino acids of the group consisting of D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and each Z is independently It is an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

於一些實施例中,第一肽(FP)包含如式(XXIII):(Z 6)C(Z 6)C(Z 2) (SEQ ID NO: 139)之胺基酸序列,其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。 In some embodiments, the first peptide (FP) includes an amino acid sequence of formula (XXIII): (Z 6 )C (Z 6 ) C (Z 2 ) (SEQ ID NO: 139), where each Z is independent Ground is an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

於一些實施例中,該掩蔽部分(MM)包含如式(XXIV):(Z 6)C(Z 8)C(Z 2) (SEQ ID NO: 140)之胺基酸序列,其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。於一些實施例中,可活化抗體包含掩蔽部分(MM),該掩蔽部分包含選自由X mCPDHPYPCXX (SEQ ID NO:181)、X mCDAFYPYCXX (SEQ ID NO:182)、X mCDSHYPYCXX (SEQ ID NO:183)、及X mCVPYYYACXX (SEQ ID NO:184)組成之群之序列,其中m為2至10,且其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸。於一些實施例中,可活化抗體包含掩蔽部分(MM),該掩蔽部分包含序列EVGSYNFVADSCPDHPYPCSA (SEQ ID NO:189)、EVGSYIVHHSDCDAFYPYCDS (SEQ ID NO:190)、EVGSYYSAYPACDSHYPYCNS (SEQ ID NO:191)、EVGSYPNPSSDCVPYYYACAY (SEQ ID NO:192)、EVGSYYSAYPACDSHYPYCQS (SEQ ID NO:193)、EVGSYPQPSSDCVPYYYACAY (SEQ ID NO:195)、或EVGSYPNPASDCVPYYYACAY (SEQ ID NO:196)。於一些實施例中,該MM包含EDCVPYYYACAY (SEQ ID NO:213)、EVGSSDCVPYYYACAY (SEQ ID NO:214)、EDCDAFYPYCDS (SEQ ID NO:215)、或EVGHSDCDAFYPYCDS (SEQ ID NO:216)之序列。 In some embodiments, the masking moiety (MM) includes an amino acid sequence of formula (XXIV): (Z 6 )C (Z 8 ) C (Z 2 ) (SEQ ID NO: 140), where each Z is independent Ground is an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, the activatable antibody includes a masking moiety (MM) selected from the group consisting of Xm CPDHPYPCXX (SEQ ID NO:181), Xm CDAFYPYCXX ( SEQ ID NO:182), NO:183), and Xm CVPYYYACXX (SEQ ID NO:184), where m is 2 to 10, and each Amino acids of the group consisting of H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the sequence EVGSYNFVADSCPDHPYPCSA (SEQ ID NO:189), EVGSYIVHHSDCDAFYPYCDS (SEQ ID NO:190), EVGSYYSAYPACDSHYPYCNS (SEQ ID NO:191), EVGSYPNPSSDCVPYYYACAY ( SEQ ID NO:192), EVGSYYSAYPACDSHYPYCQS (SEQ ID NO:193), EVGSYPQPSSDCVPYYYACAY (SEQ ID NO:195), or EVGSYPNPASDCVPYYYACAY (SEQ ID NO:196). In some embodiments, the MM includes the sequence of EDCVPYYYACAY (SEQ ID NO:213), EVGSSDCVPYYYACAY (SEQ ID NO:214), EDCDAFYPYCDS (SEQ ID NO:215), or EVGHSDCDAFYPYCDS (SEQ ID NO:216).

於一些實施例中,該掩蔽部分(MM)包含選自NFVADSCPDHPYPCSA (SEQ ID NO: 141)、IVHHSDCDAFYPYCDS (SEQ ID NO: 142)、YSAYPACDSHYPYCNS (SEQ ID NO: 143)、PNPSSDCVPYYYACAY (SEQ ID NO: 144)、YSAYPACDSHYPYCQS (SEQ ID NO: 145)、PQPSSDCVPYYYACAY (SEQ ID NO: 146)、及PNPASDCVPYYYACAY (SEQ ID NO: 147)之胺基酸序列。In some embodiments, the masking portion (MM) includes selected from NFVADSCPDHPYPCSA (SEQ ID NO: 141), IVHHSDCDAFYPYCDS (SEQ ID NO: 142), YSAYPACDSHYPYCNS (SEQ ID NO: 143), PNPSSDCVPYYYACAY (SEQ ID NO: 144) , YSAYPACDSHYPYCQS (SEQ ID NO: 145), PQPSSDCVPYYYACAY (SEQ ID NO: 146), and PNPASDCVPYYYACAY (SEQ ID NO: 147) amino acid sequences.

於一些實施例中,本文中所述之掩蔽部分(MM)中之任一者可另包含一或多個另外胺基酸序列(例如,一或多個多肽標籤)。適宜另外胺基酸序列之實例可包括(不限於)純化標籤(諸如his-標籤、flag-標籤、麥芽糖結合蛋白及谷胱甘肽-S-轉移酶標籤)、檢測標籤(諸如可光度學檢測之標籤(例如,紅色或綠色螢光蛋白等))、具有可檢測之酵素活性之標籤(例如,鹼性磷酸酶等)、含有分泌序列、前導序列及/或穩定序列之標籤、蛋白酶裂解位點(例如,弗林蛋白酶(furin)裂解位點、TEV裂解位點、凝血酶裂解位點)及類似者。於一些實施例中,一或多個另外胺基酸序列係在掩蔽部分(MM)之N端。於一些實施例中,另外胺基酸序列包含序列EVGSY (SEQ ID NO: 148)或由該序列組成。In some embodiments, any of the masking moieties (MM) described herein may further comprise one or more additional amino acid sequences (eg, one or more polypeptide tags). Examples of suitable additional amino acid sequences may include, without limitation, purification tags (such as his-tag, flag-tag, maltose-binding protein and glutathione-S-transferase tags), detection tags (such as photometrically detectable tags (e.g., red or green fluorescent protein, etc.)), tags with detectable enzyme activity (e.g., alkaline phosphatase, etc.), tags containing secretory sequences, leader sequences and/or stabilizing sequences, protease cleavage sites sites (eg, furin cleavage site, TEV cleavage site, thrombin cleavage site) and the like. In some embodiments, one or more additional amino acid sequences are N-terminal to the masking moiety (MM). In some embodiments, the additional amino acid sequence includes or consists of the sequence EVGSY (SEQ ID NO: 148).

於一些實施例中,在活化之前(例如,在用於可裂解部分(CM)內裂解之一或多種蛋白酶處理之前,在經歷pH (局部)變化(增加或減少)之前,在溫度變化(增加或減少)之前,在與第二分子(諸如小分子或蛋白質配位體)接觸之前等),該掩蔽部分結合至靶結合部分(TBM)且抑制可活化抗體結合至其靶,但是於活化後(例如,於用於可裂解部分(CM)內裂解之一或多種蛋白酶處理後,於經歷pH (局部)變化(增加或減少)後,於溫度變化(增加或減少)後,於與第二分子(諸如小分子或蛋白質配位體)接觸後等)不結合至TBM及/或抑制可活化抗體結合至其靶。於一些實施例中,當CM未經裂解時,該掩蔽部分(MM)抑制可活化抗體結合至其靶,但是當CM經裂解時,不抑制可活化抗體結合至其靶。於一些實施例中,該掩蔽部分(MM)具有結合至TBM之解離常數,其大於可活化抗體對其靶之解離常數(當以活性形式時) (例如,至少大約1.5倍、至少大約2倍、至少大約2.5倍、至少大約3倍、至少大約3.5倍、至少大約4倍、至少大約4.5倍、至少大約5倍、至少大約10倍、至少大約100倍、至少大約500倍等)。 可裂解部分 (CM) In some embodiments, a change in temperature (increase or reduced), prior to contact with a second molecule (such as a small molecule or protein ligand, etc.), the masking moiety binds to the target binding moiety (TBM) and inhibits the activatable antibody from binding to its target, but upon activation (For example, after treatment with one or more proteases for cleavage within the cleavable moiety (CM), after undergoing a (local) change (increase or decrease) in pH, after a change (increase or decrease) in temperature, and after a second Molecules (such as small molecules or protein ligands, etc.) do not bind to the TBM and/or inhibit binding of the activatable antibody to its target. In some embodiments, the masking moiety (MM) inhibits the activatable antibody from binding to its target when the CM is not cleaved, but does not inhibit the activatable antibody from binding to its target when the CM is cleaved. In some embodiments, the masking moiety (MM) has a dissociation constant for binding to TBM that is greater than the dissociation constant of the activatable antibody for its target when in active form (e.g., at least about 1.5-fold, at least about 2-fold , at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 10 times, at least about 100 times, at least about 500 times, etc.). Cleavable fraction (CM)

於一些實施例中,本發明係關於包含可裂解部分(CM)之可活化抗體。於一些實施例中,該可裂解部分(CM)藉由以下裂解及/或破壞:用可裂解部分(CM)內裂解之一或多種蛋白酶處理,藉由pH變化(增加或減少)、藉由溫度變化(增加或減少)、及/或藉由與第二分子(諸如小分子或蛋白質配位體)接觸等。In some embodiments, the invention relates to activatable antibodies comprising a cleavable moiety (CM). In some embodiments, the cleavable moiety (CM) is cleaved and/or destroyed by treatment with one or more proteases that cleave within the cleavable moiety (CM), by a pH change (increase or decrease), by Temperature change (increase or decrease), and/or by contact with a second molecule (such as a small molecule or protein ligand), etc.

於一些實施例中,該可裂解部分(CM)包含至少第一裂解位點(CS 1) (例如,第一蛋白酶裂解位點)。於一些實施例中,該第一裂解位點為第一蛋白酶裂解位點。可使用藉由此項技術中已知之任何蛋白酶(例如,已知與包含CM之可活化抗體之靶共定位之蛋白酶)識別及/或裂解之任何適宜蛋白酶裂解位點,包括例如,藉由以下識別及/或裂解之蛋白酶裂解位點:尿激酶型纖維蛋白溶酶原活化因子(uPA)、基質金屬蛋白酶(例如,MMP-1、MMP-2、MMP-3、MMP-7、MMP-8、MMP-9、MMP-10、MMP-11、MMP-12、MMP-13、MMP-14、MMP-15、MMP-16、MMP-17、MMP-19、MMP-20、MMP-23、MMP-24、MMP-26、及/或MMP-27)、菸草蝕紋病毒(TEV)蛋白酶、胞漿素、凝血酶、PSA、PSMA、ADAMS/ADAMTS (例如,ADAM 8、ADAM 9、ADAM10、ADAM12、ADAM15、ADAM17/TACE、ADAMDEC1、ADAMTS1、ADAMTS4、及/或ADAMTS5)、卡斯蛋白酶(例如,卡斯蛋白酶-1、卡斯蛋白酶-2、卡斯蛋白酶-3、卡斯蛋白酶-4、卡斯蛋白酶-5、卡斯蛋白酶-6、卡斯蛋白酶-7、卡斯蛋白酶-8、卡斯蛋白酶-9、卡斯蛋白酶-10、卡斯蛋白酶-11、卡斯蛋白酶-12、卡斯蛋白酶-13、及/或卡斯蛋白酶-14)、天冬胺酸蛋白酶(例如,RACE及/或腎素(Renin));天冬胺酸組織蛋白酶(例如,組織蛋白酶D及/或組織蛋白酶E)、半胱胺酸組織蛋白酶(例如,組織蛋白酶B、組織蛋白酶C、組織蛋白酶K、組織蛋白酶L、組織蛋白酶S、組織蛋白酶V/L2、及/或組織蛋白酶X/Z/P)、半胱胺酸蛋白酶(例如,Cruzipain、Legumain及/或Otubain-2)、KLK (例如,KLK4、KLK5、KLK6、KLK7、KLK8、KLK10、KLK11、KLK13、及/或KLK14)、金屬蛋白酶(例如,Meprin、奈溶酶(Neprilysin)、PSMA、及/或BMP-1)、絲胺酸蛋白酶(例如,經活化之蛋白C、組織蛋白酶A、組織蛋白酶G、胃促胰酶(Chymase)、及/或凝血因子蛋白酶(諸如FVIIa、FIXa、Fxa、FXIa、FXIIa))、彈性蛋白酶、粒酶B、胍基苯甲酸酶(guanidino-benzoatase)、HtrA1、人類嗜中性白血球彈性蛋白酶、乳鐵蛋白(lactoferrin)、marapsin、NS3/4A、PACE4、tPA、類胰蛋白酶(tryptase)、II型跨膜絲胺酸蛋白酶(TTSP) (例如,DESC1、DPP-4、FAP、第二型穿膜絲胺酸蛋白酶(Hepsin)、間質蛋白酶(Matriptase)-2、MT-SP1/間質蛋白酶、TMPRSS2、TMPRSS3及/或TMPRSS4)等。於一些實施例中,第一蛋白酶裂解位點為選自以下之蛋白酶之裂解位點:uPA、MMP-1、MMP-2、MMP-3、MMP-8、MMP-9、MMP-14、TEV蛋白酶、胞漿素、凝血酶、因子X、PSA、PSMA、組織蛋白酶D、組織蛋白酶K、組織蛋白酶S、ADAM10、ADAM12、ADAMTS、卡斯蛋白酶-1、卡斯蛋白酶-2、卡斯蛋白酶-3、卡斯蛋白酶-4、卡斯蛋白酶-5、卡斯蛋白酶-6、卡斯蛋白酶-7、卡斯蛋白酶-8、卡斯蛋白酶-9、卡斯蛋白酶-10、卡斯蛋白酶-11、卡斯蛋白酶-12、卡斯蛋白酶-13、卡斯蛋白酶-14、及TACE。於一些實施例中,第一蛋白酶裂解位點為選自uPA、MMP-2、MMP-9、及/或TEV蛋白酶之蛋白酶之裂解位點。於一些實施例中,蛋白酶裂解包含選自SGRSA (SEQ ID NO: 149)、PLGLAG (SEQ ID NO: 150)及ENLYFQG (SEQ ID NO: 151)之胺基酸序列。 In some embodiments, the cleavable moiety (CM) includes at least a first cleavage site (CS 1 ) (eg, a first protease cleavage site). In some embodiments, the first cleavage site is a first protease cleavage site. Any suitable protease cleavage site that is recognized and/or cleaved by any protease known in the art (e.g., a protease known to colocalize with the target of an activatable antibody comprising CM) may be used, including, for example, by Protease cleavage sites for recognition and/or cleavage: urokinase plasminogen activator (uPA), matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8 , MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP -24, MMP-26, and/or MMP-27), tobacco etch virus (TEV) protease, cytoplasmin, thrombin, PSA, PSMA, ADAMS/ADAMTS (e.g., ADAM 8, ADAM 9, ADAM10, ADAM12 , ADAM15, ADAM17/TACE, ADAMDEC1, ADAMTS1, ADAMTS4, and/or ADAMTS5), caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase Caspsin-5, Caspsin-6, Caspsin-7, Caspsin-8, Caspsin-9, Caspsin-10, Caspsin-11, Caspsin-12, Caspsin-12 -13, and/or Caspsin-14), aspartate proteases (e.g., RACE and/or Renin); aspartate cathepsins (e.g., cathepsin D and/or cathepsin E ), cysteine cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V/L2, and/or cathepsin X/Z/P), Cystine proteases (e.g., Cruzipain, Legumain, and/or Otubain-2), KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and/or KLK14), metalloproteases (e.g., Meprin , Neprilysin, PSMA, and/or BMP-1), serine proteases (such as activated protein C, cathepsin A, cathepsin G, chymase, and/or Coagulation factor proteases (such as FVIIa, FIXa, Fxa, FXIa, FXIIa), elastase, granzyme B, guanidino-benzoatase, HtrA1, human neutrophil elastase, lactoferrin ), marapsin, NS3/4A, PACE4, tPA, tryptase, type II transmembrane serine protease (TTSP) (e.g., DESC1, DPP-4, FAP, type II transmembrane serine protease) (Hepsin), Matriptase-2, MT-SP1/Matriptase, TMPRSS2, TMPRSS3 and/or TMPRSS4), etc. In some embodiments, the first protease cleavage site is a protease cleavage site selected from the following: uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV Proteases, Cytosins, Thrombin, Factor 3. Caspsin-4, Caspsin-5, Caspsin-6, Caspsin-7, Caspsin-8, Caspsin-9, Caspsin-10, Caspsin-11, Caspsin-12, Caspsin-13, Caspsin-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from uPA, MMP-2, MMP-9, and/or TEV protease. In some embodiments, protease cleavage includes an amino acid sequence selected from the group consisting of SGRSA (SEQ ID NO: 149), PLGLAG (SEQ ID NO: 150), and ENLYFQG (SEQ ID NO: 151).

於一些實施例中,可活化抗體包含掩蔽部分(MM)及可裂解部分(CM),該可裂解部分包含如式(XXV):EVGSY(Z 6)C(Z 6)C(Z 2)SGRSA (SEQ ID NO: 152)之胺基酸序列,其中各Z獨立地為選自D、A、Y、S、T、N、I、L、F、V、H、及P之胺基酸。 In some embodiments, the activatable antibody includes a masking moiety (MM) and a cleavable moiety (CM), the cleavable moiety comprising formula (XXV): EVGSY(Z 6 )C(Z 6 )C(Z 2 )SGRSA The amino acid sequence of (SEQ ID NO: 152), wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

於一些實施例中,可活化抗體包含掩蔽部分(MM)及可裂解部分(CM),該可裂解部分包含如式(XXVI):EVGSY(Z 6)C(X 6)C(Z 2)SGRSA (SEQ ID NO: 153)之胺基酸序列,其中各X獨立地為選自A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y之胺基酸,且其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。 In some embodiments, the activatable antibody includes a masking moiety (MM) and a cleavable moiety (CM), the cleavable moiety comprising formula (XXVI): EVGSY(Z 6 )C(X 6 )C(Z 2 )SGRSA (SEQ ID NO: 153) amino acid sequence, wherein each X is independently selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R , S, T, V, W, and Y amino acids, and each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P A group of amino acids.

於一些實施例中,可活化抗體包含掩蔽部分(MM)及可裂解部分(CM),該可裂解部分包含如式(XXVII):EVGSY(Z 6)C(Z 8)C(Z 2)SGRSA (SEQ ID NO: 154)之胺基酸序列,其中各Z獨立地為選自D、A、Y、S、T、N、I、L、F、V、H、及P之胺基酸。 In some embodiments, the activatable antibody includes a masking moiety (MM) and a cleavable moiety (CM), the cleavable moiety comprising formula (XXVII): EVGSY(Z 6 )C(Z 8 )C(Z 2 )SGRSA The amino acid sequence of (SEQ ID NO: 154), wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

於一些實施例中,可活化抗體包含掩蔽部分(MM)及可裂解部分(CM),該可裂解部分包含如式(XXVIII):EVGSY(Z 6)C(X 8)C(Z 2)SGRSA (SEQ ID NO: 155)之胺基酸序列,其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸,且其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。 In some embodiments, the activatable antibody includes a masking moiety (MM) and a cleavable moiety (CM), the cleavable moiety comprising formula (XXVIII): EVGSY(Z 6 )C(X 8 )C(Z 2 )SGRSA (SEQ ID NO: 155) amino acid sequence, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R , S, T, V, W, and Y, and each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and amino acids of the group consisting of P.

於一些實施例中,該可裂解部分(CM)另包含第一連接子(L 1)。於一些實施例中,該第一連接子(L 1)為第一裂解位點(CS 1) (例如,第一蛋白酶裂解位點)之C端。於一些實施例中,該可裂解部分(CM)自N端至C端包含(CS 1)-L 1之結構。 In some embodiments, the cleavable moiety (CM) further includes a first linker (L 1 ). In some embodiments, the first linker (L 1 ) is the C-terminus of the first cleavage site (CS 1 ) (eg, the first protease cleavage site). In some embodiments, the cleavable moiety (CM) includes the structure of (CS 1 )-L 1 from N-terminus to C-terminus.

可使用此項技術中已知之任何適宜連接子(例如,可撓性連接子),包括例如:甘胺酸聚合物(G)n,其中n為至少1 (例如,至少1、至少2、至少3、至少4、至少5、至少6、至少7、至少8、至少9、至少10等)之整數;甘胺酸-絲胺酸聚合物(GS)n,其中n為至少1 (例如,至少1、至少2、至少3、至少4、至少5、至少6、至少7、至少8、至少9、至少10等)之整數,諸如GGGGS (SEQ ID NO: 156)、SGGS (SEQ ID NO: 157)、GGSG (SEQ ID NO: 158)、GGSGG (SEQ ID NO: 159)、GSGSG (SEQ ID NO: 160)、GSGGG (SEQ ID NO: 161)、GGGSG (SEQ ID NO: 162)、及/或GSSSG (SEQ ID NO: 163));甘胺酸-丙胺酸聚合物;丙胺酸-絲胺酸聚合物;及類似者。連接子序列可為任何長度,諸如約1個胺基酸(例如,甘胺酸或絲胺酸)至約20個胺基酸(例如,20個胺基酸甘胺酸聚合物或甘胺酸-絲胺酸聚合物)、約1個胺基酸至約15個胺基酸、約3個胺基酸至約12個胺基酸、約4個胺基酸至約10個胺基酸、約5個胺基酸至約9個胺基酸、約6個胺基酸至約8個胺基酸等。於一些實施例中,連接子為長度約1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、或20個胺基酸中之任一者。於一些實施例中,連接子包含選自SEQ ID NO: 159至163之胺基酸序列。於一些實施例中,連接子包含SEQ ID NO: 156或157之胺基酸序列。Any suitable linker known in the art (e.g., a flexible linker) may be used, including, for example: glycine polymer (G)n, where n is at least 1 (e.g., at least 1, at least 2, at least 3. An integer of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.); glycine-serine polymer (GS) n, where n is at least 1 (for example, at least 1. An integer of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.), such as GGGGS (SEQ ID NO: 156), SGGS (SEQ ID NO: 157 ), GGSG (SEQ ID NO: 158), GGSGG (SEQ ID NO: 159), GGSSG (SEQ ID NO: 160), GSGGG (SEQ ID NO: 161), GGGSG (SEQ ID NO: 162), and/or GSSSG (SEQ ID NO: 163)); glycine-alanine polymers; alanine-serine polymers; and the like. The linker sequence can be of any length, such as about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., 20 amino acids glycine polymer or glycine -serine polymer), about 1 amino acid to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, About 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, etc. In some embodiments, the linker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or Any of 20 amino acids. In some embodiments, the linker comprises an amino acid sequence selected from SEQ ID NO: 159 to 163. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 156 or 157.

於一些實施例中,該可裂解部分(CM)另包含至少第二裂解位點(例如,至少第二、至少第三、至少第四、至少第五等)。於一些實施例中,該可裂解部分(CM)另包含第二裂解位點(CS 2)。於一些實施例中,該第二裂解位點為第二蛋白酶裂解位點。該第二蛋白酶裂解位點可為藉由上述蛋白酶中之任一者識別及/或裂解之任何適宜蛋白酶裂解位點。於一些實施例中,該第一(CS 1)及第二(CS 2)裂解位點為藉由相同蛋白酶識別及/或裂解之蛋白酶裂解位點。於一些實施例中,該第一(CS 1)及第二(CS 2)裂解位點為藉由不同蛋白酶識別及/或裂解之蛋白酶裂解位點(例如,該第一蛋白酶裂解位點藉由uPA識別及/或裂解,及該第二蛋白酶裂解位點藉由MMP-2識別及/或裂解;該第一蛋白酶裂解位點藉由uPA識別及/或裂解,及該第二蛋白酶裂解位點藉由MMP-9識別及/或裂解;該第一蛋白酶裂解位點藉由uPA識別及/或裂解,及該第二蛋白酶裂解位點藉由TEV蛋白酶識別及/或裂解等)。於一些實施例中,該至少第二裂解位點(CS 2)為第一連接子(L 1)之C端。於一些實施例中,該可裂解部分(CM)自N端至C端包含(CS 1)-L 1-(CS 2)之結構。 In some embodiments, the cleavable moiety (CM) further comprises at least a second cleavage site (eg, at least second, at least third, at least fourth, at least fifth, etc.). In some embodiments, the cleavable moiety (CM) further includes a second cleavage site ( CS2 ). In some embodiments, the second cleavage site is a second protease cleavage site. The second protease cleavage site can be any suitable protease cleavage site recognized and/or cleaved by any of the proteases described above. In some embodiments, the first (CS 1 ) and second (CS 2 ) cleavage sites are protease cleavage sites recognized and/or cleaved by the same protease. In some embodiments, the first (CS 1 ) and second (CS 2 ) cleavage sites are protease cleavage sites recognized and/or cleaved by different proteases (e.g., the first protease cleavage site is uPA recognizes and/or cleaves, and the second protease cleavage site is recognized and/or cleaved by MMP-2; the first protease cleavage site is recognized and/or cleaved by uPA, and the second protease cleavage site Recognized and/or cleaved by MMP-9; the first protease cleavage site is recognized and/or cleaved by uPA, and the second protease cleavage site is recognized and/or cleaved by TEV protease, etc.). In some embodiments, the at least second cleavage site (CS 2 ) is the C-terminus of the first linker (L 1 ). In some embodiments, the cleavable moiety (CM) includes the structure of (CS 1 )-L 1 -(CS 2 ) from N-terminus to C-terminus.

於一些實施例中,該可裂解部分(CM)另包含至少第二連接子(例如,至少第二、至少第三、至少第四、至少第五等)。於一些實施例中,該可裂解部分(CM)另包含第二連接子(L 2)。該第二連接子(L 2)可為上述任何適宜連接子。於一些實施例中,該第二連接子包含選自SEQ ID NO: 156至163之胺基酸序列。於一些實施例中,該第一(L 1)及第二(L 2)連接子係相同(例如,兩種連接子包含SEQ ID NO: 156或157之序列)。於一些實施例中,該第一(L 1)及第二(L 2)連接子係不同(例如,該第一連接子(L 1)包含SEQ ID NO: 156之胺基酸序列,及該第二連接子(L 2)包含SEQ ID NO: 157之胺基酸序列等)。於一些實施例中,該至少第二連接子(L 2)為第二裂解位點(CS 2)之C端。於一些實施例中,該可裂解部分(CM)自N端至C端包含(CS 1)-L 1-(CS 2)-L 2之結構。 示例性 MM-CM 序列 In some embodiments, the cleavable moiety (CM) further includes at least a second linker (eg, at least second, at least third, at least fourth, at least fifth, etc.). In some embodiments, the cleavable moiety (CM) further includes a second linker (L 2 ). The second linker (L 2 ) can be any suitable linker described above. In some embodiments, the second linker comprises an amino acid sequence selected from SEQ ID NO: 156 to 163. In some embodiments, the first (L 1 ) and second (L 2 ) linkers are identical (eg, both linkers comprise the sequence of SEQ ID NO: 156 or 157). In some embodiments, the first (L 1 ) and second (L 2 ) linkers are different (for example, the first linker (L 1 ) includes the amino acid sequence of SEQ ID NO: 156, and the The second linker (L 2 ) contains the amino acid sequence of SEQ ID NO: 157, etc.). In some embodiments, the at least second linker (L 2 ) is the C-terminus of the second cleavage site (CS 2 ). In some embodiments, the cleavable moiety (CM) includes the structure of (CS 1 )-L 1 -(CS 2 )-L 2 from N-terminus to C-terminus. Exemplary MM-CM sequence

於一些實施例中,本發明之可活化抗體自N端至C端包含(FP)-(PCS 1)-L 1-(PCS 2)-L 2之結構。於一些實施例中,可活化抗體包含如式(XXIX),EVGSYX 1X 2X 3X 4X 5X 6CX 7X 8X 9X 10X 11X 12CX 13X 14SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 164)之胺基酸序列,其中X1為A、D、I、N、P、或Y,X2為A、F、N、S、或V,X3為A、H、L、P、S、V、或Y,X4為A、H、S、或Y,X5為A、D、P、S、V、或Y,X6為A、D、L、S、或Y,X7為D、P、或V,X8為A、D、H、P、S、或T,X9為A、D、F、H、P、或Y,X10為L、P、或Y,X11為F、P、或Y,X12為A、P、S、或Y,X13為A、D、N、S、T、或Y,且X14為A、S、或Y。於一些實施例中,本發明之可活化抗體包含以下之胺基酸序列:EVGSYDALHYACPPDYYACYYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 165)、EVGSYNSYHAYCPHPLYPCTASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 166)、EVGSYASSAVLCVTAYFSCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 167)、EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 168)、EVGSYNFVADSCPDHPYPCSASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 169)、EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 170)、EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 171)、EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 172)、EVGSYYSAYPACDSHYPYCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 173)、EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 174)、EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 175)、EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 176)、EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 177)、EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 178)、及/或EVGSYPNPASDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 179)。於一些實施例中,本發明之多肽自N端至C端包含(FP)-(PCS 1)-L 1-(PCS 2)-L 2-(TBM)之結構。 In some embodiments, the activatable antibody of the present invention includes the structure of (FP)-(PCS 1 )-L 1 -(PCS 2 )-L 2 from the N-terminus to the C-terminus. In some embodiments , the activatable antibody comprises Formula ( XXIX ) , EVGSYX 1 164) Amino acid sequence, where X1 is A, D, I, N, P, or Y, X2 is A, F, N, S, or V, and X3 is A, H, L, P, S, V , or Y, X4 is A, H, S, or Y, X5 is A, D, P, S, V, or Y, X6 is A, D, L, S, or Y, X7 is D, P, or V, X8 is A, D, H, P, S, or T, X9 is A, D, F, H, P, or Y, X10 is L, P, or Y, X11 is F, P, or Y, X12 is A, P, S, or Y, X13 is A, D, N, S, T, or Y, and X14 is A, S, or Y. In some embodiments, the activatable antibodies of the invention comprise the following amino acid sequences: EVGSYDALHYACPPDYYACYYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 165), EVGSYNSYHAYCPHPLYPCTASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 166), EVGSYASSAVLCVTAYFSCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 1 67), EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 168), EVGSYNFVADSCPDHPYPCSASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 169), EVGSYIVHHSDCDAFYPYCDSSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 170), EVGSYIVHHSDCDAFYPYCDSSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 171), EVGSYYSAYPACDSHYPYCN SSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 172), EVGSYYSAYPACDSHYPYCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 173) , EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 174), EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 175), EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 176), EVGSYYSAYPACDSHYPYCNSAGRSAGGGG SPLGLAGSGGS (SEQ ID NO: 177), EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 178), and/or EVGSYPNPASDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 179). In some embodiments, the polypeptide of the invention includes the structure of (FP)-(PCS 1 )-L 1 -(PCS 2 )-L 2 -(TBM) from N-terminus to C-terminus.

於一些實施例中,可活化抗體包含胺基酸序列SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:220)、SGRSAGGGGTPLGLAGSGGS (SEQ ID NO:221)、或SGRSAPLGLA (SEQ ID NO:222)。於一些實施例中,可活化抗體包含EV(Zn)C(X 8)C(Z 2)SGRSA (SEQ ID NO:217)、EDC(Z 6)C(Z 2)SGRSA (SEQ ID NO:218)、或EDC(Z 6)C(Z 2)PLGLA (SEQ ID NO:219)之序列,其中各X獨立地為選自由A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y組成之群之胺基酸,其中n為1至11且其中各Z獨立地為選自由D、A、Y、S、T、N、I、L、F、V、H、及P組成之群之胺基酸。 靶結合部分 (TBM) In some embodiments, the activatable antibody comprises the amino acid sequence SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:220), SGRSAGGGGTPLGLAGSGGS (SEQ ID NO:221), or SGRSAPLGLA (SEQ ID NO:222). In some embodiments, the activatable antibody includes EV(Zn)C( X8 )C( Z2 )SGRSA (SEQ ID NO:217), EDC( Z6 )C( Z2 )SGRSA (SEQ ID NO:218 ), or the sequence of EDC(Z 6 )C(Z 2 )PLGLA (SEQ ID NO: 219), wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, Amino acids of the group consisting of L, M, N, P, Q, R, S, T, V, W, and Y, where n is 1 to 11 and each Z is independently selected from D, A, Y , S, T, N, I, L, F, V, H, and P amino acids. target binding moiety (TBM)

於一些實施例中,本發明係關於包含靶結合部分(TBM)之可活化抗體。於一些實施例中,該靶結合部分(TBM)包含抗體輕鏈可變區及/或抗體重鏈可變區。於一些實施例中,該靶結合部分(TBM)包含抗體輕鏈可變區。於一些實施例中,該靶結合部分(TBM)包含抗體重鏈可變區。於一些實施例中,該靶結合部分(TBM)包含抗體輕鏈可變區及抗體重鏈可變區。In some embodiments, the invention relates to activatable antibodies comprising a target binding moiety (TBM). In some embodiments, the target binding moiety (TBM) includes an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region. In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region. In some embodiments, the target binding moiety (TBM) includes an antibody light chain variable region and an antibody heavy chain variable region.

於一些實施例中,該靶結合部分(TBM)包含全長抗體輕鏈及/或全長抗體重鏈。抗體輕鏈可為κ或λ輕鏈。抗體重鏈可為任何類別,諸如IgG、IgM、IgE、IgA、或IgD。於一些實施例中,抗體重鏈為IgG類別,諸如IgG1、IgG2、IgG3、或IgG4子類別。可使用此項技術中已知方法將本文中所述之抗體重鏈自一種類別或子類別轉變成另一種類別或子類別。In some embodiments, the target binding moiety (TBM) includes a full-length antibody light chain and/or a full-length antibody heavy chain. The antibody light chain can be a kappa or lambda light chain. Antibody heavy chains can be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is of the IgG class, such as the IgG1, IgG2, IgG3, or IgG4 subclass. The antibody heavy chains described herein can be converted from one class or subclass to another class or subclass using methods known in the art.

本文中所述之靶結合部分(TBM)中之任一者或多者可併入:本文中所述之HVR序列中之任一者(例如,如上 A中所示之重鏈可變區HVR序列中之一者、兩者或三者,及/或輕鏈可變區HVR序列中之一者、兩者或三者);本文中所述之重鏈可變區序列及/或輕鏈可變區序列中之任一者(例如,如上 B中所示之重鏈可變區序列及/或輕鏈可變區序列);及/或本文中所述之抗體中之任一者。 Any one or more of the target binding moieties (TBMs) described herein may be incorporated into: any of the HVR sequences described herein (e.g., the heavy chain variable region as shown in Table A above One, two, or three of the HVR sequences, and/or one, two, or three of the light chain variable region HVR sequences); the heavy chain variable region sequences and/or light chain variable region sequences described herein Any of the chain variable region sequences (e.g., the heavy chain variable region sequence and/or the light chain variable region sequence as shown in Table B above); and/or any of the antibodies described herein By.

於一些實施例中,該靶結合部分(TBM)包含本文中所述之抗CTLA4抗體中之一或多者之序列,該等抗體包括關於HVR、可變區(VL、VH)及/或輕鏈及重鏈(例如,IgG1、IgG2、IgG4)之特異性胺基酸序列所述之抗體。於一些實施例中,該靶結合部分(TBM)包含包含以下之抗體輕鏈可變區:包含胺基酸序列RASQSVRGRFLA (SEQ ID NO: 58)之HVR-L1,包含胺基酸序列DASNRATGI (SEQ ID NO: 66)之HVR-L2,及/或包含胺基酸序列YCQQSSSWPPT (SEQ ID NO: 75)之HVR-L3。於一些實施例中,該靶結合部分(TBM)包含包含SEQ ID NO: 100之胺基酸序列或具有與SEQ ID NO:100之序列至少90% (例如,95%、96%、97%、98%或99%)序列同一性之序列之抗體輕鏈可變區。於一些實施例中,該靶結合部分(TBM)包含包含以下之抗體重鏈可變區:包含胺基酸序列YSISSGYHWSWI (SEQ ID NO: 23)之HVR-H1,包含胺基酸序列LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35)之HVR-H2,及/或包含胺基酸序列ARSYVYFDY (SEQ ID NO: 45)之HVR-H3。於一些實施例中,該靶結合部分(TBM)包含包含SEQ ID NO: 87之胺基酸序列或具有與SEQ ID NO:87之序列至少90% (例如,95%、96%、97%、98%或99%)序列同一性之序列之抗體重鏈可變區。於一些實施例中,該靶結合部分(TBM)包含:a)抗體輕鏈可變區,其包含包含胺基酸序列RASQSVRGRFLA (SEQ ID NO: 58)之HVR-L1,包含胺基酸序列DASNRATGI (SEQ ID NO: 66)之HVR-L2,及/或包含胺基酸序列YCQQSSSWPPT (SEQ ID NO: 75)之HVR-L3;及b)抗體重鏈可變區,其包含包含胺基酸序列YSISSGYHWSWI (SEQ ID NO: 23)之HVR-H1,包含胺基酸序列LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35)之HVR-H2,及/或包含胺基酸序列ARSYVYFDY (SEQ ID NO: 45)之HVR-H3。於一些實施例中,該靶結合部分(TBM)包含包含SEQ ID NO: 100之胺基酸序列之抗體輕鏈可變區,及包含SEQ ID NO: 87之胺基酸序列之抗體重鏈可變區。 可活化結合多肽性質 In some embodiments, the target binding moiety (TBM) comprises the sequence of one or more of the anti-CTLA4 antibodies described herein, including those related to HVR, variable regions (VL, VH) and/or light Antibodies described by the specific amino acid sequences of their chains and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising: HVR-L1 comprising the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 58), comprising the amino acid sequence DASNRATGI (SEQ ID NO: 66) HVR-L2, and/or HVR-L3 comprising the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 75). In some embodiments, the target binding moiety (TBM) comprises an amino acid sequence comprising SEQ ID NO: 100 or having at least 90% (e.g., 95%, 96%, 97%, The antibody light chain variable region has a sequence identity of 98% or 99%). In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region comprising: HVR-H1 comprising the amino acid sequence YSISSGYHWSWI (SEQ ID NO: 23), comprising the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ HVR-H2 (ID NO: 35), and/or HVR-H3 comprising the amino acid sequence ARSYVYFDY (SEQ ID NO: 45). In some embodiments, the target binding moiety (TBM) comprises an amino acid sequence comprising SEQ ID NO: 87 or having at least 90% (e.g., 95%, 96%, 97%, The antibody heavy chain variable region has a sequence identity of 98% or 99%). In some embodiments, the target binding moiety (TBM) comprises: a) an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 58), comprising the amino acid sequence DASNRATGI HVR-L2 of (SEQ ID NO: 66), and/or HVR-L3 comprising the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 75); and b) an antibody heavy chain variable region comprising an amino acid sequence HVR-H1 of YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 comprising the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), and/or HVR-H2 comprising the amino acid sequence ARSYVYFDY (SEQ ID NO: 45) H3. In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 100, and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 87. Change area. Activatable binding peptide properties

於一些實施例中,本發明之可活化結合多肽(即,可活化抗體)包含:(a)掩蔽部分(MM),(b)可裂解部分,及(c)靶結合部分。於一些實施例中,該掩蔽部分(MM)結合至可活化抗體之靶結合部分(TBM)及相較於缺少掩蔽部分之對應結合多肽結合至CTLA4 (例如,人類CTLA4)及/或相較於親本抗體結合至CTLA4 (例如,人類CTLA4)減少或抑制可活化結合部分結合至CTLA4 (例如,人類CTLA4)。In some embodiments, an activatable binding polypeptide (ie, activatable antibody) of the invention includes: (a) a masking moiety (MM), (b) a cleavable moiety, and (c) a target binding moiety. In some embodiments, the masking moiety (MM) binds to the target binding moiety (TBM) of the activatable antibody and binds to CTLA4 (e.g., human CTLA4) compared to a corresponding binding polypeptide lacking the masking moiety and/or compared to Binding of the parent antibody to CTLA4 (e.g., human CTLA4) reduces or inhibits binding of the activatable binding moiety to CTLA4 (e.g., human CTLA4).

於一些實施例中,「可活化」結合多肽係指結合多肽,當於抑制、掩蔽及/或未裂解狀態時,其展示結合至CTLA4之第一水平,及於未抑制、未掩蔽及/或裂解狀態時其展示結合至CTLA4之第二水平,其中CTLA4結合之第二水平高於CTLA4結合之第一水平。於一些實施例中,於可裂解部分內(例如,藉由一或多種蛋白酶)裂解後藉由可活化結合多肽獲取CTLA4係更高。In some embodiments, an "activatable" binding polypeptide refers to a binding polypeptide that exhibits a first level of binding to CTLA4 when in the inhibited, masked, and/or uncleaved state, and that exhibits a first level of binding to CTLA4 when in the uninhibited, unmasked, and/or In the cleaved state it exhibits a second level of binding to CTLA4, wherein the second level of CTLA4 binding is higher than the first level of CTLA4 binding. In some embodiments, acquisition of CTLA4 by activatable binding polypeptide is higher after cleavage within the cleavable moiety (eg, by one or more proteases).

於一些實施例中,當多肽與CTLA4 (例如人類CTLA4)之結合親和力於可活化抗體活化後相較於在可活化抗體活化之前(例如,於藉由於可裂解部分(CM)內裂解之一或多種蛋白酶處理活化後,於藉由pH變化(增加或減少)活化後,於藉由溫度變化(增加或減少)活化後,於藉由與第二分子(諸如小分子)接觸活化後等)增加至少約2倍(例如,至少約2倍、至少約2.5倍、至少約3倍、至少約3.5倍、至少約4倍、至少約4.5倍、至少約5倍、至少約5.5倍、至少約6倍、至少約6.5倍、至少約7倍、至少約7.5倍、至少約8倍、至少約8.5倍、至少約9倍、至少約9.5倍、至少約10倍、至少約25倍、至少約50倍、至少約75倍、至少約100倍、至少約250倍、至少約500倍、至少約750倍、或至少約1000倍、或更多)時,一般認為本發明之可活化抗體為「可活化」結合多肽。於一些實施例中,若於「活化」後可活化抗體之EC 50減少至少約2倍(例如,至少約2倍、至少約2.5倍、至少約3倍、至少約3.5倍、至少約4倍、至少約4.5倍、至少約5倍、至少約5.5倍、至少約6倍、至少約6.5倍、至少約7倍、至少約7.5倍、至少約8倍、至少約8.5倍、至少約9倍、至少約9.5倍、至少約10倍、至少約25倍、至少約50倍、至少約75倍、至少約100倍、至少約250倍、至少約500倍、至少約750倍、或至少約1000倍、或更多) (例如,如藉由ELISA或FACS檢定所量測;參見以下實例),則一般認為本發明之可活化抗體係「可活化」。於一些實施例中,若於用於可裂解部分(CM)內裂解之蛋白酶處理後多肽之EC 50減少至少約2倍(例如,如藉由ELISA或FACS檢定所量測;參見以下實例),則一般認為本發明之可活化抗體係「可活化」。 In some embodiments, the binding affinity of the polypeptide to CTLA4 (e.g., human CTLA4) is greater after activation of the activatable antibody than before activation of the activatable antibody (e.g., by one of the or After activation by various protease treatments, after activation by pH changes (increase or decrease), after activation by temperature changes (increase or decrease), after activation by contact with second molecules (such as small molecules, etc.), increase At least about 2 times (e.g., at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, at least about 10 times, at least about 25 times, at least about 50 times times, at least about 75 times, at least about 100 times, at least about 250 times, at least about 500 times, at least about 750 times, or at least about 1000 times, or more), the activatable antibody of the present invention is generally considered to be "can Activation" binding peptide. In some embodiments, if the EC 50 of the activatable antibody is reduced by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold) after "activation" , at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times , at least about 9.5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 250 times, at least about 500 times, at least about 750 times, or at least about 1000 times times, or more) (e.g., as measured by ELISA or FACS assay; see Examples below), an activatable antibody system of the invention is generally considered to be "activatable." In some embodiments, if the EC 50 of the polypeptide is reduced by at least about 2-fold upon treatment with a protease for cleavage within the cleavable moiety (CM) (e.g., as measured by ELISA or FACS assay; see Examples below), The activatable antibody system of the present invention is generally considered to be "activatable".

於一些實施例中,當掩蔽部分(MM)結合至可活化抗體之靶結合部分(TBM)時,該可活化抗體對CTLA4之K D係高於當掩蔽部分(MM)不結合至靶結合部分(TBM)時(例如於可活化抗體「活化」後(諸如於蛋白酶處理以於可裂解部分(CM)內裂解後))及/或高於親本抗體對CTLA4之K D約2 (例如,約2、約2.5、約3、約3.5、約4、約4.5、約5、約5.5、約6、約6.5、約7、約7.5、約8、約8.5、約9、約9.5、約10、約25、約50、約75、約100、約250、約500、約750、或約1000或更多)倍。量測親和力之方法係此項技術中已知,包括例如藉由以下實例中所述之方法。 In some embodiments, when the masking moiety (MM) binds to the target binding moiety (TBM) of the activatable antibody, the KD of the activatable antibody for CTLA4 is higher than when the masking moiety (MM) does not bind to the target binding moiety. (TBM) (e.g., after "activation" of the activatable antibody (e.g., after protease treatment to cleave within the cleavable moiety (CM))) and/or is approximately 2 higher than the K D of the parent antibody for CTLA4 (e.g., About 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 , about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more) times. Methods for measuring affinity are known in the art, including, for example, by the methods described in the Examples below.

於一些實施例中,當掩蔽部分結合至可活化抗體之靶結合部分時,該可活化抗體對CTLA4之K D相對於當掩蔽部分不結合至靶結合部分時(例如,於可活化抗體「活化」後(諸如於蛋白酶處理以於可裂解部分(CM)內裂解後))及/或相對於親本抗體對CTLA4之K D減少至少約25% (例如,至少約25%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、至少約95%、至少約99%)。量測親和力之方法係此項技術中已知,包括例如,藉由以下實例中所述之方法。 In some embodiments, the K of the activatable antibody for CTLA4 when the masking moiety is bound to the target-binding moiety of the activatable antibody is relative to that when the masking moiety is not bound to the target-binding moiety (e.g., when the activatable antibody is "activated" (such as after protease treatment to cleave within the cleavable moiety (CM)) and/or the KD for CTLA4 is reduced by at least about 25% (e.g., at least about 25%, at least about 30%) relative to the parent antibody , at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%). Methods for measuring affinity are known in the art, including, for example, by the methods described in the Examples below.

於一些實施例中,該掩蔽部分空間阻礙可活化抗體結合至CTLA4及/或變構阻礙可活化抗體結合至CTLA4。於一些實施例中,該掩蔽部分不包含可活化抗體及/或親本抗體之天然結合搭檔之胺基酸序列。In some embodiments, the masking moiety sterically blocks the binding of the activatable antibody to CTLA4 and/or allosterically blocks the binding of the activatable antibody to CTLA4. In some embodiments, the masking portion does not include amino acid sequences that activate the natural binding partners of the antibody and/or the parent antibody.

於一些實施例中,掩蔽部分對靶結合部分之解離常數高於可活化抗體對CTLA4之解離常數(當活化時)。於一些實施例中,掩蔽部分對靶結合部分之解離常數係高於可活化抗體對CTLA4之解離常數(當活化時)約2 (例如,約2、約2.5、約3、約3.5、約4、約4.5、約5、約5.5、約6、約6.5、約7、約7.5、約8、約8.5、約9、約9.5、約10、約25、約50、約75、約100、約250、約500、約750、或約1000或更多)倍。於一些實施例中,掩蔽部分對靶結合部分之解離常數約等於可活化抗體對CTLA4之解離常數(當活化時)。In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is higher than the dissociation constant of the activatable antibody for CTLA4 (when activated). In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is about 2 (e.g., about 2, about 2.5, about 3, about 3.5, about 4) higher than the dissociation constant of the activatable antibody for CTLA4 when activated. , about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more) times. In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is approximately equal to the dissociation constant of the activatable antibody for CTLA4 (when activated).

本文中所述之可活化抗體可經進一步修飾。於一些實施例中,該等可活化抗體連接至另外分子實體。另外分子實體之實例包括醫藥劑、肽或蛋白質、檢測劑或標籤、及抗體。The activatable antibodies described herein can be further modified. In some embodiments, the activatable antibodies are linked to another molecular entity. Examples of additional molecular entities include pharmaceutical agents, peptides or proteins, detection agents or tags, and antibodies.

於一些實施例中,本發明之可活化抗體連接至醫藥劑。醫藥劑之實例包括細胞毒性劑或其他癌症治療劑及放射性同位素。細胞毒性劑之特定實例包括紫杉醇、細胞鬆弛素B、短桿菌肽D、溴化乙錠、依米丁、絲裂黴素、依託泊苷、替尼泊苷、長春新鹼、長春鹼、秋水仙素、多柔比星、柔紅黴素、二羥基蒽醌二酮、米托蒽醌、光神黴素、放線菌素D、1-去氫睾酮、糖皮質激素、普魯卡因、丁卡因、利多卡因、普萘洛爾、及嘌呤黴素及其類似物或同系物。治療劑亦包括例如抗代謝劑(例如,胺甲喋呤、6-巰基嘌呤、6-硫鳥嘌呤、阿糖胞苷、5-氟尿嘧啶、達卡巴嗪)、烷基化劑(例如,二氯甲基二乙胺、噻替派、苯丁酸氮芥、美法侖、卡莫司汀(BSNU)及洛莫司汀(CCNU)、環磷醯胺、白消安、二溴甘露醇、鏈脲佐菌素、絲裂黴素C及順-二氯二胺鉑(II) (DDP)順鉑)、蒽環黴素(例如,柔紅黴素(先前稱作道諾黴素)及多柔比星)、抗生素(例如,更生黴素(先前稱作放線菌素)、博來黴素、光神黴素及安麯黴素(AMC))、及抗有絲分裂劑(例如,長春新鹼及長春鹼)。可與抗體共軛用於診斷或治療使用之放射性同位素之實例包括(但不限於) 碘 131、銦 111、釔 90及鑥 177。用於將多肽連接至醫藥劑之方法係此項技術中已知,諸如使用各種連接子技術。連接子類型之實例包括腙、硫醚、酯、二硫醚及含肽連接子。將治療劑連接至抗體之連接子及方法之進一步討論參見例如,Saito等人, Adv. Drug Deliv. Rev.55:199-215 (2003);Trail等人, Cancer Immunol. Immunother.52:328-337 (2003);Payne, Cancer Cell3:207-212 (2003);Allen, Nat. Rev. Cancer2:750-763 (2002);Pastan及Kreitman, Curr. Opin. Investig. Drugs3:1089-1091 (2002);Senter及Springer (2001) Adv. Drug Deliv. Rev.53:247-264。 V. 產生 CTLA4 抗體及 / 或精度 / 背景依賴性可活化抗體之核酸、載體、宿主細胞及重組方法 In some embodiments, the activatable antibodies of the invention are linked to pharmaceutical agents. Examples of pharmaceutical agents include cytotoxic or other cancer therapeutic agents and radioactive isotopes. Specific examples of cytotoxic agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, Narcissus, doxorubicin, daunorubicin, dihydroxyanthraquinonedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, Tetracaine, lidocaine, propranolol, and puromycin and their analogs or congeners. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., dichloride Methyl diethylamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, Streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly known as daunorubicin) and Doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and antromycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine -131 , indium -111 , yttrium -90 , and gallium -177 . Methods for linking polypeptides to pharmaceutical agents are known in the art, such as the use of various linker technologies. Examples of linker types include hydrazone, thioether, ester, disulfide, and peptide-containing linkers. For further discussion of linkers and methods of attaching therapeutic agents to antibodies, see, for example, Saito et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail et al., Cancer Immunol. Immunother. 52:328- 337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091 (2002); Senter and Springer (2001) Adv. Drug Deliv. Rev. 53:247-264. V. Nucleic acids, vectors, host cells and recombinant methods for producing CTLA4 antibodies and / or precision / context-dependent activatable antibodies

本發明之另一態樣提供經單離核酸分子,其包含編碼本文中所提供之結合分子(例如,抗體或可活化抗體)之胺基酸序列之核苷酸序列。藉由核苷酸序列編碼之胺基酸序列可為抗體之任何部分(諸如HVR、包含1、2或3個HVR之序列、重鏈可變區、輕鏈可變區),或可為全長重鏈或全長輕鏈。本發明之核酸可為例如DNA或RNA,且可含或可不含內含子序列。通常,核酸為cDNA分子。Another aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence of a binding molecule (eg, an antibody or activatable antibody) provided herein. The amino acid sequence encoded by the nucleotide sequence can be any part of the antibody (such as HVR, a sequence containing 1, 2 or 3 HVRs, heavy chain variable region, light chain variable region), or can be full length Heavy chain or full-length light chain. The nucleic acid of the invention may be, for example, DNA or RNA, and may or may not contain intronic sequences. Typically, the nucleic acid is a cDNA molecule.

於一些實施例中,本發明提供經單離核酸分子,其包含編碼選自由以下組成之群之胺基酸序列之核苷酸序列或由該核苷酸序列組成:(1)本文中所述之說明性抗體之HVR-H1、HVR-H2、HVR-H3、HVR-L1、HVR-L2及/或HVR-L3之胺基酸序列;(2)本文中所述之說明性抗體之重鏈可變區及/或輕鏈可變區;或(3)說明性抗體之全長重鏈或全長輕鏈。In some embodiments, the invention provides isolated nucleic acid molecules comprising or consisting of a nucleotide sequence encoding an amino acid sequence selected from the group consisting of: (1) as described herein The amino acid sequence of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and/or HVR-L3 of the illustrative antibodies; (2) The heavy chain of the illustrative antibodies described herein The variable region and/or the light chain variable region; or (3) the full-length heavy chain or the full-length light chain of the illustrative antibody.

於一些實施例中,核酸分子包含編碼如SEQ ID NO: 18至107中之任一者中所述之胺基酸序列之核苷酸序列或由該核苷酸序列組成。In some embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding an amino acid sequence as set forth in any one of SEQ ID NOs: 18 to 107.

於一些實施例中,核酸分子包含下 C中所述之核苷酸序列或由該核苷酸序列組成。 C :抗 CTLA4 可變區多核苷酸序列 抗體名稱: VH VL TY21585 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCACCTTCTCCGACTACGCTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGACTCGAGTGGATCGGTATCATCTCCCCATCTAGCGGTTCTACTAACTACGCCCAGAAGTTCCAGGGTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGAGACATTCACTCTGGTTCTTCTGGTTACTACTACGGTTTCGACGTCTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 108) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTGAGTCTGTGGACTTCTTCGGTATCTCTTTCCTGGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCCAGCACTACACCTCTTCGCCACCAGTGTACACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 121) TY21586 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACTCTATCACCTCTGGTTACTACTGGGCCTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGGTGTCTTCCATCTCTGGTTCCGGTTCTACTACCTACTACGCCGACTCTGTCAAGGGCCGTTTCACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGAGATGGTTTCGGCTACTTCGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 109) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCTCTGCCTCTTCTAGCGTGAGCTACGTGTACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTTCTCTGGAATCTGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCGTGCAGGGTCTTCAGACCCCTTGGACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 122) TY21587 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCACCTTCTCCGACTACGGTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTGAAATCTACCACTCTGGTTCTACCTACTACTCTCCATCTCTGAAGTCTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGAGACGTTGCCCCTGGTTCTTCTGGTTACTACGACGGTTTCGACTTCTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 110) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGGGTATTGGCTCTTCCCTGGCTTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCCAGCAGTACGACCAATGGCCACCTTGGACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 123) TY21588 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACTCTATCTCCTCTGGTTACCACTGGGACTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGGTGTCTGGTATCTCTGGTTACGGTGGTTCTACCTACTACGCCGACTCTGTCAAGGGCCGTTTCACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGACACAGTTATTACGGTTCCGGTAATTTCGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 111) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTGAGTCTGTGGACTTCTTCGGTAAGTCTTTCCTGCACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTAACCTGGAAACCGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCCAGCAGTCCTACTCCTGGCCTCCGACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 124) TY21589 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCACCTTCTCCGACTACTGGATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTTGGATCTCCCCATCTGGCGGTGGTACTAAGTACGCCCAGAAGTTCCAGGGTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGAGGGGCTTACGAATTTGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 112) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGTCTGTGAGCAGCCGTTTCCTGGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCCAGCAGTCCTACCCCACCCCTCTTACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 125) TY21580 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACTCTATCTCCTCTGGTTACCACTGGAGCTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGCTGGCCCGGATCGACTGGGACGATGACAAGTACTACTCTACCTCTCTGAAGTCTCGTCTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGATCGTACGTGTACTTCGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 113) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGTCTGTGCGCGGCCGTTTCCTGGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCCAGCAGTCCTCCTCCTGGCCTCCGACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 126) TY21591 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCTCTCTGTCTACCGGCGGTGTGGCTGTGAGCTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTGAAATCTACCACTCTGGTTCTACCTACTACTCTCCATCTCTGAAGTCTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCCGTCGTATCGCCACCGCTACTTACTTCGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 114) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGACCGTGTTCTCTCGTTACCTGGCTTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCCAGCAGTCCTACTACTGGCCACCTTGGACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 127) TY21686 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCTCTCTGTCTACCGGCGGTGTGGCTGTGGGCTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGGTGTCTGCTATCTCTGGTTACGGTTCTACTACCTACTACGCCGACTCTGTCAAGGGCCGTTTCACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGATTGCCATACTCCGCCTACGCTTTCGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 115) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGGGTGTGTCTTCTTACCTGGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGCCGCCTCTACCTTGCAGTCTGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTACTACTGCCAGCACCACTACGGCACCCCACTGACCTTCGGTCAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 128) TY21687 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCACCTTCTCCGGCTACGCTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTATCATCTCCCCATCTGGCGGTGGTACTAAGTACGCCCAGAAGTTCCAGGGTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGACACCCATTCGCCTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 116) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGTCTGTGGACTTCTACGGTATCTCTTTCCTGGACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCCAGCAGTACGTCTCTTCGCCACCAGAGTACACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 129) TY21689 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACACCTTCTCCGGCTACGGTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTGAAATCTACCACTCTGGTTCTACCTACTACTCTCCATCTCTGAAGTCTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGAAGAATTGACGCCTTCGACATCTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 117) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGTCTGTGGACTTCGACGGTTTCTCTTTCCTGCACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTTCTCTGGAATCTGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCCAGCAGCGTGACTCCTGGCCTTACACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 130) TY21680 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACACCTTCTCCGGCTACGCTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTATCATCTCCCCATCTGGCGGTGGTACTAAGTACGCCCAGAAGTTCCAGGGTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGACTCTATGACGTTGCCTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 118) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGTCTGTGGACTTCCACGGTAAGTCTTTCCTGCACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTTCTCTGGAATCTGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCGAGCAATCCCTGGAAGTCCCATTCACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 131) TY21691 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCACCTTCTCCGACTACGCTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTATCATCTCCCCATCTGGCGGTTCTACTAAGTACGCCCAGAAGTTCCAGGGTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGACTCGGTTACGGGTACTTCGACGTCTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 119) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGTCTGTGGACTTCTACGGTATCTCTTTCCTGCACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTTCTCTGGAATCTGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGCGTGCAGGCTCTTCAGTTGCCTCTTACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 132) TY21692 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACTCTATCACCTCTGGTCACTACTGGAGCTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTGACATCTCCCACTCTGGTTCTACCTACTACTCTCAATCTCTGAAGTCTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGCGCGCGTGGTAGTAGGACCGGCTACTTCGACTATTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 120) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGTCTATCTCTTCTTACCTGAACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTAACCTGGAAACCGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTACTACTGCCAGCACCACTACGGCACCCCACTGACCTTCGGTCAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 133) In some embodiments, the nucleic acid molecule comprises or consists of the nucleotide sequence described in Table C below. Table C : Anti- CTLA4 variable region polynucleotide sequence Antibody name: VH : VL : TY21585 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCACCTTTCCGACTACGCTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGACTCGAGTGGATCGGTATCATCTCCCCATCTAGCGGTTCTACTAACTACGCCCAGAAGTTCCAGGGTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTA AGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGAGACATTCACTCTGGTTTCTTCTGGTTACTACTACGGTTTCGACGTCTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 108) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTGAGTCTGTGGACTTCTTCGGTATCTCTTTCCTGGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAG ACTTCGCAACTTATTACTGCCAGCACTACACCTCTTCGCCACCAGTGTACACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 121) TY21586 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACTCTATCACCTCTGGTTACTACTGGGCCTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGGTGTCTTCCATCTCTGGTTCCGGTTCTACTACCTACTACGCCGACTCTGTCAAGGGCCGTTTCACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTA AGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGAGATGGTTTCGGCTACTTCGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 109) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCTCTGCCTCTTCTAGCGTGAGCTACGTGTACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTTCTCTGGAATCTGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATT ACTGCGTGCAGGGTCTTCAGACCCCTTGGACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 122) TY21587 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCACCTTTCCGACTACGGTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTGAAATCTACCACTCTGGTTCTACCTACTACTCTCCATCTCTGAAGTCTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAG AGCTGAGGACACTGCCGTCTATTATTGCGCCAGAGACGTTGCCCCTGGTTCTTCTGGTTACTACGACGGTTTCGACTTCTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 110) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCTCTCCAGGGTATTGGCTTCTTCCCTGGCTTGGTATCAACAGAAACCAGGAAAAGTCCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTT ATTACTGCCAGCAGTACGACCAATGGCCACCTTGGACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 123) TY21588 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACTCTATCTCCTCTGGTTACCACTGGGACTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGGTGTCTGGTATCTCTGGTTACGGTGGTTCTACCTACTACGCCGACTCTGTCAAGGGCCGTTTCACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCT TAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGACACAGTTATTACGGTTCCGGTAATTTCGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 111) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTGAGTCTGTGGACTTCTTCGGTAAGTCTTTCCTGCACTGGTATCAACAGAAACCAGGAAAAGTCCCGAAGCTTCTGATCTACGACGCCTCTAACCTGGAAACCGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAG ACTTCGCAACTTATTACTGCCAGCAGTCCTACTCCTGGCCTCCGACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 124) TY21589 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCACCTTTCCGACTACTGGATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTTGGATCTCCCCATCTGGCGGTGGTACTAAGTACGCCCAGAAGTTCCAGGGTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTA AGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGAGGGGCTTACGAATTTGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 112) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTTCCAGTCTGTGAGCAGCCGTTTCCTGGCCTGGTATCAACAGAAACCAGGAAAAGTCCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCA ACTTATTACTGCCAGCAGTCCTACCCCACCCCTCTTACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 125) TY21580 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACTCTATCTCCTCTGGTTACCACTGGAGCTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGCTGGCCCGGATCGACTGGGACGATGACAAGTACTACTCTACCTCTCTGAAGTCTCGTCTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAG CTGAGGACACTGCCGTCTATTATTGCGCCAGATCGTACGTGTACTTCGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 113) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGTCTGTGCGCGGCCGTTTCCTGGCCTGGTATCAACAGAAACCAGGAAAAGTCCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCA ACTTATTACTGCCAGCAGTCCTCCTCCTGGCCTCCGACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 126) TY21591 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCTCTCTGTCTACCGGCGGTGTGGCTGTGAGCTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTGAAATCTACCACTCTGGTTCTACCTACTACTCTCCATCTCTGAAGTCTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCT TAAGAGCTGAGGACACTGCCGTCTATTATTGCGCCCGTCGTATCGCCACCGCTACTTACTTCGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 114) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGACCGTGTTCTCTCGTTACCTGGCTTGGTATCAACAGAAACCAGGAAAAGTCCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCA ACTTATTACTGCCAGCAGTCCTACTGGCCACCTTGGACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 127) TY21686 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCTCTCTGTCTACCGGCGGTGGCTGTGGGCTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGGTGTCTGCTATCTCTGGTTACGGTTCTACTACCTACTACGCCGACTCTGTCAAGGGCCGTTTCACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAG CTTAAGAGCTGAGGACACTGCCGCTATTATTGCGCCAGATTGCCATACTCCGCCTACGCTTTCGACTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 115) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGGGTGTGTCTTCTTACCTGGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGCCGCCTCTACCTTGCAGTCTGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTACT ACTGCCAGCACCACTACGGCACCCCACTGACCTTCGGTCAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 128) TY21687 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCACCTTTCCGGCTACGCTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTATCATCTCCCCATCTGGCGGTGGTACTAAGTACGCCCAGAAGTTCCAGGGTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTA AGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGACACCCATTCGCCTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 116) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTTCCAGTGGACTTCTACGGTATCTCTTTCCTGGACTGGTATCAACAGAAACCAGGAAAAGTCCCGAAGCTTCTGATCTACGACGCCTCTAACCGTGCCACCGGTATCCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAG ACTTCGCAACTTATTACTGCCAGCAGTACGTCTCTTCGCCACCAGAGTACACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 129) TY21689 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACACCTTTCCGGCTACGGTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTGAAATCTACCACTCTGGTTCTACCTACTACTCTCCATCTCTGAAGTCTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAG AGCTGAGGACACTGCCGTCTATTATTGCGCCAGAAGAATTGACGCCTTCGACATCTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG ( SEQ ID NO: 117) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTTCCAGTCTGTGGACTTCGACGGTTTCTCTTTCCTGCACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTTCTGGAATCTGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAG ACTTCGCAACTTATTACTGCCAGCAGCGTGACTCCTGGCCTTACACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 130) TY21680 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACACCTTTCCGGCTACGCTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTATCATCTCCCCATCTGGCGGTGGTACTAAGTACGCCCAGAAGTTCCAGGGTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTA AGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGACTCTATGACGTTGCCTACTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 118) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTTCCAGTGGACTTCCACGGTAAGTCTTTCCTGCACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTTCTGGAATCTGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAG ACTTCGCAACTTATTACTGCGAGCAATCCCTGGAAGTCCCATTCACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 131) TY21691 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATTCACCTTTCCGACTACGCTATTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTATCATCTCCCCATCTGGCGGTTCTACTAAGTACGCCCAGAAGTTCCAGGGTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTA AGAGCTGAGGACACTGCCGTCTATTATTGCGCCAGACTCGGTTACGGGTACTTCGACGTCTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 119) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCCTCTCAGTGGACTTCTACGGTATCTCTTTCCTGCACTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATCTACGACGCCTCTTCTGGAATCTGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAG ACTTCGCAACTTATTACTGCGTGCAGGCTCTTCAGTTGCCTCTTACCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 132) TY21692 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCCGGATACTCTATCACCTCTGGTCACTACTGGAGCTGGATTCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGATCGGTGACATCTCCCACTCTGGTTCTACCTACTACTCTCAATCTCTGAAGTCTCGTGTGACTATAAGTCGCGACAATTCGAAAAACACACTGTACCTACAACTGAACAGCTTAAGAG CTGAGGACACTGCCGTCTATTATTGCGCGCGTGGTAGTAGGACCGGCTACTTCGACTATTGGGGTCAAGGAACACTAGTCACCGTCTCCTCG (SEQ ID NO: 120) GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGTCCGCCTCTGTGGGCGATCGGGTCACCATCACCTGCCGTGCTCTCCAGTCTATCTCTTCTTACCTGAACTGGTATCAACAGAAACCAGGAAAAGTCCCGAAGCTTCTGATCTACGACGCCTCTAACCTGGAAACCGGTGTGCCATCTCGCTTCTCTGGATCCGGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTT ACTACTGCCAGCACCACTACGGCACCCCACTGACCTTCGGTCAGGGTACCAAGGTGGAGATCAAACGA (SEQ ID NO: 133)

可使用任何適宜分子生物技術獲得本發明之核酸。針對藉由雜交瘤表現之抗體,編碼藉由雜交瘤製備之抗體之輕鏈及重鏈之cDNA可藉由PCR擴增或cDNA選殖技術獲得。針對獲自免疫球蛋白基因庫之抗體(例如,使用噬菌體展示技術),編碼該抗體之核酸可自該庫恢復。The nucleic acids of the invention may be obtained using any suitable molecular biology technique. For antibodies expressed by hybridomas, cDNA encoding the light chain and heavy chain of the antibody prepared by hybridomas can be obtained by PCR amplification or cDNA selection technology. For antibodies obtained from an immunoglobulin gene library (eg, using phage display technology), the nucleic acid encoding the antibody can be recovered from the library.

可藉由將編碼V H之DNA以可操作方式連接至編碼重鏈恆定區(CH1、CH2及CH3)之另一DNA分子將編碼V H區之經單離DNA轉變成全長重鏈基因。人類重鏈恆定區基因之序列係此項技術中已知(參見例如,Kabat等人(1991) NIH公開案第91-3242號)及包含此等區之DNA片段可藉由標準PCR擴增獲得。重鏈恆定區可為IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恆定區。針對Fab片段重鏈基因,可將編碼V H之DNA以可操作方式連接至僅編碼重鏈CH1恆定區之另一DNA分子。 Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991) NIH Publication No. 91-3242) and DNA fragments containing these regions can be obtained by standard PCR amplification . The heavy chain constant region may be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For the Fab fragment heavy chain gene, the DNA encoding VH can be operably linked to another DNA molecule encoding only the CH1 constant region of the heavy chain.

可藉由將編碼V L之DNA以可操作方式連接至編碼輕鏈恆定區CL之另一DNA分子將編碼V L區之經單離DNA轉變成全長輕鏈基因(以及Fab輕鏈基因)。人類輕鏈恆定區基因之序列係此項技術中已知(參見例如,Kabat等人(1991) NIH公開案第91-3242號)及包含此等區之DNA片段可藉由標準PCR擴增獲得。輕鏈恆定區可為κ或λ恆定區。 Isolated DNA encoding the V region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the V region to another DNA molecule encoding the light chain constant region CL . The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al. (1991) NIH Publication No. 91-3242) and DNA fragments containing these regions can be obtained by standard PCR amplification . The light chain constant region may be a kappa or lambda constant region.

為創建scFv基因,將編碼V H-及V L-之DNA片段以可操作方式連接至編碼可撓性連接子(例如,編碼胺基酸序列(Gly 4-Ser) 3)之另一片段,使得該等V H及V L序列可表現為鄰接單鏈蛋白,其中該等V L及V H區藉由可撓性連接子接合(參見例如,Bird等人, Science242:423-426 (1988);Huston等人, Proc. Natl. Acad. Sci. USA85:5879-5883 (1988);及McCafferty等人, Nature348:552-554 (1990))。 To create a scFv gene, a DNA segment encoding VH- and VL- is operably linked to another segment encoding a flexible linker (e.g., encoding the amino acid sequence ( Gly4 -Ser) 3 ), This allows the VH and VL sequences to appear as contiguous single-chain proteins in which the VL and VH regions are joined by flexible linkers (see, e.g., Bird et al., Science 242:423-426 (1988) ); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990)).

本發明另提供包含本文中所述之核酸分子之載體。於一些實施例中,該載體為表現載體或顯示載體(例如,病毒顯示載體、細菌顯示載體、酵母顯示載體、昆蟲顯示載體、哺乳動物顯示載體等)。核酸分子可編碼輕鏈或重鏈之部分(諸如CDR或HVR;輕鏈或重鏈可變區)、全長輕鏈或重鏈、包含重鏈或輕鏈之部分或全長之多肽、或抗體衍生物或抗原結合片段之胺基酸序列。於一些實施例中,該載體為可用於表現結合分子(諸如抗體或其抗原結合片段)之表現載體。於一些實施例中,本文中提供載體,其中第一載體包含編碼如本文中所述之重鏈可變區之多核苷酸序列,及第二載體包含編碼如本文中所述之輕鏈可變區之多核苷酸序列。於一些實施例中,單個載體包含編碼如本文中所述之重鏈可變區及如本文中所述之輕鏈可變區之多核苷酸。The invention further provides vectors comprising the nucleic acid molecules described herein. In some embodiments, the vector is an expression vector or a display vector (eg, a viral display vector, a bacterial display vector, a yeast display vector, an insect display vector, a mammalian display vector, etc.). The nucleic acid molecule may encode a portion of a light or heavy chain (such as a CDR or HVR; a light or heavy chain variable region), a full-length light or heavy chain, a polypeptide comprising a portion or full length of a heavy or light chain, or an antibody-derived The amino acid sequence of the substance or antigen-binding fragment. In some embodiments, the vector is an expression vector useful for expressing binding molecules, such as antibodies or antigen-binding fragments thereof. In some embodiments, vectors are provided herein, wherein a first vector comprises a polynucleotide sequence encoding a heavy chain variable region as described herein, and a second vector comprises a polynucleotide sequence encoding a light chain variable region as described herein. The polynucleotide sequence of the region. In some embodiments, a single vector includes a polynucleotide encoding a heavy chain variable region as described herein and a light chain variable region as described herein.

為表現本發明之結合分子,將編碼部分或全長輕鏈及重鏈之DNA插入表現載體中使得DNA分子以可操作方式連接至轉錄及轉譯控制序列。於此上下文中,術語「以可操作方式連接」意指將抗體基因連接至載體使得該載體內之轉錄及轉譯控制序列提供其調節DNA分子之轉錄及轉譯所需功能。選擇與使用之表現宿主細胞相容之表現載體及表現控制序列。可將抗體輕鏈基因及抗體重鏈基因插入分開載體中,或可將兩種基因插入相同表現載體中。藉由任何適宜方法(例如,連接抗體基因片段及載體上之互補限制位點,或基於同源重組之DNA連接)將抗體基因插入表現載體中。可使用本文中所述之抗體之輕鏈及重鏈可變區以藉由將其插入已編碼所需同型及子類別之重鏈恆定區及輕鏈恆定區之表現載體中使得V H片段以可操作方式連接至該載體內之C H片段及V L片段以可操作方式連接至該載體內之C L片段來創造任何抗體同型及子類別之全長抗體基因。此外或或者,重組表現載體可編碼促進來自宿主細胞之抗體鏈之分泌的信號肽。可將抗體鏈基因選殖至載體使得信號肽於框架內連接至抗體鏈基因之胺基端。該信號肽可為免疫球蛋白信號肽或異源信號肽(即,來自非免疫球蛋白之信號肽)。 To express the binding molecules of the invention, DNA encoding partial or full-length light and heavy chains is inserted into an expression vector such that the DNA molecules are operably linked to transcription and translation control sequences. In this context, the term "operably linked" means that the antibody gene is linked to a vector such that the transcription and translation control sequences within the vector provide the functions required to regulate the transcription and translation of the DNA molecule. Select expression vectors and expression control sequences that are compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. The antibody gene is inserted into the expression vector by any suitable method (for example, ligating the antibody gene fragment and complementary restriction sites on the vector, or DNA ligation based on homologous recombination). The light and heavy chain variable regions of the antibodies described herein can be used to render the VH fragments by inserting them into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype and subclass. The CH fragments and VL fragments operably linked to the vector are operably linked to the CL fragment within the vector to create full-length antibody genes for any antibody isotype and subclass. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that promotes secretion of antibody chains from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin).

除了抗體序列外,本發明之表現載體通常攜帶控制宿主細胞中之抗體序列之表現的調節序列。術語「調節序列」意欲包括控制抗體鏈基因之轉錄或轉譯之啟動子、增強子及其他表現控制元件(例如,多腺苷酸化信號)。此等調節序列述於例如Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990))中。熟習此項技術者應瞭解,表現載體之設計(包括調節序列之選擇)可取決於如待轉形之宿主細胞之選擇、所需蛋白質之表現水平等之此等因素。用於哺乳動物宿主細胞表現之調節序列之實例包括指導哺乳動物細胞中高水平蛋白質表現之病毒元件,諸如源自巨細胞病毒(CMV)、猿腎病毒(Simian Virus) 40 (SV40)、腺病毒(例如腺病毒主要後期啟動子(AdMLP))及多瘤病毒(polyoma)之啟動子及/或增強子。或者,可使用非病毒調節序列,諸如泛素(ubiquitin)啟動子或β-球蛋白啟動子。更進一步,調節元件由來自不同來源(諸如SR啟動子系統)之序列組成,該啟動子系統含有來自SV40早期啟動子之序列及人類T細胞白血病病毒1型之長末端重複(Takebe, Y.等人(1988) Mol. Cell. Biol.8:466-472)。 In addition to the antibody sequences, expression vectors of the invention typically carry regulatory sequences that control the expression of the antibody sequences in the host cell. The term "regulatory sequences" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Those skilled in the art will understand that the design of an expression vector, including the selection of regulatory sequences, may depend on such factors as the selection of host cells to be transformed, the expression level of the desired protein, and the like. Examples of regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as those derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus ( For example, the adenovirus major late promoter (AdMLP) and the promoter and/or enhancer of polyoma virus (polyoma). Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or the beta-globin promoter. Furthermore, the regulatory elements are composed of sequences from different sources such as the SR promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. Human (1988) Mol. Cell. Biol. 8:466-472).

除了抗體鏈基因及調節序列外,表現載體可攜帶另外序列,諸如調節載體於宿主細胞中之複製(例如,複製起源)之序列及可選擇標記基因。可選擇標記基因促進已引入載體之宿主細胞之選擇(參見例如,美國專利案第4,399,216號、第4,634,665號及第5,179,017號,所有由Axel等人)。例如,已引入載體之宿主細胞上之可選擇標記基因通常賦予對藥物(諸如G418、潮黴素(hygromycin)或胺甲喋呤)之耐性。可選擇標記基因包括二氫葉酸還原酶(DHFR)基因(用於含有胺甲喋呤之dhfr-宿主細胞選擇/擴增)及neo基因(用於G418選擇)。In addition to the antibody chain genes and regulatory sequences, the expression vector can carry additional sequences, such as sequences that regulate replication of the vector in the host cell (eg, origin of replication) and selectable marker genes. The selectable marker gene facilitates selection of the host cell into which the vector has been introduced (see, eg, U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, a selectable marker gene that has been introduced into the host cell of the vector typically confers resistance to drugs such as G418, hygromycin, or methotrexate. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for dhfr-host cell selection/amplification containing methotrexate) and the neo gene (for G418 selection).

用於表現輕鏈及重鏈,藉由任何適宜技術將編碼重鏈及輕鏈之表現載體轉染至宿主細胞中。術語「轉染」之各種形式意欲涵蓋用於將外源DNA引入原核或真核宿主細胞中通常所用之廣泛各種技術,例如,電穿孔、磷酸鈣沉澱、DEAE-葡聚糖轉染及類似者。雖然可於原核或真核宿主細胞中表現本發明之抗體,真核細胞及通常哺乳動物宿主細胞中之抗體之表現係最典型。For expression of light and heavy chains, expression vectors encoding heavy and light chains are transfected into host cells by any suitable technique. Various forms of the term "transfection" are intended to cover a wide variety of techniques commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. . Although the antibodies of the invention can be expressed in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells and generally mammalian host cells is most typical.

本發明進一步提供含有本發明提供之核酸分子之宿主細胞。宿主細胞實質上可為任何細胞,針對該細胞表現載體係可得。其可為例如高等真核宿主細胞(諸如哺乳動物細胞)、低等真核宿主細胞(諸如酵母細胞),且可為原核細胞(諸如細菌細胞)。將重組核酸引入宿主細胞中之方法係此項技術中已知,包括例如,藉由磷酸鈣轉染、DEAE、葡聚糖介導之轉染、電穿孔或噬菌體感染。The invention further provides host cells containing the nucleic acid molecules provided by the invention. The host cell can be essentially any cell for which a vector system is available. It can be, for example, a higher eukaryotic host cell (such as a mammalian cell), a lower eukaryotic host cell (such as a yeast cell), and can be a prokaryotic cell (such as a bacterial cell). Methods for introducing recombinant nucleic acids into host cells are known in the art and include, for example, by calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.

用於轉形之適宜原核宿主包括大腸桿菌 枯草芽胞桿菌( Bacillus subtilis)、鼠傷寒沙門氏桿菌( Salmonella typhimurium)及假單胞菌( Pseudomonas) 鏈黴素( Streptomyces)及葡萄球菌( Staphylococcus)屬內之各種物種。 Suitable prokaryotic hosts for transformation include Escherichia coli , Bacillus subtilis , Salmonella typhimurium and Pseudomonas , Streptomyces and Staphylococcus Various species within the genus.

用於轉形之適宜真核宿主包括酵母、昆蟲(例如,S2細胞)及哺乳動物細胞。用於表現本發明之結合分子之哺乳動物宿主細胞包括例如中國倉鼠卵巢(CHO)細胞(包括dhfr-CHO細胞,述於Urlaub及Chasin, Proc. Natl. Acad. Sci. USA77:4216-4220 (1980);Sharp, J. Mol. Biol.159:601-621 (1982)中)、NS0骨髓瘤細胞、COS細胞、HEK293F細胞、HEK293T細胞及Sp2細胞。特定言之,併與NS0骨髓瘤或CHO細胞使用,另一表現系統為於WO 87/04462、WO 89/01036及EP 338,841中所揭示之GS (麩胺醯胺合成酶)基因表現系統。於一些實施例中,於CHO細胞中產生本發明之抗體。於一些實施例中,本發明之抗體經修飾,且不包含C端離胺酸殘基(例如,本文中所述之抗體重鏈之C端離胺酸殘基經移除(諸如在抗體產生之前或在抗體產生期間))。當將編碼抗體基因之表現載體引入哺乳動物宿主細胞中時,抗體藉由培養宿主細胞持續足以允許抗體於宿主細胞中表現或將抗體分泌至宿主細胞生長之培養基中之時間段產生。可使用此項技術中已知之任何適宜蛋白質純化方法(例如,蛋白A層析法及/或離子交換層析法)將抗體自培養基回收。 VI. 組合物 Suitable eukaryotic hosts for transformation include yeast, insect (eg, S2 cells), and mammalian cells. Mammalian host cells for expressing binding molecules of the invention include, for example, Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 ( 1980); Sharp, J. Mol. Biol. 159:601-621 (1982)), NS0 myeloma cells, COS cells, HEK293F cells, HEK293T cells and Sp2 cells. Specifically, and used with NSO myeloma or CHO cells, another expression system is the GS (glutamine synthetase) gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. In some embodiments, the antibodies of the invention are produced in CHO cells. In some embodiments, the antibodies of the invention are modified and do not include a C-terminal lysine residue (e.g., the C-terminal lysine residue of the antibody heavy chain described herein is removed (such as in antibody production). before or during antibody production)). When an expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the medium in which the host cell is grown. Antibodies can be recovered from the culture medium using any suitable protein purification method known in the art (eg, protein A chromatography and/or ion exchange chromatography). VI. Composition

於其他態樣中,本發明提供含有本發明提供之結合分子(例如,抗體或可活化抗體)之組合物。於一態樣中,該組合物為包含結合分子(例如,抗體或可活化抗體)及醫藥上可接受之載劑之醫藥組合物。該等組合物可藉由此項技術中已知之習知方法製備。In other aspects, the invention provides compositions containing binding molecules (eg, antibodies or activatable antibodies) provided by the invention. In one aspect, the composition is a pharmaceutical composition comprising a binding molecule (eg, an antibody or activatable antibody) and a pharmaceutically acceptable carrier. These compositions can be prepared by conventional methods known in the art.

於一些實施例中,本發明提供包含本發明提供之結合分子(例如,抗體或可活化抗體)及醫藥上可接受之載劑之組合物,其中該結合分子包含包含本文中所揭示之HVR胺基酸序列之可變域,且其中與存在於該組合物中之結合分子(例如,抗體或可活化抗體)之總量相比,該組合物包含不超過約11%、10%、8%、5%、3%、或2%之在該胺基酸序列之天冬醯胺處糖基化的該結合分子(例如,抗體或可活化抗體)。於另一實施例中,與存在於該組合物中之結合分子(例如,抗體或可活化抗體)之總量相比,該組合物包含至少約2%之該在該胺基酸序列之天冬醯胺處糖基化的結合分子(例如,抗體或可活化抗體)。In some embodiments, the invention provides compositions comprising a binding molecule (e.g., an antibody or activatable antibody) provided by the invention and a pharmaceutically acceptable carrier, wherein the binding molecule comprises an HVR amine disclosed herein variable domains of amino acid sequences, and wherein the composition comprises no more than about 11%, 10%, 8% compared to the total amount of binding molecules (e.g., antibodies or activatable antibodies) present in the composition , 5%, 3%, or 2% of the binding molecule (e.g., antibody or activatable antibody) glycosylated at asparagine of the amino acid sequence. In another embodiment, the composition comprises at least about 2% of the amino acid sequence present in the composition compared to the total amount of binding molecules (e.g., antibodies or activatable antibodies) present in the composition. A binding molecule (eg, an antibody or activatable antibody) that is glycosylated at asparagine.

術語「醫藥上可接受之載劑」係指適用於調配物中用於遞送結合分子之任何非活性物質。載劑可為抗黏劑、黏合劑、塗層、崩解劑、填料或稀釋劑、防腐劑(諸如抗氧化劑、抗細菌劑或抗真菌劑)、甜味劑、延遲吸收劑、潤濕劑、乳化劑、緩衝劑及類似者。適宜醫藥上可接受之載劑之實例包括水、乙醇、多元醇(諸如甘油、丙二醇、聚乙二醇及類似者)、右旋糖、植物油(諸如橄欖油)、鹽水、緩衝劑、緩衝鹽水、及等滲劑(諸如糖、聚醇、山梨醇及氯化鈉)。The term "pharmaceutically acceptable carrier" refers to any inactive material suitable for use in a formulation for delivery of a binding molecule. The carrier may be an anti-adhesive agent, a binder, a coating, a disintegrant, a filler or diluent, a preservative (such as an antioxidant, antibacterial or antifungal agent), a sweetener, an absorption delaying agent, a wetting agent , emulsifiers, buffers and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), dextrose, vegetable oils (such as olive oil), saline, buffers, buffered saline , and isotonic agents (such as sugar, polyols, sorbitol and sodium chloride).

組合物可呈任何適宜形式,諸如液體、半固體及固體劑型。液體劑型之實例包括溶液(例如,可注射及可輸注溶液)、微乳液、脂質體、分散液或懸浮液。固體劑型之實例包括錠劑、丸劑、膠囊、微膠囊及粉末。適用於遞送結合分子(例如,抗體或可活化抗體)之組合物之特定形式為用於注射或輸注之無菌液體(諸如溶液、懸浮液或分散液)。無菌溶液可藉由將所需量之抗體併入適宜載劑中,接著滅菌微過濾來製備。一般而言,分散液藉由將結合分子(例如,抗體或可活化抗體)併入含有基本分散介質及其他載劑之無菌媒劑中來製備。於用於製備無菌液體之無菌粉末之情況下,製備方法包括真空乾燥及冷凍乾燥(凍乾)以產生活性成分加上來自其先前經無菌過濾之溶液之任何另外所需成分之粉末。組合物之各種劑型可藉由此項技術中已知之習知技術製備。The compositions may be in any suitable form, such as liquid, semi-solid and solid dosage forms. Examples of liquid dosage forms include solutions (eg, injectable and infusible solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. Particular forms of compositions suitable for delivery of binding molecules (eg, antibodies or activatable antibodies) are sterile liquids (such as solutions, suspensions or dispersions) for injection or infusion. Sterile solutions can be prepared by incorporating the required amount of antibody into a suitable vehicle, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating a binding molecule (eg, an antibody or activatable antibody) into a sterile vehicle that contains a basic dispersion medium and other carriers. In the case of sterile powders for the preparation of sterile liquids, methods of preparation include vacuum drying and freeze-drying (lyophilization) to produce a powder of the active ingredient plus any additional required ingredients from its previously sterile-filtered solution. Various dosage forms of the compositions can be prepared by conventional techniques known in the art.

包含於組合物中之結合分子(例如,抗體或可活化抗體)之相對量將取決於許多因素(諸如使用之特異性結合分子及載劑、劑型、及所需釋放及藥效學特性)變化。單一劑型中之結合分子(例如,抗體或可活化抗體)之量一般將為產生治療效應之量,但是亦可為更少量。一般而言,此量將為相對於劑型之總重量約0.01%至約99%、約0.1%至約70%、或約1%至約30%之範圍。The relative amounts of binding molecules (e.g., antibodies or activatable antibodies) included in the composition will vary depending on many factors, such as the specific binding molecules and carriers used, the dosage form, and the desired release and pharmacodynamic properties. . The amount of binding molecule (eg, antibody or activatable antibody) in a single dosage form will generally be that amount that produces a therapeutic effect, but may be smaller. Generally, this amount will range from about 0.01% to about 99%, from about 0.1% to about 70%, or from about 1% to about 30%, relative to the total weight of the dosage form.

除了結合分子(例如,抗體或可活化抗體)外,一或多種另外治療劑可包含於組合物中。另外治療劑之實例述於下文中。待包含於組合物中之另外治療劑之適宜量可由熟習此項技術者容易選擇,且將取決於許多因素(諸如使用之特定劑及載劑、劑型、及所需釋放及藥效動力學特性)變化。包含於單一劑型中之另外治療劑之量一般將為產生治療效應之劑之量,但是亦可為更少量。In addition to the binding molecule (eg, antibody or activatable antibody), one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described below. Appropriate amounts of additional therapeutic agents to be included in the composition can be readily selected by one skilled in the art and will depend on many factors, such as the specific agents and carriers used, the dosage form, and the desired release and pharmacodynamic properties. ) changes. The amount of additional therapeutic agent included in a single dosage form will generally be that amount of agent that produces a therapeutic effect, but smaller amounts can also be used.

本文中所述之結合分子(例如,抗體或可活化抗體)及/或組合物(例如,醫藥組合物)中之任一者可用於製備藥劑(例如,用於治療有需要受試者之癌症或延遲該癌症進展之藥劑)。 VII. 結合分子及醫藥組合物之用途 Any of the binding molecules (e.g., antibodies or activatable antibodies) and/or compositions (e.g., pharmaceutical compositions) described herein can be used to prepare a medicament (e.g., for treating cancer in a subject in need thereof) or agents that delay the progression of the cancer). VII. Use of binding molecules and pharmaceutical compositions

本發明提供之結合分子(例如,抗體或可活化抗體)及醫藥組合物可用於治療、診斷或其他目的,諸如調節免疫反應、治療癌症、增強其他癌症療法之功效、增強疫苗功效、或治療自體免疫性疾病。因此,於其他態樣中,本發明提供使用結合分子(例如,抗體或可活化抗體)或醫藥組合物之方法。於一態樣中,本發明提供一種治療哺乳動物之病症之方法,其包括對需要治療之哺乳動物投與有效量之本發明提供之結合分子(例如,抗體或可活化抗體)或組合物。結合分子(例如,抗體或可活化抗體)可為CTLA4抗體(例如,人類抗人類CTLA4抗體)或CTLA4可活化抗體。於一些實施例中,該哺乳動物為人類。The binding molecules (eg, antibodies or activatable antibodies) and pharmaceutical compositions provided by the invention can be used for therapeutic, diagnostic or other purposes, such as modulating immune responses, treating cancer, enhancing the efficacy of other cancer therapies, enhancing vaccine efficacy, or treating autoimmune diseases. immune diseases. Accordingly, among other aspects, the invention provides methods of using binding molecules (eg, antibodies or activatable antibodies) or pharmaceutical compositions. In one aspect, the invention provides a method of treating a condition in a mammal, which includes administering to a mammal in need of treatment an effective amount of a binding molecule (eg, an antibody or activatable antibody) or a composition provided by the invention. The binding molecule (eg, antibody or activatable antibody) can be a CTLA4 antibody (eg, human anti-human CTLA4 antibody) or CTLA4 activatable antibody. In some embodiments, the mammal is a human.

於一些實施例中,該病症為癌症。可利用本發明提供之方法、用途、組合物或藥劑治療或預防各種癌症。此等癌症之實例包括肺癌,諸如支氣管癌(例如,鱗狀細胞癌、小細胞癌、大細胞癌及腺癌)、肺泡細胞癌、支氣管腺瘤、軟骨錯構瘤(非癌)、及肉瘤(癌);心臟癌症,諸如黏液瘤、纖維瘤及橫紋肌瘤;骨癌,諸如骨軟骨瘤、軟骨瘤(chondromas)、軟骨母細胞瘤、軟骨黏液樣纖維瘤、骨樣骨瘤、巨細胞腫瘤、軟骨肉瘤、多發性骨髓瘤、骨肉瘤、纖維肉瘤、惡性纖維組織細胞瘤、尤因氏瘤(Ewing's tumor) (尤因氏肉瘤)、及網狀細胞肉瘤;腦癌,諸如神經膠質瘤(例如,多形性膠質母細胞瘤)、間變性星形細胞瘤、星形細胞瘤、少突膠質細胞瘤、髓母細胞瘤、脊索瘤、神經鞘膜瘤(Schwannomas)、室管膜瘤、腦膜瘤、垂體腺瘤、松果體瘤、骨瘤、血管母細胞瘤、顱咽管瘤、軟骨瘤、生殖細胞瘤、畸胎瘤、皮樣囊腫及血管瘤;消化系統癌症,諸如平滑肌瘤、表皮樣癌、腺癌、平滑肌肉瘤、胃腺癌、腸脂肪瘤、腸神經纖維瘤、腸纖維瘤、大腸息肉及結腸直腸癌;肝癌,諸如肝細胞腺瘤、血管瘤、肝細胞癌、纖維板層癌、膽管癌、肝母細胞瘤及血管肉瘤;腎臟癌症,諸如腎腺癌、腎細胞癌、腎上腺樣瘤及腎盂移行細胞癌;膀胱癌;血液癌,諸如急性淋巴細胞性(淋巴母細胞性)白血病、急性骨髓性(髓細胞性、骨髓性、髓母細胞性、髓單細胞性)白血病、慢性淋巴細胞性白血病(例如,塞紮裡氏(Sezary)症候群及毛細胞白血病)、慢性髓細胞性(類骨髓性、骨髓性、粒細胞性)白血病、霍奇金氏(Hodgkin's)淋巴瘤、非霍奇金氏淋巴瘤、B細胞淋巴瘤、蕈樣肉芽腫及骨髓增生性病症(包括諸如真性紅細胞增多症、骨髓纖維化、血小板增多症及慢性髓細胞性白血病之骨髓增生性病症);皮膚癌,諸如基底細胞癌、鱗狀細胞癌、黑色素瘤、卡波西氏(Kaposi's)肉瘤及佩吉特氏病(Paget's disease);頭頸癌;眼相關癌症,諸如眼癌及眼內黑色素癌;男性生殖系統癌症,諸如良性前列腺增生、前列腺癌及睾丸癌(例如,精原細胞瘤、畸胎瘤、胚胎癌及絨膜癌);乳癌;女性生殖系統癌症,諸如子宮癌(子宮內膜癌)、子宮頸癌、卵巢癌、外陰癌、陰道癌、輸卵管癌及葡萄胎;甲狀腺癌(包括乳頭狀、濾泡性、間變性或髓樣癌);嗜鉻細胞瘤(腎上腺);副甲狀腺之非癌生長;胰癌;及血液癌,諸如白血病、骨髓瘤、非霍奇金氏淋巴瘤及霍奇金氏淋巴瘤。In some embodiments, the condition is cancer. Various cancers can be treated or prevented using the methods, uses, compositions or agents provided by the present invention. Examples of such cancers include lung cancer, such as bronchial carcinoma (eg, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, cartilaginous hamartoma (non-cancerous), and sarcoma (cancer); heart cancers, such as myxoma, fibroma, and rhabdomyomas; bone cancers, such as osteochondromas, chondromas, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, giant cell tumors , chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; brain cancers such as glioma ( For example, glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannomas, ependymoma, Meningiomas, pituitary adenomas, pineal tumors, osteomas, hemangioblastomas, craniopharyngiomas, enchondromas, germinomas, teratomas, dermoid cysts, and hemangiomas; digestive system cancers, such as smooth muscle tumors, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colorectal polyps and colorectal cancer; liver cancer, such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, Fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancers, such as renal adenocarcinoma, renal cell carcinoma, adrenaloid tumors, and transitional cell carcinoma of the renal pelvis; bladder cancer; blood cancers, such as acute lymphocytic (lymphoblastic) cellular) leukemia, acute myeloid (myeloid, myeloid, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), Chronic myeloid (myeloid, myeloid, granulocytic) leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, mycosis fungoides and myeloproliferative disorders (Including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, and chronic myelogenous leukemia); Skin cancers, such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's ) Sarcomas and Paget's disease; head and neck cancers; eye-related cancers, such as ocular cancer and intraocular melanoma; male reproductive system cancers, such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., spermatogonia tumors, teratomas, embryonal carcinomas and choriocarcinomas); breast cancer; cancers of the female reproductive system, such as uterine cancer (endometrial cancer), cervical cancer, ovarian cancer, vulvar cancer, vaginal cancer, fallopian tube cancer and mole; Thyroid cancer (including papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal gland); noncancerous growths of the parathyroid gland; pancreatic cancer; and blood cancers, such as leukemia, myeloma, non-Hodgkin's disease King's lymphoma and Hodgkin's lymphoma.

於另一態樣中,本發明提供一種增強哺乳動物之免疫反應之方法,其包括對該哺乳動物投與有效量之本發明所提供之結合分子(例如抗體或可活化抗體)或組合物。於一些實施例中,該結合分子為CTLA4抗體或其抗原結合片段且該哺乳動物為人類。於一些實施例中,該結合分子為CTLA4可活化抗體且該哺乳動物為人類。術語「增強免疫反應」或其文法變型意指刺激、引起、增加、提高或增強哺乳動物之免疫系統之任何反應。該免疫反應可為細胞反應(即細胞介導,諸如細胞毒性T淋巴細胞介導)或體液反應(即抗體介導),且可為一級或二級免疫反應。增強免疫反應之實例包括活化PBMCs及/或T細胞(包括增加一或多種細胞激素(諸如IL-2及/或IFNγ)之分泌)。免疫反應之增強可使用熟習此項技術者已知之許多活體外或活體內量測評估,該等量測包括(但不限於)細胞毒性T淋巴細胞檢定、細胞激素釋放、腫瘤消退、具有腫瘤動物之存活、抗體產生、免疫細胞增殖、細胞表面標記物之表現,及細胞毒性。通常,當相較於未經處理之哺乳動物或未使用所述方法處理之哺乳動物之免疫反應時,本發明之方法增強哺乳動物之免疫反應。In another aspect, the present invention provides a method for enhancing the immune response of a mammal, which includes administering to the mammal an effective amount of a binding molecule (eg, an antibody or an activatable antibody) or a composition provided by the present invention. In some embodiments, the binding molecule is a CTLA4 antibody or antigen-binding fragment thereof and the mammal is a human. In some embodiments, the binding molecule is a CTLA4-activatable antibody and the mammal is a human. The term "enhancing immune response" or its grammatical variations means any response that stimulates, causes, increases, enhances or enhances the immune system of a mammal. The immune response can be a cellular response (ie, cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (ie, antibody-mediated), and can be a primary or secondary immune response. Examples of enhanced immune responses include activation of PBMCs and/or T cells (including increased secretion of one or more cytokines, such as IL-2 and/or IFNγ). Enhancement of the immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, cytokine release, tumor regression, tumor-bearing animals survival, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Generally, the methods of the present invention enhance the immune response of a mammal when compared to the immune response of an untreated mammal or a mammal that has not been treated with the method.

於實踐治療方法中,結合分子(例如抗體或可活化抗體)可單獨投與作為單藥療法或與一或多種另外治療劑或療法組合投與。因此,於另一態樣中,本發明提供一種組合療法,其包含結合分子(例如抗體或可活化抗體)與一或多種另外療法或治療劑組合用於分開、依序或同時投與。術語「另外治療劑」可係指除了本發明提供之結合分子(例如抗體或可活化抗體)外之任何治療劑。於一特定態樣中,本發明提供一種用於治療哺乳動物之癌症之組合療法,其包括對該哺乳動物投與有效量之本文中所提供之結合分子(例如抗體或可活化抗體)與一或多種另外治療劑組合。於另一實施例中,該哺乳動物為人類。In practicing treatment methods, binding molecules (eg, antibodies or activatable antibodies) may be administered alone as monotherapy or in combination with one or more additional therapeutic agents or therapies. Accordingly, in another aspect, the invention provides a combination therapy comprising a binding molecule (eg, an antibody or activatable antibody) in combination with one or more additional therapies or therapeutic agents for separate, sequential or simultaneous administration. The term "additional therapeutic agent" may refer to any therapeutic agent other than the binding molecules (eg, antibodies or activatable antibodies) provided herein. In a specific aspect, the invention provides a combination therapy for treating cancer in a mammal, comprising administering to the mammal an effective amount of a binding molecule (eg, an antibody or activatable antibody) provided herein and an or combinations of additional therapeutic agents. In another embodiment, the mammal is a human.

廣泛多種癌症治療劑可與本發明提供之結合分子(例如抗體或可活化抗體)組合使用。一般技術者將認知可與本發明之方法及結合分子(例如抗體或可活化抗體)組合使用之其他癌症療法之存在及開發且該等療法將不限於本文中所述療法之形式。可用於治療癌症之組合療法中之另外治療劑類別之實例包括(1)化療劑,(2)免疫治療劑,及(3)激素治療劑。於一些實施例中,該另外治療為病毒基因療法、免疫檢查點抑制劑、靶療法、放射療法、疫苗接種療法及/或化療。A wide variety of cancer therapeutics can be used in combination with the binding molecules (eg, antibodies or activatable antibodies) provided herein. One of ordinary skill will recognize the existence and development of other cancer therapies that can be used in combination with the methods and binding molecules (eg, antibodies or activatable antibodies) of the present invention and such therapies will not be limited to the forms of therapies described herein. Examples of additional classes of therapeutic agents that may be used in combination therapies to treat cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonal therapeutic agents. In some embodiments, the additional treatment is viral gene therapy, immune checkpoint inhibitors, target therapy, radiation therapy, vaccination therapy, and/or chemotherapy.

術語「化療劑」係指可造成癌細胞死亡或干涉癌細胞之生長、分裂、修復及/或功能之化學或生物物質。化療劑之實例包括揭示於WO 2006/129163及US 20060153808中之彼等,其揭示內容以引用的方式併入本文中。特定化療劑之實例包括:(1)烷基化劑,諸如苯丁酸氮芥(LEUKERAN)、環磷醯胺(CYTOXAN)、異環磷醯胺(IFEX)、鹽酸氮芥(mechlorethamine hydrochloride) (MUSTARGEN)、噻替呱(THIOPLEX)、鏈脲黴素(ZANOSAR)、卡莫司汀(BICNU, GLIADEL WAFER)、洛莫司汀(CEENU)及達卡巴嗪(DTIC-DOME);(2)生物鹼或植物長春化生物鹼,包括細胞毒性抗生素,諸如多柔比星(ADRIAMYCIN)、表柔比星(epirubicin) (ELLENCE, PHARMORUBICIN)、柔紅黴素(CERUBIDINE, DAUNOXOME)、奈莫柔比星(nemorubicin)、伊達比星(idarubicin) (IDAMYCIN PFS, ZAVEDOS)、米托蒽醌(DHAD, NOVANTRONE)、更生黴素(放線菌素D,COSMEGEN)、光神黴素(MITHRACIN)、絲裂黴素(MUTAMYCIN)及博來黴素(BLENOXANE)、酒石酸長春瑞濱(vinorelbine) (NAVELBINE)、長春鹼(VELBAN)、長春新鹼(ONCOVIN)及長春地辛(vindesine) (ELDISINE);(3)抗代謝劑,諸如卡培他濱(capecitabine) (XELODA)、阿糖胞苷(CYTOSAR-U)、氟達拉濱(fludarabine) (FLUDARA)、吉西他濱(gemcitabine) (GEMZAR)、羥基脲(HYDRA)、胺甲喋呤(FOLEX, MEXATE, TREXALL)、奈拉濱(nelarabine) (ARRANON)、曲麥克特(trimetrexate) (NEUTREXIN)及培美曲塞(pemetrexed) (ALIMTA);(4)嘧啶拮抗劑,諸如5-氟尿嘧啶(5-FU);卡培他濱(XELODA)、雷替曲塞(raltitrexed) (TOMUDEX)、替加氟(tegafur)-尿嘧啶(UFTORAL)及吉西他濱(GEMZAR);(5)紫杉烷,諸如多西他奇(docetaxel) (TAXOTERE)、紫杉醇(paclitaxel) (TAXOL);(6)鉑藥物,諸如順鉑(PLATINOL)及卡鉑(carboplatin) (PARAPLATIN)及奧沙利鉑(oxaliplatin) (ELOXATIN);(7)拓撲異構酶抑制劑,諸如依立替康(irinotecan) (CAMPTOSAR)、托泊替康(topotecan) (HYCAMTIN)、依託泊苷(ETOPOPHOS, VEPESSID, TOPOSAR)及替尼泊苷(VUMON);(8)表鬼臼毒素(epipodophyllotoxin) (鬼臼毒素衍生物),諸如依託泊苷(ETOPOPHOS, VEPESSID, TOPOSAR);(9)葉酸衍生物,諸如甲醯四氫葉酸(leucovorin) (WELLCOVORIN);(10)亞硝基脲,諸如卡莫司汀(BiCNU)、洛莫司汀(CeeNU);(11)受體酪胺酸激酶抑制劑,包括表皮生長因子受體(EGFR)、血管內皮生長因子(VEGF)、胰島素受體、類胰島素生長因子受體(IGFR)、肝細胞生長因子受體(HGFR)及血小板源性生長因子受體(PDGFR),諸如吉非替尼(gefitinib) (IRESSA)、埃羅替尼(erlotinib) (TARCEVA)、硼替佐米(bortezomib) (VELCADE)、甲磺酸伊馬替尼(imatinib mesylate) (GLEEVEC)、基非替尼(genefitinib)、拉帕替尼(lapatinib)、索拉非尼(sorafenib)、沙利度胺、舒尼替尼(sunitinib) (SUTENT)、阿西替尼(axitinib)、利妥昔單抗(RITUXAN, MABTHERA)、曲妥單抗(HERCEPTIN)、西妥昔單抗(ERBITUX)、貝伐單抗(bevacizumab) (AVASTIN)及蘭尼單抗(ranibizumab) (LUCENTIS)、lym-1 (ONCOLYM)、揭示於WO2002/053596中之類胰島素生長因子-1受體(IGF-1R)之抗體;(12)血管生長抑制劑,諸如貝伐單抗(AVASTIN)、蘇拉明(suramin) (GERMANIN)、血管抑素(angiostatin)、SU5416、沙利度胺及基質金屬蛋白酶抑制劑(諸如巴馬司他(batimastat)及馬馬司他(marimastat)),及揭示於WO2002055106中之彼等;及(13)蛋白酶抑制劑,諸如硼替佐米(VELCADE)。The term "chemotherapeutic agent" refers to a chemical or biological substance that causes the death of cancer cells or interferes with the growth, division, repair and/or function of cancer cells. Examples of chemotherapeutic agents include those disclosed in WO 2006/129163 and US 20060153808, the disclosures of which are incorporated herein by reference. Examples of specific chemotherapeutic agents include: (1) Alkylating agents such as chlorambucil (LEUKERAN), cyclophosphamide (CYTOXAN), ifosfamide (IFEX), mechlorethamine hydrochloride ( MUSTARGEN), THIOPLEX, streptozotocin (ZANOSAR), carmustine (BICNU, GLIADEL WAFER), lomustine (CEENU) and dacarbazine (DTIC-DOME); (2) Biological Alkaloids or plant vinculizing alkaloids, including cytotoxic antibiotics such as doxorubicin (ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin (CERUBIDINE, DAUNOXOME), nemorubicin (nemorubicin), idarubicin (IDAMYCIN PFS, ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE), dactinomycin (actinomycin D, COSMEGEN), mithramycin (MITHRACIN), mitomycete MUTAMYCIN and BLENOXANE, vinorelbine tartrate (NAVELBINE), vinblastine (VELBAN), vincristine (ONCOVIN) and vindesine (ELDISINE); (3) Antimetabolites such as capecitabine (XELODA), cytarabine (CYTOSAR-U), fludarabine (FLUDARA), gemcitabine (GEMZAR), hydroxyurea (HYDRA) , methotrexate (FOLEX, MEXATE, TREXALL), nelarabine (ARRANON), trimetrexate (NEUTREXIN) and pemetrexed (ALIMTA); (4) Pyrimidine antagonists , such as 5-fluorouracil (5-FU); capecitabine (XELODA), raltitrexed (TOMUDEX), tegafur-uracil (UFTORAL) and gemcitabine (GEMZAR); (5 ) Taxanes, such as docetaxel (TAXOTERE), paclitaxel (TAXOL); (6) Platinum drugs, such as cisplatin (PLATINOL) and carboplatin (PARAPLATIN) and oxalidine Platinum (oxaliplatin) (ELOXATIN); (7) Topoisomerase inhibitors, such as irinotecan (CAMPTOSAR), topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID, TOPOSAR) and teniposide (VUMON); (8) epipodophyllotoxin (podophyllotoxin derivatives), such as etoposide (ETOPOPHOS, VEPESSID, TOPOSAR); (9) folic acid derivatives, such as methylphenidate Hydrofolate (leucovorin) (WELLCOVORIN); (10) Nitrosoureas, such as carmustine (BiCNU), lomustine (CeeNU); (11) Receptor tyrosine kinase inhibitors, including epidermal growth factor Receptor (EGFR), vascular endothelial growth factor (VEGF), insulin receptor, insulin-like growth factor receptor (IGFR), hepatocyte growth factor receptor (HGFR) and platelet-derived growth factor receptor (PDGFR), such as Gefitinib (IRESSA), erlotinib (TARCEVA), bortezomib (VELCADE), imatinib mesylate (GLEEVEC), gefitinib (genefitinib), lapatinib (lapatinib), sorafenib (sorafenib), thalidomide, sunitinib (sunitinib) (SUTENT), axitinib (axitinib), rituximab ( RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab (ERBITUX), bevacizumab (AVASTIN) and ranibizumab (LUCENTIS), lym-1 (ONCOLYM) , antibodies similar to insulin growth factor-1 receptor (IGF-1R) disclosed in WO2002/053596; (12) vascular growth inhibitors, such as bevacizumab (AVASTIN), suramin (GERMANIN) , angiostatin, SU5416, thalidomide and matrix metalloproteinase inhibitors (such as batimastat and marimastat), and those disclosed in WO2002055106; and ( 13) Protease inhibitors such as bortezomib (VELCADE).

術語「免疫治療劑」係指可增強哺乳動物之免疫反應之化學或生物物質。免疫治療劑之實例包括:卡介苗( bacillusCalmette-Guerin) (BCG);細胞激素,諸如干擾素;疫苗,諸如MyVax個人化免疫療法、Onyvax-P、Oncophage、GRNVAC1、Favld、Provenge、GVAX、Lovaxin C、BiovaxID、GMXX及NeuVax;及抗體,諸如阿侖單抗(alemtuzumab) (CAMPATH)、貝伐單抗(AVASTIN)、西妥昔單抗(ERBITUX)、吉妥珠單抗奧佐米星(gemtuzumab ozogamicin) (MYLOTARG)、替伊莫單抗(ibritumomab tiuxetan) (ZEVALIN)、帕尼單抗(panitumumab) (VECTIBIX)、利妥昔單抗(RITUXAN, MABTHERA)、曲妥珠單抗(HERCEPTIN)、托西莫單抗(tositumomab) (BEXXAR)、伊匹單抗(YERVOY)、曲美目單抗(tremelimumab)、CAT-3888、OX40受體之促效劑抗體(諸如揭示於WO2009/079335中者)、CD40受體之促效劑抗體(諸如揭示於WO2003/040170中者)、及TLR-9促效劑(諸如揭示於WO2003/015711、WO2004/016805及WO2009/022215中者)。 The term "immunotherapeutic agent" refers to a chemical or biological substance that enhances the immune response of a mammal. Examples of immunotherapeutic agents include: bacillus Calmette-Guerin (BCG); cytokines such as interferon; vaccines such as MyVax Personalized Immunotherapy, Onyvax-P, Oncophage, GRNVAC1, Favld, Provenge, GVAX, Lovaxin C , BiovaxID, GMXX and NeuVax; and antibodies such as alemtuzumab (CAMPATH), bevacizumab (AVASTIN), cetuximab (ERBITUX), gemtuzumab ozogamicin (gemtuzumab) ozogamicin) (MYLOTARG), ibritumomab tiuxetan (ZEVALIN), panitumumab (VECTIBIX), rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), Tositumomab (BEXXAR), ipilimumab (YERVOY), tremelimumab, CAT-3888, agonist antibodies of the OX40 receptor (such as those disclosed in WO2009/079335 ), agonist antibodies to the CD40 receptor (such as those disclosed in WO2003/040170), and TLR-9 agonists (such as those disclosed in WO2003/015711, WO2004/016805 and WO2009/022215).

術語「激素治療劑」係指抑制或消除激素產生或抑制或抵消激素對癌細胞生長及/或存活之影響之化學或生物物質。適用於本文中方法之此等劑之實例包括揭示於US20070117809中之彼等。特定激素治療劑之實例包括他莫西芬(tamoxifen) (NOLVADEX)、托瑞米芬(toremifene) (Fareston)、氟維司群(fulvestrant) (FASLODEX)、阿納托唑(anastrozole) (ARIMIDEX)、依西美坦(exemestane) (AROMASIN)、來曲唑(letrozole) (FEMARA)、乙酸甲地孕酮(megestrol acetate) (MEGACE)、戈舍瑞林(goserelin) (ZOLADEX)及亮脯利特(leuprolide) (LUPRON)。本發明之結合分子亦可與諸如以下之非藥物激素療法組合使用:(1)移除所有或部分器官或腺之手術方法,該等器官或腺參與激素產生,諸如卵巢、睾丸、腎上腺及垂體,及(2)輻射治療,其中患者之器官或腺經歷足以抑制或消除靶向激素之產生之量之輻射。The term "hormone therapeutic agent" refers to a chemical or biological substance that inhibits or eliminates the production of hormones or inhibits or counteracts the effects of hormones on the growth and/or survival of cancer cells. Examples of such agents suitable for use in the methods herein include those disclosed in US20070117809. Examples of specific hormonal therapeutic agents include tamoxifen (NOLVADEX), toremifene (Fareston), fulvestrant (FASLODEX), anastrozole (ARIMIDEX), exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate (MEGACE), goserelin (ZOLADEX), and leuprolide ( leuprolide) (LUPRON). The binding molecules of the present invention may also be used in combination with non-pharmacological hormone therapies such as: (1) surgical procedures that remove all or part of organs or glands involved in hormone production, such as the ovaries, testicles, adrenal glands, and pituitary glands , and (2) radiation therapy, in which the patient's organs or glands undergo radiation in an amount sufficient to inhibit or eliminate the production of the targeted hormone.

於一些實施例中,該另外治療劑為下列中之一或多者:鉑美特、瑞復美、來那度胺、泊馬度胺、沙利度胺、DNA烷基化含鉑衍生物、順鉑、5-氟尿嘧啶、環磷醯胺、抗CD137抗體、抗PD-1抗體、抗PD-L1抗體、抗CD20抗體、抗CD40抗體、抗DR5抗體、抗CD1d抗體、抗TIM3抗體、抗SLAMF7抗體、抗KIR受體抗體、抗OX40抗體、抗HER2抗體、抗ErbB-2抗體、抗EGFR抗體、西妥昔單抗、利妥昔單抗、曲妥珠單抗、派姆單抗、放射療法、單劑量輻射、分次輻射、焦點輻射、全器官輻射、IL-12、IFNα、GM-CSF、嵌合抗原受體、授受性轉移之T細胞、抗癌疫苗、及溶瘤病毒。In some embodiments, the additional therapeutic agent is one or more of the following: platinum, revolumab, lenalidomide, pomalidomide, thalidomide, DNA alkylated platinum-containing derivatives , cisplatin, 5-fluorouracil, cyclophosphamide, anti-CD137 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti- SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, Radiation therapy, single-dose radiation, fractionated radiation, focal radiation, whole-organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptors, receptive transferred T cells, anti-cancer vaccines, and oncolytic viruses.

用於治療癌症之組合療法亦包括結合分子(例如,抗體或可活化抗體)與移除腫瘤之手術之組合。可在手術之前、期間或之後對哺乳動物投與結合分子(例如,抗體或可活化抗體)。Combination therapies for treating cancer also include combinations of binding molecules (eg, antibodies or activatable antibodies) and surgery to remove tumors. The binding molecule (eg, antibody or activatable antibody) can be administered to the mammal before, during, or after surgery.

用於治療癌症之組合療法亦包括結合分子(例如,抗體或可活化抗體)與輻射療法(諸如電離(電磁)放射療法(例如,X-射線或γ射線)及粒子束輻射療法(例如,高線性能量輻射))之組合。輻射源可係哺乳動物之外部或內部。可在輻射療法之前、期間或之後對哺乳動物投與結合分子(例如,抗體或可活化抗體)。Combination therapies for the treatment of cancer also include binding molecules (e.g., antibodies or activatable antibodies) with radiation therapy, such as ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) and particle beam radiation therapy (e.g., high Combination of linear energy radiation)). The source of radiation can be external or internal to the mammal. The binding molecule (eg, antibody or activatable antibody) can be administered to the mammal before, during, or after radiation therapy.

本發明提供之結合分子(例如,抗體或可活化抗體)及組合物可經由任何適宜投與之經腸途徑或非經腸途徑投與。術語投與之「經腸途徑」係指經由胃腸道之任何部分投與。經腸途徑之實例包括經口、經黏膜、經頰、及經直腸途徑、或經胃內途徑。投與之「非經腸途徑」係指除了經腸途徑外之投與途徑。投與之非經腸途徑之實例包括靜脈內、肌肉內、皮內、腹膜內、腫瘤內、膀胱內、動脈內、鞘內、囊內、眶內、心內、經氣管、關節內、囊下、蛛網膜下、脊椎內、硬膜外及胸骨內、皮下或局部投與。本發明之結合分子(例如,抗體或可活化抗體)及組合物可使用任何適宜方法投與,諸如藉由口服、鼻飼管、胃造口管、注射、輸注、可移植輸注泵及滲透泵。投與之適宜途徑及方法可取決於諸如以下之許多因素變化:正在使用之特異性結合分子(例如,抗體或可活化抗體)、所需吸收速率、使用之特定調配物或劑型、正在治療之病症之類型或嚴重度、特異性作用位點及患者病狀,且可藉由熟習此項技術者容易選擇。The binding molecules (eg, antibodies or activatable antibodies) and compositions provided herein may be administered via any suitable route of administration, enteral or parenteral. The term administration "enteral route" refers to administration through any part of the gastrointestinal tract. Examples of enteral routes include oral, transmucosal, transbuccal, and transrectal routes, or intragastric routes. Administration "Parental route" refers to a route of administration other than the enteral route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrathecal, intrasaccular, intraorbital, intracardiac, transtracheal, intraarticular, intrasaccular Subcutaneous, subarachnoid, intraspinal, epidural and intrasternal, subcutaneous or topical administration. The binding molecules (eg, antibodies or activatable antibodies) and compositions of the invention may be administered using any suitable method, such as by oral administration, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. Appropriate routes and methods of administration may vary depending on many factors such as: the specific binding molecule (e.g., antibody or activatable antibody) being used, the desired rate of absorption, the particular formulation or dosage form being used, the condition being treated The type or severity of the disease, the specific site of action, and the patient's condition can be easily selected by those skilled in the art.

術語結合分子(例如,抗體或可活化抗體)之「有效量」可係指有效用於預期治療目的之量。例如,於增強免疫反應之背景下,「有效量」可為有效刺激、引起、增加、提高或增強哺乳動物之免疫系統之任何反應的任何量。於治療疾病之背景下,「有效量」可為足以造成正在治療之哺乳動物中之任何所需或有益效果的任何量。具體而言,於治療癌症中,所需或有益效果之實例包括抑制癌細胞之進一步生長或擴散、癌細胞之死亡、抑制癌症再發生、減少與癌症相關之疼痛、或提高哺乳動物之存活。結合分子(例如,抗體或可活化抗體)之治療上有效量範圍通常為約0.001至約500 mg/kg,及更通常約0.01至約100 mg/kg哺乳動物之體重。例如,該量可為約0.3 mg/kg、1 mg/kg、3 mg/kg、5 mg/kg、10 mg/kg、50 mg/kg、或100 mg/kg哺乳動物之體重。於一些實施例中,結合分子(例如,抗體或可活化抗體)之治療上有效量係於約0.01至30 mg/kg哺乳動物之體重之範圍內。於一些其他實施例中,結合分子(例如,抗體或可活化抗體)之治療上有效量係於約0.05至15 mg/kg哺乳動物之體重之範圍內。待投與之精確劑量水平可由熟習此項技術者容易確定且將取決於許多因素,諸如待治療之病症之類型及嚴重度、採用之特定結合分子(例如,抗體或可活化抗體)、投與途徑、投與時間、治療持續時間、採用之特定另外療法、正在治療之患者之年齡、性別、體重、病狀、一般健康及先前醫療史、及醫療技術中熟知之類似因素。The term "effective amount" of a binding molecule (eg, antibody or activatable antibody) may refer to an amount effective for the intended therapeutic purpose. For example, in the context of enhancing an immune response, an "effective amount" may be any amount effective to stimulate, elicit, increase, enhance or enhance any response of the mammalian immune system. In the context of treating a disease, an "effective amount" may be any amount sufficient to cause any desired or beneficial effect in the mammal being treated. Specifically, in treating cancer, examples of desirable or beneficial effects include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of recurrence of cancer, reduction of pain associated with cancer, or improvement of survival of a mammal. A therapeutically effective amount of a binding molecule (eg, antibody or activatable antibody) generally ranges from about 0.001 to about 500 mg/kg, and more typically from about 0.01 to about 100 mg/kg of the mammalian body weight. For example, the amount may be about 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg, or 100 mg/kg of the mammal's body weight. In some embodiments, a therapeutically effective amount of a binding molecule (eg, antibody or activatable antibody) is in the range of about 0.01 to 30 mg/kg body weight of the mammal. In some other embodiments, the therapeutically effective amount of binding molecule (eg, antibody or activatable antibody) is in the range of about 0.05 to 15 mg/kg body weight of the mammal. The precise dosage level to be administered can be readily determined by one skilled in the art and will depend on many factors, such as the type and severity of the condition to be treated, the specific binding molecule employed (e.g., antibody or activatable antibody), the administration Route, time of administration, duration of treatment, specific additional therapies used, age, gender, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical art.

通常在多種場合投與結合分子(例如,抗體或可活化抗體)或組合物。單一劑量之間之間隔可為例如每日、每週、每月、每三個月或每年。示例性治療方案需要以下投與:每週一次、每兩週一次、每三週一次、每四週一次、每月一次、每三個月一次或每三至六個月一次。結合分子(例如,抗體或可活化抗體)之典型劑量方案包括1 mg/kg體重或3 mg/kg體重,經由靜脈內投與,使用下列給藥時程表中之一者:(i)每四週六個劑量,然後每三個月;(ii)每三週;(iii) 3 mg/kg體重一次,接著每三週1 mg/kg體重。 VIII. 套組 Binding molecules (eg, antibodies or activatable antibodies) or compositions are typically administered on a variety of occasions. Intervals between single doses may be, for example, daily, weekly, monthly, every three months or yearly. Exemplary treatment regimens require the following administration: once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Typical dosage regimens for binding molecules (e.g., antibodies or activatable antibodies) include 1 mg/kg body weight or 3 mg/kg body weight administered intravenously using one of the following dosing schedules: (i) every Six doses at four weeks, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight, then 1 mg/kg every three weeks. VIII. Set

於另一態樣中,本文中提供一種套組,其包含本發明之結合分子(例如,抗體或可活化抗體)及/或組合物。於一些實施例中,該套組另包含一種包裝插入物,其包含結合分子(例如,抗體或可活化抗體)及/或組合物之使用說明。於一些實施例中,該套組另包含一或多種緩衝劑,例如,用於儲存、轉移、投與或以其他方式使用結合分子(例如,抗體或可活化抗體)及/或組合物。於一些實施例中,該套組另包含用於儲存結合分子(例如,抗體或可活化抗體)及/或組合物之一或多種容器。In another aspect, provided herein is a kit comprising a binding molecule (eg, antibody or activatable antibody) and/or composition of the invention. In some embodiments, the kit further includes a packaging insert containing the binding molecule (eg, antibody or activatable antibody) and/or instructions for use of the composition. In some embodiments, the kit further includes one or more buffers, eg, for storage, transfer, administration or other use of the binding molecule (eg, antibody or activatable antibody) and/or composition. In some embodiments, the kit further includes one or more containers for storing binding molecules (eg, antibodies or activatable antibodies) and/or compositions.

認為上述書面描述足以使熟習此項技術者能實踐本發明。僅出於說明目的提供下列實例,且不意欲以任何方式限制本發明之範圍。的確,除了本文中所示及所述之彼等外之本發明之各種修改將自上述描述對熟習此項技術者變得顯而易見且落於隨附申請專利範圍之範圍內。 實例 實例 1 :特異性結合至人類 CTLA4 之一級 Fab 之產生 It is believed that the foregoing written description is sufficient to enable any person skilled in the art to practice the invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Examples Example 1 : Generation of primary Fab that specifically binds to human CTLA4

採用專有噬菌粒庫(參見PCT公開案號PCT/CN2017/098333,其全文以引用的方式併入本文中;亦參見PCT公開案號PCT/CN2017/098299,其全文以引用的方式併入本文中)篩選人類CTLA4抗原。進行總共3至5輪篩選。於最後輪篩選後,進行單群落上清液ELISA以識別特異性識別人類CTLA4 (參見例如,UniProt寄存編號P16410)之主要命中。將該等主要命中定義為其ELISA信號為背景至少兩倍之彼等。然後將該等命中定序,及將獨特純系於大腸桿菌中表現及純化。其對人類CTLA4之親和力藉由ForteBio Octet RED96 Systems量測。簡言之,使用AHC感測器(抗人類IgG Fc捕獲浸及讀取生物感測器)捕獲重組人類CTLA4-Fc (Sino Biological, 11159-H03H),及浸入含有經純化Fab之孔中,該等Fab於動態緩衝液(含0.02% Tween20、0.1% BSA之PBS緩衝液)中稀釋至10 µg/mL。將獲取之ForteBio數據用Data Acquisition軟體7.1處理,及將動力學數據擬合至1:1朗繆爾(Langmuir)結合模型。將候選之清單用ELISA陽性命中及獨特序列二者精簡至234個Fab命中。遵照K D反應信號R> 0.1,R 2> 0.9之標準,將清單進一步精簡至所關注之43個命中。此等命中之親和力及動力學參數(減去背景)示於下 1中。 1 :選定 Fab 對人類 CTLA4 之親和力 命中ID K D(M) kon(1/Ms) koff(1/s) B18153 5.96E-09 8.93E+04 5.32E-04 B15719 6.93E-09 3.21E+05 2.22E-03 B15746 7.15E-09 3.07E+05 2.20E-03 B15734 7.63E-09 3.05E+05 2.33E-03 B15710 8.01E-09 3.51E+05 2.81E-03 B13874 1.06E-08 1.19E+05 1.26E-03 B13898 1.07E-08 1.09E+05 1.16E-03 B15728 1.18E-08 4.24E+05 4.99E-03 B15705 1.20E-08 1.94E+05 2.33E-03 B15716 1.21E-08 3.91E+04 4.71E-04 B15672 1.23E-08 6.83E+04 8.39E-04 B15738 1.24E-08 3.76E+05 4.67E-03 B15694 1.28E-08 1.52E+05 1.94E-03 B15754 1.35E-08 2.76E+05 3.71E-03 B15749 1.38E-08 2.69E+05 3.72E-03 B15740 1.45E-08 1.78E+05 2.58E-03 B15489 1.46E-08 6.08E+04 8.87E-04 B13880 1.59E-08 1.04E+05 1.66E-03 B15699 1.66E-08 4.42E+05 7.35E-03 B15745 1.73E-08 2.58E+05 4.47E-03 B18174 1.82E-08 5.16E+04 9.38E-04 B15739 1.86E-08 4.24E+05 7.86E-03 B15721 2.21E-08 5.31E+05 1.17E-02 B15673 2.44E-08 6.95E+04 1.70E-03 B15737 2.75E-08 4.77E+05 1.31E-02 B15696 2.79E-08 1.44E+05 4.03E-03 B15491 2.98E-08 6.24E+04 1.86E-03 B15724 3.01E-08 5.52E+05 1.66E-02 B15741 3.08E-08 2.09E+05 6.44E-03 B15757 3.72E-08 1.05E+05 3.91E-03 B15759 5.36E-08 2.46E+05 1.32E-02 B15756 5.55E-08 1.20E+05 6.63E-03 B15722 6.12E-08 7.09E+04 4.34E-03 B15750 6.23E-08 9.37E+04 5.84E-03 B15702 6.92E-08 3.61E+05 2.50E-02 B14242 7.58E-08 3.36E+05 2.55E-02 B13878 8.59E-08 3.08E+04 2.64E-03 B15717 1.05E-07 1.77E+05 1.85E-02 B15187 1.37E-07 2.73E+04 3.75E-03 B15695 1.52E-07 1.81E+05 2.76E-02 B15189 1.70E-07 2.04E+04 3.47E-03 B15762 1.85E-07 4.60E+04 8.49E-03 B15730 2.59E-07 3.45E+04 8.93E-03 Using a proprietary phagemid library (see PCT Publication No. PCT/CN2017/098333, the entire text of which is incorporated herein by reference; see also PCT Publication No. PCT/CN2017/098299, the entire text of which is incorporated by reference Herein) Screening for human CTLA4 antigen. Conduct a total of 3 to 5 rounds of screening. After the final round of screening, a single-population supernatant ELISA was performed to identify major hits that specifically recognized human CTLA4 (see, eg, UniProt Accession No. P16410). Primary hits were defined as those whose ELISA signal was at least twice background. The hits were then sequenced, and the unique lines were expressed and purified in E. coli. Its affinity for human CTLA4 was measured by ForteBio Octet RED96 Systems. Briefly, recombinant human CTLA4-Fc (Sino Biological, 11159-H03H) was captured using an AHC sensor (Anti-Human IgG Fc Capture and Read Biosensor) and immersed into wells containing purified Fab. The Fab was diluted to 10 µg/mL in dynamic buffer (PBS buffer containing 0.02% Tween20, 0.1% BSA). The acquired ForteBio data were processed with Data Acquisition software 7.1, and the kinetic data were fitted to a 1:1 Langmuir binding model. The list of candidates was refined to 234 Fab hits using both ELISA positive hits and unique sequences. According to the criteria of K D reaction signal R>0.1 and R2 >0.9, the list was further streamlined to 43 hits of interest. The affinity and kinetic parameters (background subtracted) for these hits are shown in Table 1 below. Table 1 : Affinity of selected Fabs for human CTLA4 Hit ID : K D (M) : kon(1/Ms) : koff(1/s) : B18153 5.96E-09 8.93E+04 5.32E-04 B15719 6.93E-09 3.21E+05 2.22E-03 B15746 7.15E-09 3.07E+05 2.20E-03 B15734 7.63E-09 3.05E+05 2.33E-03 B15710 8.01E-09 3.51E+05 2.81E-03 B13874 1.06E-08 1.19E+05 1.26E-03 B13898 1.07E-08 1.09E+05 1.16E-03 B15728 1.18E-08 4.24E+05 4.99E-03 B15705 1.20E-08 1.94E+05 2.33E-03 B15716 1.21E-08 3.91E+04 4.71E-04 B15672 1.23E-08 6.83E+04 8.39E-04 B15738 1.24E-08 3.76E+05 4.67E-03 B15694 1.28E-08 1.52E+05 1.94E-03 B15754 1.35E-08 2.76E+05 3.71E-03 B15749 1.38E-08 2.69E+05 3.72E-03 B15740 1.45E-08 1.78E+05 2.58E-03 B15489 1.46E-08 6.08E+04 8.87E-04 B13880 1.59E-08 1.04E+05 1.66E-03 B15699 1.66E-08 4.42E+05 7.35E-03 B15745 1.73E-08 2.58E+05 4.47E-03 B18174 1.82E-08 5.16E+04 9.38E-04 B15739 1.86E-08 4.24E+05 7.86E-03 B15721 2.21E-08 5.31E+05 1.17E-02 B15673 2.44E-08 6.95E+04 1.70E-03 B15737 2.75E-08 4.77E+05 1.31E-02 B15696 2.79E-08 1.44E+05 4.03E-03 B15491 2.98E-08 6.24E+04 1.86E-03 B15724 3.01E-08 5.52E+05 1.66E-02 B15741 3.08E-08 2.09E+05 6.44E-03 B15757 3.72E-08 1.05E+05 3.91E-03 B15759 5.36E-08 2.46E+05 1.32E-02 B15756 5.55E-08 1.20E+05 6.63E-03 B15722 6.12E-08 7.09E+04 4.34E-03 B15750 6.23E-08 9.37E+04 5.84E-03 B15702 6.92E-08 3.61E+05 2.50E-02 B14242 7.58E-08 3.36E+05 2.55E-02 B13878 8.59E-08 3.08E+04 2.64E-03 B15717 1.05E-07 1.77E+05 1.85E-02 B15187 1.37E-07 2.73E+04 3.75E-03 B15695 1.52E-07 1.81E+05 2.76E-02 B15189 1.70E-07 2.04E+04 3.47E-03 B15762 1.85E-07 4.60E+04 8.49E-03 B15730 2.59E-07 3.45E+04 8.93E-03

接下來,藉由ELISA測定各種Fab命中之物種交叉反應性。簡言之,在4℃下,將100 μL 1.25 μg/mL抗人類IgG (Fab特異性)抗體(Sigma, I5260)塗覆在Maxisorp微定量盤(Thermo Scientific 446469)上過夜。於阻斷後,添加100 μL Fab命中(2 μg/mL)及培育1小時。於洗滌孔3至4次後,添加與人類FC片段融合之人類、食蟹獼猴或小鼠CTLA4抗原之連續稀釋及培育1小時。於洗滌後,將經HRP標記之山羊抗人類FC用PBS 1:2000稀釋,及添加至各孔用於一小時培育。將板洗滌三次及在室溫下用TMB受質培育3至5分鐘。於停止反應後,量測450 nm處之吸光度。下 2中概述所測試Fab各者之物種交叉反應性。有趣的是,此分析識別具有變化交叉反應性之Fab:結果指示命中B13873、B15700、B15704、B15706、B15709、B15711、B15712、B15715、B15720、B15725、B15723、B15731、B15732、B15735、B15736、B15744、B15760、B16083、及B15188結合至人類、猴及小鼠CTLA4;命中B15188、B15190、B15701、B15729、B15733、B15742、B15747、B15743、B15751、B15752、B15753及B18157結合至人類及猴CTLA4,且弱結合至小鼠CTLA4;命中B13878、B14242、B15189、B15491、B15673、B15694、B15696、B15699、B15702、B15705、B15710、B15716、B15717、B15719、B15721、B15722、B15724、B15728、B15734、B15737、B15738、B15739、B15740、B15745、B15746、B15749、B15750、B15754、B15756、B15757、B15759及B15762結合至人類CTLA4,但是不結合至小鼠CTLA4;命中B15688結合至人類及小鼠CTLA4,但是不結合至猴CTLA4;命中B13874、B13880、B13898、B15187、B15489、B15672、B15695、B15730、B15741、B18153及B18174結合至人類CTLA4,但是不結合至猴或小鼠CTLA4。 2 :與人類、猴及小鼠 CTLA4 Fab 交叉反應性 命中ID 結合人類CTLA? 結合猴CTLA4? 結合小鼠CTLA4? B13873 B15688 B15700 B15704 B15706 B15709 B15711 B15712 B15715 B15720 B15723 B15725 B15731 B15732 B15735 B15736 B15744 B15760 B16083 B15188 B15190 B15701 B15729 B15733 B15742 B15743 B15747 B15751 B15752 B15753 B18157 B13874 B13878 B13880 B13898 B14242 B15187 B15189 B15489 B15491 B15672 B15673 B15694 B15695 B15696 B15699 B15702 B15705 B15710 B15716 B15717 B15719 B15721 B15722 B15724 B15728 B15730 B15734 B15737 B15738 B15739 B15740 B15741 B15745 B15746 B15749 B15750 B15754 B15756 B15757 B15759 B15762 B18153 B18174 實例 2 IgG 轉變及表現 Next, species cross-reactivity of various Fab hits was determined by ELISA. Briefly, 100 μL of 1.25 μg/mL anti-human IgG (Fab specific) antibody (Sigma, I5260) was coated on a Maxisorp microplate (Thermo Scientific 446469) overnight at 4°C. After blocking, add 100 μL of Fab hit (2 μg/mL) and incubate for 1 hour. After washing the wells 3 to 4 times, add serial dilutions of human, cynomolgus or mouse CTLA4 antigen fused to human FC fragments and incubate for 1 hour. After washing, HRP-labeled goat anti-human FC was diluted 1:2000 with PBS and added to each well for one hour incubation. The plates were washed three times and incubated with TMB substrate for 3 to 5 minutes at room temperature. After stopping the reaction, measure the absorbance at 450 nm. The species cross-reactivity for each of the Fabs tested is summarized in Table 2 below. Interestingly, this analysis identified Fabs with varying cross-reactivity: results indicated hits B13873, B15700, B15704, B15706, B15709, B15711, B15712, B15715, B15720, B15725, B15723, B15731, B15732, B15735, B15736 ,B15744, B15760, B16083, and B15188 bind to human, monkey, and mouse CTLA4; hits B15188, B15190, B15701, B15729, B15733, B15742, B15747, B15743, B15751, B15752, B15753, and B18157 bind to human and monkey CTLA4 , and weakly combined to mouse CTLA4; hits B13878, B14242, B15189, B15491, B15673, B15694, B15696, B15699, B15702, B15705, B15710, B15716, B15717, B15719, B15721, B15722, B1572 4. B15728, B15734, B15737, B15738, B15739, B15740, B15745, B15746, B15749, B15750, B15754, B15756, B15757, B15759, and B15762 bind to human CTLA4, but not mouse CTLA4; hit B15688 binds to human and mouse CTLA4, but not monkey CTLA4; hit B13874, B13880, B13898, B15187, B15489, B15672, B15695, B15730, B15741, B18153, and B18174 bind to human CTLA4, but not to monkey or mouse CTLA4. Table 2 : Fab cross-reactivity with human, monkey and mouse CTLA4 Hit ID : Combined with human CTLA? Binding monkey CTLA4? Binding mouse CTLA4? B13873 yes yes yes B15688 yes no yes B15700 yes yes yes B15704 yes yes yes B15706 yes yes yes B15709 yes yes yes B15711 yes yes yes B15712 yes yes yes B15715 yes yes yes B15720 yes yes yes B15723 yes yes yes B15725 yes yes yes B15731 yes yes yes B15732 yes yes yes B15735 yes yes yes B15736 yes yes yes B15744 yes yes yes B15760 yes yes yes B16083 yes yes yes B15188 yes yes weak B15190 yes yes weak B15701 yes yes weak B15729 yes yes weak B15733 yes yes weak B15742 yes yes weak B15743 yes yes weak B15747 yes yes weak B15751 yes yes weak B15752 yes yes weak B15753 yes yes weak B18157 yes yes weak B13874 yes no no B13878 yes yes no B13880 yes no no B13898 yes no no B14242 yes yes no B15187 yes no no B15189 yes yes no B15489 yes no no B15491 yes yes no B15672 yes no no B15673 yes yes no B15694 yes yes no B15695 yes no no B15696 yes yes no B15699 yes yes no B15702 yes yes no B15705 yes yes no B15710 yes yes no B15716 yes yes no B15717 yes yes no B15719 yes yes no B15721 yes yes no B15722 yes yes no B15724 yes yes no B15728 yes yes no B15730 yes no no B15734 yes yes no B15737 yes yes no B15738 yes yes no B15739 yes yes no B15740 yes yes no B15741 yes no no B15745 yes yes no B15746 yes yes no B15749 yes yes no B15750 yes yes no B15754 yes yes no B15756 yes yes no B15757 yes yes no B15759 yes yes no B15762 yes yes no B18153 yes no no B18174 yes no no Example 2 : IgG transformation and expression

然後將來自以上實例1之經精簡命中中之13者轉變成人類IgG1抗體用於詳細生物物理及功能表征( 3)。將Fab命中B15709、B15716、B15722、B15732、B15740、B15744、B15756、B15700、B15711、B15717、B15735、B15736及B16083之重鏈及輕鏈分開選殖至哺乳動物表現載體pTT5-SPB。亦將參考抗體之重鏈及輕鏈選殖至pTT5-SPB。 3 :選殖為 IgG1 抗體之 Fab 命中 抗體(AB) 名稱: Fab 命中ID 同型: TY21585 B15709 hIgG1 TY21586 B15716 hIgG1 TY21587 B15722 hIgG1 TY21588 B15732 hIgG1 TY21589 B15740 hIgG1 TY21580 B15744 hIgG1 TY21591 B15756 hIgG1 TY21687 B15700 hIgG1 TY21688 B15711 hIgG1 TY21689 B15717 hIgG1 TY21680 B15735 hIgG1 TY21691 B15736 hIgG1 TY21692 B16083 hIgG1 TAC2114 參考 hIgG1 Thirteen of the refined hits from Example 1 above were then converted into human IgG1 antibodies for detailed biophysical and functional characterization ( Table 3 ). The heavy and light chains of Fab hits B15709, B15716, B15722, B15732, B15740, B15744, B15756, B15700, B15711, B15717, B15735, B15736 and B16083 were separately cloned into the mammalian expression vector pTT5-SPB. The heavy and light chains of the reference antibody were also cloned into pTT5-SPB. Table 3 : Fab hits selected as IgG1 antibodies Antibody (AB) name: Fab hit ID : Same type: TY21585 B15709 hIgG1 TY21586 B15716 hIgG1 TY21587 B15722 hIgG1 TY21588 B15732 hIgG1 TY21589 B15740 hIgG1 TY21580 B15744 hIgG1 TY21591 B15756 hIgG1 TY21687 B15700 hIgG1 TY21688 B15711 hIgG1 TY21689 B15717 hIgG1 TY21680 B15735 hIgG1 TY21691 B15736 hIgG1 TY21692 B16083 hIgG1 TAC2114 refer to hIgG1

按照製造商之方案,將編碼抗體重鏈及輕鏈之質粒對短暫轉染至293F細胞。收集利用編碼抗體TY21585、TY21586、TY21587、TY21588、TY21589、TY21580、或TY21591之質粒轉染之細胞之上清液,藉由離心及過濾澄清,及將所得IgG用標準蛋白A親和層析法(MabSelect SuRe,GE Healthcare)純化。將蛋白質溶離及中和,及緩衝交換至20 mM PB緩衝液(20 mM NaH 2PO 4,150 mM NaCl,pH 7.0)中。藉由UV-分光光度法測定蛋白質濃度,及在變性、還原及非還原條件下藉由SDS-PAGE或SEC-HPLC分析IgG純度。 Plasmid pairs encoding antibody heavy and light chains were transiently transfected into 293F cells according to the manufacturer's protocol. The supernatants of cells transfected with plasmids encoding antibodies TY21585, TY21586, TY21587, TY21588, TY21589, TY21580, or TY21591 were collected, clarified by centrifugation and filtration, and the resulting IgG was subjected to standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare) purification. Proteins were eluted, neutralized, and buffer exchanged into 20 mM PB buffer (20 mM NaH 2 PO 4 , 150 mM NaCl, pH 7.0). Protein concentration was determined by UV-spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing and non-reducing conditions.

收集利用編碼抗體TY21687、TY21689、TY21680、TY21691、或TY21692之質粒轉染之細胞之上清液,藉由離心及過濾澄清,及將所得IgG用標準蛋白A親和層析法(MabSelect SuRe,GE Healthcare)純化。將蛋白質溶離及中和,及緩衝交換至20 mM組胺酸緩衝液(20 mM組胺酸,3.5 mL 6M NaCl,pH 5.5)中。藉由UV-分光光度法測定蛋白質濃度,及在變性、還原及非還原條件下藉由SDS-PAGE或SEC-HPLC分析IgG純度。 實例 3 :經 IgG 轉變之抗體之活體外功能表征 The supernatants of cells transfected with plasmids encoding antibodies TY21687, TY21689, TY21680, TY21691, or TY21692 were collected, clarified by centrifugation and filtration, and the resulting IgG was subjected to standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare ) purification. Proteins were eluted, neutralized, and buffer exchanged into 20 mM histidine buffer (20 mM histidine, 3.5 mL 6M NaCl, pH 5.5). Protein concentration was determined by UV-spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing and non-reducing conditions. Example 3 : In vitro functional characterization of IgG- converted antibodies

根據製造商之指導方針,使用Biacore™ T200儀器(Biacore AB, Uppsala, Sweden)藉由表面電漿子共振(SPR)分析檢查抗體對人類、猴及小鼠CTLA4之結合親和力及動力學( 4)。根據胺偶合套組(GE Biacore #BR-1000-50)之說明,藉由將其胺基偶合在感測器晶片之羧化表面上將來自人類抗體捕獲套組(GE BR-1008-39)之抗人類IgG (Fc)抗體固定在CM5晶片上。使用經固定之抗人類IgG (Fc)抗體捕獲抗體TY21585、TY21586、TY21580、TY21591、TY21687、TY21689、TY21680、TY21691、TY21692及TAC2114。TAC2114具有與商業抗體伊匹單抗相同之胺基酸序列。在六種不同濃度(3.13、6.25、12.5、25、50及100 nM於運行緩衝液中稀釋)下量測結合,及使用30 μL/min之流率。使用之運行緩衝液為HBS-EP (100 mM HEPES、1.5M氯化鈉、0.05%表面活性劑P20,pH 7.6)。根據製造商之指導方針,使用Biacore T200評價軟體(Biacore AB)將締合及解離曲線擬合至1: 1朗繆爾結合模型。如下 4中所示,所有測試抗體能結合人類及猴CTLA4,及所有抗體(除了TY21591、TY21689及TAC2114)亦能結合至小鼠CTLA4。 4 IgG1 抗體與人類、猴及小鼠 CTLA4 之結合親和力    K D(nM) 抗體名稱: 人類CTLA4 猴CTLA4 小鼠CTLA4 TY21585 8.15 8.26 1.71 TY21586 7.80 15.00 303 TY21580 2.58 0.44 0.51 TY21591 9.68 3.27 NC TY21687 1.82 1.52 2.76 TY21689 0.95 0.91 NC TY21680 1.48 1.29 0.67 TY21691 2.08 2.44 1.46 TY21692 3.95 2.76 1.12 TAC2114 6.68 1.98 NC NC=非交叉反應性。 The binding affinity and kinetics of the antibodies to human, monkey, and mouse CTLA4 were examined by surface plasmon resonance (SPR) analysis using a Biacore™ T200 instrument (Biacore AB, Uppsala, Sweden) according to the manufacturer's guidelines ( Table 4 ). Antibody capture kit (GE BR-1008-39) from the Human Antibody Capture Kit (GE Biacore #BR-1000-50) was prepared by coupling its amine groups onto the carboxylated surface of the sensor chip according to the instructions of the Amine Coupling Kit (GE Biacore #BR-1000-50). Anti-human IgG (Fc) antibodies were immobilized on CM5 chips. Antibodies TY21585, TY21586, TY21580, TY21591, TY21687, TY21689, TY21680, TY21691, TY21692 and TAC2114 were captured using immobilized anti-human IgG (Fc) antibodies. TAC2114 has the same amino acid sequence as the commercial antibody ipilimumab. Binding was measured at six different concentrations (3.13, 6.25, 12.5, 25, 50 and 100 nM diluted in running buffer) and using a flow rate of 30 μL/min. The running buffer used was HBS-EP (100 mM HEPES, 1.5M sodium chloride, 0.05% surfactant P20, pH 7.6). Association and dissociation curves were fitted to a 1:1 Langmuir binding model using Biacore T200 evaluation software (Biacore AB) according to the manufacturer's guidelines. As shown in Table 4 below, all antibodies tested were able to bind human and monkey CTLA4, and all antibodies (except TY21591, TY21689, and TAC2114) were also able to bind to mouse CTLA4. Table 4 : Binding affinity of IgG1 antibodies to human, monkey and mouse CTLA4 K D (nM) Antibody name: Human CTLA4 : Monkey CTLA4 : Mouse CTLA4 : TY21585 8.15 8.26 1.71 TY21586 7.80 15.00 303 TY21580 2.58 0.44 0.51 TY21591 9.68 3.27 NC TY21687 1.82 1.52 2.76 TY21689 0.95 0.91 NC TY21680 1.48 1.29 0.67 TY21691 2.08 2.44 1.46 TY21692 3.95 2.76 1.12 TAC2114 6.68 1.98 NC NC = non-cross-reactive.

接下來測試某些IgG抗體結合至可溶性人類( 1A ,表 5A)或犬( 1B ,表 5B) CTLA4之能力。製備1 μg/mL與人類FC片段融合之人類CTLA4,或與His標籤融合之犬CTLA4及用於在2至8℃下塗覆ELISA板過夜。於阻斷後,添加100 μL經連續稀釋之IgG抗體及在37℃下培育1小時。將板洗滌四次及然後在37℃下用HRP-抗人類IgG (Fab特異性) (1:6000稀釋)培育1小時。將板再次洗滌四次,及在室溫下用TMB受質培育15分鐘。於停止反應後量測450 nm處之吸光度。藉由Graphpad Prism 6利用非線性擬合分析數據。如 1A B 5A B中所示,所有測試抗體結合至人類CTLA4及除了抗體TY21586及TAC2114外亦結合至犬CTLA4。有趣的是,TY21580以最高親和力結合人類及狗CTLA4二者,各自為0.27及0.49之K D 5A :針對人類 CTLA4 ELISA 抗體名稱: LogEC 50 EC 50 K D M nM nM TY21585 -9.256 5.552E-10 0.5552 0.56 TY21586 -8.896 1.27E-09 1.2710 1.27 TY21580 -9.571 2.685E-10 0.2685 0.27 TY21591 -9.080 8.309E-10 0.8309 0.83 TY21687 -9.495 3.201E-10 0.3201 0.32 TAC2114 -9.482 3.296E-10 0.3296 0.33 5B :針對犬 CTLA4 ELISA 抗體名稱: LogEC 50 EC 50 K D M nM nM TY21585 -8.219 6.045E-09 6.0450 6.05 TY21586 ND ND ND ND TY21580 -9.313 4.86E-10 0.4860 0.49 TY21591 -9.151 7.066E-10 0.7066 0.71 TY21687 -9.128 7.451E-10 0.7451 0.75 TAC2114 ND ND ND ND ND=未測定。 Certain IgG antibodies were next tested for their ability to bind to soluble human ( Figure 1A , Table 5A ) or canine ( Figure 1B , Table 5B ) CTLA4. Prepare 1 μg/mL of human CTLA4 fused to a human FC fragment, or canine CTLA4 fused to a His tag, and use to coat ELISA plates overnight at 2 to 8°C. After blocking, 100 μL of serially diluted IgG antibodies were added and incubated at 37°C for 1 hour. Plates were washed four times and then incubated with HRP-anti-human IgG (Fab specific) (1:6000 dilution) for 1 hour at 37°C. The plates were washed four more times and incubated with TMB substrate for 15 minutes at room temperature. After stopping the reaction, measure the absorbance at 450 nm. Data were analyzed using nonlinear fitting with Graphpad Prism 6. As shown in Figures 1A - B and Tables 5A - B , all tested antibodies bound to human CTLA4 and except for antibodies TY21586 and TAC2114 also bound to canine CTLA4. Interestingly, TY21580 bound both human and dog CTLA4 with the highest affinity, with K D of 0.27 and 0.49 respectively. Table 5A : ELISA for human CTLA4 Antibody name: LogEC 50 : EC 50 : K D M nM nM TY21585 -9.256 5.552E-10 0.5552 0.56 TY21586 -8.896 1.27E-09 1.2710 1.27 TY21580 -9.571 2.685E-10 0.2685 0.27 TY21591 -9.080 8.309E-10 0.8309 0.83 TY21687 -9.495 3.201E-10 0.3201 0.32 TAC2114 -9.482 3.296E-10 0.3296 0.33 Table 5B : ELISA for canine CTLA4 Antibody name: LogEC 50 : EC 50 : K D M nM nM TY21585 -8.219 6.045E-09 6.0450 6.05 TY21586 ND ND ND ND TY21580 -9.313 4.86E-10 0.4860 0.49 TY21591 -9.151 7.066E-10 0.7066 0.71 TY21687 -9.128 7.451E-10 0.7451 0.75 TAC2114 ND ND ND ND ND=not determined.

亦評估抗體對在HEK293F細胞表面上短暫表現之人類及小鼠CTLA4之親和力( 2)。簡言之,將HEK293F細胞自亦編碼EGFP之雙順反子IRES載體用表現全長人類、猴或小鼠CTLA4之質粒轉染,及使用EGFP表現識別經轉染細胞。於48小時後,將哺乳動物細胞懸浮液(2 x 10 5/孔)轉染至微量離心(Eppendorf)管,離心,遺棄上清液,將細胞用1 mL PBSA再懸浮(至密度為4×10 6個細胞/mL),及添加至96孔板。將測試抗體之3倍連續稀釋(15 μg/mL、5 μg/mL、1.67 μg/mL、0.55 μg/mL、0.185 μg/mL、0.062 μg/mL、及0.0309 μg/mL,加上0 μg/mL空白對照)吸量管移入96孔板,在冰上培育1小時(避光),將細胞用預冷卻之1 x PBSA緩衝液洗滌,及隨後在冰上用Alexa Fluor® 647結合之小鼠抗人類FC抗體培育30分鐘。然後將細胞洗滌一次,之後藉由流動式細胞測量術(Beckman® CytoFlex)分析。如 2中所示,所有測試抗體能結合HEK293F細胞表面上表現之人類CTLA4,及除了TY21589外,所有抗體結合至小鼠細胞表面暴露之小鼠CTLA4。TY21580以低nM親和力結合至細胞表面上表現之人類及小鼠CTLA4,然而抗體TY21585及TY21586以高nM親和力結合至細胞表面上表現之人類CTLA4。 The antibody's affinity for human and mouse CTLA4 transiently expressed on the surface of HEK293F cells was also evaluated ( Figure 2 ). Briefly, HEK293F cells were transfected with plasmids expressing full-length human, monkey, or mouse CTLA4 from a bicistronic IRES vector also encoding EGFP, and EGFP expression was used to identify transfected cells. After 48 hours, the mammalian cell suspension (2 x 10 5 /well) was transfected into a microcentrifuge tube (Eppendorf), centrifuged, the supernatant was discarded, and the cells were resuspended in 1 mL PBSA (to a density of 4× 10 6 cells/mL) and added to a 96-well plate. 3-fold serial dilutions of test antibodies (15 μg/mL, 5 μg/mL, 1.67 μg/mL, 0.55 μg/mL, 0.185 μg/mL, 0.062 μg/mL, and 0.0309 μg/mL, plus 0 μg/ mL blank control) was pipetted into a 96-well plate, incubated on ice for 1 hour (protected from light), cells were washed with pre-cooled 1x PBSA buffer, and then incubated with Alexa Fluor® 647-conjugated mice on ice. Anti-human FC antibodies were incubated for 30 minutes. Cells were then washed once and analyzed by flow cytometry (Beckman® CytoFlex). As shown in Figure 2 , all antibodies tested were able to bind to human CTLA4 expressed on the surface of HEK293F cells, and with the exception of TY21589, all antibodies bound to mouse CTLA4 exposed on the surface of mouse cells. TY21580 binds to human and mouse CTLA4 expressed on the cell surface with low nM affinity, whereas antibodies TY21585 and TY21586 bind to human CTLA4 expressed on the cell surface with high nM affinity.

亦使用ForteBio red 96儀器(Pall, USA)測試抗體TY21580、TY21687、TY21680及TY21691對大鼠CTLA4蛋白之結合親和力及動力學。使用SA感測器(Pall, 185019)以固定與人類FC融合之經生物素化大鼠CTLA4蛋白,及然後使感測器與在15 μg/mL (用補充有0.02% Tween 20及0.1% BSA之KB緩衝液、PBS緩衝液稀釋)之濃度下之IgG-轉變之命中接觸300秒,然後於KB緩衝液中解離300秒。根據製造商之指導方針,使用ForteBio資料分析7.1 (Pall, USA)將締合及解離曲線擬合至1:1朗繆爾結合模型。如 6中所示,所有測試抗體能結合至大鼠CTLA4。 6 :測試抗體對大鼠 CTLA4 之結合親和力 抗體名稱: K D(nM) TY21580 0.38 TY21687 0.78 TY21680 0.21 TY21691 0.58 IgG 與經活化 T 細胞之結合 ForteBio red 96 instrument (Pall, USA) was also used to test the binding affinity and kinetics of antibodies TY21580, TY21687, TY21680 and TY21691 to rat CTLA4 protein. An SA sensor (Pall, 185019) was used to immobilize biotinylated rat CTLA4 protein fused to human FC, and the sensor was then incubated with 0.02% Tween 20 and 0.1% BSA at 15 μg/mL. The IgG-converted hits were contacted for 300 seconds at a concentration of KB buffer, PBS buffer (diluted in PBS buffer) and then dissociated in KB buffer for 300 seconds. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio Data Analysis 7.1 (Pall, USA) according to the manufacturer's guidelines. As shown in Table 6 , all tested antibodies were able to bind to rat CTLA4. Table 6 : Binding affinity of test antibodies to rat CTLA4 Antibody name: K D (nM) : TY21580 0.38 TY21687 0.78 TY21680 0.21 TY21691 0.58 Binding of IgG to activated T cells

接下來,測試IgG結合至經活化人類、猴及小鼠T細胞之能力。將人類PBMC使用Histopaque-1077 (Sigma)藉由密度梯度離心自健康供體(#106)之血液新鮮單離。將人類T細胞使用人類T細胞濃化套組(StemCell Technologies)自PBMC單離,接著用抗CD3及抗CD28抗體刺激。簡言之,在4℃下將抗CD3抗體(純系:OKT3,BioLegend)以0.2 μg含於200 μL中/孔塗覆於96孔板過夜。於洗滌後,將懸浮於含10% FBS及1% Penn/Strep之RPMI-1640中之T細胞添加至該板中。將5x10E5個T細胞含於200 μL中添加至96孔板之各孔。然後添加1 μL抗人類CD28抗體(純系:28.2,BD)至5 μg/mL之最終濃度。將T細胞培育96小時,及然後藉由流動式細胞測量術分析測定TY21580與T細胞之結合( 3)。在37℃下將T細胞用經APC標記之TY21580或人類IgG1 (同型對照)染色2小時。於洗滌後,在CytoFLEX流動式細胞測量儀(Beckman Coulter)上分析細胞及用FlowJo軟體分析數據。如 3中所示,TY21580結合至經活化CD4+及CD8+人類T細胞,而對照IgG顯示不結合。此外,APC-TY21580顯示不結合至休止T細胞(不顯示數據)。 Next, the IgGs were tested for their ability to bind to activated human, monkey and mouse T cells. Human PBMC were freshly isolated from the blood of a healthy donor (#106) by density gradient centrifugation using Histopaque-1077 (Sigma). Human T cells were isolated from PBMC using a human T cell enrichment kit (StemCell Technologies) and then stimulated with anti-CD3 and anti-CD28 antibodies. Briefly, anti-CD3 antibody (clone: OKT3, BioLegend) was coated on a 96-well plate at 0.2 μg in 200 μL/well overnight at 4°C. After washing, T cells suspended in RPMI-1640 containing 10% FBS and 1% Penn/Strep were added to the plate. Add 5x10E5 T cells in 200 μL to each well of a 96-well plate. Then add 1 μL of anti-human CD28 antibody (clone: 28.2, BD) to a final concentration of 5 μg/mL. T cells were incubated for 96 hours, and binding of TY21580 to T cells was then determined by flow cytometric analysis ( Figure 3 ). T cells were stained with APC-labeled TY21580 or human IgG1 (isotype control) for 2 hours at 37°C. After washing, cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter) and data were analyzed using FlowJo software. As shown in Figure 3 , TY21580 bound to activated CD4+ and CD8+ human T cells, whereas control IgG showed no binding. Furthermore, APC-TY21580 was shown not to bind to resting T cells (data not shown).

將猴PBMC使用Histopaque-1077 (Sigma)藉由密度梯度離心自初始食蟹獼猴之血液新鮮單離。將猴T細胞使用泛T細胞單離套組非人類靈長類動物(Miltenyi Biotec)自PBMC單離,接著用抗CD3及抗CD28抗體刺激。簡言之,在4℃下將抗CD3抗體(純系:SP34,BD)以0.2 μg含於200 μL中/孔塗覆於96孔板過夜。於洗滌後,將懸浮於含10% FBS及1% Penn/Strep之RPMI-1640中之T細胞添加至該板中。將2x10E5個T細胞含於200 μL中添加至96孔板之各孔。然後添加1 μL抗人類CD28抗體(純系:28.2,BD)至5 μg/mL之最終濃度。將T細胞培育72小時,及藉由流動式細胞測量術分析測定TY21580與T細胞之結合( 3)。在37℃下將T細胞用經APC標記之TY21580或人類IgG1 (同型對照)染色2小時。於洗滌後,在CytoFLEX流動式細胞測量儀(Beckman Coulter)上分析細胞及用FlowJo軟體分析數據。如 3中所示,TY21580結合至經活化CD4+及CD8+猴T細胞,而對照IgG顯示不結合。此外,APC-TY21580顯示不結合至休止T細胞(不顯示數據) Monkey PBMC were freshly isolated from naive cynomolgus monkey blood by density gradient centrifugation using Histopaque-1077 (Sigma). Monkey T cells were isolated from PBMC using a pan-T cell isolation kit from non-human primates (Miltenyi Biotec), followed by stimulation with anti-CD3 and anti-CD28 antibodies. Briefly, anti-CD3 antibody (clone: SP34, BD) was coated on a 96-well plate at 0.2 μg in 200 μL/well overnight at 4°C. After washing, T cells suspended in RPMI-1640 containing 10% FBS and 1% Penn/Strep were added to the plate. Add 2x10E5 T cells in 200 μL to each well of a 96-well plate. Then add 1 μL of anti-human CD28 antibody (clone: 28.2, BD) to a final concentration of 5 μg/mL. T cells were incubated for 72 hours, and binding of TY21580 to T cells was determined by flow cytometry analysis ( Figure 3 ). T cells were stained with APC-labeled TY21580 or human IgG1 (isotype control) for 2 hours at 37°C. After washing, cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter) and data were analyzed using FlowJo software. As shown in Figure 3 , TY21580 bound to activated CD4+ and CD8+ monkey T cells, whereas control IgG showed no binding. Furthermore, APC-TY21580 was shown not to bind to resting T cells (data not shown)

使用自成年BALB/c小鼠脾單離之小鼠T細胞誘導CTLA-4表現。使用來自新鮮小鼠脾之脾細胞以利用EasySep™小鼠T細胞單離套組(StemCell Technologies)單離T細胞,接著用抗小鼠CD3及抗CD28抗體刺激。簡言之,在4℃下將抗小鼠CD3ε抗體(Biolegend)以0.2 μg含於200 μL中/孔塗覆於96孔板過夜。於洗滌後,將懸浮於含10% FBS及1% Penn/Strep之RPMI-1640中之小鼠T細胞以5x10E5個T細胞含於200 μL中添加至該板之各孔中。然後添加1 μL抗小鼠CD28抗體(eBioscience)至5 μg/mL之最終濃度。將小鼠T細胞培育72小時及然後藉由流動式細胞測量術分析測定TY21580與T細胞之結合( 3)。在37℃下將T細胞用經APC標記之TY21580或人類IgG1 (同型對照)染色2小時。於洗滌後,在CytoFLEX流動式細胞測量儀(Beckman Coulter)上分析細胞及用FlowJo軟體分析數據。如 3中所示,TY21580結合至經活化CD4+及CD8+ T細胞,而對照IgG顯示不結合。 抗體對人類 CTLA4 之結合選擇性 CTLA-4 expression was induced using mouse T cells isolated from the spleen of adult BALB/c mice. T cells were isolated using the EasySep™ Mouse T Cell Isolation Kit (StemCell Technologies) using splenocytes from fresh mouse spleens, followed by stimulation with anti-mouse CD3 and anti-CD28 antibodies. Briefly, anti-mouse CD3ε antibody (Biolegend) was coated on a 96-well plate at 0.2 μg in 200 μL/well overnight at 4°C. After washing, mouse T cells suspended in RPMI-1640 containing 10% FBS and 1% Penn/Strep were added to each well of the plate at 5x10E5 T cells in 200 μL. Then 1 μL of anti-mouse CD28 antibody (eBioscience) was added to a final concentration of 5 μg/mL. Mouse T cells were incubated for 72 hours and the binding of TY21580 to T cells was then determined by flow cytometry analysis ( Figure 3 ). T cells were stained with APC-labeled TY21580 or human IgG1 (isotype control) for 2 hours at 37°C. After washing, cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter) and data were analyzed using FlowJo software. As shown in Figure 3 , TY21580 bound to activated CD4+ and CD8+ T cells, whereas control IgG showed no binding. Antibody binding selectivity for human CTLA4

接下來檢查抗體選擇性。將人類CTLA4、PD1、LAG3、Tim3、B7H3、CD95、TNFR1、OX40、CD40、PD-L1、BLTA、VISTA、PDL2、ICOS及B7H4在HEK293F細胞表面上短暫過度表現。將經轉染細胞於預冷卻之1 x PBSA緩衝液(1.76 mM KH2PO4、10.14 mM Na 2HPO4 12H 2O、2.68 mM KCl、136.89 mM NaCl及1% BSA)中洗滌,然後在冰上用100 nM測試抗體培育1小時。將細胞用染色緩衝液洗滌一次,及添加Alexa Fluor® 647結合之小鼠抗人類FC抗體及在冰上培育30分鐘,避光。將樣品用染色緩衝液洗滌一次,之後藉由流動式細胞測量術分析。於人類CTLA4、PD1、LAG3、Tim3及B7-H3中測試TY21585、TY21586、TY21580、TY21687、TY21689、TY21680及TY21691 ( 4A);於人類CD95、TNFR1、OX40及CD40中進一步測試TY21585、TY21586、TY21580 ( 4B);此外,於人類PD-L1、BLTA、VISTA、PDL2、ICOS及B7-H4中測試TY21586、TY21580 ( 4C)。如 4A C中所示,所有測試抗體特異性結合至人類CTLA4,且不結合至任何其他測試抗原(或利用空載體轉染之親本細胞)。 藉由 ELISA 之配位體競爭結合 Next check the antibody selectivity. Human CTLA4, PD1, LAG3, Tim3, B7H3, CD95, TNFR1, OX40, CD40, PD-L1, BLTA, VISTA, PDL2, ICOS, and B7H4 were transiently overexpressed on the surface of HEK293F cells. The transfected cells were washed in pre-chilled 1× PBSA buffer (1.76 mM KH2PO4, 10.14 mM Na 2 HPO4 12H 2 O, 2.68 mM KCl, 136.89 mM NaCl, and 1% BSA), and then incubated on ice with 100 nM test antibody was incubated for 1 hour. The cells were washed once with staining buffer, and Alexa Fluor® 647-conjugated mouse anti-human FC antibody was added and incubated on ice for 30 minutes, protected from light. Samples were washed once with staining buffer before analysis by flow cytometry. TY21585, TY21586, TY21580, TY21687, TY21689, TY21680 and TY21691 were tested in human CTLA4, PD1, LAG3, Tim3 and B7-H3 ( Figure 4A ); TY21585, TY21586, TY21580 were further tested in human CD95, TNFR1, OX40 and CD40 ( Figure 4B ); In addition, TY21586 and TY21580 were tested in human PD-L1, BLTA, VISTA, PDL2, ICOS and B7-H4 ( Figure 4C ). As shown in Figure 4A to C , all test antibodies specifically bound to human CTLA4 and did not bind to any other test antigen (or parental cells transfected with empty vector). Ligand competitive binding by ELISA

然後藉由ELISA測試抗體阻斷CTLA4結合至其同源配位體CD80及CD86之能力。將重組人類CTLA4 (與人類Fc及His標籤融合)於碳酸鹽緩衝液pH 9.4中稀釋至1 μg/mL,及在4℃下塗覆在Maxisorp板上過夜。在37℃下將板用補充有2% (w/v)脫脂乳之PBS阻斷1小時。於洗滌後,將50 μL經生物素化CD80 (4 μg/mL)及50 μL各種濃度之測試抗體(範圍自200 μg/mL至1.56 μg/mL之2倍連續稀釋液)依次添加至各孔及在37℃下培育1小時。將板洗滌四次,及將100 μL HRP-中性親和素(neutravidin) (1:1000)添加至各孔及在37℃下培育1小時。將板如先前所述洗滌,及添加50 μL TMB受質溶液及在室溫下培育5分鐘,之後藉由50 μL硫酸(2M)停止反應。如 5A B中所示,除了TY21589,所有測試抗體阻斷CTLA4結合至CD80。 The antibodies were then tested for their ability to block CTLA4 binding to its cognate ligands CD80 and CD86 by ELISA. Recombinant human CTLA4 (fused to human Fc and His tags) was diluted to 1 μg/mL in carbonate buffer pH 9.4 and plated onto Maxisorp plates overnight at 4°C. Plates were blocked with PBS supplemented with 2% (w/v) skim milk for 1 hour at 37°C. After washing, 50 μL of biotinylated CD80 (4 μg/mL) and 50 μL of test antibodies at various concentrations (2-fold serial dilutions ranging from 200 μg/mL to 1.56 μg/mL) were added to each well. and incubate at 37°C for 1 hour. The plate was washed four times and 100 μL of HRP-neutravidin (1:1000) was added to each well and incubated at 37°C for 1 hour. The plate was washed as previously described, and 50 μL of TMB substrate solution was added and incubated at room temperature for 5 minutes before the reaction was stopped by 50 μL of sulfuric acid (2M). As shown in Figure 5A to B , all tested antibodies except TY21589 blocked CTLA4 binding to CD80.

將與人類Fc融合之重組人類CD86於碳酸鹽緩衝液pH 9.4中稀釋至1 μg/mL,及在4℃下塗覆在Maxisorp板上過夜。在37℃下將板用補充有2% (w/v)脫脂乳之PBS阻斷1小時。於洗滌後,將50 μL與人類FC及His標籤融合之經生物素化人類CTLA4 (2.8 μg/mL)及50 μL各種濃度之測試抗體(範圍自100 μg/mL至0.78 μg/mL之2倍連續稀釋液)依次添加至各孔及在37℃下培育1小時。將板洗滌四次,及將100 μL HRP-中性親和素(1:1000)添加至各孔及在37℃下培育1小時。將板如先前所述洗滌,及添加50 μL TMB受質溶液及在室溫下培育5分鐘,之後藉由50 μL硫酸(2M)停止反應。如 5C D中所示,所有測試抗體阻斷CTLA4結合至CD86。 藉由流動式細胞測量術之配位體競爭結合 Recombinant human CD86 fused to human Fc was diluted to 1 μg/mL in carbonate buffer pH 9.4 and plated on Maxisorp plates overnight at 4°C. Plates were blocked with PBS supplemented with 2% (w/v) skim milk for 1 hour at 37°C. After washing, 50 μL of biotinylated human CTLA4 (2.8 μg/mL) fused to human FC and His tags and 50 μL of test antibodies at various concentrations (ranging from 100 μg/mL to 2 times 0.78 μg/mL) were added. Serial dilutions) were added to each well and incubated at 37°C for 1 hour. The plate was washed four times and 100 μL of HRP-neutravidin (1:1000) was added to each well and incubated at 37°C for 1 hour. The plate was washed as previously described, and 50 μL of TMB substrate solution was added and incubated at room temperature for 5 minutes before the reaction was stopped by 50 μL of sulfuric acid (2M). As shown in Figure 5C to D , all tested antibodies blocked CTLA4 binding to CD86. Ligand competitive binding by flow cytometry

亦藉由流動式細胞測量術測試抗體阻斷CTLA4結合至其同源配位體CD80及CD86之能力。將編碼全長人類CTLA4之質粒於HEK293F細胞中短暫表現。將細胞用染色緩衝液(包含1.76 mM KH 2PO 4、10.14 mM Na 2HPO 4·12H 2O、2.68 mM KCl、136.89 mM NaCl及1% BSA之PBSA緩衝液)洗滌,及再懸浮於含100 nM測試抗體之染色緩衝液中。於冰上培育60分鐘後,將100 nM經生物素化人類CD80-Fc-Bio或CD86-Fc-Bio添加至各孔及在冰上再培育1小時。將細胞用染色緩衝液洗滌一次,及添加100 μL含Alexa fluor 633結合之鏈黴抗生物素(streptavidin)之染色緩衝液及在冰上培育30分鐘,避光。將細胞洗滌一次,及藉由CytoFlex流動式細胞測量術分析。如 6A中所示,所有測試抗體以濃度依賴性方式阻斷CTLA4結合至CD80。TY21588顯示最強阻斷能力,其次TY21580及TAC2114具有顯著阻斷;及TY21585、TY21587、TY21589、TY21591具有較不有效阻斷。TY21589顯示少至無阻斷。如 6B中所示,所有測試抗體以濃度依賴性方式阻斷CTLA4結合至CD86。TY21588、TY21589、TY21580、TY21591及TAC2114顯示最強阻斷能力;TY21585及TY21587具有較不有效阻斷。 結合至 FcγR The ability of the antibodies to block CTLA4 binding to its cognate ligands CD80 and CD86 was also tested by flow cytometry. The plasmid encoding full-length human CTLA4 was transiently expressed in HEK293F cells. The cells were washed with staining buffer (PBSA buffer containing 1.76 mM KH 2 PO 4 , 10.14 mM Na 2 HPO 4 ·12H 2 O, 2.68 mM KCl, 136.89 mM NaCl, and 1% BSA) and resuspended in 100 nM test antibody in staining buffer. After incubation on ice for 60 minutes, 100 nM biotinylated human CD80-Fc-Bio or CD86-Fc-Bio was added to each well and incubated on ice for an additional hour. The cells were washed once with staining buffer, and 100 μL of staining buffer containing Alexa fluor 633-conjugated streptavidin was added and incubated on ice for 30 minutes, protected from light. Cells were washed once and analyzed by CytoFlex flow cytometry. As shown in Figure 6A , all tested antibodies blocked CTLA4 binding to CD80 in a concentration-dependent manner. TY21588 showed the strongest blocking ability, followed by TY21580 and TAC2114 with significant blocking; and TY21585, TY21587, TY21589, and TY21591 with less effective blocking. TY21589 showed little to no blocking. As shown in Figure 6B , all tested antibodies blocked CTLA4 binding to CD86 in a concentration-dependent manner. TY21588, TY21589, TY21580, TY21591 and TAC2114 showed the strongest blocking ability; TY21585 and TY21587 had less effective blocking. Binds to FcγR

接下來測試TY21586、TY21580及TAC2114對CD16a (176Phe) (Sino Biological Inc,10389-H08H)、CD16a (176Val,10389-H08H1)、CD32a (Sino Biological Inc,10374-H08H)、CD32b (Sino Biological Inc,10259-H08H)及CD64 (Sino Biological Inc,10256-H08H)之結合親和力。根據製造商之指導方針,使用Biacore™ T200儀器(Biacore AB,Uppsala, Sweden)藉由表面電漿子共振(SPR)分析檢查蛋白質結合。根據胺偶合套組(GE Biacore #BR-1000-50)之說明,藉由將蛋白質L之胺基偶合在感測器晶片之羧化表面上將蛋白質L (Sino Biological Inc. 11044-H07E)固定在CM5晶片上。使用經固定之抗人類IgG (Fc)抗體捕獲TY21586、TY21580及TAC2114。將於FcγR蛋白之運行緩衝液中稀釋之連續濃度(12.5、25、50、25、100及200 nM)在30 μL/min之速率下注射。所用之運行緩衝液為HBS-EP (100 mM HEPES、1.5M氯化鈉、0.05%表面活性劑P20,pH 7.6)。根據製造商之指導方針,使用Biacore T200評價軟體(Biacore AB, Uppsala, Sweden)將締合及解離曲線擬合至1: 1朗繆爾結合模型。如下 7中所示,TY21586及TY21580顯示相較於參考抗體(TAC2114) 相似之結合至FcγR之親和力。 7 :結合至 FcγR 之抗體 K D(nM) 抗體名稱 CD16a (176Phe) CD16a (176Val) CD32a CD32b CD64 TY21586 143.000 453.000 884.000 1340.000 0.214 TY21580 145.000 624.000 884.000 1110.000 0.202 TAC2114 237.000 577.000 706.000 735.000 0.255 結合至 FcRn Next, TY21586, TY21580 and TAC2114 were tested against CD16a (176Phe) (Sino Biological Inc, 10389-H08H), CD16a (176Val, 10389-H08H1), CD32a (Sino Biological Inc, 10374-H08H), CD32b (Sino Biological Inc, 10259 -H08H) and CD64 (Sino Biological Inc, 10256-H08H). Protein binding was examined by surface plasmon resonance (SPR) analysis using a Biacore™ T200 instrument (Biacore AB, Uppsala, Sweden) according to the manufacturer's guidelines. Protein L (Sino Biological Inc. 11044-H07E) was immobilized by coupling its amine groups to the carboxylated surface of the sensor chip according to the instructions of the amine coupling kit (GE Biacore #BR-1000-50). on a CM5 wafer. TY21586, TY21580 and TAC2114 were captured using immobilized anti-human IgG (Fc) antibodies. Sequential concentrations (12.5, 25, 50, 25, 100, and 200 nM) of FcγR protein diluted in running buffer were injected at a rate of 30 μL/min. The running buffer used was HBS-EP (100 mM HEPES, 1.5M sodium chloride, 0.05% surfactant P20, pH 7.6). Association and dissociation curves were fitted to a 1:1 Langmuir binding model using Biacore T200 evaluation software (Biacore AB, Uppsala, Sweden) according to the manufacturer's guidelines. As shown in Table 7 below, TY21586 and TY21580 showed similar affinity for binding to FcγR compared to the reference antibody (TAC2114). Table 7 : Antibodies that bind to FcγR K D (nM) Antibody name CD16a (176Phe) CD16a (176Val) CD32a CD32b CD64 TY21586 143.000 453.000 884.000 1340.000 0.214 TY21580 145.000 624.000 884.000 1110.000 0.202 TAC2114 237.000 577.000 706.000 735.000 0.255 Binds to FcRn

根據製造商之指導方針,使用Biacore™ T200儀器(Biacore AB, Uppsala, Sweden)藉由表面電漿子共振(SPR)分析檢查測試抗體對重組人類FcRn之結合親和力。根據胺偶合套組(GE Biacore #BR-1000-50)之說明,藉由將人類FcRn蛋白之胺基偶合在感測器晶片之羧化表面上將人類FcRn蛋白(Sino Biological Inc. 11044-H07E)固定在CM5晶片上。將100 nM各抗體於運行緩衝液(50 mM NaPO4、150 mM NaCl及0.05% (v/v)表面活性劑20,pH 6.0)中稀釋,及將樣品在30 μL/min之速率下注射120秒。如 7中所示,抗體TY21585、TY21580、TY21591、TY21687及TY21691展示較TAC2114更高之結合至FcRn之結合%,其指示相較於參考抗體(TAC2114),Biacore晶片上之IgG-FcRn複合物可經歷使該複合物穩定之構形變化。抗體TY21586、TY21587、TY21589、TY21689及TY21680顯示低的結合%。 人類 PBMC 活化 The binding affinity of the test antibodies to recombinant human FcRn was checked by surface plasmon resonance (SPR) analysis using a Biacore™ T200 instrument (Biacore AB, Uppsala, Sweden) according to the manufacturer's guidelines. The human FcRn protein (Sino Biological Inc. 11044-H07E) was coupled to the carboxylated surface of the sensor chip by coupling the amine group of the human FcRn protein to the carboxylated surface of the sensor chip according to the instructions of the amine coupling kit (GE Biacore #BR-1000-50). ) is fixed on the CM5 chip. Dilute 100 nM of each antibody in running buffer (50 mM NaPO4, 150 mM NaCl, and 0.05% (v/v) Surfactant 20, pH 6.0) and inject the sample at 30 μL/min for 120 sec. . As shown in Figure 7 , antibodies TY21585, TY21580, TY21591, TY21687, and TY21691 exhibited a higher % binding to FcRn than TAC2114, indicating that the IgG-FcRn complex on the Biacore wafer was stronger compared to the reference antibody (TAC2114). Can undergo conformational changes that stabilize the complex. Antibodies TY21586, TY21587, TY21589, TY21689 and TY21680 showed low binding %. Human PBMC activation

初步研究顯示TY21580不刺激人類T細胞活化或增殖。因為T細胞上之CTLA4活性與第一信號(TCR/CD3)及涉及B7-CD28/CTLA-4之第二信號相關,選擇人類PBMC,及測定TY21580在低濃度抗CD3之存在下之活性。在4℃下將抗CD3抗體(OKT-3)塗覆在96孔板上過夜。於洗滌後,將1 x 10 5個經新鮮單離之人類PBMC添加至各孔,接著在不同濃度下添加測試製品。於使用人類IL-2 ELISA Ready-SET-Go (Invitrogen)套組刺激48小時後量測IL-2之誘導。使用人類IFNγ ELISA Ready-SET-Go (Invitrogen)套組量測上清液中之IFNγ。如 8A 9中所示,在抗CD3之存在下抗體TY21580顯著增加人類PBMC活化,而TY21580單獨不具有活性。 樹突狀細胞 MLR 檢定 Preliminary studies show that TY21580 does not stimulate human T cell activation or proliferation. Because CTLA4 activity on T cells correlates with the first signal (TCR/CD3) and the second signal involving B7-CD28/CTLA-4, human PBMC were selected and the activity of TY21580 in the presence of low concentrations of anti-CD3 was determined. Anti-CD3 antibody (OKT-3) was coated on 96-well plates overnight at 4°C. After washing, 1 x 10 5 freshly isolated human PBMC were added to each well, followed by addition of test article at different concentrations. IL-2 induction was measured 48 hours after stimulation with the human IL-2 ELISA Ready-SET-Go (Invitrogen) kit. IFNγ in the supernatant was measured using the Human IFNγ ELISA Ready-SET-Go (Invitrogen) kit. As shown in Figures 8A and 9 , antibody TY21580 significantly increased human PBMC activation in the presence of anti-CD3, whereas TY21580 alone was inactive. Dendritic cell MLR assay

於三個供體對:D42/D109、D32/D104及D104/D42中使用單核細胞衍生之DC及CD4+ T淋巴細胞進行DC-MLR檢定( 10)。為得到DC細胞,將PBMC藉由密度梯度離心自健康供體單離,及使用陽性選擇商業套組(StemCell)將CD14+單核細胞自PBMC純化。藉由於補充有10%熱滅活FBS、1%盤尼西林(penicillin)/鏈黴素(streptomycin)、20 ng/mL rhGM-CSF及20 ng/mL rhIL-4之RPMI-1640中活體外培養6天使CD14+單核細胞偏向於DC。在第3天用新鮮培養基更換培養基。在第6天於補充有10%熱滅活FBS、1%盤尼西林/鏈黴素及50 ng/mL rhTNF-α之RPMI-1640培養基中誘導DC成熟24小時。將CD4+ T細胞藉由來自另一健康供體之陰性單離物純化。將測試製品滴定為對應濃度(如 10中所示)。將所收集之DC (1 x 10 4)與含有或不含有經滴定測試製品之異基因CD4+ T細胞(1 x 10 5)共培養。將抗PD1抗體用作DC-MLR檢定之陽性對照。於共培養後之第5天,使用人類IFNγ Ready-SET-Go ELISA套組藉由ELISA於上清液中量測IFNγ。如 10中所示,抗體TY21580於使用人類CD4+ T細胞及DC之DC-MLR檢定中顯示弱活性。 抗體 TY21580 ADCC 活性 DC-MLR assay was performed using monocyte-derived DC and CD4+ T lymphocytes in three donor pairs: D42/D109, D32/D104 and D104/D42 ( Figure 10 ). To obtain DC cells, PBMC were isolated from healthy donors by density gradient centrifugation, and CD14+ monocytes were purified from PBMC using a positive selection commercial kit (StemCell). By in vitro culture for 6 days in RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 20 ng/mL rhGM-CSF and 20 ng/mL rhIL-4 CD14+ monocytes are biased toward DCs. Replace the medium with fresh medium on day 3. DC maturation was induced for 24 hours on day 6 in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, and 50 ng/mL rhTNF-α. CD4+ T cells were purified by negative isolates from another healthy donor. The test articles were titrated to corresponding concentrations ( as shown in Figure 10 ). Collected DCs (1 x 10 4 ) were co-cultured with allogeneic CD4+ T cells (1 x 10 5 ) with or without titrated test article. Anti-PD1 antibody was used as a positive control for DC-MLR assay. On day 5 after co-culture, IFNγ was measured in the supernatant by ELISA using the Human IFNγ Ready-SET-Go ELISA Kit. As shown in Figure 10 , antibody TY21580 showed weak activity in the DC-MLR assay using human CD4+ T cells and DCs. ADCC activity of antibody TY21580

將過度表現人類CTLA-4之HEK293F細胞用作靶細胞以評價TY21580介導之ADCC活性。使用人類NK單離套組(StemCell)將人類NK細胞自人類PBMC新鮮單離。將1 x 10 5個NK細胞及1 x 10 4個HEK293F/hCTLA-4細胞(E:T比率10:1)與不同濃度之抗體混合。於培育4小時後,量測LDH以測定ADCC活性。然後使用下列公式計算溶胞%:溶胞% = [(實驗釋放) – Ave (靶+ NK)]/[Ave (靶最大值) – Ave (僅靶)] ×100%。如 11A B中所示,TY21580顯示較參考抗體(TAC2114)更強ADCC活性。同型對照顯示無任何ADCC活性。 HEK293F cells overexpressing human CTLA-4 were used as target cells to evaluate TY21580-mediated ADCC activity. Human NK cells were freshly isolated from human PBMC using the Human NK Isolation Kit (StemCell). 1 x 10 5 NK cells and 1 x 10 4 HEK293F/hCTLA-4 cells (E:T ratio 10:1) were mixed with different concentrations of antibodies. After 4 hours of incubation, LDH was measured to determine ADCC activity. Then calculate % lysis using the following formula: % lysis = [(experimental release) – Ave (target + NK)]/[Ave (target max) – Ave (target only)] × 100%. As shown in Figures 11A - B , TY21580 showed stronger ADCC activity than the reference antibody (TAC2114). Isotype controls showed no ADCC activity.

亦使用人類Treg細胞(A,供體#96;B,供體#12)及NK細胞(A,供體#99;B,供體#05)評價ADCC活性。為得到人類Treg細胞,將人類PBMC自健康供體新鮮單離,及使用EASYSEP™人類調節T細胞濃化套組(StemCell Technologies)陰性選擇Treg細胞。在IL-2之存在下將經濃化人類Treg細胞進一步藉由CD3/CD28刺激擴增,及藉由CD25及FOXP3染色及FACS分析證實。為得到人類NK細胞,將人類PBMC自另一健康供體新鮮單離,及使用人類NK單離套組(StemCell Technologies)單離NK細胞。在37℃下將人類Treg細胞用10 µM鈣黃綠素-AM (Invitrogen)標記30分鐘。於洗滌三次後,將經標記之Treg細胞與不同濃度之測試製品混合,接著添加NK細胞。將1 x 10 5個NK細胞及1 x 10 4個經標記之人類Treg細胞添加至96孔板之孔中,及混合以使E:T比率10:1。於培育4小時後,量測上清液中之鈣黃綠素-AM濃度以使用下列公式測定ADCC活性:溶胞% = [(實驗釋放) – Ave (靶+ NK)]/[Ave (靶最大值) – Ave (僅靶)] ×100%。如 12A B中所示,抗體TY21580顯示較參考抗體(TAC2114)更強ADCC活性。同型對照顯示無ADCC活性。 TY21580 CDC 活性 ADCC activity was also evaluated using human Treg cells (A, donor #96; B, donor #12) and NK cells (A, donor #99; B, donor #05). To obtain human Treg cells, human PBMC were freshly isolated from healthy donors, and Treg cells were negatively selected using the EASYSEP™ Human Regulatory T Cell Enrichment Kit (StemCell Technologies). Enriched human Treg cells were further expanded by CD3/CD28 stimulation in the presence of IL-2 and confirmed by CD25 and FOXP3 staining and FACS analysis. To obtain human NK cells, human PBMC were freshly isolated from another healthy donor, and NK cells were isolated using a human NK isolation kit (StemCell Technologies). Human Treg cells were labeled with 10 µM calcein-AM (Invitrogen) for 30 min at 37°C. After washing three times, the labeled Treg cells were mixed with different concentrations of the test product, and then NK cells were added. Add 1 x 10 5 NK cells and 1 x 10 4 labeled human Treg cells to the wells of a 96-well plate and mix to achieve an E:T ratio of 10:1. After 4 hours of incubation, the calcein-AM concentration in the supernatant was measured to determine ADCC activity using the following formula: % lysis = [(experimental release) – Ave (target + NK)]/[Ave (target maximum) ) – Ave (target only)] ×100%. As shown in Figures 12A - B , antibody TY21580 showed stronger ADCC activity than the reference antibody (TAC2114). Isotype control showed no ADCC activity. CDC activity of TY21580

在37℃下將過度表現人類CTLA-4之HEK293F細胞用10 µM鈣黃綠素-AM (Invitrogen)標記30分鐘。向96孔板之孔中,將不同濃度之抗體與1x10 4個經標記之細胞及5%正常人類血清補體(NHSC, Quidel)混合。於培育5小時後,量測上清液中之鈣黃綠素-AM以測定CDC活性( 13)。 HEK293F cells overexpressing human CTLA-4 were labeled with 10 µM calcein-AM (Invitrogen) for 30 minutes at 37°C. Different concentrations of antibodies were mixed with 1x10 4 labeled cells and 5% normal human serum complement (NHSC, Quidel) into the wells of a 96-well plate. After 5 hours of incubation, calcein-AM in the supernatant was measured to determine CDC activity ( Figure 13 ).

將人類PBMC自健康供體(供體#57)新鮮單離。使用EasySep人類CD4 +T細胞濃化套組(StemCell)單離CD4 +T細胞,及用PMA (50 ng/mL)+離子黴素(Ionmycin)(1 µM)刺激20小時以誘導細胞表面上之CTLA-4表現。然後在37℃下將經活化之人類CD4 +T細胞用10 µM鈣黃綠素-AM (Invitrogen)標記30分鐘。向96孔板之孔中,將不同濃度之抗體與1 x 10 4個經標記之人類CD4+ T細胞及5%正常人類血清補體(NHSC, Quidel)混合。於培育5小時後,量測上清液中之鈣黃綠素-AM以測定CDC活性( 14)。TY21580顯示對HEK293F/hCTLA-4細胞或經活化之人類T細胞無CDC活性。 Human PBMC were freshly isolated from a healthy donor (Donor #57). CD4 + T cells were isolated using the EasySep Human CD4 + T Cell Enrichment Kit (StemCell) and stimulated with PMA (50 ng/mL) + Ionmycin (1 µM) for 20 hours to induce expression on the cell surface. CTLA-4 performance. Activated human CD4 + T cells were then labeled with 10 µM calcein-AM (Invitrogen) for 30 minutes at 37°C. Different concentrations of antibodies were mixed with 1 x 10 4 labeled human CD4+ T cells and 5% normal human serum complement (NHSC, Quidel) into the wells of a 96-well plate. After 5 hours of incubation, calcein-AM in the supernatant was measured to determine CDC activity ( Figure 14 ). TY21580 shows no CDC activity on HEK293F/hCTLA-4 cells or activated human T cells.

一併考慮,此等結果指示本文中所述之抗體能以高親和力及特異性結合至人類CTLA4,且此等抗體有效阻斷CTLA4與其同源配位體CD86及CD80之相互作用。亦顯示該等抗體具有與來自不同物種之CTLA4之交叉反應性。此外,結合至CTLA4可調節T細胞活化且誘導對表現CTLA4之細胞(諸如Treg)之ADCC活性。 實例 4 IgG 轉變之抗體之活體內表徵 Taken together, these results indicate that the antibodies described herein can bind to human CTLA4 with high affinity and specificity, and that these antibodies effectively block the interaction of CTLA4 with its cognate ligands CD86 and CD80. The antibodies were also shown to have cross-reactivity with CTLA4 from different species. Furthermore, binding to CTLA4 modulates T cell activation and induces ADCC activity on cells expressing CTLA4, such as Tregs. Example 4 : In vivo characterization of IgG- converted antibodies

如以上實例中所述,抗體之物種交叉反應性(人類及小鼠)允許測定抗體於多個同源腫瘤模型中之抗腫瘤效能,該等模型包括MC38及CT26結腸直腸腫瘤模型、H22肝腫瘤模型、PAN02胰腫瘤模型及3LL肺腫瘤模型。 MC38 結腸直腸腫瘤模型中之抗腫瘤功效 As described in the examples above, the species cross-reactivity of the antibodies (human and mouse) allows determination of the anti-tumor potency of the antibodies in multiple syngeneic tumor models, including MC38 and CT26 colorectal tumor models, H22 liver tumors model, PAN02 pancreatic tumor model and 3LL lung tumor model. Anti-tumor efficacy in the MC38 colorectal tumor model

將C57BL/6小鼠(n=8隻/組,雌性,6至8週齡)皮下接種MC38 (NTCC-MC38)鼠科結腸癌細胞。當建立腫瘤(80 mm 3)時,用同型對照抗體及三種不同劑量之抗體TY21580藉由腹膜內注射開始處理,一週兩次持續三週。一週兩次監測腫瘤生長並報告為隨時間之平均腫瘤體積± s.e.m. ( 15A C)。如 15A中所示,相較於同型對照抗體,TY21580展示強效活體內抗腫瘤活性,其中腫瘤在所有三種劑量下完全消退。如 15B中所示,於處理後多達六十天,10 mg/kg之TY21580組中之8/8小鼠,2.5 mg/kg之TY21580組中之7/8,0.5 mg/kg之TY21580組中之6/8保持無腫瘤。當將10 mg/kg之TY21580組中之小鼠再激發時,證明對MC38腫瘤細胞之持久免疫記憶,如 15C中所示。 CT26 結腸直腸腫瘤模型中之抗腫瘤功效 C57BL/6 mice (n=8/group, female, 6 to 8 weeks old) were inoculated subcutaneously with MC38 (NTCC-MC38) murine colon cancer cells. When tumors (80 mm 3 ) were established, treatment with isotype control antibody and three different doses of antibody TY21580 began by intraperitoneal injection twice a week for three weeks. Tumor growth was monitored twice weekly and reported as mean tumor volume ± s.e.m. over time ( Figure 15A to C ). As shown in Figure 15A , TY21580 demonstrated potent in vivo anti-tumor activity compared to the isotype control antibody, with complete tumor regression at all three doses. As shown in Figure 15B , up to sixty days after treatment, 8/8 mice in the 10 mg/kg TY21580 group, 7/8 in the 2.5 mg/kg TY21580 group, and 0.5 mg/kg TY21580 6/8 of the group remained tumor-free. When mice in the TY21580 group at 10 mg/kg were rechallenged, long-lasting immune memory against MC38 tumor cells was demonstrated, as shown in Figure 15C . Anti-tumor efficacy in CT26 colorectal tumor model

將BALB/c小鼠(n=8隻/組,雌性,7至8週齡)皮下接種CT26 (上海生物科學研究院(Shanghai Institutes for Biological Sciences))鼠科結腸癌細胞。當建立腫瘤(70 mm 3)時,用同型對照抗體及兩種不同劑量之抗體TY21580藉由腹膜內注射開始處理,一週兩次。一週兩次監測腫瘤生長並報告為隨時間之平均腫瘤體積± s.e.m.。如 16中所示,相較於同型對照抗體,TY21580展示強效活體內抗腫瘤活性,在低至0.1至1 mg/kg之劑量下具有幾乎100%抑制。 H22 肝腫瘤模型中之抗腫瘤功效 BALB/c mice (n=8/group, female, 7 to 8 weeks old) were inoculated subcutaneously with CT26 (Shanghai Institutes for Biological Sciences) murine colon cancer cells. When tumors were established (70 mm 3 ), treatment was started by intraperitoneal injection twice a week with isotype control antibody and two different doses of antibody TY21580. Tumor growth was monitored twice a week and reported as mean tumor volume ± s.e.m. over time. As shown in Figure 16 , TY21580 demonstrated potent in vivo anti-tumor activity compared to the isotype control antibody, with almost 100% inhibition at doses as low as 0.1 to 1 mg/kg. Anti-tumor efficacy in H22 liver tumor model

將BALB/c小鼠(n=5隻/組,雌性,7至8週齡)皮下接種H22 (中國典型培養物保藏中心(China Center for Type Culture Collection))鼠科肝癌細胞。當建立腫瘤(60 mm 3)時,用同型對照抗體、在三種不同劑量(0.1 mg/kg、1 mg/kg、5 mg/kg)下之TY21586、或在兩種不同劑量(0.1 mg/kg、1 mg/kg)下之抗體TY21580藉由腹膜內注射開始處理,一週兩次。一週兩次監測腫瘤生長並報告為隨時間之平均腫瘤體積± s.e.m.。如 17中所示,相較於同型對照抗體,TY21580及TY21586均以劑量依賴性方式展示強效活體內抗腫瘤活性。當在相同劑量下比較時,TY21580於此腫瘤模型中較TY21586更強效。此外,在1 mg/kg下之TY21580投與導致腫瘤消退。 於路易士肺腫瘤模型中之抗腫瘤功效 BALB/c mice (n=5/group, female, 7 to 8 weeks old) were subcutaneously inoculated with H22 (China Center for Type Culture Collection) murine hepatoma cells. When tumors (60 mm 3 ) were established, cells were treated with isotype control antibody, TY21586 at three different doses (0.1 mg/kg, 1 mg/kg, 5 mg/kg), or TY21586 at two different doses (0.1 mg/kg , 1 mg/kg) of antibody TY21580 was initiated by intraperitoneal injection twice a week. Tumor growth was monitored twice weekly and reported as mean tumor volume ± s.e.m. over time. As shown in Figure 17 , compared to the isotype control antibody, both TY21580 and TY21586 exhibited potent in vivo anti-tumor activity in a dose-dependent manner. When compared at the same dose, TY21580 was more potent than TY21586 in this tumor model. Furthermore, administration of TY21580 at 1 mg/kg resulted in tumor regression. Anti-tumor efficacy in Lewis lung tumor model

將C57BL/6小鼠(n=6隻/組,雌性,8週齡)皮下接種路易士 (JenNio Bio, Guandong, China)鼠科肺癌細胞。當建立腫瘤(70 mm 3)時,用同型對照抗體、或均在5 mg/kg之劑量下之抗體TY21580、TY21687、TY21680或TY21691藉由腹膜內注射開始處理,一週兩次。一週兩次監測腫瘤生長並報告為隨時間之平均腫瘤體積± s.e.m.。如 18中所示,相較於同型對照抗體,抗體TY21580、TY21687及TY21680顯示腫瘤生長之顯著抑制,而抗體TY21691不顯示強效抗腫瘤活性。 PAN02 胰腫瘤模型中之抗腫瘤功效 C57BL/6 mice (n=6/group, female, 8 weeks old) were subcutaneously inoculated with Lewis (JenNio Bio, Guandong, China) murine lung cancer cells. When tumors (70 mm3 ) were established, treatment was initiated by intraperitoneal injection twice a week with isotype control antibody, or antibodies TY21580, TY21687, TY21680 or TY21691, each at a dose of 5 mg/kg. Tumor growth was monitored twice a week and reported as mean tumor volume ± s.e.m. over time. As shown in Figure 18 , compared to the isotype control antibody, antibodies TY21580, TY21687 and TY21680 showed significant inhibition of tumor growth, while antibody TY21691 did not show potent anti-tumor activity. Anti-tumor efficacy in PAN02 pancreatic tumor model

將C57BL/6小鼠(n=8隻/組,雌性,6週齡)皮下接種PAN-02 (CAMS細胞培養中心)鼠科胰癌細胞。當建立腫瘤(85 mm 3)時,用同型對照抗體、或在三種不同劑量(0.5 mg/kg、2 mg/kg、0.5 mg/kg)下之抗體TY21580藉由腹膜內注射開始處理,一週兩次。一週兩次監測腫瘤生長並報告為隨時間之平均腫瘤體積± s.e.m.。如 19中所示,相較於同型對照抗體,TY21580以劑量依賴性方式顯示強效抗腫瘤活性。 抗體 TY21580 單藥療法或與抗 CD137 抗體組合於 3LL 肺腫瘤模型中之抗腫瘤功效 C57BL/6 mice (n=8/group, female, 6 weeks old) were subcutaneously inoculated with PAN-02 (CAMS Cell Culture Center) murine pancreatic cancer cells. When tumors (85 mm 3 ) were established, treatment with isotype control antibody, or antibody TY21580 at three different doses (0.5 mg/kg, 2 mg/kg, 0.5 mg/kg) was started by intraperitoneal injection twice a week. Second-rate. Tumor growth was monitored twice a week and reported as mean tumor volume ± s.e.m. over time. As shown in Figure 19 , TY21580 showed potent anti-tumor activity in a dose-dependent manner compared to the isotype control antibody. Anti-tumor efficacy of antibody TY21580 monotherapy or in combination with anti -CD137 antibody in 3LL lung tumor model

將C57BL/6小鼠(n=10隻/組,雌性,6至8週齡)皮下接種3LL (JCRB)鼠科肺癌細胞。當建立腫瘤(75 mm 3)時,用同型對照抗體、TY21580 (10 mg/kg)、抗CD137 (10 mg/kg)、或TY21580與抗CD137之組合藉由腹膜內注射開始處理,一週兩次。抗CD137為所開發之專有單株抗體,其具有結合人類及鼠科CD137二者之能力(參見PCT申請案號PCT/CN2017/098332,其全文以引用的方式併入本文中)。一週兩次監測腫瘤生長並報告為隨時間之平均腫瘤體積± s.e.m.。如 20A B中所示,相較於同型對照抗體,TY21580及抗CD137均顯示強效抗腫瘤活性,且該組合抑制腫瘤生長超過單獨任一單藥療法。 具有對 TY21580 之完全反應之小鼠之再激發 C57BL/6 mice (n=10/group, female, 6 to 8 weeks old) were inoculated subcutaneously with 3LL (JCRB) murine lung cancer cells. When tumors (75 mm3 ) were established, treatment was initiated by intraperitoneal injection twice a week with isotype control antibody, TY21580 (10 mg/kg), anti-CD137 (10 mg/kg), or a combination of TY21580 and anti-CD137 . Anti-CD137 is a proprietary monoclonal antibody developed with the ability to bind both human and murine CD137 (see PCT Application No. PCT/CN2017/098332, the entire text of which is incorporated herein by reference). Tumor growth was monitored twice a week and reported as mean tumor volume ± s.e.m. over time. As shown in Figure 20A - B , both TY21580 and anti-CD137 showed potent anti-tumor activity compared to isotype control antibodies, and the combination inhibited tumor growth more than either monotherapy alone. Rechallenge of mice with complete response to TY21580

將BALB/c小鼠(n=8隻/組,雌性,7至8週齡)皮下接種H22 (中國典型培養物保藏中心)鼠科肝癌細胞。當建立腫瘤(60 mm 3)時,用同型對照抗體、或在兩種不同劑量(1 mg/kg、10 mg/kg)下之抗體TY21580藉由腹膜內注射開始處,一週兩次持續3週理。一週兩次監測腫瘤生長並報告為隨時間之平均腫瘤體積± s.e.m.。相較於同型對照抗體,在兩種劑量下之TY21580於最後劑量幾天後導致完全腫瘤消退,及於處理60天後小鼠保持無腫瘤。然後在第60天將10 mg/kg之TY21580處理組中之小鼠用H22腫瘤細胞於相反腹脇部中皮下再激發,及監測腫瘤生長。如 21中所示,於用相同腫瘤細胞再激發後此等小鼠保持無腫瘤,表明於此等小鼠中發展特定抗腫瘤記憶。同時用相同數目之H22腫瘤細胞接種之初始小鼠建立再激發對照組,及其腫瘤快速生長。 抗體藥物動力學 BALB/c mice (n=8/group, female, 7 to 8 weeks old) were subcutaneously inoculated with H22 (China Type Culture Collection Center) murine hepatoma cells. When tumors (60 mm 3 ) were established, the isotype control antibody, or the antibody TY21580 at two different doses (1 mg/kg, 10 mg/kg) was started by intraperitoneal injection twice a week for 3 weeks. reason. Tumor growth was monitored twice a week and reported as mean tumor volume ± s.e.m. over time. Compared to the isotype control antibody, TY21580 at both doses resulted in complete tumor regression days after the final dose, and mice remained tumor-free after 60 days of treatment. Mice in the 10 mg/kg TY21580 treated group were then re-challenged subcutaneously with H22 tumor cells in the opposite flanks on day 60, and tumor growth was monitored. As shown in Figure 21 , these mice remained tumor-free after rechallenge with the same tumor cells, indicating the development of specific anti-tumor memory in these mice. At the same time, the initial mice inoculated with the same number of H22 tumor cells were used to establish a rechallenge control group, and their tumors grew rapidly. Antibody Pharmacokinetics

於BALB/c雌性小鼠(約8週齡)中進行抗體TY21585、TY21586、TY21580及TY21591之藥物動力學研究。對三隻小鼠/組藉由尾靜脈注射靜脈內注射10 mg/kg之測試抗體。於給藥後1小時、8小時、48小時、168小時、336小時及500小時時收集血液樣品(約20 μL/樣品)。自無抗體投與之三隻初始雌性小鼠收集空白對照血液。藉由ELISA測定各測試抗體之血清濃度,其中使用CTLA4-His-Fc捕獲,及使用經HRP標記之抗人類IgG (Fab特異性)抗體(Sigma)檢測。如 22中所示,TY21586展示於小鼠中與TAC2114可比較的藥物動力學,而TY21585、TY21580及TY21591更快被清除。 Pharmacokinetic studies of antibodies TY21585, TY21586, TY21580 and TY21591 were conducted in BALB/c female mice (approximately 8 weeks old). Three mice/group were injected intravenously with 10 mg/kg of test antibody via tail vein injection. Blood samples (approximately 20 μL/sample) were collected at 1 hour, 8 hours, 48 hours, 168 hours, 336 hours and 500 hours after administration. Blank control blood was collected from three naive female mice administered no antibody. Serum concentrations of each test antibody were determined by ELISA using CTLA4-His-Fc capture and detection using HRP-labeled anti-human IgG (Fab specific) antibodies (Sigma). As shown in Figure 22 , TY21586 exhibited comparable pharmacokinetics to TAC2114 in mice, while TY21585, TY21580, and TY21591 were cleared more quickly.

亦於初始食蟹獼猴中進行TY21586及TY21580之藥物動力學研究。藉由靜脈內一次全劑量注射在10 mg/kg下對一隻雌性及一隻雄性猴投與各抗體。在預劑量(0小時)及給藥後0.25小時、1小時、8小時、24小時、72小時、120小時、168小時、240小時、336小時、504小時及672小時時收集血清樣品。藉由ELISA測定TY21586及TY21580之血清濃度,其中使用CTLA4-His-Fc捕獲,及使用經HRP標記之抗人類IgG (Fab特異性)抗體(Sigma)檢測。如 23 24中所示,相較於TY21586,TY21580於猴中更快清除,可能由於此等動物中觀察到之抗藥物抗體之快速增加。 重複給藥毒性研究 Pharmacokinetic studies of TY21586 and TY21580 were also conducted in initial cynomolgus macaques. Each antibody was administered to one female and one male monkey by a single intravenous full-dose injection at 10 mg/kg. Serum samples were collected at pre-dose (0 hours) and 0.25 hours, 1 hour, 8 hours, 24 hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours and 672 hours post-dose. Serum concentrations of TY21586 and TY21580 were determined by ELISA using CTLA4-His-Fc capture and detection using HRP-labeled anti-human IgG (Fab specific) antibodies (Sigma). As shown in Figures 23 and 24 , TY21580 was cleared more rapidly in monkeys than TY21586, possibly due to the rapid increase in anti-drug antibodies observed in these animals. Repeated dose toxicity studies

於正常BALB/c小鼠中進行TY21580之重複給藥毒性。在第1天、第4天、第7天及第11天腹膜內(10 mL/kg)投與媒劑對照或抗體TY21580 (在25 mg/kg或50 mg/kg下)。各組包含五隻雌性小鼠及五隻雄性小鼠(五週齡)。每日監測小鼠異常行為及症狀,及每日量測食物攝取及體重。在第14天,將動物安樂死用於驗屍後檢查及其他分析。自各動物收集血液,其中每組收集之多達六種血液樣品(三種雄性、三種雌性)用於血液學(RBC、血小板、WBC、WBC差分)及/或血液生物化學(ALT、AST、GLB、ALP及LDH等)分析。收集來自各小鼠之下列器官並稱重:心臟、肺、胸腺、肝、脾、腎、睾丸及卵巢。將每組來自6種動物(三隻雄性、三隻雌性)之肝樣品固定於FFPE中。製備用於肝組織之FFPE塊,切片及H&E染色用於組織病理學分析。Repeated dose toxicity of TY21580 was performed in normal BALB/c mice. Vehicle control or antibody TY21580 (at 25 mg/kg or 50 mg/kg) was administered intraperitoneally (10 mL/kg) on days 1, 4, 7, and 11. Each group included five female mice and five male mice (five weeks old). Abnormal behaviors and symptoms of mice were monitored daily, and food intake and body weight were measured daily. On day 14, animals were euthanized for post-mortem examination and other analyses. Blood was collected from each animal, with up to six blood samples per group (three male, three female) collected for hematology (RBC, platelets, WBC, WBC differential) and/or blood biochemistry (ALT, AST, GLB, ALP and LDH, etc.) analysis. The following organs from each mouse were collected and weighed: heart, lungs, thymus, liver, spleen, kidneys, testes, and ovaries. Liver samples from each group of six animals (three males, three females) were fixed in FFPE. FFPE blocks of liver tissue were prepared, sectioned, and H&E stained for histopathological analysis.

在整個研究之生命週期期間,未觀察到異常行為,或非預定之動物死亡。相較於媒劑對照,TY21580不影響動物之食物攝取及體重。驗屍後檢查亦不顯示處理組小鼠在兩種劑量水平下之任何明顯病變,除了脾重量於TY21580處理組中增加( 25A B)。血液學分析不顯示任何顯著變化,如由用TY21580處理之小鼠中測試之血液生物化學參數所指示。於來自小鼠之肝之組織病理學切片中未發現明顯異常( 26)。總之,TY21580於此研究中良好耐受,其中於小鼠中未觀察到顯著毒性。 No abnormal behavior or unintended animal deaths were observed throughout the lifespan of the study. Compared to the vehicle control, TY21580 did not affect the animals' food intake and body weight. Post-mortem examination also did not reveal any obvious lesions in the treated mice at both dose levels, except for an increase in spleen weight in the TY21580-treated group ( Figures 25A - B ). Hematological analysis did not show any significant changes as indicated by the blood biochemical parameters tested in mice treated with TY21580. No obvious abnormalities were found in histopathological sections of livers from mice ( Figure 26 ). Overall, TY21580 was well tolerated in this study, with no significant toxicity observed in mice.

一併考慮,此等結果指示本文中所述之CTLA4抗體對小鼠極安全,具有強效抗腫瘤活性,且可誘導對腫瘤細胞之持久免疫記憶。 實例 5 :抗體可展性特性 Taken together, these results indicate that the CTLA4 antibodies described herein are extremely safe in mice, have potent antitumor activity, and can induce long-lasting immune memory on tumor cells. Example 5 : Antibody developability properties

用於可展性評估,將經純化TY21586及TY21580交換至儲備緩衝液(20 mM組胺酸,pH 5.5)中。於儲備緩衝液中進行所有實驗,包括溶解度、在加速應力條件下之穩定性及示差掃描螢光(DSF)測試。針對所有SEC-HPLC分析,使用TSKgel管柱(Tosoh Bioscience G3000SWxl)。 抗體溶解度 For spreadability assessment, purified TY21586 and TY21580 were exchanged into stock buffer (20 mM histidine, pH 5.5). All experiments, including solubility, stability under accelerated stress conditions, and differential scanning fluorescence (DSF) testing, were performed in stock buffer. For all SEC-HPLC analyses, TSKgel columns (Tosoh Bioscience G3000SWxl) were used. Antibody solubility

將含有抗體TY21586或TY21580之樣品於儲備緩衝液中在大於100 mg/mL之濃度下調配,及測試高分子量(HMW)蛋白質聚集體之量( 8)。然後於儲備緩衝液中將抗體調整至約12 mg/mL。然後將樣品(各12 μg)通過SEC-HPLC檢定以檢測高分子量蛋白質聚集體。如 27中所示,在高濃度(100 mg/mL以上)下調配之抗體30分鐘未觀察到HMW聚集體之顯著增加。 8 :抗體溶解度 抗體名稱 濃度(mg/mL) 聚集(HMW %) TY21586 197.8 0 TY21580 126.0 +0.10 在加速應力條件下之抗體穩定性 Samples containing antibody TY21586 or TY21580 were formulated in stock buffer at concentrations greater than 100 mg/mL and tested for the amount of high molecular weight (HMW) protein aggregates ( Table 8 ). Antibodies were then adjusted to approximately 12 mg/mL in stock buffer. Samples (12 μg each) were then assayed by SEC-HPLC to detect high molecular weight protein aggregates. As shown in Figure 27 , no significant increase in HMW aggregates was observed for antibodies formulated at high concentrations (above 100 mg/mL) for 30 minutes. Table 8 : Antibody solubility Antibody name Concentration(mg/mL) Aggregation (HMW %) TY21586 197.8 0 TY21580 126.0 +0.10 Antibody stability under accelerated stress conditions

亦在加速應力條件下檢查抗體穩定性。此等實驗之結果概述於 9 28中。TY21586及TY21580於6個凍(-80℃)熔(室溫)循環後保持穩定。於50℃下7天後,HMW聚集體或低分子量(LMW)片段存在小變化。於長期时程實驗(40℃持續多達28天)中,TY21586及TY21580保持穩定,且HMW聚集體或LMW片段無顯著增加。 9 :在加速應力條件下之 HMX% 變化 抗體名稱 凍熔 6 個循環 50 7 d 50 7 d LMW% 40 28 d 40 28 d LMW% TY21586 5.34 0 2.14 -0.22 1.37 TY21580 0.49 0 2.02 0.02 0.43 Antibody stability was also examined under accelerated stress conditions. The results of these experiments are summarized in Table 9 and Figure 28 . TY21586 and TY21580 remain stable after 6 freeze (-80°C) and melt (room temperature) cycles. After 7 days at 50°C, there are small changes in HMW aggregates or low molecular weight (LMW) fragments. In long-term time course experiments (up to 28 days at 40°C), TY21586 and TY21580 remained stable with no significant increase in HMW aggregates or LMW fragments. Table 9 : HMX% changes under accelerated stress conditions Antibody name 6 cycles of freeze-thaw 50 7 days 50 7d LMW% 40 28 days 40 28 d LMW% TY21586 5.34 0 2.14 -0.22 1.37 TY21580 0.49 0 2.02 0.02 0.43

此外,熱穩定性(如由示差掃描螢光(DSF)所量測)顯示TY21586及TY21580二者在高達至少約55℃下係穩定。轉變中點(Tm) (幾乎所有蛋白質域之展開轉變發生所在之特徵溫度)示於下 10中。 10 :藉由 DSF 之熱穩定性 抗體名稱 Tm 開始( ℃) Tm1 ( ℃) Tm2 ( ℃) TY21586-16Z01 55 67.26 76.40 TY21580-16Z01 55 67.64 76.50 Furthermore, thermal stability, as measured by differential scanning fluorescence (DSF), shows that both TY21586 and TY21580 are stable up to at least about 55°C. The transition midpoint (Tm) (the characteristic temperature at which the unfolding transition of nearly all protein domains occurs) is shown in Table 10 below. Table 10 : Thermal stability by DSF Antibody name Tm starts ( ℃) Tm1( ℃) Tm2 ( ℃) TY21586-16Z01 55 67.26 76.40 TY21580-16Z01 55 67.64 76.50

最後,發現於離心後抗體TY21586及TY21580之最高可達成濃度各自係超過 197.8 mg/mL及126.0 mg/mL。Finally, it was found that the highest achievable concentrations of antibodies TY21586 and TY21580 after centrifugation were over 197.8 mg/mL and 126.0 mg/mL, respectively.

一併考慮,此等結果指示甚至在無調配物最佳化下,CTLA4抗體TY21586及TY21580具有優異可展性特性。 實例 6 :識別 TY21580 衍生之 CTLA4 可活化抗體之自阻斷肽的方法 Taken together, these results indicate that CTLA4 antibodies TY21586 and TY21580 have excellent spreadability properties even without formulation optimization. Example 6 : Method for identifying self-blocking peptides of TY21580 -derived CTLA4- activatable antibodies

本文中描述一種新穎系統,其已經設計及執行以有效發現具有良好可展性之掩蔽部分。於此系統中,靶抗體片段Fab ( 29)或scFv ( 30)首先在酵母表面展示,及經證實功能結合至其抗原。然後將改良之肽庫直接融合至CTLA4抗體(TY21580)之輕鏈之N端,及構築在酵母表面上展示融合蛋白之酵母庫。然後該酵母庫經歷若干輪基於FACS之篩選:首先將具有與抗原低結合之酵母純系濃化,然後將經濃化酵母純系用蛋白酶處理以移除N端肽,及選擇具有與抗原高結合之純系( 29 30)。於4至5輪分選後,自此等純系提取質粒及通過DNA定序證實掩蔽肽序列。 實例 7 :設計 CTLA4 可活化抗體之受限肽庫 (CPL) This paper describes a novel system that has been designed and implemented to efficiently discover masked parts with good malleability. In this system, the target antibody fragment Fab ( Figure 29 ) or scFv ( Figure 30 ) is first displayed on the yeast surface, and functional binding to its antigen is confirmed. The improved peptide library was then directly fused to the N-terminus of the light chain of the CTLA4 antibody (TY21580), and a yeast library displaying the fusion protein on the yeast surface was constructed. The yeast library then undergoes several rounds of FACS-based screening: yeast lines with low binding to the antigen are first concentrated, then the concentrated yeast lines are treated with protease to remove the N-terminal peptide, and yeast lines with high binding to the antigen are selected. Pure line ( Figures 29 and 30 ). After 4 to 5 rounds of sorting, plasmids were extracted from these pure lines and the masking peptide sequence was confirmed by DNA sequencing. Example 7 : Design of a constrained peptide library (CPL) of CTLA4- activatable antibodies

設計四種示例性受限肽庫(CPL) ( 11)。 11 :經設計之 CPL CPL 名稱: 胺基酸序列: CPL010 EVGSY(Z 6)C(Z 6)C(Z 2)SGRSA (SEQ ID NO: 152) CPL011 EVGSY(Z 6)C(X 6)C(Z 2)SGRSA (SEQ ID NO: 153) CPL012 EVGSY(Z 6)C(Z 8)C(Z 2)SGRSA (SEQ ID NO: 154) CPL013 EVGSY(Z 6)C(X 8)C(Z 2)SGRSA (SEQ ID NO: 155) 各X獨立地為選自由以下組成之群之胺基酸:A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、及Y;各Z獨立地為選自由以下組成之群之胺基酸:D、A、Y、S、T、N、I、L、F、V、H、及P Four exemplary constrained peptide libraries (CPLs) were designed ( Table 11 ). Table 11 : Designed CPL CPL name: Amino acid sequence: CPL010 EVGSY(Z 6 )C(Z 6 )C(Z 2 )SGRSA (SEQ ID NO: 152) CPL011 EVGSY(Z 6 )C(X 6 )C(Z 2 )SGRSA (SEQ ID NO: 153) CPL012 EVGSY(Z 6 )C(Z 8 )C(Z 2 )SGRSA (SEQ ID NO: 154) CPL013 EVGSY(Z 6 )C(X 8 )C(Z 2 )SGRSA (SEQ ID NO: 155) Each X is independently an amino acid selected from the group consisting of: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; each Z is independently an amino acid selected from the group consisting of: D, A, Y, S, T, N, I, L, F, V, H, and P

其核心處為序列Z 6CX 6CZ 2(SEQ ID NO: 137)或Z6CX8CZ2 (SEQ ID NO: 138),及兩個固定半胱胺酸殘基形成二硫鍵以限制肽構形。於經合成之寡核苷酸中,於所有地方除了環內部採用簡併密碼子NHC,其中於CPL011及CPL013中亦採用NNK密碼子。與NNK或NNS密碼子相比,NHC密碼子編碼12個殘基( 12),其包含顯著多樣性,但是缺少化學不穩定殘基甲硫胺酸、色胺酸及半胱胺酸。此外,與NNK或NNS密碼子相比減少之理論多樣性使能構築具由更好覆蓋率之庫。 12 NHC 密碼子 NHC AAC ACC ATC TAC TCC TTC GAC GCC GTC CAC CCC CTC 胺基酸: N T I Y S F D A V H P L Its core is the sequence Z 6 CX 6 CZ 2 (SEQ ID NO: 137) or Z6CX8CZ2 (SEQ ID NO: 138), and two fixed cysteine residues form a disulfide bond to restrict the peptide configuration. In the synthesized oligonucleotide, the degenerate codon NHC is used everywhere except inside the loop, where the NNK codon is also used in CPL011 and CPL013. Compared to the NNK or NNS codons, the NHC codon encodes 12 residues ( Table 12 ), which contains significant diversity but lacks the chemically unstable residues methionine, tryptophan and cysteine. Furthermore, the reduced theoretical diversity compared to NNK or NNS codons enables the construction of libraries with better coverage. Table 12 : NHC codons NHC : AAC ACC ATC TAC TCC TTC GAC GCC GTC CAC CCC CTC Amino acids: N T I Y S F D A V H P L

於此等掩蔽肽序列後為不變裂解肽序列 (SGRSAGGGGSPLGLAGSGGS, SEQ ID NO: 180),該序列含有兩個蛋白酶識別位點:針對蛋白酶尿激酶型纖維蛋白溶酶原活化因子(uPA)之SGRSA (SEQ ID NO: 149),及針對蛋白酶基質金屬蛋白酶-2 (MMP-2)及基質金屬蛋白酶-9 (MMP-9)之PLGLAG (SEQ ID NO: 150)。此等識別位點已於靶向劑之活體內腫瘤細胞特異性活化中由許多組使用 (參見例如,Ke等人(1997) J Biol Chem 272(33):20456-62;Gerspach等人(2006) Cancer Immunol Immunother 55(12):1590-600;及Jiang等人(2004) Proc Natl Acad Sci USA 101(51):17867-72)。在基於酵母之篩選期間,將MMP-9識別序列用菸草蝕刻病毒(TEV)蛋白酶識別序列(ENLYFQG, SEQ ID NO: 151)置換,這由於TEV蛋白酶之可得性及特異性。 Following these masking peptide sequences is the invariant cleavage peptide sequence (SGRSAGGGGSPLGLAGSGGS, SEQ ID NO: 180), which contains two protease recognition sites: SGRSA for the protease urokinase-type plasminogen activator (uPA) (SEQ ID NO: 149), and PLGLAG (SEQ ID NO: 150) against the proteases matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). These recognition sites have been used by many groups in the specific activation of tumor cells in vivo by targeting agents (see, e.g., Ke et al. (1997) J Biol Chem 272(33):20456-62; Gerspach et al. (2006) ) Cancer Immunol Immunother 55(12):1590-600; and Jiang et al. (2004) Proc Natl Acad Sci USA 101(51):17867-72). During the yeast-based screen, the MMP-9 recognition sequence was replaced with the Tobacco Etch Virus (TEV) protease recognition sequence (ENLYFQG, SEQ ID NO: 151) due to the availability and specificity of the TEV protease.

將CPL及不變裂解肽以scFv或Fab形式融合至靶抗體(TY21580)之輕鏈之N端,該靶抗體被連接至酵母表面展示之Aga2蛋白。替代TEV蛋白酶識別位點之納入於識別正確類型之掩蔽肽序列中係重要的,即,在蛋白酶裂解之前阻斷抗原結合,及於蛋白酶裂解後使能抗原結合。下述實例證明,初始於酵母中顯示之可活化抗體之裂解-活化機理於哺乳動物細胞中表現之全IgG分子中複製。 實例 8 :構築及驗證靶向 CTLA4 TY21580 衍生之可活化抗體 功能靶抗體在酵母表面上之展示 CPL and invariant cleavage peptide were fused to the N-terminus of the light chain of the target antibody (TY21580) in the form of scFv or Fab, and the target antibody was linked to the Aga2 protein displayed on the yeast surface. The incorporation of alternative TEV protease recognition sites is important in identifying the correct type of masking peptide sequence, i.e., blocking antigen binding before protease cleavage, and enabling antigen binding after protease cleavage. The following examples demonstrate that the cleavage-activation mechanism of activatable antibodies originally demonstrated in yeast is replicated in full IgG molecules expressed in mammalian cells. Example 8 : Construction and verification of TY21580-derived activatable antibody targeting CTLA4 function . Display of target antibody on yeast surface

使用低副本數目基於CEN/ARS之載體以在釀酒酵母( S. cerevisiae)中之可誘導GAL1-10啟動子之控制下表現靶抗體(靶向人類 CTLA4之抗體TY21580)。scFv之表面展示通過在GAL1啟動子之控制下在其C端融合之Aga2蛋白達成,類似於先前公開之排列(Boder及Wittrup (1997) Nat Biotechnol 15(6):553-7)。針對Fab,其表面展示通過在GAL1啟動子之控制下融合至重鏈(融合VH及CH1)之N端之Aga2蛋白達成,而輕鏈(融合VL及CL)係在GAL10啟動子之控制下。該等Fab通過其與膜錨定之重鏈之締合在酵母表面上展示。 A low copy number CEN/ARS-based vector was used to express the target antibody (antibody TY21580 targeting human CTLA4) under the control of the inducible GAL1-10 promoter in S. cerevisiae . Surface display of scFv was achieved by fusion of the Aga2 protein at its C-terminus under the control of the GAL1 promoter, similar to a previously published arrangement (Boder and Wittrup (1997) Nat Biotechnol 15(6):553-7). For Fab, its surface display is achieved through the Aga2 protein fused to the N-terminus of the heavy chain (fused to VH and CH1) under the control of the GAL1 promoter, while the light chain (fused to VL and CL) is under the control of the GAL10 promoter. These Fabs are displayed on the yeast surface through their association with membrane-anchored heavy chains.

Fab或scFv之表面展示藉由用識別融合親和力標籤之抗體染色來驗證,及使用經生物素化人類CTLA4檢查在酵母上展示之Fab或scFv之功能性。簡言之,於半乳糖介質中誘導48小時後,收集酵母細胞(1x10^6),用PBSA緩衝液洗滌一次,及然後在室溫下用10 nM經生物素化抗原培育1小時。然後將酵母細胞用PBSA緩衝液洗滌兩次,及用PE結合之鏈黴抗生物素(1:500稀釋) (eBioscience #2-4317-87)在4℃下培育30分鐘。然後藉由流動式細胞測量術分析酵母細胞。如 31A B中所示,靶向CTLA4之Fab ( 31A)及scFv ( 31B)均在酵母表面上成功展示,且均能強結合至其抗原。 構築含 CPL 之酵母庫 Surface display of Fab or scFv is verified by staining with antibodies that recognize the fusion affinity tag, and functionality of Fab or scFv displayed on yeast is checked using biotinylated human CTLA4. Briefly, after 48 hours of induction in galactose medium, yeast cells (1x10^6) were collected, washed once with PBSA buffer, and then incubated with 10 nM biotinylated antigen for 1 hour at room temperature. Yeast cells were then washed twice with PBSA buffer and incubated with PE-conjugated streptavidin (1:500 dilution) (eBioscience #2-4317-87) for 30 minutes at 4°C. The yeast cells were then analyzed by flow cytometry. As shown in Figures 31A to B , both CTLA4-targeting Fab ( Figure 31A ) and scFv ( Figure 31B ) were successfully displayed on the yeast surface, and both were able to bind strongly to its antigen. Construction of yeast library containing CPL

通過5個PCR循環將編碼CPL之經合成寡核苷酸與編碼裂解肽之寡核苷酸融合。PCR反應之組成為:1X PrimeSTAR緩衝液、2.5 mM dNTP、各100 μM F-引物及R-引物,及100 μM模板1 (CPL寡核苷酸)及模板2 (編碼裂解肽之寡核苷酸)各者、及2.5 U PrimeSTAR HS DNA聚合酶。使用之PCR程序為:1) 96℃持續5分鐘之1個循環;2) 96℃ (15秒),60℃ (15秒),72℃ (6秒)之5個循環;及3) 72℃持續3分鐘之1個循環。使用核酸外切酶I消化單股DNA,之後通過凝膠電泳純化PCR產物。然後將經純化之PCR產物用BamHI及KpnI消化,及選殖至用相同兩種限制酵素消化之細菌過濾載體中。於過濾載體中,將CPL及裂解肽放在細菌分泌信號肽之下游,及缺少信號序列之β-內醯胺酶(lactamase)之上游。在胺比西林(ampicillin)板上選擇之功能性β-內醯胺酶指示CPL及裂解肽之框架內融合,從而消除引入經合成間併寡核苷酸中之任何框架外誤差(N-1或N-2)。此外,亦自池減少一些差摺疊序列。將連接產物轉形至電活性細菌細胞,及CPL庫之多樣性一般係在5x10^9與1x10^10之間。個別純系之定序指示通過此方法達成極高框架內速率(於許多情況下,幾乎100%)。The synthesized oligonucleotide encoding CPL was fused to the oligonucleotide encoding the cleavage peptide by 5 cycles of PCR. The PCR reaction consisted of: 1X PrimeSTAR buffer, 2.5 mM dNTPs, 100 μM each of F-primer and R-primer, and 100 μM Template 1 (CPL oligonucleotide) and Template 2 (oligonucleotide encoding the cleavage peptide) ) each, and 2.5 U PrimeSTAR HS DNA polymerase. The PCR programs used were: 1) 1 cycle at 96°C for 5 minutes; 2) 5 cycles at 96°C (15 seconds), 60°C (15 seconds), 72°C (6 seconds); and 3) 72°C One cycle lasting 3 minutes. Single-stranded DNA was digested using exonuclease I, followed by purification of the PCR product by gel electrophoresis. The purified PCR product was then digested with BamHI and KpnI and selected into a bacterial filter vector digested with the same two restriction enzymes. In the filter carrier, CPL and cleavage peptide are placed downstream of the bacterial secretion signal peptide and upstream of the β-lactamase lacking the signal sequence. Functional beta-lactamases selected on ampicillin plates direct in-frame fusion of CPL and cleavage peptides, thereby eliminating any out-of-frame errors introduced into the synthetic synthesized oligonucleotides (N-1 or N-2). In addition, some poorly folded sequences are also reduced from the pool. The ligation product is transformed into electroactive bacterial cells, and the diversity of the CPL library is generally between 5x10^9 and 1x10^10. Sequencing of individual pure lines indicates extremely high in-frame rates (in many cases, almost 100%) achieved by this method.

為製備含CPL之酵母庫,自細菌庫提取質粒,及用作編碼CPL及裂解肽之DNA片段之PCR擴增的模板。將經擴增之PCR片段通過凝膠電泳純化,及與表現融合至Aga2之靶抗體之線性化質粒一起轉形至電活化酵母細胞。PCR片段及質粒之兩端之同源序列確保酵母細胞內部之有效同源重組。經構築之酵母庫之多樣性一般為1x10^9至2x10^9。 CTLA4 抗體之掩蔽肽之基於 FACS 之篩選 To prepare a yeast library containing CPL, plasmids were extracted from bacterial libraries and used as templates for PCR amplification of DNA fragments encoding CPL and cleavage peptides. The amplified PCR fragment was purified by gel electrophoresis and transformed into electroactivated yeast cells together with a linearized plasmid expressing the target antibody fused to Aga2. The homologous sequences at both ends of the PCR fragment and plasmid ensure efficient homologous recombination within yeast cells. The diversity of the constructed yeast bank is generally 1x10^9 to 2x10^9. FACS- based screening of masking peptides for CTLA4 antibodies

使用來自CPL酵母庫之總計1x10^8個酵母細胞篩選對靶抗體之掩蔽肽。針對通過MoFlo XDP之各輪分選,收集於半乳糖介質中誘導之酵母細胞,用PBSA緩衝液洗滌一次,及然後用10 nM (於後來輪中減少至1 nM)經生物素化之抗原在室溫下培育1小時。然後將酵母細胞用PBSA緩衝液洗滌兩次,及用PE共軛之鏈黴抗生物素(1:500稀釋) (eBioscience #2-4317-87)在4℃下培育30分鐘。於用PBSA緩衝液再洗滌兩次後,將酵母細胞調整至2-3 OD/mL,及經歷分選。如 32中所示,於第1輪中,使用10 nM經生物素化之CTLA4-Fc,及將弱結合物濃化。於葡萄糖介質中生長後,將來自第1輪之酵母細胞於半乳糖介質中誘導及用AcTEV蛋白酶(6U/OD細胞) (Thermo Fisher Scientific #12575015)在30℃下處理2小時,及將強結合物純化。自第3輪分選開始,將經生物素化之CTLA4-Fc之濃度降低至1 nM,及收集弱結合物。在第4輪,亦將酵母細胞之溶離份用AcTEV平行處理,以驗證蛋白酶裂解介導之靶抗體之活化。如 32中所示,顯而易見AcTEV裂解導致強結合至抗原之細胞群體之急劇增加,表明該篩選策略係有效。將來自第5輪分選之單純系平板接種在選擇性培養基上,及個別生長以進一步證實裂解介導之經活化抗原結合。 A total of 1x10^8 yeast cells from the CPL yeast library were used to screen masking peptides against target antibodies. For each round of sorting by MoFlo Incubate at room temperature for 1 hour. Yeast cells were then washed twice with PBSA buffer and incubated with PE-conjugated streptavidin (1:500 dilution) (eBioscience #2-4317-87) for 30 minutes at 4°C. After two more washes with PBSA buffer, the yeast cells were adjusted to 2-3 OD/mL and subjected to sorting. As shown in Figure 32 , in round 1, 10 nM biotinylated CTLA4-Fc was used, and weak binders were concentrated. After growth in glucose medium, yeast cells from round 1 were induced in galactose medium and treated with AcTEV protease (6U/OD cells) (Thermo Fisher Scientific #12575015) for 2 hours at 30°C and strong binding material purification. Beginning with the 3rd round of sorting, the concentration of biotinylated CTLA4-Fc was reduced to 1 nM, and weak binders were collected. In round 4, yeast cell lysates were also treated with AcTEV in parallel to verify protease cleavage-mediated activation of target antibodies. As shown in Figure 32 , it is apparent that AcTEV lysis results in a dramatic increase in the population of cells that bind strongly to the antigen, indicating that this screening strategy is effective. Pure lines from round 5 of sorting were plated on selective media and grown individually to further confirm lysis-mediated activated antigen binding.

33A B中所示,在掩蔽肽之存在下,呈scFv ( 33A)或Fab ( 33B)形式之選定CTLA4可活化抗體純系展示與抗原之少結合。然而,當將酵母細胞用TEV蛋白酶處理以移除掩蔽肽時,與抗原之結合急劇增加。將TEV識別位點併入裂解肽中,與應用TEV蛋白酶組合驗證選定純系顯著增加掩蔽肽選擇之成功率。 As shown in Figures 33A - B , selected CTLA4 activatable antibody clones in scFv ( Figure 33A ) or Fab ( Figure 33B ) format displayed minimal binding to the antigen in the presence of the masking peptide. However, when yeast cells were treated with TEV protease to remove the masking peptide, binding to the antigen increased dramatically. Incorporating the TEV recognition site into the cleaved peptide, combined with the use of TEV protease to verify the selected pure line, significantly increases the success rate of masking peptide selection.

為識別掩蔽肽序列,自選定酵母純系(Generay #GK2002-200)提取穿梭質粒,及轉形至活性大腸桿菌細胞。製備質粒,及將編碼掩蔽肽之區域定序及比對。如所預期,可將此等序列分成若干組,指示通過分選輪之清楚濃化。四組掩蔽肽序列與不變裂解肽序列一起列於 13中。 13 :掩蔽肽序列 樣品ID 肽名稱: 掩蔽+ 裂解肽序列: TY22401 B13189 EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 168) TY22402 B13180 EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 170) TY22403 B13192 EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 172) TY22404 B13197 EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 174) IgG 轉變及表現 To identify the masked peptide sequence, shuttle plasmids were extracted from the selected yeast pure line (Generay #GK2002-200) and transformed into viable E. coli cells. Plasmids are prepared, and the region encoding the masking peptide is sequenced and aligned. As expected, these sequences can be divided into groups indicating clear enrichment by the sorting wheel. The four sets of masked peptide sequences are listed in Table 13 along with the invariant cleavage peptide sequences. Table 13 : Masked peptide sequences Sample ID : Peptide name: Masked + cleavage peptide sequence: TY22401 B13189 EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 168) TY22402 B13180 EVGSYIVHHSDCDAFYPYCDSSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 170) TY22403 B13192 EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 172) TY22404 B13197 EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 174) IgG changes and manifestations

將列於 13中之四組掩蔽肽,以及源自其中之二者(B13192及B13197)以消除潛在糖基化位點之另外四個掩蔽肽序列( 14)轉變成IgG1。 14 :另外掩蔽肽序列 樣品 ID 掩蔽 + 裂解肽序列: TY22563 EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 177) TY22564 EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 178) TY22565 EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 179) TY22566 EVGSYPNPASDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 180) The four sets of masking peptides listed in Table 13 , as well as four additional masking peptide sequences ( Table 14 ) derived from two of them (B13192 and B13197) to eliminate potential glycosylation sites, were converted to IgG1. Table 14 : Additional masked peptide sequences Sample ID : Masked + cleavage peptide sequence: TY22563 EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 177) TY22564 EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 178) TY22565 EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 179) TY22566 EVGSYPNPASDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 180)

將重鏈及輕鏈分開選殖至哺乳動物表現載體pCDNA3.3 (Thermo Fisher Scientific,目錄號K830001),及將掩蔽肽及不變裂解肽以與在酵母表面上展示相同之方式融合至輕鏈之N端。親本CTLA4抗體(TY21580)之VH及VL序列列於以下: 抗CTLA4重鏈可變區(SEQ ID NO: 87): EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS 抗CTLA4輕鏈可變區(SEQ ID NO: 100): DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR. The heavy and light chains were cloned separately into the mammalian expression vector pCDNA3.3 (Thermo Fisher Scientific, Cat. No. K830001), and the masking peptide and the invariant cleavage peptide were fused to the light chain in the same manner as for display on the yeast surface. The N end. The VH and VL sequences of the parent CTLA4 antibody (TY21580) are listed below: Anti-CTLA4 heavy chain variable region (SEQ ID NO: 87): EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS Anti-CTLA4 light chain variable region (SEQ ID NO: 100): DIQLTQSPSSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR.

將質粒對短暫轉染至HEK293F細胞中。於6天後,收集上清液,藉由離心及過濾澄清,及將IgG用標準蛋白A親和層析法(MabSelect SuRe,GE Healthcare)純化。將IgG溶離及中和,及緩衝交換至PB緩衝液(20 mM磷酸鈉、150 mM NaCl,pH 7.0)中。藉由UV-分光光度法測定蛋白質濃度,及在變性、還原及非還原條件下藉由SDS-PAGE或SEC-HPLC分析IgG純度。重要的是,可活化抗體於HEK293細胞中之表現水平類似於其親本抗體,且其於蛋白A樹脂後之純化產率亦相似,表明掩蔽肽及裂解肽之存在對哺乳動物細胞中之抗體表現不具有負面影響。 掩蔽效率之量測 Plasmid pairs were transiently transfected into HEK293F cells. After 6 days, the supernatant was collected, clarified by centrifugation and filtration, and the IgG was purified using standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). The IgG was eluted, neutralized, and buffer exchanged into PB buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). Protein concentration was determined by UV-spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing and non-reducing conditions. Importantly, the activatable antibody was expressed in HEK293 cells at a level similar to that of its parent antibody, and its purification yield after protein A resin was also similar, indicating that the presence of masking peptides and cleavage peptides is beneficial to the antibody in mammalian cells. Performance has no negative impact. Measurement of masking efficiency

使用ForteBio Octet RED96系統(Pall, USA)快速評估掩蔽肽之功效。簡言之,將可活化抗體(及其親本抗體,TY21580)於KB緩衝液(補充有0.02% Tween 20及0.1% BSA之PBS緩衝液)中稀釋至30 μg/mL,及藉由抗人類IgG捕獲(AHC)生物感測器(Pall, USA)平行捕獲。然後允許將感測器與His標記之CTLA4蛋白質(25 nM)締合300秒,及然後於KB緩衝液中再締合300秒。根據製造商之指導方針使用ForteBio數據分析7.1 (Pall, USA)將締合及解離曲線擬合至1:1朗繆爾結合模型。如 34A B中所示,利用可活化抗體達成之反應顯著低於針對親本抗體達成之反應,表明掩蔽肽有效阻斷抗體與其抗原之結合。然而,在四種可活化抗體中,TY22401係較不有效,與來自以下討論之ELISA檢定之結果一致。 The efficacy of masking peptides was quickly evaluated using the ForteBio Octet RED96 system (Pall, USA). Briefly, activatable antibodies (and their parent antibody, TY21580) were diluted to 30 μg/mL in KB buffer (PBS buffer supplemented with 0.02% Tween 20 and 0.1% BSA) and incubated with anti-human Parallel capture using an IgG capture (AHC) biosensor (Pall, USA). The sensor was then allowed to associate with His-tagged CTLA4 protein (25 nM) for 300 seconds and then in KB buffer for an additional 300 seconds. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio Data Analysis 7.1 (Pall, USA) according to the manufacturer's guidelines. As shown in Figures 34A - B , the response achieved with the activatable antibody was significantly lower than the response achieved against the parent antibody, indicating that the masking peptide effectively blocks the binding of the antibody to its antigen. However, of the four activatable antibodies, the TY22401 line was less effective, consistent with results from the ELISA assay discussed below.

將重組人類CTLA4-Fc於PBS中稀釋至1 μg/mL及在4℃下塗覆在Maxisorp板上過夜。在37℃下將板用補充有3%脫脂乳之PBS阻斷1小時。於洗滌後,將100 μL抗體之3倍連續稀釋添加至各孔。於37℃下培育1小時後,將板洗滌四次,及將100 μL HRP結合之抗人類IgG (Fab特異性) (1:6000稀釋)添加至各孔。將板在37℃下培育1小時,洗滌四次,及然後將50 μL TMB受質溶液添加至各孔,及將板在室溫下培育。於用50 μL H 2SO 4/孔停止反應後,量測450 nm處之吸光度。藉由使用GraphPad Prism 6軟體之非對稱S形(五參數邏輯方程)模型擬合ELISA數據來評價EC 50。進行可活化抗體TY22401、TY22402及TY22404之實驗兩次,導致針對此等可活化抗體各者獲得兩個經計算之掩蔽效率。各可活化抗體之掩蔽效率藉由可活化抗體之結合之EC 50除以親本抗體(TY21580)之EC 50來計算。如 35A B 15中所示,相較於親本抗體,所有可活化抗體顯示與其抗原之急劇減少之結合,及經計算之掩蔽效率範圍自48至2213。掩蔽效率之差異可由EC 50值之量測及數據擬合中之變化造成,且各可活化抗體之掩蔽效率可落入經計算之範圍內(例如,可活化抗體TY22402之掩蔽效率係在377與2213之間)。此等結果指示當於哺乳動物細胞中表現時,自CPL識別之多個掩蔽肽維持其掩蔽效率且作為全IgG分子之部分。 15 :在蛋白酶裂解之前之可活化抗體 ELISA 樣品ID LogEC 50 EC 50 M nM R 2 掩蔽效率: 數據批次1 TY21580 -9.665 2.161E-10 0.216 0.999 1.0 TY22401 -7.623 2.382E-08 23.82 0.997 110 TY22402 -6.321 4.779E-07 477.9 0.997 2213 TY22404 -6.749 178.4E-07 178.4 0.998 826    數據批次2 TY21580 -9.478 3.324E-10 0.3324 0.998 1.0 TY22401 -7.800 1.586E-08 15.86 0.994 48 TY22402 -6.902 1.254E-07 125.4 0.998 377 TY22404 -6.892 1.281E-07 128.1 0.998 385          TY21580 -9.48 3.3E-10 0.33    1.0 TY22563 -7.32 4.771E-08 47.71    143.5 TY22564 -7.41 3.898E-08 38.98    117.3 TY22565 -6.68 2.099E-07 209.9    631.5 TY22566 -6.79 1.6264E-07 162.6    489.2 掩蔽肽之移除恢復抗體活性 Recombinant human CTLA4-Fc was diluted to 1 μg/mL in PBS and plated on Maxisorp plates overnight at 4°C. Plates were blocked with PBS supplemented with 3% skim milk for 1 hour at 37°C. After washing, 100 μL of 3-fold serial dilutions of antibody were added to each well. After incubation for 1 hour at 37°C, the plates were washed four times and 100 μL of HRP-conjugated anti-human IgG (Fab specific) (1:6000 dilution) was added to each well. The plate was incubated for 1 hour at 37°C, washed four times, and then 50 μL of TMB substrate solution was added to each well and the plate was incubated at room temperature. After stopping the reaction with 50 μL H 2 SO 4 /well, measure the absorbance at 450 nm. EC50 was evaluated by fitting ELISA data using an asymmetric sigmoid (five-parameter logistic equation) model using GraphPad Prism 6 software. Experiments with activatable antibodies TY22401, TY22402 and TY22404 were performed twice, resulting in two calculated masking efficiencies for each of these activatable antibodies. The masking efficiency of each activatable antibody was calculated by dividing the EC 50 of binding of the activatable antibody by the EC 50 of the parent antibody (TY21580). As shown in Figures 35A - B and Table 15 , all activatable antibodies showed sharply reduced binding to their antigens compared to the parental antibodies, with calculated masking efficiencies ranging from 48 to 2213. Differences in masking efficiency can be caused by changes in the measurement of EC50 values and data fitting, and the masking efficiency of each activatable antibody can fall within the calculated range (for example, the masking efficiency of activatable antibody TY22402 is between 377 and between 2213). These results indicate that multiple masking peptides recognized from CPL maintain their masking efficiency and are part of the full IgG molecule when expressed in mammalian cells. Table 15 : ELISA of activatable antibodies prior to protease cleavage Sample ID : LogEC 50 : EC 50 M : nM : R2 : Masking efficiency: Data batch 1 TY21580 -9.665 2.161E-10 0.216 0.999 1.0 TY22401 -7.623 2.382E-08 23.82 0.997 110 TY22402 -6.321 4.779E-07 477.9 0.997 2213 TY22404 -6.749 178.4E-07 178.4 0.998 826 Data batch 2 TY21580 -9.478 3.324E-10 0.3324 0.998 1.0 TY22401 -7.800 1.586E-08 15.86 0.994 48 TY22402 -6.902 1.254E-07 125.4 0.998 377 TY22404 -6.892 1.281E-07 128.1 0.998 385 TY21580 -9.48 3.3E-10 0.33 1.0 TY22563 -7.32 4.771E-08 47.71 143.5 TY22564 -7.41 3.898E-08 38.98 117.3 TY22565 -6.68 2.099E-07 209.9 631.5 TY22566 -6.79 1.6264E-07 162.6 489.2 Removal of masking peptide restores antibody activity

將經純化之可活化抗體用識別裂解序列之蛋白酶處理,及然後測試以確定掩蔽肽之移除是否恢復其活性。作為實例,將20 μg TY22404 (0.5 mg/mL)於反應緩衝液(50 mM Tris-HCl,0.01% Tween 20,pH 8.5)中用1 μg重組人類uPA (Acrobiosystems, # PLU-H5229)處理;或將TY22404於反應緩衝液(50 mM Tris,150 mM NaCl,5 mM CaCl 2,20 μM ZnCl 2,pH 7.5)中用5或10單位之重組人類MMP-9 (BioVision, # 7867-500)處理。在37℃下進行反應21小時。藉由SDS-PAGE分析證實掩蔽肽自輕鏈移除( 36A)。然後如上所述藉由ELISA量測掩蔽效率。如 36B 及表 16中所示,於移除掩蔽肽後,可活化抗體於其結合至抗原方面變得與親本抗體不可區分。 16 :於蛋白酶裂解後之可活化抗體 ELISA 樣品ID LogEC 50 EC 50 nM 掩蔽效率: TY21580 -9.35 0.447 1.0 TY22404 -7.01 96.8 216 TY22404-uPA -9.40 0.402 0.9 TY22404-MMP-9 -9.39 0.412 0.9 可活化抗體可展性特性 The purified activatable antibody is treated with a protease that recognizes the cleavage sequence and then tested to determine whether removal of the masking peptide restores its activity. As an example, 20 μg of TY22404 (0.5 mg/mL) was treated with 1 μg of recombinant human uPA (Acrobiosystems, #PLU-H5229) in reaction buffer (50 mM Tris-HCl, 0.01% Tween 20, pH 8.5); or TY22404 was treated with 5 or 10 units of recombinant human MMP-9 (BioVision, #7867-500) in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl 2 , 20 μM ZnCl 2 , pH 7.5). The reaction was carried out at 37°C for 21 hours. Removal of the masking peptide from the light chain was confirmed by SDS-PAGE analysis ( Figure 36A ). Masking efficiency was then measured by ELISA as described above. As shown in Figure 36B and Table 16 , upon removal of the masking peptide, the activatable antibody became indistinguishable from the parent antibody in terms of its binding to antigen. Table 16 : Activatable antibody ELISA after protease cleavage Sample ID : LogEC 50 : EC 50 nM : Masking efficiency: TY21580 -9.35 0.447 1.0 TY22404 -7.01 96.8 216 TY22404-uPA -9.40 0.402 0.9 TY22404-MMP-9 -9.39 0.412 0.9 Activable antibody developability properties

出於製造目的,關鍵的是所發現之可活化抗體具有良好可展性特性。利用於哺乳動物細胞中表現之經純化之可活化抗體進行若干不同測試。將可活化抗體於於20 mM組胺酸,pH 5.5中調整至1 mg/mL,及使用具有Waters 2996 UV檢測器及TSKgel g3000 SWXL管柱(300 mm × 7.8 mm)之Waters 2695 (Tosoh Bioscience)使用分析型尺寸排阻層析法進行抗體質量分析。針對各檢定,注射10 μg抗體,及於緩衝液(200 mM磷酸鈉,在pH 7.0下)中在0.5 mL/min之流率下進行分級分離。For manufacturing purposes, it is critical that the activated antibodies found have good developability properties. Several different tests were performed using purified activatable antibodies expressed in mammalian cells. Activatable antibodies were adjusted to 1 mg/mL in 20 mM histidine, pH 5.5, and a Waters 2695 (Tosoh Bioscience) with a Waters 2996 UV detector and a TSKgel g3000 SWXL column (300 mm × 7.8 mm) was used. Antibody mass analysis using analytical size exclusion chromatography. For each assay, 10 μg of antibody was injected and fractionated in buffer (200 mM sodium phosphate at pH 7.0) at a flow rate of 0.5 mL/min.

進行三個加速應力測試:在50℃下培育可活化抗體7天,在40℃下培育可活化抗體28天,及六個凍熔循環。藉由將100 μL樣品(1 mg/mL含於20 mM組胺酸,pH 5.5中)在-80℃下冷凍30分鐘,接著在室溫下解凍60分鐘進行凍熔測試。如 37A C中所示,所有可活化抗體保持穩定,且於50℃下儲存7天或40℃下儲存28天後展示小的聚集。於六個凍熔循環之後,其顯示輕微惡化;然而,主要單體峰保持約95%,指示此等可活化抗體在此等加速應力測試下極其穩定。不希望受理論束縛,值得注意的是,可活化抗體尚未通過廣泛緩衝液最佳化處理,及因此,可活化抗體之穩定性可利用最佳化緩衝液及賦形劑進一步改善。 Three accelerated stress tests were performed: incubation of activatable antibodies at 50°C for 7 days, incubation of activatable antibodies at 40°C for 28 days, and six freeze-thaw cycles. Freeze-thaw testing was performed by freezing 100 μL of sample (1 mg/mL in 20 mM histidine, pH 5.5) at -80°C for 30 minutes, followed by thawing at room temperature for 60 minutes. As shown in Figure 37A - C , all activatable antibodies remained stable and exhibited small aggregation after storage at 50°C for 7 days or 40°C for 28 days. After six freeze-thaw cycles, they showed slight deterioration; however, the main monomer peak remained approximately 95%, indicating that these activatable antibodies are extremely stable under these accelerated stress tests. Without wishing to be bound by theory, it is worth noting that the activatable antibodies have not been extensively buffer optimized, and therefore, the stability of the activatable antibodies can be further improved using optimized buffers and excipients.

接下來,將可活化抗體於20 mM組胺酸,pH 5.5中濃縮至大於150 mg/mL ( 17)。未觀察到可活化抗體沉澱,且樣品之黏度相當容易控制。然後將濃縮之可活化抗體稀釋至20 mg/mL或1 mg/mL用於分析高分子量(HMW)物質。如 38 及表 17中所示,未觀察到HMW物質之明顯增加,表明此等可活化抗體於所測試之緩衝液中極其可溶且穩定,多達高濃度。 17 :可活化抗體之濃度 >150 mg/mL 樣品ID 起始濃度(mg/mL) 高濃度(mg/mL) TY22401 10.9 187.2 TY22402 8.4 160.0 Next, activatable antibodies were concentrated to greater than 150 mg/mL in 20 mM histidine, pH 5.5 ( Table 17 ). No precipitation of activatable antibodies was observed, and the viscosity of the sample was fairly easy to control. The concentrated activatable antibody is then diluted to 20 mg/mL or 1 mg/mL for analysis of high molecular weight (HMW) substances. As shown in Figure 38 and Table 17 , no significant increase in HMW species was observed, indicating that these activatable antibodies are extremely soluble and stable in the buffers tested, up to high concentrations. Table 17 : Concentration of activatable antibodies >150 mg/mL Sample ID : Starting concentration (mg/mL) : High concentration (mg/mL) : TY22401 10.9 187.2 TY22402 8.4 160.0

為研究可活化抗體在低pH下之穩定性,將經純化之可活化抗體(在10 mg/mL下含於20 mM組胺酸,pH 5.5中)用檸檬酸滴定至1 mg/mL,及將pH調整至3.7並保持在室溫下持續30及60分鐘。之後,將樣品用1M Tris-鹼中和至pH 7.0。如上所述,利用ForteBio量測可活化抗體之掩蔽效率。如 39中所示,掩蔽效率於低pH培育30或60分鐘後仍未改變,表明掩蔽肽於低pH培育後保持其阻斷功效。 To study the stability of activatable antibodies at low pH, purified activatable antibodies (10 mg/mL in 20 mM histidine, pH 5.5) were titrated with citric acid to 1 mg/mL, and Adjust pH to 3.7 and keep at room temperature for 30 and 60 minutes. Afterwards, the samples were neutralized to pH 7.0 with 1 M Tris-base. As described above, ForteBio was used to measure the masking efficiency of activatable antibodies. As shown in Figure 39 , the masking efficiency remained unchanged after 30 or 60 minutes of low pH incubation, indicating that the masking peptide maintained its blocking efficacy after low pH incubation.

一併考慮,資料指示所發現之可活化抗體在各種應力條件下保持穩定,及因此,其具有良好可展性特性。 實例 9 :靶向 CTLA4 之可活化抗體之活體外及活體內表徵 Taken together, the data indicate that the activated antibodies discovered remain stable under various stress conditions and, therefore, have good malleability properties. Example 9 : In vitro and in vivo characterization of activatable antibodies targeting CTLA4

應瞭解,T細胞上之CTLA-4活性係與第一信號(TCR/CD3)及涉及B7-CD28/CTLA-4之第二信號相關。 活體外功能表征 It should be understood that CTLA-4 activity on T cells is related to a first signal (TCR/CD3) and a second signal involving B7-CD28/CTLA-4. In vitro functional characterization

此處在低濃度之抗CD3抗體之存在下評價靶向CTLA4之可活化抗體對人類PBMC活化之活性。使用Histopaque-1077 (Sigma)藉由密度梯度離心將人類PBMC自健康供體(#44)之血液新鮮單離。在4℃下將抗CD3 (OKT-3)抗體塗覆在96孔板上過夜。於洗滌後,將1x10^5個經新鮮單離之人類PBMC添加至各孔,接著添加不同濃度之測試製品。於使用人類IL-2 ELISA Ready-SET-Go (Invitrogen)套組刺激48小時後量測IL-2之誘導。使用人類IFN-γ ELISA Ready-SET-Go (Invitrogen)套組量測上清液中之IFN-γ。如 40A B中所證明,在高濃度下,TY22404誘導IL-2產生,及TY22401誘導IFN-γ產生。然而,可活化抗體之活性顯著低於親本TY21580抗體之活性。 Here the activity of activatable antibodies targeting CTLA4 on human PBMC activation was evaluated in the presence of low concentrations of anti-CD3 antibodies. Human PBMCs were freshly isolated from the blood of a healthy donor (#44) by density gradient centrifugation using Histopaque-1077 (Sigma). Anti-CD3 (OKT-3) antibodies were coated on 96-well plates overnight at 4°C. After washing, 1x10^5 freshly isolated human PBMCs were added to each well, followed by different concentrations of test article. IL-2 induction was measured 48 hours after stimulation with the human IL-2 ELISA Ready-SET-Go (Invitrogen) kit. IFN-γ in the supernatant was measured using the Human IFN-γ ELISA Ready-SET-Go (Invitrogen) kit. As demonstrated in Figures 40A - B , at high concentrations, TY22404 induced IL-2 production, and TY22401 induced IFN-γ production. However, the activity of the activatable antibody was significantly lower than that of the parent TY21580 antibody.

接下來,測試可活化抗體之抗體依賴性細胞毒性活性及與親本抗體TY21580比較。使用ADCC報告基因檢定評價可活化抗體之ADCC活性。將過度表現人類CTLA4之HEK293F細胞(HEK293F/hCTLA-4細胞)用作靶細胞;將過度表現CD16a及NFAT-Luc之Jurkat細胞系(Jurkat/CD16a細胞)用作效應細胞。將1x10^5個Jurkat/CD16a細胞及1x10^4個HEK293F/hCTLA-4細胞(E:T比率10:1)與不同濃度之抗體混合。於培育6小時後,將100 µL One-Glo試劑添加至細胞,及將細胞裂解10分鐘。移除上清液用於使用SpectraMax i3x板讀取器之發光量測。如 41中所示,可活化抗體顯示較親本抗體TY21580若干對數更低ADCC活性。TY22401之ADCC活性較TY22402及TY22404之ADCC活性更高。一併考慮,活體外資料指示經更好掩蔽之可活化抗體具有較少ADCC活性。 Next, the antibody-dependent cytotoxic activity of the activatable antibodies was tested and compared with the parent antibody TY21580. The ADCC activity of activatable antibodies is evaluated using the ADCC reporter assay. HEK293F cells that overexpress human CTLA4 (HEK293F/hCTLA-4 cells) were used as target cells; Jurkat cell lines that overexpressed CD16a and NFAT-Luc (Jurkat/CD16a cells) were used as effector cells. 1x10^5 Jurkat/CD16a cells and 1x10^4 HEK293F/hCTLA-4 cells (E:T ratio 10:1) were mixed with different concentrations of antibodies. After 6 hours of incubation, 100 µL One-Glo reagent was added to the cells and the cells were lysed for 10 minutes. The supernatant was removed and used for luminescence measurements using a SpectraMax i3x plate reader. As shown in Figure 41 , the activatable antibody showed several log lower ADCC activity than the parent antibody TY21580. The ADCC activity of TY22401 is higher than that of TY22402 and TY22404. Taken together, the in vitro data indicate that better masked activatable antibodies have less ADCC activity.

接下來於多個同源小鼠腫瘤模型(包括MC38結腸直腸腫瘤模型、CT26結腸直腸腫瘤模型、H22肝腫瘤模型及3LL肺腫瘤模型)中評價可活化抗體之抗腫瘤活性及與親本抗體TY21580之抗腫瘤活性比較。 MC38 結腸直腸腫瘤模型中之抗腫瘤功效 Next, the anti-tumor activity of the activatable antibody was evaluated in multiple syngeneic mouse tumor models (including MC38 colorectal tumor model, CT26 colorectal tumor model, H22 liver tumor model, and 3LL lung tumor model) and compared with the parent antibody TY21580. Comparison of anti-tumor activity. Anti-tumor efficacy in the MC38 colorectal tumor model

將C57BL/6小鼠(n=8隻/組,雌性,6至8週齡)皮下接種MC38 (NTCC-MC38)鼠科結腸癌細胞。當建立腫瘤(70 mm 3)時,用同型對照抗體、親本抗體TY21580、或三種可活化抗體中之一者藉由腹膜內注射開始處理,一週兩次。一週兩次監測腫瘤生長,評估隨時間之平均腫瘤體積± s.e.m. ( 42A)及個別腫瘤生長曲線( 42B)。如 42A B中所示,所有三種可活化抗體於MC38同源小鼠腫瘤模型中顯示與親本抗體TY21580可比較之強效抗腫瘤活性。 CT26 結腸直腸腫瘤模型中之抗腫瘤功效 C57BL/6 mice (n=8/group, female, 6 to 8 weeks old) were inoculated subcutaneously with MC38 (NTCC-MC38) murine colon cancer cells. When tumors (70 mm3 ) were established, treatment was initiated by intraperitoneal injection twice a week with isotype control antibody, parental antibody TY21580, or one of three activatable antibodies. Tumor growth was monitored twice a week, and mean tumor volume ± s.e.m. over time ( Figure 42A ) and individual tumor growth curves ( Figure 42B ) were assessed. As shown in Figures 42A - B , all three activatable antibodies showed potent anti-tumor activity comparable to the parent antibody TY21580 in the MC38 syngeneic mouse tumor model. Anti-tumor efficacy in CT26 colorectal tumor model

將BALB/c小鼠(n=8隻/組,雌性,7至8週齡)皮下接種CT26 (上海生物科學研究院)鼠科結腸癌細胞。當建立腫瘤(100 mm 3)時,用同型對照抗體、親本抗體TY21580、或三種可活化抗體中之一者在5 mg/kg下藉由腹膜內注射開始處理,一週兩次。一週兩次監測腫瘤生長並報告為隨時間之平均腫瘤體積± s.e.m.。如 43中所示,所有三種可活化抗體於CT26同源小鼠腫瘤模型中顯示與親本抗體TY21580可比較之強效抗腫瘤活性。 H22 肝腫瘤模型中之抗腫瘤功效 BALB/c mice (n=8/group, female, 7 to 8 weeks old) were subcutaneously inoculated with CT26 (Shanghai Institute of Biological Sciences) murine colon cancer cells. When tumors were established (100 mm3 ), treatment was initiated with isotype control antibody, parental antibody TY21580, or one of three activatable antibodies at 5 mg/kg by intraperitoneal injection twice a week. Tumor growth was monitored twice weekly and reported as mean tumor volume ± s.e.m. over time. As shown in Figure 43 , all three activatable antibodies showed potent anti-tumor activity comparable to the parent antibody TY21580 in the CT26 syngeneic mouse tumor model. Anti-tumor efficacy in H22 liver tumor model

將BALB/c小鼠(n=8隻/組,雌性,7至8週齡)皮下接種H22 (中國典型培養物保藏中心)鼠科肝癌細胞。當建立腫瘤(100 mm 3)時,用同型對照抗體、親本抗體TY21580、或三種可活化抗體中之一者在5 mg/kg下藉由腹膜內注射開始處理,一週兩次。一週兩次監測腫瘤生長並報告為隨時間之平均腫瘤體積± s.e.m.。如 44中所示,所有三種可活化抗體於H22同源小鼠腫瘤模型中顯示與親本抗體TY21580可比較之強效抗腫瘤活性。 3LL 肺癌模型中之抗腫瘤功效 BALB/c mice (n=8/group, female, 7 to 8 weeks old) were subcutaneously inoculated with H22 (China Type Culture Collection Center) murine hepatoma cells. When tumors were established (100 mm 3 ), treatment was initiated with isotype control antibody, parental antibody TY21580, or one of three activatable antibodies by intraperitoneal injection at 5 mg/kg twice a week. Tumor growth was monitored twice a week and reported as mean tumor volume ± s.e.m. over time. As shown in Figure 44 , all three activatable antibodies showed potent anti-tumor activity comparable to the parent antibody TY21580 in the H22 syngeneic mouse tumor model. Anti-tumor efficacy in 3LL lung cancer model

將C57BL/6小鼠(n=10隻/組,雌性,6至8週齡)皮下接種3LL (JCRB)鼠科肺癌細胞。當建立腫瘤(75 mm 3)時,用同型對照抗體、親本抗體TY21580、或三種可活化抗體中之一者藉由腹膜內注射開始處理,一週兩次。一週兩次監測腫瘤生長,評估隨時間之平均腫瘤體積± s.e.m. ( 45A)及個別腫瘤生長曲線( 45B)。如 45A B中所示,所有三種可活化抗體於3LL同源小鼠腫瘤模型中顯示與親本抗體TY21580可比較之強效抗腫瘤活性。 藥物動力學分析 C57BL/6 mice (n=10/group, female, 6 to 8 weeks old) were inoculated subcutaneously with 3LL (JCRB) murine lung cancer cells. When tumors (75 mm3 ) were established, treatment was initiated by intraperitoneal injection twice a week with isotype control antibody, parental antibody TY21580, or one of three activatable antibodies. Tumor growth was monitored twice a week, and mean tumor volume ± s.e.m. over time ( Figure 45A ) and individual tumor growth curves ( Figure 45B ) were assessed. As shown in Figures 45A - B , all three activatable antibodies showed potent anti-tumor activity comparable to the parent antibody TY21580 in the 3LL syngeneic mouse tumor model. Pharmacokinetic analysis

於BALB/c雌性小鼠(約8週齡)中進行藥物動力學研究。對三隻小鼠/組腹膜內注射10 mg/kg之測試製品。於給藥後3小時、6小時、24小時、48小時、96小時及168小時時收集血液樣品(約50 µL/樣品)。自無抗體投與之三隻初始雌性小鼠收集空白對照血液。藉由ELISA測定各測試抗體之血清濃度,其中使用抗人類IgG Fc捕獲,及使用經HRP標記之抗人類IgG (Fab特異性)抗體(Sigma)檢測( 46A C)。相較於針對親本抗體TY21580收集之先前資料,可活化抗體TY22401 ( 46A)、TY22402 ( 46B)及TY22404 ( 46C)具有慢得多的清除時間及更長半衰期。TY22401具有196小時之半衰期,且在168小時之藥物濃度係約55 µg/mL。TY22402具有134小時之半衰期,且在168小時之藥物濃度係約40 µg/mL。TY22404具有254小時之半衰期,且在168小時之藥物濃度係約45 µg/mL。相比之下,親本抗體TY21580具有107小時之半衰期,且在168小時之藥物濃度係約17 µg/mL。 重複給藥毒性研究 Pharmacokinetic studies were performed in BALB/c female mice (approximately 8 weeks old). Three mice/group were injected intraperitoneally with 10 mg/kg of the test article. Blood samples (approximately 50 µL/sample) were collected at 3 hours, 6 hours, 24 hours, 48 hours, 96 hours and 168 hours after administration. Blank control blood was collected from three naive female mice administered no antibody. Serum concentrations of each test antibody were determined by ELISA using anti-human IgG Fc capture and detection using HRP-labeled anti-human IgG (Fab specific) antibodies (Sigma) ( Figure 46A to C ). Activatable antibodies TY22401 ( Figure 46A ), TY22402 ( Figure 46B ) and TY22404 ( Figure 46C ) had much slower clearance times and longer half-lives compared to previous data collected against the parent antibody TY21580. TY22401 has a half-life of 196 hours, and the drug concentration at 168 hours is approximately 55 µg/mL. TY22402 has a half-life of 134 hours, and the drug concentration at 168 hours is approximately 40 µg/mL. TY22404 has a half-life of 254 hours, and the drug concentration at 168 hours is approximately 45 µg/mL. In comparison, the parent antibody TY21580 has a half-life of 107 hours, and the drug concentration at 168 hours is approximately 17 µg/mL. Repeated dose toxicity studies

當評價TY21580對NOD小鼠之糖尿病發病年齡之影響時,發現TY21580之高劑量可導致NOD但是非正常BALB/c小鼠之動物死亡。此處使用NOD小鼠模型評價可活化抗體相較於TY21580之安全性。在第0、3、7及12天將NOD小鼠(n=5隻/組,雌性,6週齡)用同型對照抗體、親本抗體TY21580、或三種可活化抗體中之一者在50 mg/kg下藉由腹膜內注射處理。於TY21580處理組中,於第三次給藥後1隻動物死亡,及於第四次給藥後3隻動物死亡。如 47中所示,在研究終止時用同型對照或三種可活化抗體中之任一者處理之所有動物係活著且健康良好。此等資料指示可活化抗體於小鼠中具有可接受之安全性/毒性特性,且於NOD小鼠中,可活化抗體較親本抗體TY21580安全得多。 實例 10 :靶向 CTLA4 之可活化抗體之另外活體內表徵 When evaluating the effect of TY21580 on the age of onset of diabetes in NOD mice, it was found that high doses of TY21580 could cause death in NOD but not normal BALB/c mice. Here, the NOD mouse model was used to evaluate the safety of activatable antibodies compared to TY21580. On days 0, 3, 7 and 12, NOD mice (n=5/group, female, 6 weeks old) were treated with isotype control antibody, parental antibody TY21580, or one of the three activatable antibodies at 50 mg. /kg by intraperitoneal injection. In the TY21580 treatment group, 1 animal died after the third dose, and 3 animals died after the fourth dose. As shown in Figure 47 , all animals treated with isotype control or any of the three activatable antibodies were alive and in good health at the end of the study. These data indicate that the activatable antibody has an acceptable safety/toxicity profile in mice and that the activatable antibody is significantly safer than the parent antibody TY21580 in NOD mice. Example 10 : Additional in vivo characterization of activatable antibodies targeting CTLA4

於親本抗體TY21580之先前研究中,發現TY21580之重複給藥導致雌性及雄性正常BALB/c小鼠二者中之增加之脾大小。除此之外,TY21580不顯示對其他評價參數之任何顯著副作用,該等參數包括許多器官之重量、肝組織病理學、血液學及血液生物化學。因此,評價若干可活化抗體對脾大小之影響且與親本抗體TY21580比較。In a previous study of the parent antibody TY21580, it was found that repeated administration of TY21580 resulted in increased spleen size in both female and male normal BALB/c mice. In addition, TY21580 did not show any significant side effects on other parameters evaluated, including weights of many organs, liver histopathology, hematology and blood biochemistry. Therefore, the effect of several activatable antibodies on spleen size was evaluated and compared with the parent antibody TY21580.

於正常BALB/c小鼠中如下進行可活化抗體之重複給藥毒性:在第1、4、7及11天腹膜內投與50 mg/kg之同型對照抗體、TY21580親本抗體、或可活化抗體TY22402、TY22566或TY22401 (10 mL/kg)。各組包含五隻雌性小鼠(五週齡)。每日監測小鼠異常行為及症狀,及每日量測食物攝取及體重。在第14天,將動物安樂死用於驗屍後檢查及其他分析。有趣的是,雖然可活化抗體TY22402 ( 48A)或TY22566 ( 48B)之投與相較於同型對照稍微增加小鼠之脾大小,此等可活化抗體相較於親本抗體TY21580之投與顯示對脾大小顯著更少影響。可活化抗體TY22401之投與相較於同型對照顯著增加脾大小,但是較使用親本抗體TY21580觀察到者仍在較低程度( 48C)。 Repeated dose toxicity of activatable antibodies was performed in normal BALB/c mice by intraperitoneal administration of 50 mg/kg of isotype control antibody, TY21580 parental antibody, or activatable antibody on days 1, 4, 7, and 11 Antibody TY22402, TY22566, or TY22401 (10 mL/kg). Each group contained five female mice (five weeks old). Abnormal behaviors and symptoms of mice were monitored daily, and food intake and body weight were measured daily. On day 14, animals were euthanized for post-mortem examination and other analyses. Interestingly, although administration of activatable antibodies TY22402 ( Fig. 48A ) or TY22566 ( Fig. 48B ) slightly increased spleen size in mice compared to isotype controls, administration of these activatable antibodies compared to parental antibody TY21580 showed significantly less effect on spleen size. Administration of activatable antibody TY22401 significantly increased spleen size compared to isotype control, but to a lesser extent than that observed with parental antibody TY21580 ( Figure 48C ).

因為CTLA4在Treg細胞上構成表現,評價可活化抗體TY22402、TY22566或TY22401對全血及脾二者中之Treg細胞、CD4 +T細胞及CD8 +T細胞之影響及與親本抗體TY21580比較。將來自全血之單核細胞或脾細胞染色及使用下列抗體閘控:抗CD45-BV421、抗CD3-AF488、抗CD4-BV510、抗CD8a-PerCP-cy5.5、抗CD25-APC及抗FoxP3-PE。將Treg細胞定義為CD45 +CD3 +CD4 +CD25 +Foxp3 +。如 18中所示,相較於同型對照,親本抗體TY21580增加脾中之Treg細胞之百分比;然而,可活化抗體TY22402及TY22566不影響脾Treg之百分比。於全血中,當與同型對照相比時,TY21580、TY22402及TY22566稍微增加Treg細胞之百分比。可活化抗體TY22401增加脾及全血中之Treg細胞之百分比。TY21580、TY22402、TY22566或TY22401不顯著改變CD4 +及CD8 +T細胞之百分比(不顯示數據)。 18 顯示可活化抗體對脾 Treg 及血液 Treg 細胞之影響之 FACS 分析 組: 樣品: 脾: CD4 +T 細胞中之 Treg % 血液: CD4 +T 細胞中之 Treg % 同型對照 (50 mg/kg BIW) 6-1 10.50 1.71 6-2 8.25 0.70 6-3 8.04 0.67 6-4 6.81 0.90 平均值 8.40 1.00 SD 1.54 0.49 TY21580 (50 mg/kg BIW) 5-1 11.55 2.14 5-2 8.52 1.56 5-3 10.64 1.84 5-4 11.69 1.40 平均值 10.60 1.74 SD 1.47 0.32 TY22402 (50 mg/kg BIW) 3-1 7.10 1.42 3-2 7.08 1.00 3-3 10.77 1.70 3-4 10.25 1.98 平均值 8.80 1.53 SD 1.99 0.42 TY22566 (50 mg/kg BIW) 4-1 11.31 3.07 4-2 6.42 1.04 4-3 11.31 2.70 4-4 5.26 1.48 平均值 8.58 2.07 SD 3.19 0.97 TY22401 (50 mg/kg BIW) 2-1 10.39 2.23 2-2 11.12 4.09 2-3 10.63 1.71 2-4 11.96 2.76 平均值 11.03 2.70 SD 0.69 1.02 實例 11 :另外可活化抗體可展性檢定 Because CTLA4 constitutes expression on Treg cells, the effects of activatable antibodies TY22402, TY22566, or TY22401 on Treg cells, CD4 + T cells, and CD8 + T cells in both whole blood and spleen were evaluated and compared with the parental antibody TY21580. Monocytes or splenocytes from whole blood were stained and gated with the following antibodies: anti-CD45-BV421, anti-CD3-AF488, anti-CD4-BV510, anti-CD8a-PerCP-cy5.5, anti-CD25-APC, and anti-FoxP3 -PE. Treg cells were defined as CD45 + CD3 + CD4 + CD25 + Foxp3 + . As shown in Figure 18 , the parental antibody TY21580 increased the percentage of Treg cells in the spleen compared to the isotype control; however, the activatable antibodies TY22402 and TY22566 did not affect the percentage of splenic Treg cells. In whole blood, TY21580, TY22402 and TY22566 slightly increased the percentage of Treg cells when compared to isotype control. The activating antibody TY22401 increases the percentage of Treg cells in spleen and whole blood. TY21580, TY22402, TY22566, or TY22401 did not significantly alter the percentages of CD4 + and CD8 + T cells (data not shown). Table 18 : FACS analysis showing the effect of activatable antibodies on splenic Treg and blood Treg cells Group: Sample: Spleen: Treg % of CD4 + T cells Blood: Treg % in CD4 + T cells Isotype control (50 mg/kg , BIW) 6-1 10.50 1.71 6-2 8.25 0.70 6-3 8.04 0.67 6-4 6.81 0.90 average value 8.40 1.00 SD 1.54 0.49 TY21580 (50 mg/kg , BIW) 5-1 11.55 2.14 5-2 8.52 1.56 5-3 10.64 1.84 5-4 11.69 1.40 average value 10.60 1.74 SD 1.47 0.32 TY22402 (50 mg/kg , BIW) 3-1 7.10 1.42 3-2 7.08 1.00 3-3 10.77 1.70 3-4 10.25 1.98 average value 8.80 1.53 SD 1.99 0.42 TY22566 (50 mg/kg , BIW) 4-1 11.31 3.07 4-2 6.42 1.04 4-3 11.31 2.70 4-4 5.26 1.48 average value 8.58 2.07 SD 3.19 0.97 TY22401 (50 mg/kg , BIW) 2-1 10.39 2.23 2-2 11.12 4.09 2-3 10.63 1.71 2-4 11.96 2.76 average value 11.03 2.70 SD 0.69 1.02 Example 11 : Additional Activatable Antibody Spreadability Assay

利用於哺乳動物細胞中表現之經純化之可活化抗體進行若干不同測試以測定其可展性特性。進行三種加速應力測試。首先,將可活化抗體TY22401、TY22402或TY22566在50℃下培育7天,及藉由SEC測定其穩定性並與同型對照比較( 49)。於50℃下培育7天後針對可活化抗體觀察到高分子量(HMW)聚集體或低分子量(LMW)片段之僅稍微增加(若有的話) ( 19)。 19 :在 50 7 天之可活化抗體穩定性 樣品: HMW % ( 對照 ) LMW % ( 對照 ) HMW % (50 7 ) LMW % (50 ,7 ) TY22566 1.35 0.33 1.24 2.78 TY22401 2.15 0.20 2.13 1.97 TY22402 2.46 0 0.45 2.57 Several different assays were performed using purified activatable antibodies expressed in mammalian cells to determine their spreadability properties. Three accelerated stress tests were performed. First, the activatable antibodies TY22401, TY22402 or TY22566 were incubated at 50°C for 7 days, and their stability was determined by SEC and compared with the isotype control ( Figure 49 ). Only slight, if any, increase in high molecular weight (HMW) aggregates or low molecular weight (LMW) fragments was observed for activatable antibodies after 7 days of incubation at 50°C ( Table 19 ). Table 19 : Stability of activatable antibodies at 50 °C for 7 days Sample: HMW % ( control ) LMW % ( Control ) HMW % (50 , 7 days ) LMW % (50 , 7 days ) TY22566 1.35 0.33 1.24 2.78 TY22401 2.15 0.20 2.13 1.97 TY22402 2.46 0 0.45 2.57

接下來,將可活化抗體TY22401、TY22402或TY22566在40℃下培育7、14、21或28天,及藉由SEC測定其穩定性並與同型對照比較( 50)。於40℃下培育各種時間點後針對可活化抗體觀察到高分子量(HMW)聚集體或低分子量(LMW)片段之僅稍微增加(若有的話) ( 20)。 20 :在 40 28 天之可活化抗體穩定性 樣品: HMW % ( 對照 ) LMW % ( 對照 ) HMW % (40 28 ) LMW % (40 28 ) TY22566 1.02 0.00 1.86 1.64 TY22401 1.57 0.00 2.03 1.19 TY22402 1.02 0.00 1.86 1.64 Next, the activatable antibodies TY22401, TY22402, or TY22566 were incubated at 40°C for 7, 14, 21, or 28 days, and their stability was determined by SEC and compared with isotype controls ( Figure 50 ). Only slight, if any, increases in high molecular weight (HMW) aggregates or low molecular weight (LMW) fragments were observed for activatable antibodies after various time points of incubation at 40°C ( Table 20 ). Table 20 : Stability of activatable antibodies at 40 °C for 28 days Sample: HMW % ( control ) LMW % ( Control ) HMW % (40 , 28 days ) LMW % (40 , 28 days ) TY22566 1.02 0.00 1.86 1.64 TY22401 1.57 0.00 2.03 1.19 TY22402 1.02 0.00 1.86 1.64

此外,使可活化抗體TY22401、TY22402或TY22566經歷六個凍熔循環。藉由在-80℃下將100 μL樣品(1 mg/mL含於20 mM組胺酸,pH 5.5中)冷凍30分鐘,接著在室溫下解凍60分鐘進行凍熔測試,及藉由SEC量測穩定性並與同型對照比較( 51)。於此等凍熔循環後針對可活化抗體觀察到高分子量(HMW)聚集體之僅稍微增加(若有的話) ( 21)。 21 :於 6 個凍熔循環後之可活化抗體穩定性 樣品: HMW % ( 對照 ) HMW % (6 個循環 ) TY22566 1.35 4.36 TY22401 2.15 4.96 TY22402 2.46 3.48 Additionally, activatable antibody TY22401, TY22402, or TY22566 was subjected to six freeze-thaw cycles. Freeze-thaw testing was performed by freezing 100 μL samples (1 mg/mL in 20 mM histidine, pH 5.5) at -80°C for 30 minutes, followed by thawing at room temperature for 60 minutes, and by SEC measurements. Test stability and compare with isotype control ( Figure 51 ). Only slight, if any, increase in high molecular weight (HMW) aggregates was observed for activatable antibodies after these freeze-thaw cycles ( Table 21 ). Table 21 : Stability of activatable antibodies after 6 freeze-thaw cycles Sample: HMW % ( control ) HMW % (6 cycles ) TY22566 1.35 4.36 TY22401 2.15 4.96 TY22402 2.46 3.48

接下來,將可活化抗體於20 mM組胺酸,pH 5.5中濃縮至大於115 mg/mL。然後將經濃縮之可活化抗體稀釋至20 mg/mL用於分析高分子量(HMW)物質。如 52 及表 22中所示,未觀察到HMW物質之明顯增加,表明此等可活化抗體於所測試之緩衝液中極其可溶且穩定,多達高濃度。 22 :可活化抗體之濃度 >150 mg/mL 樣品: 經濃縮 (mg/mL) HMW % ( 對照 ) HMW % ( 經濃縮 ) TY22566 125.73 2.35 3.69 TY22401 115.98 1.47 1.80 TY22402 128.87 1.82 4.04 Next, the activatable antibody is concentrated to >115 mg/mL in 20 mM histidine, pH 5.5. The concentrated activatable antibody was then diluted to 20 mg/mL for analysis of high molecular weight (HMW) species. As shown in Figure 52 and Table 22 , no significant increase in HMW species was observed, indicating that these activatable antibodies are extremely soluble and stable in the buffers tested, up to high concentrations. Table 22 : Concentration of activatable antibodies >150 mg/mL Sample: Concentrated (mg/mL) HMW % ( control ) HMW % ( concentrated ) TY22566 125.73 2.35 3.69 TY22401 115.98 1.47 1.80 TY22402 128.87 1.82 4.04

一併考慮,資料指示所發現之可活化抗體在各種應力條件下保持穩定,及因此,甚至在無調配物最佳化下,其具有良好可展性特性( 53)。 實例12:TY21580及伊匹單抗對CTLA4之抗原決定基結合及交叉反應性 Taken together, the data indicate that the found activatable antibodies remain stable under various stress conditions and, therefore, have good spreadability properties even without formulation optimization ( Figure 53 ). Example 12: Epitope binding and cross-reactivity of TY21580 and ipilimumab to CTLA4

人類CTLA4之F片、FG環路及G片含有對CTLA4與其配位體CD80及CD86相互作用重要之大多數接觸殘基( 54 ;圖 55A 55B)。例如,人類CTLA-4/CD86介面藉由CTLA4之前β-片上之殘基E33、R35、T53、E97、M99、Y100、P101、P102、P103、Y104、Y105、L106形成(Schwartz等人(2001) Nature 410(6828): 604-608)及CTLA4之FG環路中之殘基之丙胺酸置換(99MYPPPYY105)減少或廢除與CD80之結合(Stamper等人(2001) Nature 410(6828): 608-611)。 The F-sheet, FG loop, and G-sheet of human CTLA4 contain most of the contact residues important for the interaction of CTLA4 with its ligands CD80 and CD86 ( Figure 54 ; Figures 55A and 55B ). For example, the human CTLA-4/CD86 interface is formed by residues E33, R35, T53, E97, M99, Y100, P101, P102, P103, Y104, Y105, L106 on the β-sheet preceding CTLA4 (Schwartz et al. (2001) Nature 410(6828): 604-608) and alanine substitution (99MYPPPYY105) of residues in the FG loop of CTLA4 reduce or abolish binding to CD80 (Stamper et al. (2001) Nature 410(6828): 608-611 ).

伊匹單抗(抗CTLA4抗體)亦在CTLA4之前β-片上在F及G股(殘基I93、K95、E97、L106及I108)、FG環路(殘基99MYPPPY104)及CC’環路(殘基L39、V46及I93;位於FG環路之對側)處與CTLA4相互作用。先前研究顯示人類CTLA4之殘基S20、R35、R40、Q76、D88、K95、E97、Y104、L106及I108之突變導致伊匹單抗結合之顯著損失,其中殘基R35、K95、E97、Y104及I108之突變顯示CTLA4結合之嚴重損失。此外,顯示伊匹單抗於伊匹單抗-CTLA4晶體結構中直接接觸CTLA4之殘基R35、K95、E97、Y104、L106及I108 (Ramagopal等人(2017) Proc Natl Acad Sci USA 114(21): 4223-4232)。此外,晶體結構已揭示伊匹單抗抗原決定基部分佔據CTLA4之CD80/CD86結合位點,具有較受體-配位體介面(針對CTLA-4/CD80 1,255 Å2及針對CTLA-4/CD86 1,212 Å2)更大介面面積(針對CTLA4/伊匹單抗1,880 Å2) (Ramagopal等人(2017) Proc Natl Acad Sci USA 114(21): 4223-4232;Lee等人(2016) Nat Commun 7(13354);He等人(2017) Oncotarget 8:67129-67139)。Ipilimumab (an anti-CTLA4 antibody) also targets the F and G strands (residues I93, K95, E97, L106 and I108), the FG loop (residue 99MYPPPY104) and the CC' loop (residues 99MYPPPY104) on the CTLA4 beta-sheet. Bases L39, V46 and I93; located on the opposite side of the FG loop) interact with CTLA4. Previous studies have shown that mutations in residues S20, R35, R40, Q76, D88, K95, E97, Y104, L106 and I108 of human CTLA4 lead to a significant loss of ipilimumab binding, among which residues R35, K95, E97, Y104 and Mutation of I108 shows severe loss of CTLA4 binding. Furthermore, ipilimumab was shown to directly contact residues R35, K95, E97, Y104, L106 and I108 of CTLA4 in the ipilimumab-CTLA4 crystal structure (Ramagopal et al. (2017) Proc Natl Acad Sci USA 114(21) : 4223-4232). In addition, the crystal structure has revealed that the ipilimumab epitope partially occupies the CD80/CD86 binding site of CTLA4 and has a relatively high receptor-ligand interface (1,255 Å2 for CTLA-4/CD80 and 1,212 Å for CTLA-4/CD86 Å2) Larger interface area (1,880 Å2 for CTLA4/ipilimumab) (Ramagopal et al. (2017) Proc Natl Acad Sci USA 114(21): 4223-4232; Lee et al. (2016) Nat Commun 7(13354) ; He et al. (2017) Oncotarget 8:67129-67139).

研究抗CTLA4抗體TY21580及伊匹單抗之抗原決定基結合特徵。為縮小重要CTLA4抗原決定基基序及結合至CTLA4之TY21580及伊匹單抗之殘基,採用系統方法。首先,使用低副本數目基於CEN/ARS之載體以在用於釀酒酵母中之細胞表面展示之可誘導GAL1-10啟動子之控制下表現小鼠CTLA4、人類CTLA4或各種人類CTLA4突變蛋白質(Boder及Wittrup (1997) Nat Biotechnol 15(6):553-7)。於突變CTLA4載體中,將人類CTLA4之特定基序或殘基突變成丙胺酸或小鼠 CTLA4之對應殘基。將TY21580或伊匹單抗添加至展示CTLA4之細胞中及通過流動式細胞測量術評估抗體結合。Study the epitope binding characteristics of anti-CTLA4 antibody TY21580 and ipilimumab. A systematic approach was used to narrow down important CTLA4 epitope motifs and the residues of TY21580 and ipilimumab that bind to CTLA4. First, low copy number CEN/ARS-based vectors were used to express mouse CTLA4, human CTLA4, or various human CTLA4 mutant proteins under the control of the inducible GAL1-10 promoter for cell surface display in Saccharomyces cerevisiae (Boder and Wittrup (1997) Nat Biotechnol 15(6):553-7). In the mutated CTLA4 vector, specific motifs or residues of human CTLA4 are mutated into alanine or the corresponding residues of mouse CTLA4. TY21580 or ipilimumab was added to CTLA4-displaying cells and antibody binding was assessed by flow cytometry.

用於比較,來自CTLA4-CD80 (PDBID:1I8L)、CTLA4-CD86 (PDBID:1I85)及CTLA4-伊匹單抗(PDBID: 5XJ3, 5TRU)晶體結構(於來自分子間介面之C-α原子之4 Å內)之CTLA4上之接觸殘基示於 54中。將藉由其相互作用C-α原子定義之接觸殘基於源自其對應X-射線結構之其人類序列中標記為灰色殘基。 For comparison, from CTLA4-CD80 (PDBID: 1I8L), CTLA4-CD86 (PDBID: 1I85) and CTLA4-ipilimumab (PDBID: 5XJ3, 5TRU) crystal structures (between C-α atoms from the intermolecular interface The contact residues on CTLA4 (within 4 Å) are shown in Figure 54 . Contact residues defined by their interacting C-alpha atoms are marked as gray residues in their human sequences derived from their corresponding X-ray structures.

56A 56B中所示,TY21580及伊匹單抗之交叉反應性之差異係驚人的,其中TY21580結合至人類及小鼠CTLA4二者,及伊匹單抗結合至人類CTLA4但是不結合至小鼠CTLA4。用於比較,使人類CD80 ( 56C)、人類CD86 ( 56D)及小鼠CD86 ( 56E)與人類及小鼠CTLA4二者相互作用。 As shown in Figures 56A and 56B , the difference in cross-reactivity between TY21580 and ipilimumab is striking, with TY21580 binding to both human and mouse CTLA4, and ipilimumab binding to human CTLA4 but not to Mouse CTLA4. For comparison, human CD80 ( Fig. 56C ), human CD86 ( Fig. 56D ), and mouse CD86 ( Fig. 56E ) were interacted with both human and mouse CTLA4.

為進一步研究伊匹單抗及TY21580對CTLA4之抗原決定基結合特性,將人類CTLA4之各種基序突變及評估結合。TY21580、伊匹單抗、人類CD80、人類CD86及小鼠CD86維持對ADS42AAA CTLA4突變體相較於野生型CTLA4相似結合能力( 56A 56E)。然而,當將人類CTLA4 CC’環路基序(42ADSQVT47)突變為小鼠CTLA4 CC’環路基序(42TNDQMT47)時,TY21580相較於結合至突變體之伊匹單抗、人類CD80、人類CD86及小鼠CD86兩倍更強結合至突變體( 56A 56E)。一致觀察到CD80及CD86不直接結合至此CC’環路基序(參見例如, 54)。雖然伊匹單抗與人類CTLA4中之此基序直接接觸( 54;Ramagopal等人(2017) Proc Natl Acad Sci USA 114(21): 4223-4232),人類CTLA4結合對CC’環路之突變變化之影響係極小(參見 56B中之人類WT相對於ADSQVT42TNDQMT)。一併考慮,此等結果表明CC’環路基序不顯著促進伊匹單抗或TY21580對人類CTLA4之結合親和力。 In order to further study the epitope binding properties of ipilimumab and TY21580 on CTLA4, various motifs of human CTLA4 were mutated and evaluated. TY21580, ipilimumab, human CD80, human CD86, and mouse CD86 maintained similar binding abilities to ADS42AAA CTLA4 mutants compared to wild-type CTLA4 ( Figures 56A to 56E ). However, when the human CTLA4 CC' loop motif (42ADSQVT47) was mutated into the mouse CTLA4 CC' loop motif (42TNDQMT47), TY21580 showed better binding to the mutant ipilimumab, human CD80, human CD86, and small Murine CD86 bound twice more strongly to the mutant ( Figures 56A to 56E ). It was consistently observed that CD80 and CD86 do not bind directly to this CC' loop motif (see, eg, Figure 54 ). Although ipilimumab directly contacts this motif in human CTLA4 ( Figure 54 ; Ramagopal et al. (2017) Proc Natl Acad Sci USA 114(21):4223-4232), human CTLA4 binds mutations to the CC' loop The impact of the change was minimal (see human WT vs. ADSQVT42TNDQMT in Figure 56B ). Taken together, these results indicate that the CC' loop motif does not significantly contribute to the binding affinity of ipilimumab or TY21580 to human CTLA4.

來自抗原決定基定位資料之最有趣觀察中之一者為當自人類CTLA4之殘基105至108引入突變時,結合至CTLA4之伊匹單抗與TY21580之間之驚人差異。例如,當將YL105AA及LGI106AAA引入人類CTLA4時,伊匹單抗失去其結合CTLA4之能力( 56B),與 54中所示之對伊匹單抗結合至人類CTLA4重要之已知接觸胺基酸一致。此外,如 56A 56C 56E中所示,TY21580、人類CD80、人類CD86及小鼠CD86對CTLA4 YL105AA突變體之結合親和力減弱。雖然人類CD80、人類CD86及小鼠CD86與LGI106AAA之結合親和力亦減弱,TY21580與LGI106AAA之結合親和力顯著增強。此與伊匹單抗與LGI106AAA之結合親和力之總損失形成對比。 One of the most interesting observations from the epitope mapping data is the surprising difference between ipilimumab and TY21580 binding to CTLA4 when mutations are introduced from residues 105 to 108 of human CTLA4. For example, when YL105AA and LGI106AAA were introduced into human CTLA4, ipilimumab lost its ability to bind CTLA4 ( Figure 56B ), with the known contact amine groups shown in Figure 54 being important for ipilimumab binding to human CTLA4. Acid consistent. Furthermore, as shown in Figures 56A and 56C - 56E , the binding affinity of TY21580, human CD80, human CD86, and mouse CD86 to the CTLA4 YL105AA mutant was reduced. Although the binding affinity of human CD80, human CD86 and mouse CD86 to LGI106AAA is also weakened, the binding affinity of TY21580 to LGI106AAA is significantly enhanced. This is in contrast to the overall loss of binding affinity of ipilimumab to LGI106AAA.

用於CTLA4藉由TY21580及伊匹單抗之結合位點之抗原決定基定位,將突變引入人類CTLA4之特異性位點。針對誘變選擇之位點為CTLA4藉由伊匹單抗、CD80及CD86之已知接觸位點(基於其晶體結構,參見 54),該等位點在人類與小鼠CTLA4序列之間具有大的序列變化。例如, 55A 55B顯示CD80及CD86與人類CTLA4之複合結構之正視圖,其中突出I108且顯示為球。I108為與伊匹單抗接觸之最關鍵位點中之一者,然而其遠離CD80及CD86接觸區。G股含有接觸伊匹單抗、CD80及CD86之大多數殘基,該等殘基在人類與小鼠CTLA4之間顯著不同,如 54中所示。CC’環路基序亦含有與伊匹單抗之接觸殘基,該等殘基在人類與小鼠CTLA4之間不同,然而, 56A 56E中之結果指示引入CC’環路基序之突變體不顯著促進伊匹單抗、TY21580、CD80或CD86對CTLA4之整體結合。因此,為進一步評估G股基序對伊匹單抗、TY21580、CD80及CD86之結合貢獻,將人類CTLA4之G股(105YLGIG109)突變為小鼠CTLA4 (105FVGMG109)。 It is used to map the epitope of CTLA4 through the binding site of TY21580 and ipilimumab, and introduce mutations into the specific site of human CTLA4. The sites selected for mutagenesis were known contact sites of CTLA4 via ipilimumab, CD80 and CD86 (based on their crystal structures, see Figure 54 ), which are shared between human and mouse CTLA4 sequences. Large sequence changes. For example, Figures 55A and 55B show front views of complex structures of CD80 and CD86 with human CTLA4, with I108 highlighted and shown as a sphere. I108 is one of the most critical sites of contact with ipilimumab, however it is far from the CD80 and CD86 contact areas. The G-strand contains most of the residues that contact ipilimumab, CD80, and CD86, which are significantly different between human and mouse CTLA4, as shown in Figure 54 . The CC' loop motif also contains contact residues with ipilimumab that differ between human and mouse CTLA4, however, the results in Figures 56A to 56E indicate that mutants introducing the CC' loop motif Does not significantly promote the overall binding of ipilimumab, TY21580, CD80 or CD86 to CTLA4. Therefore, to further evaluate the contribution of the G-share motif to the binding of ipilimumab, TY21580, CD80 and CD86, the G-share of human CTLA4 (105YLGIG109) was mutated into mouse CTLA4 (105FVGMG109).

56B中所示,伊匹單抗遭受對YLGIG105FVGMG突變體之結合親和力之嚴重損失。相反,當引入此突變時,TY21580、人類CD80、人類CD86及小鼠CD86保持結合能力( 56A 56C 56E)。此指示TY21580具有與伊匹單抗不同之結合抗原決定基,由於人類與小鼠CTLA4之間之G股結合中之此差異。 As shown in Figure 56B , ipilimumab suffered a severe loss of binding affinity to the YLGIG105FVGMG mutant. In contrast, TY21580, human CD80, human CD86 and mouse CD86 retained binding ability when this mutation was introduced ( Figures 56A , 56C to 56E ). This indicates that TY21580 has a different binding epitope than ipilimumab due to this difference in G-strand binding between human and mouse CTLA4.

此外,人類CTLA4之G股中之其他胺基酸經對應小鼠序列或其他胺基酸置換以探測其與伊匹單抗、TY21580、CD80及CD86之相互作用。已知I108為用於伊匹單抗結合之極其重要胺基酸。與此期望一致,伊匹單抗失去其與I108A、I108S、I108E、I108K及I108R CTLA4突變體之結合親和力( 56B)。相反,I108突變體(I108A、I108S、I108E、I108K及I108R)對TY21580、CD80或CD86之結合親和力不具有影響,突出TY21580、CD80及CD86之結合抗原決定基相較於伊匹單抗(如由I108,伊匹單抗之最關鍵結合位點中之一者所示)之重要差異(Ramagopal等人(2017) Proc Natl Acad Sci USA 114(21): 4223-4232)。 55A 55B中之複合結構顯示I108的確物理上遠離CTLA4與其配位體CD80及CD86之間之結合位點,及因此可對CD80/CD86與CTLA4之間之結合不具有顯著影響。吾人之觀察證實TY21580之結合抗原決定基更類似於CD80/CD86,不同於伊匹單抗之抗原決定基。因此,認為TY21580特異性結合至包含胺基酸殘基Y105及L106,但是非人類CTLA4之I108之抗原決定基。 實例13:TY21580及伊匹單抗對CTLA4配位體結合阻斷之影響 In addition, other amino acids in the G-strand of human CTLA4 were replaced with corresponding mouse sequences or other amino acids to detect its interactions with ipilimumab, TY21580, CD80 and CD86. I108 is known to be an extremely important amino acid for ipilimumab binding. Consistent with this expectation, ipilimumab lost its binding affinity to the I108A, I108S, I108E, I108K and I108R CTLA4 mutants ( Figure 56B ). In contrast, the I108 mutants (I108A, I108S, I108E, I108K and I108R) had no effect on the binding affinity of TY21580, CD80 or CD86, highlighting the binding epitopes of TY21580, CD80 and CD86 compared to ipilimumab (e.g. by ipilimumab). I108, one of the most critical binding sites of ipilimumab) (Ramagopal et al. (2017) Proc Natl Acad Sci USA 114(21): 4223-4232). The composite structures in Figures 55A and 55B show that I108 is indeed physically far away from the binding site between CTLA4 and its ligands CD80 and CD86, and therefore may not have a significant impact on the binding between CD80/CD86 and CTLA4. Our observations confirm that the binding epitope of TY21580 is more similar to CD80/CD86 and different from the epitope of ipilimumab. Therefore, it is believed that TY21580 specifically binds to an epitope comprising amino acid residues Y105 and L106, but not I108 of human CTLA4. Example 13: Effect of TY21580 and ipilimumab on CTLA4 ligand binding blockade

通過基於ELISA之實驗測試TY21580及伊匹單抗阻斷CTLA4結合至其同源配位體CD80及CD86之能力,如實例3中所述( 藉由 ELISA 之配位體競爭結合)。簡言之,於一實驗中,將ELISA微定量盤塗覆重組人類CTLA4蛋白(1 μg/mL)及將經生物素化之CTLA4配位體(在1 μg/mL下之CD80或在2 μg/mL下之CD86)以及TY21580、伊匹單抗或同型對照抗體之連續稀釋添加至各孔( 57A 57B)。於另一實驗中,將ELISA微定量盤塗覆重組CD80或CD86蛋白(1 μg/mL)及將經生物素化之人類CTLA4 (0.2 μg/mL或1 μg/mL)以及TY21580、伊匹單抗或同型對照抗體之連續稀釋添加至各孔( 57C 57D)。於兩個實驗中,使用經HRP標記之中性親和素檢測人類CTLA4與CD80或CD86之間之結合水平,如實例3中所述( 藉由 ELISA 之配位體競爭結合)。 The ability of TY21580 and ipilimumab to block CTLA4 binding to its cognate ligands CD80 and CD86 was tested by ELISA-based experiments as described in Example 3 ( ligand competition binding by ELISA ) . Briefly, in one experiment, ELISA microplates were coated with recombinant human CTLA4 protein (1 μg/mL) and biotinylated CTLA4 ligand (CD80 at 1 μg/mL or CD80 at 2 μg /mL) and serial dilutions of TY21580, ipilimumab, or isotype control antibodies were added to each well ( Figures 57A and 57B ). In another experiment, an ELISA microplate was coated with recombinant CD80 or CD86 protein (1 μg/mL) and biotinylated human CTLA4 (0.2 μg/mL or 1 μg/mL), as well as TY21580, ipilimumab. Serial dilutions of anti- or isotype control antibodies were added to each well ( Figures 57C and 57D ). In two experiments, HRP-labeled neutral avidin was used to detect the binding level between human CTLA4 and CD80 or CD86, as described in Example 3 ( ligand competition for binding by ELISA ).

57A 57B中所示,當將CTLA4固定在微定量盤上時,TY21580及伊匹單抗以劑量依賴性方式阻斷CD80及CD86結合至CTLA4,然而同型對照抗體不顯示阻斷活性,指示檢定特異性。如 57C 57D中所示,當將CD80或CD86固定在微定量盤上時,TY21580及伊匹單抗再次以劑量依賴性方式阻斷CD80及CD86結合至CTLA4,而同型對照抗體不顯示阻斷活性。如 23中所示,雖然TY21580及伊匹單抗均阻斷配位體結合至CTLA4,當將人類CTLA4固定在微定量盤上時相較於當將CD80或CD86固定在微定量盤上時,TY21580及伊匹單抗之劑量依賴性阻斷活性係驚人不同。此等結果指示TY21580及伊匹單抗展示可比較之配位體阻斷活性,在CTLA4固定條件下具有相似IC50。然而,在CD80或CD86配位體固定條件下,伊匹單抗展示對CD80及CD86二者較TY21580強得多的配位體阻斷活性,如圖 57C 57D 23中所示。然而,另外實驗表明在飽和濃度下添加抗CTLA4抗體後CTLA4與其配位體之相互作用之完全阻斷對腫瘤排斥不必要,意指TY21580在不完全阻斷CD80或CD86下仍可活化T細胞且具有強效抗腫瘤功效。事實上,TY21586顯示CD80及CD86之完全阻斷,類似於伊匹單抗,如 58中所示。然而,TY21586及伊匹單抗具有較測試之其他抗體低得多的ADCC報告基因活性( 59),及因此於經由ADCC效應耗盡Treg細胞中較不有效(亦參見Tang等人(2018) Cell Biosci. 8(30):1-3)。不希望受理論束縛,據信CD28依賴性T細胞活化(例如,藉由阻斷CTLA4活性)對有效腫瘤排斥係重要的,然而T細胞之過度刺激可係有害。因此,針對TY21580觀察到之較弱配位體阻斷可為超過伊匹單抗之較強配位體阻斷活性之優點。 23 TY21580 及伊匹單抗對 CTLA4 配位體結合阻斷之 IC50 測試抗體 IC 50(nM) 板結合CTLA4 溶液中之CTLA4 溶液中之CD80 溶液中之CD86 板結合CD80 板結合CD86 TY21580 3.809 0.4806 59.08 5.583 伊匹單抗 4.116 0.5873 1.789 1.429 As shown in Figures 57A and 57B , TY21580 and ipilimumab blocked CD80 and CD86 binding to CTLA4 in a dose-dependent manner when CTLA4 was immobilized on microplates, whereas the isotype control antibody showed no blocking activity. Indicates assay specificity. As shown in Figures 57C and 57D , when CD80 or CD86 was immobilized on microplates, TY21580 and ipilimumab again blocked CD80 and CD86 binding to CTLA4 in a dose-dependent manner, whereas the isotype control antibody showed no resistance. off activity. As shown in Table 23 , although TY21580 and ipilimumab both block ligand binding to CTLA4, when human CTLA4 is immobilized on microtiter plates compared to when CD80 or CD86 are immobilized on microtiter plates , the dose-dependent blocking activities of TY21580 and ipilimumab are surprisingly different. These results indicate that TY21580 and ipilimumab exhibit comparable ligand blocking activity with similar IC50 under CTLA4-fixed conditions. However, under conditions where CD80 or CD86 ligands were immobilized, ipilimumab exhibited much stronger ligand blocking activity for both CD80 and CD86 than TY21580, as shown in Figures 57C and 57D and Table 23 . However, additional experiments show that complete blocking of the interaction between CTLA4 and its ligand after adding anti-CTLA4 antibodies at saturating concentrations is not necessary for tumor rejection, meaning that TY21580 can still activate T cells without completely blocking CD80 or CD86 and It has strong anti-tumor effect. In fact, TY21586 showed complete blockade of CD80 and CD86, similar to ipilimumab, as shown in Figure 58 . However, TY21586 and ipilimumab had much lower ADCC reporter activity than other antibodies tested ( Figure 59 ), and were therefore less effective in depleting Treg cells via the ADCC effect (see also Tang et al. (2018) Cell Biosci. 8(30):1-3). Without wishing to be bound by theory, it is believed that CD28-dependent T cell activation (eg, by blocking CTLA4 activity) is important for effective tumor rejection, whereas overstimulation of T cells can be detrimental. Therefore, the weaker ligand blocking observed for TY21580 may be an advantage over the stronger ligand blocking activity of ipilimumab. Table 23 : IC50 of TY21580 and ipilimumab for blocking CTLA4 ligand binding Test antibody IC 50 (nM) Plate binding CTLA4 CTLA4 in solution CD80 in solution CD86 in solution plate binding CD80 plate binding CD86 TY21580 3.809 0.4806 59.08 5.583 ipilimumab 4.116 0.5873 1.789 1.429

雖然此等差異之潛在機理目前不清楚,結果指示TY21580與伊匹單抗之性質之間存在固有差異。一併考慮,此等結果表明TY21580可充當配位體阻斷劑,破壞CD80及CD86結合至其抑制性受體CTLA4。不希望受理論束縛,認為通過弱配位體阻斷將CD80及CD86自CTLA4固存解除允許此等配位體在T細胞活化期間通過共刺激受體CD28信號傳導,其可導致在T細胞免疫活化期間增加之有效性及安全性。 實例 14 TY21580 及伊匹單抗對 CD28 路徑激活之 CTLA4 阻斷之影響 Although the underlying mechanisms for these differences are currently unclear, the results indicate that there are inherent differences between the properties of TY21580 and ipilimumab. Taken together, these results indicate that TY21580 can act as a ligand blocker, disrupting the binding of CD80 and CD86 to their inhibitory receptor CTLA4. Without wishing to be bound by theory, it is believed that demobilization of CD80 and CD86 from CTLA4 sequestration by weak ligand blockade allows these ligands to signal through the costimulatory receptor CD28 during T cell activation, which may result in increased risk in T cell immunity. Increased effectiveness and safety during activation. Example 14 : Effect of TY21580 and ipilimumab on CTLA4 blockade of CD28 pathway activation

使用由Promega開發之基於細胞之CTLA4阻斷生物檢定(目錄號JA3001及JA3005),評估TY21580及伊匹單抗之CTLA4阻斷功能。此檢定涉及在TY21580或伊匹單抗之存在或不存在下將兩種經遺傳工程改造之細胞系共培養,然後使用Bio-Glo™螢光素酶檢定系統量測由螢光素酶報告基因產生之生物發光信號。經共培養之兩種細胞系為(1) CTLA4效應細胞–表現人類CTLA4及藉由對TCR/CD28活化回應之初始啟動子驅動之螢光素酶報告基因之Jurkat T細胞;及(2) APC/Raji細胞 –內源表現CTLA4配位體CD80及CD86且另外表現經設計以抗原獨立性方式活化TCR之細胞表面蛋白之Raji細胞。當將此等兩種細胞類型共培養時,CTLA4與CD28對CD80及CD86結合競爭,其抑制CD28路徑之活化及導致低啟動子介導之發光。添加抗CTLA4抗體至此系統中可阻斷CTLA4與其配位體CD80及CD86之相互作用,導致更高TCR/CD28活化及隨後啟動子介導之發光。 The CTLA4 blocking function of TY21580 and ipilimumab was evaluated using a cell-based CTLA4 blocking bioassay developed by Promega (catalog numbers JA3001 and JA3005). This assay involves co-culture of two genetically engineered cell lines in the presence or absence of TY21580 or ipilimumab, followed by measurement of a luciferase reporter gene using the Bio-Glo™ Luciferase Assay System The bioluminescent signal produced. The two cell lines co-cultured were (1) CTLA4 effector cells – Jurkat T cells expressing human CTLA4 and a luciferase reporter gene driven by an initial promoter in response to TCR/CD28 activation; and (2) APCs /Raji cells – Raji cells that endogenously express the CTLA4 ligands CD80 and CD86 and additionally express cell surface proteins designed to activate TCR in an antigen-independent manner. When these two cell types are co-cultured, CTLA4 and CD28 compete for CD80 and CD86 binding, which inhibits activation of the CD28 pathway and results in low promoter-mediated luminescence. Adding anti-CTLA4 antibodies to this system blocks the interaction of CTLA4 with its ligands CD80 and CD86, resulting in higher TCR/CD28 activation and subsequent promoter-mediated luminescence.

58中所示,所有測試抗體以劑量依賴性方式激活TCR/CD28路徑,如藉由隨著抗體濃度增加增加報告基因信號(即,發光)所證明。相比之下,同型對照抗體不顯示活性。此等結果證明藉由此等不同抗CTLA4抗體之CTLA4功能阻斷活性之範圍。雖然該等抗體之EC50有點相似( 24),藉由此等抗體刺激之功能信號傳導活化之量級指示伊匹單抗及TY21586為所測試之最強效CTLA4阻斷劑,接著為TY21680、TY21580及TY21687 ( 58)。作為出自所測試抗體之CTLA4阻斷劑,TY21691具有最弱活性。此等結果證明所有測試抗體可以劑量依賴性方式阻斷CTLA4與CD80及CD86之相互作用,導致下游TCR/CD28路徑活性。不希望受理論束縛,結果表明所有測試抗體可增加T細胞活化,其可有利於治療癌症。如上所討論,較弱配位體阻斷劑(諸如TY21580)可於治療癌症中特別有效,因為其相較於強結合劑減低之過度刺激T細胞之風險。 24 :抗 CTLA4 抗體對 TCR/CD28 路徑激活之 EC50 伊匹單抗 TY21580 TY21586 TY21687 TY21680 TY21691 EC 50(μg/mL) 1.62 5.06 4.66 5.01 8.21 2.64 實例15:抗CTLA4抗體之ADCC活性 As shown in Figure 58 , all tested antibodies activated the TCR/CD28 pathway in a dose-dependent manner, as demonstrated by increased reporter signal (i.e., luminescence) with increasing antibody concentration. In contrast, isotype control antibodies showed no activity. These results demonstrate the range of CTLA4 function blocking activity by these different anti-CTLA4 antibodies. Although the EC50s of these antibodies are somewhat similar ( Table 24 ), the magnitude of functional signaling activation stimulated by these antibodies indicates that ipilimumab and TY21586 are the most potent CTLA4 blockers tested, followed by TY21680, TY21580 and TY21687 ( Figure 58 ). TY21691 had the weakest activity as a CTLA4 blocker from the antibodies tested. These results demonstrate that all tested antibodies can block the interaction of CTLA4 with CD80 and CD86 in a dose-dependent manner, resulting in downstream TCR/CD28 pathway activity. Without wishing to be bound by theory, the results indicate that all tested antibodies increase T cell activation, which may be beneficial in treating cancer. As discussed above, weaker ligand blockers (such as TY21580) may be particularly effective in treating cancer due to their reduced risk of overstimulating T cells compared to strong binders. Table 24 : EC50 of anti -CTLA4 antibody on TCR/CD28 pathway activation ipilimumab TY21580 TY21586 TY21687 TY21680 TY21691 EC 50 (μg/mL) 1.62 5.06 4.66 5.01 8.21 2.64 Example 15: ADCC activity of anti-CTLA4 antibodies

測試及比較伊匹單抗、TY21580、TY21586、TY21687、TY21680及TY21691之抗體依賴性細胞毒性(ADCC)活性。使用ADCC報告基因檢定評價可活化抗體之ADCC活性。將過度表現人類CTLA4之HEK293F細胞(HEK293F/hCTLA4細胞)用作靶細胞;將過度表現CD16及NFAT-Luc之Jurkat細胞系(Jurkat/CD16a細胞)用作效應細胞。在經連續稀釋之抗CTLA4抗體之存在或不存在下,將1.2 x 10 5個Jurkat/CD16a細胞及2x10 4個HEK293F/hCTLA4細胞(E:T比率6:1)於96孔組織培養微定量盤中混合。於培育6小時後,將One-Glo試劑添加至細胞,及將細胞裂解。為量測報告基因活性,移除上清液用於使用SpectraMax i3x板讀取器之發光量測。將人類IgG1同型對照抗體用作陰性對照。 The antibody-dependent cellular cytotoxicity (ADCC) activity of ipilimumab, TY21580, TY21586, TY21687, TY21680 and TY21691 was tested and compared. The ADCC activity of activatable antibodies is evaluated using the ADCC reporter assay. HEK293F cells that overexpress human CTLA4 (HEK293F/hCTLA4 cells) were used as target cells; Jurkat cell lines that overexpressed CD16 and NFAT-Luc (Jurkat/CD16a cells) were used as effector cells. 1.2 x 10 Jurkat/CD16a cells and 2 x 10 HEK293F/hCTLA4 cells (E:T ratio 6:1) were plated in a 96-well tissue culture microplate in the presence or absence of serially diluted anti-CTLA4 antibodies. Medium mix. After 6 hours of incubation, One-Glo reagent was added to the cells and the cells were lysed. To measure reporter activity, the supernatant was removed and used for luminescence measurements using a SpectraMax i3x plate reader. A human IgG1 isotype control antibody was used as a negative control.

59中所示,所有測試抗體以劑量依賴性方式顯示各種程度之ADCC信號傳導活化,然而同型對照不顯示任何ADCC活性。此等結果證明藉由此等不同抗CTLA4抗體之ADCC信號傳導/刺激活性之範圍。如由信號傳導活化之量級( 59)及EC50 ( 25)二者所證明,TY21580誘導所有所測試抗體之最強效ADCC信號傳導活性。TY21691為誘導ADCC信號傳導中之最少活性抗體。雖然早期資料證明此等抗體具有對人類CTLA4之相似結合親和力,具有單數位nM之KD,此等ADCC報告基因結果表明所測試抗體之結合抗原決定基可較其結合親和力更顯著影響ADCC活性。應注意,雖然TY21586、TY21680、TY21580、TY21687及TY21691係交叉反應性,以上結果顯示其於配位體阻斷及ADCC活性中之有效性顯著不同,表明抗體抗原決定基結合位點之細微差異可導致抗腫瘤活性之顯著差異。 25 :抗 CTLA4 抗體對 ADCC 報告基因信號傳導之 EC50   伊匹單抗 TY21580 TY21586 TY21687 TY21680 TY21691 EC50 (μg/ml) 1.08 0.174 4.69 9.5 6.76 >10 實例16:TY21580及伊匹單抗於鼠科MC38結腸直腸腫瘤模型中之抗腫瘤功效 As shown in Figure 59 , all tested antibodies showed various degrees of activation of ADCC signaling in a dose-dependent manner, whereas the isotype control did not show any ADCC activity. These results demonstrate the range of ADCC signaling/stimulatory activity by these different anti-CTLA4 antibodies. TY21580 induced the most potent ADCC signaling activity of all antibodies tested, as demonstrated by both the magnitude of signaling activation ( Figure 59 ) and the EC50 ( Table 25 ). TY21691 is the least active antibody in inducing ADCC signaling. Although early data demonstrate that these antibodies have similar binding affinities to human CTLA4, with KDs in the single digit nM, these ADCC reporter results indicate that the binding epitopes of the tested antibodies can significantly affect ADCC activity more than their binding affinities. It should be noted that although TY21586, TY21680, TY21580, TY21687 and TY21691 are cross-reactive, the above results show that their effectiveness in ligand blocking and ADCC activity is significantly different, indicating that subtle differences in antibody epitope binding sites can resulting in significant differences in antitumor activity. Table 25 : EC50 of anti- CTLA4 antibody on ADCC reporter gene signaling ipilimumab TY21580 TY21586 TY21687 TY21680 TY21691 EC50 (μg/ml) 1.08 0.174 4.69 9.5 6.76 >10 Example 16: Anti-tumor efficacy of TY21580 and ipilimumab in the murine MC38 colorectal tumor model

為測定TY21580及伊匹單抗之抗腫瘤功效,將敲入人類CTLA4之C57BL/6小鼠(n=6隻/組,雌性,5至9週齡)皮下接種MC38鼠科結腸癌細胞。當建立腫瘤(99 mm 3)時,將小鼠用同型對照抗體(1 mg/kg)、TY21580 (1 mg/kg或0.2 mg/kg)或伊匹單抗(1 mg/kg或0.2 mg/kg)藉由一週兩次腹膜內注射持續兩週處理。一週兩次監測組平均腫瘤生長( 60A)及不同組中之各小鼠之個別腫瘤生長( 60B)並報告為隨時間之平均腫瘤體積± SEM。如 60A 60B中所示,TY21580顯示在1 mg/kg (組-2)及0.2 mg/kg (組-3)之劑量下之完全抗腫瘤效應,及此等小鼠中之所有腫瘤完全被廢除,除了對一小鼠第一次給藥後第32天極小腫瘤留下。伊匹單抗亦顯示在1 mg/kg (組-4)之劑量下之完全抗腫瘤效應;然而,在第一次給藥後之第32天用伊匹單抗之較低劑量(0.2 mg/kg,組-5)處理之腫瘤之一半(3/6)自腫瘤抑制逃脫。此顯示TY21580在抗腫瘤活性方面較伊匹單抗更有效。TY21580及伊匹單抗均良好耐受,在研究期間無動物死亡,且在所測試之劑量水平下於小鼠中未觀察到顯著體重損失。總之,此等結果表明TY21580為具有較伊匹單抗更強效抗癌活性之安全且有效抗癌劑。 實例17:TY21580及伊匹單抗對鼠科MC38結腸癌模型中之腫瘤內調節T (Treg)細胞水平之影響 To determine the anti-tumor efficacy of TY21580 and ipilimumab, C57BL/6 mice with human CTLA4 knock-in (n=6/group, female, 5 to 9 weeks old) were subcutaneously inoculated with MC38 murine colon cancer cells. When tumors (99 mm 3 ) were established, mice were treated with isotype control antibody (1 mg/kg), TY21580 (1 mg/kg or 0.2 mg/kg), or ipilimumab (1 mg/kg or 0.2 mg/kg). kg) were treated by intraperitoneal injection twice a week for two weeks. Group average tumor growth ( Figure 60A) and individual tumor growth of each mouse in different groups ( Figure 60B) were monitored twice a week and reported as mean tumor volume ± SEM over time. As shown in Figures 60A and 60B , TY21580 showed complete anti-tumor effects at doses of 1 mg/kg (Group-2) and 0.2 mg/kg (Group-3), and all tumors in these mice were completely was abolished, except for one mouse with minimal tumors remaining on day 32 after the first dose. Ipilimumab also showed complete antitumor effect at a dose of 1 mg/kg (Group-4); however, on day 32 after the first dose, a lower dose of ipilimumab (0.2 mg /kg, group-5), half (3/6) of the tumors treated escaped tumor suppression. This shows that TY21580 is more effective than ipilimumab in anti-tumor activity. Both TY21580 and ipilimumab were well tolerated, with no animals dying during the study and no significant weight loss observed in mice at the dose levels tested. Taken together, these results indicate that TY21580 is a safe and effective anticancer agent with more potent anticancer activity than ipilimumab. Example 17: Effects of TY21580 and ipilimumab on intratumoral regulatory T (Treg) cell levels in the murine MC38 colon cancer model

於皮下MC38鼠科結腸癌同源模型中評價於用TY21580或伊匹單抗處理後CD4 +T細胞亞群中之T調節(Treg)細胞(CD4 +CD25 +)之百分比( 61A)。將荷瘤動物用TY21580或伊匹單抗在1 mg/kg (Q3d × 3個劑量)下處理。自腫瘤單離CD4 +T細胞,然後自此等CD4 +T細胞單離腫瘤浸潤淋巴細胞(TIL)及外周細胞(即,PBMC及脾細胞)作為亞群。 The percentage of T regulatory (Treg) cells (CD4 + CD25 + ) in the CD4 + T cell subset following treatment with TY21580 or ipilimumab was evaluated in the subcutaneous MC38 murine colon cancer syngeneic model ( Figure 61A ). Tumor-bearing animals were treated with TY21580 or ipilimumab at 1 mg/kg (Q3d × 3 doses). CD4 + T cells are isolated from tumors, and then tumor-infiltrating lymphocytes (TILs) and peripheral cells (ie, PBMCs and splenocytes) are isolated from these CD4 + T cells as subsets.

於TIL中,於TY21580處理後Treg細胞之百分比顯著降低(針對TY21580處理組15.2%相對於針對同型對照組41.6%, P<0.001)。於伊匹單抗處理後Treg細胞無顯著降低(針對伊匹單抗28.4%相對於針對同型對照41.6%, P=ns)。此與伊匹單抗不顯著改變或耗盡微腫瘤環境內之FOXP3+ Treg細胞之觀察一致(Sharma等人(2018) Clin. Cancer Res. online publication only, PMID 30054281;Ferrara等人(2018) Clin. Cancer Res.)。於PBMC中,於TY21580處理後Treg細胞之百分比稍微增加(針對TY21580組6.2%相對於針對同型對照組3.9%, P=0.01),但是於伊匹單抗處理後未看到此影響(針對伊匹單抗組4.8%相對於針對同型對照組3.9%)。亦參見Ha等人PNAS (2019) 116(2):609-618。於脾細胞中,TY21580及伊匹單抗處理均不具有對Treg細胞之百分比之影響(針對同型對照組8.6%;針對TY21580組7.8%;針對伊匹單抗組9.4%)。 In TIL, the percentage of Treg cells was significantly reduced after TY21580 treatment (15.2% for the TY21580-treated group vs. 41.6% for the isotype control group, P < 0.001). There was no significant decrease in Treg cells after ipilimumab treatment (28.4% for ipilimumab vs. 41.6% for isotype control, P = ns). This is consistent with the observation that ipilimumab does not significantly alter or deplete FOXP3+ Treg cells within the microtumor environment (Sharma et al. (2018) Clin. Cancer Res. online publication only, PMID 30054281; Ferrara et al. (2018) Clin. Cancer Res.). In PBMC, the percentage of Treg cells increased slightly after TY21580 treatment (6.2% for TY21580 vs. 3.9% for the isotype control group, P =0.01), but this effect was not seen after ipilimumab treatment (for ipilimumab) 4.8% in the pimumab group versus 3.9% in the isotype control group). See also Ha et al. PNAS (2019) 116(2):609-618. In splenocytes, neither TY21580 nor ipilimumab treatment had an impact on the percentage of Treg cells (8.6% for the isotype control group; 7.8% for the TY21580 group; 9.4% for the ipilimumab group).

亦評價細胞毒性T淋巴細胞(CD8 +T細胞)與Treg細胞之比率(即,CD8 +/Treg比率) ( 61B)。於TIL中,於用TY21580處理後CD8 +/Treg比率增加(針對TY21580 18.7相對於針對同型對照3.7; P=0.0517,接近0.05)。伊匹單抗處理相較於同型對照對CD8 +/Treg比率之影響不顯著,及至多較TY21580之影響弱。於PBMC及脾細胞中,於TY21580或伊匹單抗處理後CD8 +/Treg比率不顯著改變。此等結果證明TY21580具體而言於腫瘤浸潤細胞(即,TIL)中,但是非外周細胞(即,PBMC及脾細胞)中展示誘導Treg細胞耗盡及增加CD8 +/Treg比率之活性。 The ratio of cytotoxic T lymphocytes (CD8 + T cells) to Treg cells (i.e., CD8 + /Treg ratio) was also evaluated ( Figure 61B ). In TILs, the CD8 + /Treg ratio increased after treatment with TY21580 (18.7 for TY21580 vs. 3.7 for isotype control; P =0.0517, close to 0.05). The effect of ipilimumab treatment on the CD8 + /Treg ratio was not significant compared to the isotype control, and was at most weaker than the effect of TY21580. In PBMC and splenocytes, the CD8 + /Treg ratio did not change significantly after treatment with TY21580 or ipilimumab. These results demonstrate that TY21580 exhibits activity in inducing Treg cell depletion and increasing the CD8 + /Treg ratio specifically in tumor infiltrating cells (i.e., TILs), but not in peripheral cells (i.e., PBMCs and splenocytes).

TY21580抗CTLA4抗體對T細胞之調節活性提供對TY21580之活體內抗腫瘤功效之機械理解。不希望受理論束縛,結果表明TY21580於腫瘤微環境中降低免疫抑制Treg活性且增強細胞毒性T淋巴細胞(CD8 +T細胞)活性以介導抗腫瘤反應。TY21580與伊匹單抗之間於腫瘤Treg耗盡及CD8 +/Treg比率中之數量差異亦顯示TY21580展示較伊匹單抗於活體內更好抗腫瘤活性。 實例18:TY21580對鼠科CT26結腸癌模型中之腫瘤內調節T (Treg)細胞水平之影響 The regulatory activity of TY21580 anti-CTLA4 antibody on T cells provides mechanistic understanding of the in vivo anti-tumor efficacy of TY21580. Without wishing to be bound by theory, the results indicate that TY21580 reduces immunosuppressive Treg activity and enhances cytotoxic T lymphocyte (CD8 + T cell) activity in the tumor microenvironment to mediate anti-tumor responses. The quantitative differences in tumor Treg depletion and CD8 + /Treg ratio between TY21580 and ipilimumab also indicate that TY21580 exhibits better anti-tumor activity than ipilimumab in vivo. Example 18: Effect of TY21580 on intratumoral regulatory T (Treg) cell levels in the murine CT26 colon cancer model

於皮下CT26鼠科結腸癌同源模型中評價於用TY21580處理後CD4 +T細胞亞群中之T調節(Treg)細胞之百分比( 62A)。將荷瘤動物用TY21580 (在第0天及第3天5 mg/kg)處理。自腫瘤單離CD4 +T細胞,然後自此等CD4 +T細胞單離腫瘤浸潤淋巴細胞(TIL)及外周細胞(即,PBMC及淋巴結(LN)細胞)作為亞群。 The percentage of T regulatory (Treg) cells in the CD4 + T cell subset after treatment with TY21580 was evaluated in a subcutaneous CT26 murine colon cancer syngeneic model ( Figure 62A ). Tumor-bearing animals were treated with TY21580 (5 mg/kg on days 0 and 3). CD4 + T cells are isolated from tumors, and tumor-infiltrating lymphocytes (TILs) and peripheral cells (ie, PBMCs and lymph node (LN) cells) are then isolated as subsets from these CD4 + T cells.

62A中所示,TY21580相較於同型對照顯著降低TIL中之Treg細胞之百分比(針對TY21580 12.6%相對於針對同型對照38.5%, P<0.001)。然而,TY21580相較於同型對照不影響外周淋巴細胞(即,PBMC及淋巴結(LN)細胞)中之Treg細胞之百分比。於TY21580處理後PBMC中之Treg細胞之百分比為5.8%相對於於同型對照處理後4.9% ( P>0.05)。於TY21580處理後淋巴結細胞中之Treg細胞之百分比為8.1%相對於於同型對照處理後7.7% ( P>0.05)。 As shown in Figure 62A , TY21580 significantly reduced the percentage of Treg cells in TILs compared to isotype control (12.6% for TY21580 vs. 38.5% for isotype control, P < 0.001). However, TY21580 did not affect the percentage of Treg cells in peripheral lymphocytes (ie, PBMCs and lymph node (LN) cells) compared to isotype controls. The percentage of Treg cells in PBMC after TY21580 treatment was 5.8% compared to 4.9% after isotype control treatment ( P > 0.05). The percentage of Treg cells in lymph node cells after TY21580 treatment was 8.1% compared to 7.7% after isotype control treatment ( P >0.05).

亦評價細胞毒性T淋巴細胞(CD8 +T細胞)與Treg細胞之比率(即,CD8 +/Treg比率)。如 62B中所示,TY21580亦相較於同型對照顯著增加TIL中之CD8 +T/Treg比率(針對TY21580 8.0%相對於針對同型對照0.6%, P<0.01)。然而,TY21580不具有對外周淋巴細胞中之CD8 +T/Treg比率之顯著影響。於PBMC中,於TY21580處理後CD8 +T/Treg比率為5.3相對於於同型對照處理後6.3 ( P>0.05)。於淋巴結細胞中,於TY21580處理後CD8 +T/Treg比率為4.9相對於於同型對照處理後4.7 ( P>0.05)。 The ratio of cytotoxic T lymphocytes (CD8 + T cells) to Treg cells (i.e., CD8 + /Treg ratio) was also evaluated. As shown in Figure 62B , TY21580 also significantly increased the CD8 + T/Treg ratio in TILs compared to the isotype control (8.0% for TY21580 vs. 0.6% for the isotype control, P < 0.01). However, TY21580 did not have a significant effect on the CD8 + T/Treg ratio in peripheral lymphocytes. In PBMC, the CD8 + T/Treg ratio was 5.3 after TY21580 treatment versus 6.3 after isotype control treatment ( P > 0.05). In lymph node cells, the CD8 + T/Treg ratio was 4.9 after TY21580 treatment versus 4.7 after isotype control treatment ( P > 0.05).

此外,來自TY21580處理組之Foxp3 +CD4 +Treg細胞中之CTLA4表現水平顯著低於TIL中之同型對照組之彼等(針對TY21580 8985.2 MFI相對於針對同型對照20948.0 MFI ; 63)。然而,CTLA4表現水平於PBMC (針對TY21580 2046.7 MFI相對於針對同型對照2740.9 MFI)或淋巴結(針對TY21580 3062.0 MFI相對於針對同型對照3247.9 MFI)中不改變。TIL中之Treg細胞亦展示較外周細胞(即,PBMC及LN)中之Treg細胞高得多的CTLA4表現水平,及CTLA4表現於TY21580處理後於TIL Treg細胞中顯著較低。 Furthermore, CTLA4 expression levels in Foxp3 + CD4 + Treg cells from the TY21580-treated group were significantly lower than those in the isotype control group in TIL (8985.2 MFI for TY21580 vs. 20948.0 MFI for the isotype control; Figure 63 ). However, CTLA4 expression levels did not change in PBMC (2046.7 MFI for TY21580 vs. 2740.9 MFI for isotype control) or lymph nodes (3062.0 MFI for TY21580 vs. 3247.9 MFI for isotype control). Treg cells in TIL also displayed much higher CTLA4 expression levels than Treg cells in peripheral cells (i.e., PBMC and LN), and CTLA4 expression was significantly lower in TIL Treg cells after TY21580 treatment.

一併考慮,此等結果證明TY21580展示具體而言於腫瘤細胞(即,TIL),但是非外周細胞(即,PBMC或淋巴結細胞)中誘導Treg耗盡及增加CD8 +/Treg比率之活性。TY21580對T細胞之此等調節活性提供對TY21580之強效活體內抗腫瘤功效之機械理解。不希望受理論束縛,此等結果表明TY21580於腫瘤微環境中降低免疫抑制Treg活性且增強細胞毒性T淋巴細胞(CD8 +T細胞)活性以介導抗腫瘤反應。 實例 19 TY21580 於大型建立之 H22 肝腫瘤模型中之抗腫瘤功效 Taken together, these results demonstrate that TY21580 exhibits activity in inducing Treg depletion and increasing the CD8 + /Treg ratio specifically in tumor cells (i.e., TILs), but not peripheral cells (i.e., PBMCs or lymph node cells). These regulatory activities of TY21580 on T cells provide mechanistic understanding of the potent in vivo anti-tumor efficacy of TY21580. Without wishing to be bound by theory, these results indicate that TY21580 reduces immunosuppressive Treg activity and enhances cytotoxic T lymphocyte (CD8 + T cell) activity in the tumor microenvironment to mediate anti-tumor responses. Example 19 : Anti-tumor efficacy of TY21580 in a large established H22 liver tumor model

為測定TY21580於大型建立之腫瘤中之抗腫瘤功效,將雌性BALB/c小鼠皮下接種小鼠H22肝癌細胞。當在約500 mm 3或約800mm 3下建立相對大腫瘤時,將小鼠用TY21580在5mg/kg下藉由一週兩次(BIW)腹膜內注射持續4個劑量處理。將同型抗體用作對照。每週兩次監測組平均腫瘤生長( 64A)及不同組中之各小鼠之個別腫瘤生長( 64B 64D)並報告為隨時間之平均腫瘤體積± SEM。 To determine the anti-tumor efficacy of TY21580 in large established tumors, female BALB/c mice were subcutaneously inoculated with mouse H22 hepatoma cells. When relatively large tumors were established at about 500 mm or about 800 mm , mice were treated with TY21580 at 5 mg/kg by intraperitoneal injection twice a week (BIW) for 4 doses. Isotype antibodies were used as controls. Group mean tumor growth ( Figure 64A) and individual tumor growth for each mouse in different groups ( Figures 64B to 64D) were monitored twice weekly and reported as mean tumor volume ± SEM over time.

如針對組平均腫瘤生長之 64A及針對個別腫瘤生長之 64B 64D中所示,相較於同型對照抗體處理組( 64A 64B),於兩個TY21580處理組(即,當腫瘤達到約500 mm 3之大小時開始處理之組,如圖 64A 64C中所述,或約800mm 3時開始處理之組,如圖 64A 64D中所述)中觀察到建立之大H22腫瘤之顯著消退。此等結果證明TY21580於抑制大型建立之腫瘤中之顯著功效。 實例 20 :掩蔽肽長度對掩蔽效率之影響 As shown in Figure 64A for group average tumor growth and Figures 64B through 64D for individual tumor growth, compared to the isotype control antibody-treated group ( Figures 64A and 64B ), in both TY21580-treated groups (i.e., when tumors reached Significant establishment of large H22 tumors was observed in groups starting treatment at approximately 500 mm 3 in size, as depicted in Figures 64A and 64C , or in groups starting at approximately 800 mm 3 in size, as depicted in Figures 64A and 64D . subside. These results demonstrate the significant efficacy of TY21580 in inhibiting large established tumors. Example 20 : Effect of masking peptide length on masking efficiency

選擇兩種可活化抗體TY22402及TY22404測試掩蔽效率對掩蔽肽長度之依賴性以適合其特定應用。藉由自N端移除殘基,剩下第一半胱胺酸殘基之前之僅5或2個殘基於掩蔽肽中將TY22402及TY22404之掩蔽肽自21個殘基縮短至16或12個殘基( 26)。使此等可活化抗體表現及自哺乳動物細胞純化及如實例8中所述量測其掩蔽效率並與親本抗體TY21580比較。來自兩個實驗之結果指示可使用在第一半胱胺酸殘基之前之具有範圍自2至11個殘基之長度之不同掩蔽肽製備此等可活化抗體以調節抗體掩蔽效率( 65A 65B ;表 27 28)。此似乎表明核心掩蔽基序含有半胱胺酸環路及其緊鄰殘基,且足夠維持掩蔽效率。 26 :具有變化肽長度之掩蔽肽 樣品ID: 掩蔽+裂解肽序列(未加底線): TY22402 EVGSYIVHHSDCDAFYPYCDS SGRSAGGGGTPLGLAGSGGS(SEQ ID NO: 197) TY22775 EVGHSDCDAFYPYCDS SGRSAGGGGTPLGLAGSGGS(SEQ ID NO: 198) TY22864 EDCDAFYPYCDS SGRSAGGGGTPLGLAGSGGS(SEQ ID NO: 199) TY22404 EVGSYPNPSSDCVPYYYACAY SGRSAGGGGTPLGLAGSGGS(SEQ ID NO: 200) TY22776 EVGSSDCVPYYYACAY SGRSAGGGGTPLGLAGSGGS(SEQ ID NO: 201) TY22871 EDCVPYYYACAY SGRSAGGGGTPLGLAGSGGS(SEQ ID NO: 202) Two activatable antibodies, TY22402 and TY22404, were selected to test the dependence of masking efficiency on the length of the masking peptide to suit their specific applications. By removing residues from the N-terminus, leaving only 5 or 2 residues before the first cysteine residue in the masking peptide, the masking peptides of TY22402 and TY22404 are shortened from 21 residues to 16 or 12 residues. residues ( Table 26 ). These activatable antibodies were expressed and purified from mammalian cells and their masking efficiencies were measured as described in Example 8 and compared to the parent antibody TY21580. Results from two experiments indicate that these activatable antibodies can be prepared using different masking peptides with lengths ranging from 2 to 11 residues preceding the first cysteine residue to modulate antibody masking efficiency ( Figure 65A and 65B ; Tables 27 and 28 ). This seems to indicate that the core masking motif contains the cysteine loop and its immediately adjacent residues and is sufficient to maintain masking efficiency. Table 26 : Masking peptides with varying peptide lengths Sample ID: Masked + cleaved peptide sequence (ununderlined): TY22402 EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 197) TY22775 EVGHSDCDAFYPYCDSSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 198) TY22864 EDCDAFYPYCDSSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 199) TY22404 EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200) TY22776 EVGSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 201) TY22871 EDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 202)

27顯示 65A中之抗體之掩蔽效率。表28顯示 65B中之抗體之掩蔽效率。 27 :具有變化掩蔽肽長度之抗體之掩蔽效率 樣品ID EC50(nM) 掩蔽效率 TY21580 0.2223 TY22402 53.99 243 TY22775 37.31 168 TY22404 68.40 308 TY22776 65.90 296 28 :具有變化掩蔽肽長度之抗體之掩蔽效率 樣品ID EC50(nM) 掩蔽效率 TY21580 0.2125 TY22402 115.6 554 TY22864 117 550 TY22404 121.5 572 TY22871 88.09 414 實例 21 :裂解肽長度對掩蔽效率之影響 Table 27 shows the masking efficiency of the antibodies in Figure 65A . Table 28 shows the masking efficiency of the antibodies in Figure 65B . Table 27 : Masking efficiency of antibodies with varying masking peptide lengths Sample ID EC50(nM) Masking efficiency TY21580 0.2223 TY22402 53.99 243 TY22775 37.31 168 TY22404 68.40 308 TY22776 65.90 296 Table 28 : Masking efficiency of antibodies with varying masking peptide lengths Sample ID EC50(nM) Masking efficiency TY21580 0.2125 TY22402 115.6 554 TY22864 117 550 TY22404 121.5 572 TY22871 88.09 414 Example 21 : Effect of cleaved peptide length on masking efficiency

選擇TY22404測試掩蔽效率對裂解肽長度之依賴性以適合其特定應用。將TY22404之裂解肽縮短至各種長度( 29)。使可活化抗體表現及自哺乳動物細胞純化,及如實例8中所述量測其掩蔽效率並與親本抗體TY21580比較。如 66 及表 30中所示,結果指示可使用具有範圍自5至20個殘基之其長度之不同裂解肽製備此等可活化抗體以調節抗體掩蔽效率。掩蔽與裂解基序之間之強相關性係驚人的;當肽長度自41截短至17個胺基酸時,TY23291之掩蔽效率相較於TY22404增加至少30倍。此等結果指示可設計及工程改造若干新穎掩蔽肽。此外,可進一步探索掩蔽與裂解基序之間之偶聯。 29 :具有變化裂解肽長度之掩蔽肽 樣品ID 肽名稱 掩蔽+ 裂解肽序列( 未加底線) TY22404 EVGSYPNPSSDCVPYYYACAY SGRSAGGGGTPLGLAGSGGS(SEQ ID NO: 200) TY23286 EVGSYPNPSSDCVPYYYACAY SGRSAPLGLA(SEQ ID NO: 209) TY23289 EDCVPYYYACAY SGRSAPLGLA(SEQ ID NO: 210) TY23280 EDCVPYYYACAY SGRSA(SEQ ID NO: 211) TY23291 EDCVPYYYACAY PLGLA(SEQ ID NO: 212) TY22404 was chosen to test the dependence of masking efficiency on cleaved peptide length to suit its specific application. The cleavage peptides of TY22404 were shortened to various lengths ( Table 29 ). Activatable antibodies were expressed and purified from mammalian cells, and their masking efficiencies were measured as described in Example 8 and compared to the parent antibody TY21580. As shown in Figure 66 and Table 30 , the results indicate that these activatable antibodies can be prepared using different cleavage peptides with lengths ranging from 5 to 20 residues to modulate antibody masking efficiency. The strong correlation between masking and cleavage motifs is striking; when the peptide length is truncated from 41 to 17 amino acids, the masking efficiency of TY23291 increases at least 30-fold compared to TY22404. These results indicate that several novel masking peptides can be designed and engineered. In addition, the coupling between masking and cleavage motifs can be further explored. Table 29 : Masking peptides with varying cleavage peptide lengths Sample ID Peptide name Masked + cleaved peptide sequence ( ununderlined) : TY22404 EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200) TY23286 EVGSYPNPSSDCVPYYYACAYSGRSAPLGLA (SEQ ID NO: 209) TY23289 EDCVPYYYACAYSGRSAPLGLA (SEQ ID NO: 210) TY23280 EDCVPYYYACAYSGRSA (SEQ ID NO: 211) TY23291 EDCVPYYYACAY PLGLA (SEQ ID NO: 212)

30顯示 66中之抗體之掩蔽效率。 30 :具有變化裂解肽長度之抗體之掩蔽效率 樣品ID EC50 (nM) 掩蔽效率 TY21580 0.2505 TY22404 117.4 469 TY23286 1496 5972 TY23289 133.2 532 TY23280 2952 11784 TY23291 3656 14595 Table 30 shows the masking efficiency of the antibodies in Figure 66 . Table 30 : Masking efficiency of antibodies with varying cleavage peptide lengths Sample ID EC50 (nM) Masking efficiency TY21580 0.2505 TY22404 117.4 469 TY23286 1496 5972 TY23289 133.2 532 TY23280 2952 11784 TY23291 3656 14595

1A B顯示結合至CTLA4之抗體,如由ELISA所測定。 1A顯示指定抗體結合至人類CTLA4。 1B顯示指定抗體結合至犬CTLA4。 Figures 1A - B show antibodies binding to CTLA4 as determined by ELISA. Figure 1A shows binding of the indicated antibodies to human CTLA4. Figure IB shows binding of the indicated antibodies to canine CTLA4.

2顯示指定抗體、同型對照或媒劑(PBSA)與短暫過度表現空載體(pIRES)之HEK293F細胞或小鼠或人類CTLA4之物種交叉反應性,如由流動式細胞測量術所測定。 Figure 2 shows species cross-reactivity of indicated antibodies, isotype controls or vehicle (PBSA) with HEK293F cells transiently overexpressing empty vector (pIRES) or mouse or human CTLA4, as determined by flow cytometry.

3顯示抗體TY21580或同型對照結合至經活化人類、猴及小鼠T細胞,如由流動式細胞測量術所測定。 Figure 3 shows binding of antibody TY21580 or isotype control to activated human, monkey and mouse T cells as determined by flow cytometry.

4A C顯示對CTLA4特異性之抗體,如由流動式細胞測量術所測定。 4A顯示指定抗體或同型對照至短暫過度表現人類PD-1、CTLA4、LAG3、TIM3、B7-H3或空載體(293F)之HEK293F細胞之結合。 4B顯示指定抗體、同型對照或媒劑(PBSA)至短暫過度表現人類CD95、CD120a、OX40、CD40、CTLA4或空載體(pIRES)之HEK293F細胞之結合。 4C顯示指定抗體、同型對照或媒劑(PBSA)至短暫過度表現人類TIM3、CTLA4、PD-L1、LAG3、BTLA、VISTA、PD-L2、ICOS、B7-H4、PD-1、B7-H3或空載體(pIRES)之HEK293F細胞之結合。 Figures 4A to C show antibodies specific for CTLA4 as determined by flow cytometry. Figure 4A shows binding of indicated antibodies or isotype controls to HEK293F cells transiently overexpressing human PD-1, CTLA4, LAG3, TIM3, B7-H3 or empty vector (293F). Figure 4B shows binding of the indicated antibodies, isotype control or vehicle (PBSA) to HEK293F cells transiently overexpressing human CD95, CD120a, OX40, CD40, CTLA4 or empty vector (pIRES). Figure 4C shows transient overexpression of human TIM3, CTLA4, PD-L1, LAG3, BTLA, VISTA, PD-L2, ICOS, B7-H4, PD-1, B7-H3 by indicated antibodies, isotype controls, or vehicle (PBSA). Or the combination of empty vector (pIRES) in HEK293F cells.

5A D顯示抗體之阻斷能力,如由ELISA所測定。 5A顯示抗體TY21687、TY21689、TY21680及TY21691阻斷人類CD80結合至人類CTLA4之能力。 5B顯示抗體TAC2114、TY21585、TY21587、TY21588、TY21589、TY21580及TY21591阻斷人類CD80結合至人類CTLA4之能力。 5C顯示抗體TY21687、TY21689、TY21680及TY21691阻斷人類CD86結合至人類CTLA4之能力。 5D顯示抗體TAC2114、TY21585、TY21587、TY21588、TY21589、TY21580及TY21591阻斷人類CD86結合至人類CTLA4之能力。 Figures 5A to D show the blocking ability of antibodies as determined by ELISA. Figure 5A shows the ability of antibodies TY21687, TY21689, TY21680 and TY21691 to block the binding of human CD80 to human CTLA4. Figure 5B shows the ability of antibodies TAC2114, TY21585, TY21587, TY21588, TY21589, TY21580 and TY21591 to block the binding of human CD80 to human CTLA4. Figure 5C shows the ability of antibodies TY21687, TY21689, TY21680 and TY21691 to block the binding of human CD86 to human CTLA4. Figure 5D shows the ability of antibodies TAC2114, TY21585, TY21587, TY21588, TY21589, TY21580 and TY21591 to block the binding of human CD86 to human CTLA4.

6A B顯示抗體之阻斷能力,如由FACS所測定。 6A顯示指定抗體、同型對照或媒劑(PBSA)阻斷人類CD80結合至短暫過度表現人類CTLA4之HEK293F細胞之能力。 6B顯示指定抗體、同型對照或媒劑(PBSA)阻斷人類CD86結合至短暫過度表現人類CTLA4之HEK293F細胞之能力。 Figures 6A - B show the blocking ability of antibodies as determined by FACS. Figure 6A shows the ability of the indicated antibodies, isotype control or vehicle (PBSA) to block human CD80 binding to HEK293F cells transiently overexpressing human CTLA4. Figure 6B shows the ability of the indicated antibodies, isotype control or vehicle (PBSA) to block human CD86 binding to HEK293F cells transiently overexpressing human CTLA4.

7顯示指定抗體結合FcRn之能力,如由表面電漿子共振(SPR)所測定。 Figure 7 shows the ability of designated antibodies to bind FcRn as determined by surface plasmon resonance (SPR).

8A B顯示人類外周血單核細胞(PBMC)經抗體TY21580或同型對照活化,如由ELISA所量測。 8A顯示對來自用抗體TY21580或同型對照處理之經CD3刺激之人類PBMC之IL-2分泌的影響。 8B顯示對來自用抗體TY21580或同型對照處理之經CD3刺激之人類PBMC之IFNγ分泌的影響。 Figures 8A - B show activation of human peripheral blood mononuclear cells (PBMC) by antibody TY21580 or isotype control, as measured by ELISA. Figure 8A shows the effect on IL-2 secretion from CD3-stimulated human PBMC treated with antibody TY21580 or isotype control. Figure 8B shows the effect on IFNγ secretion from CD3-stimulated human PBMC treated with antibody TY21580 or isotype control.

9顯示在抗CD3抗體之存在或不存在下,對來自用抗體TY21580處理之人類PBMC之IL-2分泌的影響,如由ELISA所量測。 Figure 9 shows the effect in the presence or absence of anti-CD3 antibodies on IL-2 secretion from human PBMC treated with antibody TY21580, as measured by ELISA.

10顯示對來自用抗體TY21580、同型對照或抗PD-1抗體處理之與異源CD4 +T細胞共培養之人類樹突狀細胞(DC)之IFNγ分泌的影響,如由ELISA所量測。 Figure 10 shows the effect on IFNγ secretion from human dendritic cells (DC) co-cultured with allogeneic CD4 + T cells treated with antibody TY21580, isotype control or anti-PD-1 antibody, as measured by ELISA.

11A B顯示示例性抗體對短暫過度表現人類CTLA4之HEK293F細胞之抗體依賴性細胞介導之細胞毒性(ADCC)活性,如由乳酸脫氫酶(LDH)釋放檢定所測定。 11A顯示抗體TY21580或同型對照對短暫過度表現人類CTLA4且與人類自然殺手(NK)細胞培育之HEK293F細胞之ADCC活性。 11B顯示抗體TY21580、TAC2114或同型對照對短暫過度表現人類CTLA4且與人類NK細胞培育之HEK293F細胞之ADCC活性。 Figures 11A - B show the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of exemplary antibodies against HEK293F cells transiently overexpressing human CTLA4, as determined by lactate dehydrogenase (LDH) release assay. Figure 11A shows ADCC activity of antibody TY21580 or isotype control on HEK293F cells transiently overexpressing human CTLA4 and cultured with human natural killer (NK) cells. Figure 11B shows the ADCC activity of antibodies TY21580, TAC2114 or isotype control on HEK293F cells transiently overexpressing human CTLA4 and cultured with human NK cells.

12A B顯示示例性抗體對自兩個供體單離之人類Treg之ADCC活性,如由鈣黃綠素(calcein)-AM釋放檢定所測定。 12A顯示抗體TY21580、TAC2114或同型對照對與人類NK細胞培育之人類Treg細胞(來自供體#96)之ADCC活性。 12B顯示抗體TY21580、TAC2114或同型對照對與人類NK細胞培育之人類Treg細胞(來自供體#12)之ADCC活性。 Figures 12A - B show the ADCC activity of exemplary antibodies on human Tregs isolated from two donors, as determined by calcein-AM release assay. Figure 12A shows the ADCC activity of antibodies TY21580, TAC2114 or isotype control on human Treg cells (from donor #96) cultured with human NK cells. Figure 12B shows the ADCC activity of antibodies TY21580, TAC2114 or isotype control on human Treg cells (from donor #12) cultured with human NK cells.

13顯示抗體TY21580或同型對照對短暫過度表現人類CTLA4之HEK293F細胞之補體依賴性細胞毒性(CDC)活性,如由鈣黃綠素-AM釋放檢定所測定。 Figure 13 shows the complement-dependent cytotoxicity (CDC) activity of antibody TY21580 or isotype control on HEK293F cells transiently overexpressing human CTLA4, as determined by calcein-AM release assay.

14顯示抗體TY21580或同型對照對經活化人類CD4 +T細胞之CDC活性,如由鈣黃綠素-AM釋放檢定所測定。 Figure 14 shows the CDC activity of antibody TY21580 or isotype control on activated human CD4 + T cells as determined by calcein-AM release assay.

15A C顯示抗體TY21580或同型對照於MC38同源小鼠結腸直腸腫瘤模型中之活體內抗腫瘤功效。 15A顯示荷MC38建立之腫瘤之雌性C57BL/6小鼠之不同處理組之腫瘤生長曲線。數據點表示組平均值;誤差線表示SEM。 15B顯示所測試之各組之個別腫瘤生長。 15C顯示再激發研究,該等研究指示對MC3腫瘤細胞之免疫之持久記憶。 Figures 15A to C show the in vivo anti-tumor efficacy of antibody TY21580 or isotype control in the MC38 syngeneic mouse colorectal tumor model. Figure 15A shows the tumor growth curves of different treatment groups of female C57BL/6 mice bearing tumors established by MC38. Data points represent group means; error bars represent SEM. Figure 15B shows individual tumor growth for each group tested. Figure 15C shows rechallenge studies indicating long-lasting memory of immunity to MC3 tumor cells.

16顯示抗體TY21580或同型對照於CT26同源小鼠結腸直腸腫瘤模型中之活體內抗腫瘤功效。顯示荷CT26建立之腫瘤之雌性C57BL/6小鼠之不同處理組之腫瘤生長曲線。數據點表示組平均值;誤差線表示SEM。 Figure 16 shows the in vivo anti-tumor efficacy of antibody TY21580 or isotype control in CT26 syngeneic mouse colorectal tumor model. Tumor growth curves of different treatment groups of female C57BL/6 mice bearing CT26-established tumors are shown. Data points represent group means; error bars represent SEM.

17顯示抗體TY21586、TY21580或同型對照於H22同源小鼠肝腫瘤模型中之活體內抗腫瘤功效。顯示荷H22建立之腫瘤之雌性C57BL/6小鼠之不同處理組之腫瘤生長曲線。數據點表示組平均值;誤差線表示SEM。 Figure 17 shows the in vivo anti-tumor efficacy of antibodies TY21586, TY21580 or isotype control in the H22 syngeneic mouse liver tumor model. Tumor growth curves of different treatment groups in female C57BL/6 mice bearing H22-established tumors are shown. Data points represent group means; error bars represent SEM.

18顯示抗體TY21580、TY21687、TY21687、TY21691及TY21580或同型對照於路易士同源小鼠肺腫瘤模型中之活體內抗腫瘤功效。顯示荷路易士建立之腫瘤之雌性C57BL/6小鼠之不同處理組之腫瘤生長曲線。數據點表示組平均值;誤差線表示SEM。 Figure 18 shows the in vivo anti-tumor efficacy of antibodies TY21580, TY21687, TY21687, TY21691 and TY21580 or isotype control in Lewis syngeneic mouse lung tumor model. Tumor growth curves of different treatment groups of female C57BL/6 mice with tumors established in Louisville are shown. Data points represent group means; error bars represent SEM.

19顯示抗體TY21580或同型對照於PAN02同源小鼠胰腫瘤模型中之活體內抗腫瘤功效。顯示荷PAN02建立之腫瘤之雌性C57BL/6小鼠之不同處理組之腫瘤生長曲線。數據點表示組平均值;誤差線表示SEM。 Figure 19 shows the in vivo anti-tumor efficacy of antibody TY21580 or isotype control in the PAN02 syngeneic mouse pancreatic tumor model. Tumor growth curves of different treatment groups of female C57BL/6 mice bearing PAN02-established tumors are shown. Data points represent group means; error bars represent SEM.

20A B顯示抗體TY21580、抗CD137抗體或同型對照,以及TY21580 +抗CD137組合療法於3LL同源小鼠肺腫瘤模型中之活體內抗腫瘤功效。 20A顯示荷3LL建立之腫瘤之雌性C57BL/6小鼠之不同處理組之腫瘤生長曲線。數據點表示組平均值;誤差線表示SEM。 20B顯示所測試之各組之個別腫瘤生長曲線。 Figures 20A - B show the in vivo anti-tumor efficacy of antibody TY21580, anti-CD137 antibody or isotype control, and TY21580 + anti-CD137 combination therapy in the 3LL syngeneic mouse lung tumor model. Figure 20A shows the tumor growth curves of different treatment groups of female C57BL/6 mice bearing tumors established by 3LL. Data points represent group means; error bars represent SEM. Figure 20B shows individual tumor growth curves for each group tested.

21顯示再激發研究,該研究指示對H22小鼠肝腫瘤細胞之免疫之持久記憶。將於TY21580處理組中具有完全反應之小鼠在第59天在相反腹脇部用H22腫瘤細胞皮下再激發。亦同時將初始小鼠用H22腫瘤細胞接種。 Figure 21 shows rechallenge studies indicating long-lasting memory of immunity to H22 mouse liver tumor cells. Mice with complete responses in the TY21580-treated group were rechallenged subcutaneously with H22 tumor cells in the opposite flank on day 59. Naive mice were also inoculated with H22 tumor cells at the same time.

22顯示在10 mg/kg之濃度下對雌性BALB/c小鼠靜脈內投與之指定抗體之血液濃度的時間過程,如由ELISA所測定。 Figure 22 shows the time course of blood concentrations of the indicated antibodies administered intravenously to female BALB/c mice at a concentration of 10 mg/kg, as determined by ELISA.

23顯示於食蟹獼猴中在10 mg/kg之濃度下靜脈內投與之指定抗體之血液濃度的時間過程,如由ELISA所測定。 Figure 23 shows the time course of blood concentrations of the indicated antibodies administered intravenously at a concentration of 10 mg/kg in cynomolgus macaques, as determined by ELISA.

24顯示於食蟹獼猴中在10 mg/kg之濃度下靜脈內投與之指定抗體之血液濃度相較於此等猴中之抗藥物抗體(ADA)之出現的時間過程,如由ELISA所測定。 Figure 24 shows the time course of the appearance of anti-drug antibodies (ADA) in cynomolgus macaques compared with the blood concentrations of the indicated antibodies administered intravenously at a concentration of 10 mg/kg, as determined by ELISA. Determination.

25A B顯示於重複腹膜內投與抗體TY21580或媒劑對照後雄性及雌性BALB/c小鼠之平均脾重量。 25A顯示於第1、4、7及11天重複腹膜內投與抗體TY21580或媒劑對照後雄性BALB/c小鼠之平均脾重量。 25B顯示於第1、4、7及11天重複腹膜內投與抗體TY21580或媒劑對照後雌性BALB/c小鼠之平均脾重量。 Figures 25A - B show mean spleen weights of male and female BALB/c mice after repeated intraperitoneal administration of antibody TY21580 or vehicle control. Figure 25A shows the mean spleen weight of male BALB/c mice after repeated intraperitoneal administration of antibody TY21580 or vehicle control on days 1, 4, 7 and 11. Figure 25B shows the mean spleen weight of female BALB/c mice after repeated intraperitoneal administration of antibody TY21580 or vehicle control on days 1, 4, 7 and 11.

26顯示於第1、4、7及11天重複腹膜內投與抗體TY21580或媒劑對照後BALB/c小鼠之組織病理學。 Figure 26 shows histopathology of BALB/c mice after repeated intraperitoneal administration of antibody TY21580 or vehicle control on days 1, 4, 7 and 11.

27A B顯示示例性抗體於高濃度下儲存後之穩定性。 27A顯示抗體TY21586於>100 mg/mL下儲存後之尺寸排阻層析法(SEC)譜。 27B顯示抗體TY21580於>100 mg/mL下儲存後之SEC譜。 Figures 27A - B show the stability of exemplary antibodies after storage at high concentrations. Figure 27A shows the size exclusion chromatography (SEC) spectrum of antibody TY21586 after storage at >100 mg/mL. Figure 27B shows the SEC spectrum of antibody TY21580 after storage at >100 mg/mL.

28顯示示例性抗體在加速應力條件下之SEC譜。 Figure 28 shows SEC spectra of exemplary antibodies under accelerated stress conditions.

29顯示自阻斷肽使用在酵母表面上展示之抗CTLA4抗體之Fab片段之選擇過程的示意圖。 Figure 29 shows a schematic representation of the selection process for self-blocking peptides using Fab fragments of anti-CTLA4 antibodies displayed on the yeast surface.

30顯示自阻斷肽使用在酵母表面上展示之抗CTLA4抗體之scFv片段之選擇過程的示意圖。 Figure 30 shows a schematic representation of the selection process for self-blocking peptides using scFv fragments of anti-CTLA4 antibodies displayed on the yeast surface.

31A B顯示靶向酵母上之CTLA4之Fab及scFv之功能展示,如由流動式細胞測量術所測定。 31A顯示靶向酵母表面上之CTLA4之Fab之功能展示。 31B顯示靶向酵母表面上之CTLA4之scFv之功能展示。 Figures 31A - B show functional display of Fab and scFv targeting CTLA4 on yeast, as determined by flow cytometry. Figure 31A shows functional demonstration of Fab targeting CTLA4 on the yeast surface. Figure 31B shows functional demonstration of scFv targeting CTLA4 on the yeast surface.

32顯示靶向人類CTLA4之可活化抗體之示例性選擇過程。使展示融合蛋白之酵母庫經歷若干輪基於FACS之篩選。 Figure 32 shows an exemplary selection process for activatable antibodies targeting human CTLA4. A yeast library displaying the fusion protein is subjected to several rounds of FACS-based screening.

33A B顯示示例性CTLA4可活化抗體純系之CTLA4結合親和力,如由流動式細胞測量術所測定。 33A顯示以scFv形式之CTLA4可活化抗體純系(包括具有完整掩蔽肽或具有經TEV蛋白酶裂解之掩蔽肽之CTLA4可活化抗體純系B13287)相較於不具有掩蔽肽之靶抗體之scFv片段之結合親和力。 33B顯示以Fab形式之CTLA4可活化抗體純系(包括具有完整掩蔽肽或具有經TEV蛋白酶裂解之掩蔽肽之CTLA4可活化抗體純系B13189)相較於不具有掩蔽肽之靶抗體之Fab片段之CTLA4結合親和力。 Figures 33A - B show the CTLA4 binding affinities of exemplary CTLA4 activatable antibody clones, as determined by flow cytometry. Figure 33A shows the binding of scFv fragments of CTLA4-activatable antibody clones in scFv format (including CTLA4-activatable antibody clone B13287 with intact masking peptide or with masking peptide cleaved by TEV protease) compared to scFv fragments of target antibodies without masking peptides. Affinity. Figure 33B shows CTLA4 activatable antibody clones in Fab format (including CTLA4 activatable antibody clone B13189 with intact masking peptide or with masking peptide cleaved by TEV protease) compared to CTLA4 of Fab fragments of the target antibody without masking peptide Binding affinity.

34A B顯示示例性CTLA4可活化抗體TY22401、TY22403、TY22402及TY22404相較於親本抗體TY21580之掩蔽效率。 34A顯示指定可活化抗體相較於親本抗體TY21580之締合及解離曲線,如由ForteBio System所測定。 34B顯示可活化抗體相較於親本抗體TY21580之相對結合率之圖。 Figures 34A - B show the masking efficiency of exemplary CTLA4 activatable antibodies TY22401, TY22403, TY22402 and TY22404 compared to the parent antibody TY21580. Figure 34A shows the association and dissociation curves of designated activatable antibodies compared to the parent antibody TY21580, as determined by the ForteBio System. Figure 34B shows a graph showing the relative binding rates of activatable antibodies compared to the parent antibody TY21580.

35A B顯示示例性CTLA4可活化抗體對重組人類CTLA4-Fc之掩蔽效率,如由ELISA所測定。 35A顯示第一批ELISA資料,該資料指示CTLA4可活化抗體TY22401、TY22402、TY22403、TY22404相較於親本抗體TY21580結合至重組人類CTLA4-Fc。 35B顯示CTLA4可活化抗體TY22563、TY22564、TY22565、TY22566相較於親本抗體TY21580結合至重組人類CTLA4-Fc。 Figures 35A - B show the masking efficiency of exemplary CTLA4 activatable antibodies for recombinant human CTLA4-Fc, as determined by ELISA. Figure 35A shows the first ELISA data indicating that CTLA4-activatable antibodies TY22401, TY22402, TY22403, TY22404 bind to recombinant human CTLA4-Fc compared to the parent antibody TY21580. Figure 35B shows that CTLA4-activatable antibodies TY22563, TY22564, TY22565, TY22566 bind to recombinant human CTLA4-Fc compared to the parental antibody TY21580.

36A B顯示在移除掩蔽肽後CTLA4可活化抗體TY22404之活性。 36A顯示未處理、用蛋白酶uPA處理或用蛋白酶MMP-9之5或10個單位處理之可活化抗體TY22404之SDS-PAGE結果。 36B顯示未處理、用蛋白酶uPA處理或用蛋白酶MMP-9處理之可活化抗體TY22404相較於親本抗體TY21580之結合,由ELISA所測定。 Figures 36A - B show the activity of CTLA4-activatable antibody TY22404 after removal of the masking peptide. Figure 36A shows SDS-PAGE results of activatable antibody TY22404 untreated, treated with protease uPA, or treated with 5 or 10 units of protease MMP-9. Figure 36B shows binding of activatable antibody TY22404 untreated, treated with protease uPA, or treated with protease MMP-9 compared to the parent antibody TY21580, as determined by ELISA.

37A C顯示示例性可活化抗體在加速應力條件下之尺寸排阻層析法(SEC)譜。 37A顯示可活化抗體TY22402於六個凍熔循環後相較於對照條件之SEC譜。 37B顯示可活化抗體TY22402於50℃下7天後相較於對照條件之SEC譜。 37C顯示示例性可活化抗體於50℃下7天後、於40℃下儲存多達28天後、或於六個凍熔循環後相較於對照條件之SEC主峰面積百分比。 Figures 37A - C show size exclusion chromatography (SEC) spectra of exemplary activatable antibodies under accelerated stress conditions. Figure 37A shows the SEC spectrum of activatable antibody TY22402 after six freeze-thaw cycles compared to control conditions. Figure 37B shows the SEC spectrum of activatable antibody TY22402 after 7 days at 50°C compared to control conditions. Figure 37C shows the percent SEC main peak area of an exemplary activatable antibody after 7 days at 50°C, after storage at 40°C for up to 28 days, or after six freeze-thaw cycles compared to control conditions.

38顯示可活化抗體TY22401及TY22402於約8 mg/mL或>150 mg/mL下儲存後之SEC主峰面積百分比。 Figure 38 shows the SEC main peak area percentage after storage of activatable antibodies TY22401 and TY22402 at approximately 8 mg/mL or >150 mg/mL.

39顯示未經處理之可活化抗體TY21580、TY22401、TY22402及TY22566在pH 3.7下培育30分鐘,或在pH 3.7下培育1小時之掩蔽效率,如由ForteBio System所測定。 Figure 39 shows the masking efficiency of untreated activatable antibodies TY21580, TY22401, TY22402, and TY22566 incubated at pH 3.7 for 30 minutes, or at pH 3.7 for 1 hour, as determined by the ForteBio System.

40A B顯示人類外周血單核細胞(PBMC)藉由同型對照抗體、親本抗體TY21580或示例性CTLA4可活化抗體TY22401、TY22402或TY22404之活化,如由ELISA所量測。 40A顯示對來自用同型對照抗體、親本抗體TY21580及示例性CTLA4可活化抗體TY22401、TY22402或TY22404刺激之CD3引發之人類PBMC之IL-2分泌的影響。 40B顯示對來自用同型對照抗體、親本抗體TY21580及示例性CTLA4可活化抗體TY22401、TY22402或TY22404刺激之CD3引發之人類PBMC之IFNγ分泌的影響。 Figures 40A - B show activation of human peripheral blood mononuclear cells (PBMC) by isotype control antibody, parental antibody TY21580, or exemplary CTLA4 activatable antibodies TY22401, TY22402, or TY22404, as measured by ELISA. Figure 40A shows the effects on IL-2 secretion from CD3-primed human PBMC stimulated with isotype control antibody, parental antibody TY21580, and exemplary CTLA4 activatable antibodies TY22401, TY22402, or TY22404. Figure 40B shows the effect on IFNγ secretion from CD3 primed human PBMC stimulated with isotype control antibody, parental antibody TY21580 and exemplary CTLA4 activatable antibodies TY22401, TY22402 or TY22404.

41顯示同型對照抗體、親本抗體TY21580或示例性可活化抗體TY22401、TY21580或TY22404對短暫過度表現人類CTLA4之HEK293F細胞之抗體依賴性細胞介導之細胞毒性(ADCC)活性,如由ADCC報告基因檢定所測定。 Figure 41 shows the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isotype control antibody, parental antibody TY21580, or exemplary activatable antibody TY22401, TY21580, or TY22404 on HEK293F cells transiently overexpressing human CTLA4, as reported by ADCC Genetic laboratory assay.

42A B顯示親本抗體TY21580、同型對照抗體或示例性CTLA4可活化抗體TY22401、TY22402或TY22566於MC38同源小鼠結腸直腸腫瘤模型中之活體內抗腫瘤功效。 42A顯示荷MC38建立之腫瘤之雌性C57BL/6小鼠之不同處理組之腫瘤生長曲線。數據點表示組平均值;誤差線表示SEM。 42B顯示用TY21580、TY22401、TY22402及TY22566處理之組之個別腫瘤生長曲線。 Figures 42A - B show the in vivo anti-tumor efficacy of parental antibody TY21580, isotype control antibody, or exemplary CTLA4-activatable antibodies TY22401, TY22402, or TY22566 in the MC38 syngeneic mouse colorectal tumor model. Figure 42A shows the tumor growth curves of different treatment groups of female C57BL/6 mice bearing tumors established by MC38. Data points represent group means; error bars represent SEM. Figure 42B shows individual tumor growth curves for groups treated with TY21580, TY22401, TY22402 and TY22566.

43顯示同型對照抗體、親本抗體TY21580或三種可活化抗體中之一者於CT26同源小鼠結腸直腸腫瘤模型中之活體內抗腫瘤功效。顯示荷CT26建立之腫瘤之雌性C57BL/6小鼠之不同處理組之腫瘤生長曲線。數據點表示組平均值;誤差線表示SEM。 Figure 43 shows the in vivo anti-tumor efficacy of the isotype control antibody, the parental antibody TY21580, or one of the three activatable antibodies in the CT26 syngeneic mouse colorectal tumor model. Tumor growth curves of different treatment groups of female C57BL/6 mice bearing CT26-established tumors are shown. Data points represent group means; error bars represent SEM.

44顯示同型對照抗體、親本抗體TY21580或三種可活化抗體中之一者於H22同源小鼠肝腫瘤模型中之活體內抗腫瘤功效。顯示荷H22建立之腫瘤之雌性C57BL/6小鼠之不同處理組之腫瘤生長曲線。數據點表示組平均值;誤差線表示SEM。 Figure 44 shows the in vivo anti-tumor efficacy of the isotype control antibody, the parental antibody TY21580, or one of the three activatable antibodies in the H22 syngeneic mouse liver tumor model. Tumor growth curves of different treatment groups of female C57BL/6 mice bearing H22-established tumors are shown. Data points represent group means; error bars represent SEM.

45A B顯示親本抗體TY21580、同型對照抗體及示例性可活化抗體TY22401、TY22402或TY22566於3LL同源小鼠肺腫瘤模型中之活體內抗腫瘤功效。 45A顯示荷3LL建立之腫瘤之雌性C57BL/6小鼠之不同處理組之腫瘤生長曲線。數據點表示組平均值;誤差線表示SEM。 45B顯示用TY21580、TY22401、TY22402及TY22566處理之組之個別腫瘤生長曲線。 Figures 45A - B show the in vivo anti-tumor efficacy of parental antibody TY21580, isotype control antibody, and exemplary activatable antibodies TY22401, TY22402, or TY22566 in the 3LL syngeneic mouse lung tumor model. Figure 45A shows tumor growth curves of different treatment groups of female C57BL/6 mice bearing tumors established by 3LL. Data points represent group means; error bars represent SEM. Figure 45B shows individual tumor growth curves for groups treated with TY21580, TY22401, TY22402 and TY22566.

46A C顯示在10 mg/kg之濃度下對雌性BALB/c小鼠靜脈內投與之試驗品(TA)之血液濃度的時間過程,如由ELISA所測定。 46A顯示在10 mg/kg之濃度下對雌性BALB/c小鼠靜脈內投與之可活化抗體TY22401之血液濃度相較於親本抗體TY21580的時間過程。 46B顯示在10 mg/kg之濃度下對雌性BALB/c小鼠靜脈內投與之可活化抗體TY22402之血液濃度相較於親本抗體TY21580的時間過程。 46C顯示在10 mg/kg之濃度下對雌性BALB/c小鼠靜脈內投與之可活化抗體TY22404之血液濃度相較於親本抗體TY21580的時間過程。 Figures 46A to C show the time course of blood concentrations of test article (TA) administered intravenously to female BALB/c mice at a concentration of 10 mg/kg, as determined by ELISA. Figure 46A shows the time course of blood concentrations of activatable antibody TY22401 compared to the parental antibody TY21580 administered intravenously to female BALB/c mice at a concentration of 10 mg/kg. Figure 46B shows the time course of blood concentrations of activatable antibody TY22402 compared to the parental antibody TY21580 administered intravenously to female BALB/c mice at a concentration of 10 mg/kg. Figure 46C shows the time course of blood concentrations of activatable antibody TY22404 compared to the parental antibody TY21580 administered intravenously to female BALB/c mice at a concentration of 10 mg/kg.

47顯示同型對照抗體、親本抗體TY21580及示例性可活化抗體TY22566、TY22401及TY22402使用NOD小鼠模型之重複給藥毒性。針對各處理組顯示20天內之存活率百分比。 Figure 47 shows repeated dose toxicity of isotype control antibody, parental antibody TY21580 and exemplary activatable antibodies TY22566, TY22401 and TY22402 using the NOD mouse model. Percent survival over 20 days is shown for each treatment group.

48A C顯示於重複腹膜內投與指定可活化抗體後BALB/c小鼠之平均脾重量。 48A顯示於第1、4、7及11天重複腹膜內投與可活化抗體TY22402、親本抗體TY21580或同型對照後BALB/c小鼠之平均脾重量。 48B顯示於第1、4、7及11天重複腹膜內投與可活化抗體TY22566、親本抗體TY21580或同型對照後BALB/c小鼠之平均脾重量。 48C顯示於第1、4、7及11天重複腹膜內投與可活化抗體TY22401、親本抗體TY21580或同型對照後BALB/c小鼠之平均脾重量。 Figures 48A to C show the mean spleen weight of BALB/c mice after repeated intraperitoneal administration of the indicated activatable antibodies. Figure 48A shows the mean spleen weight of BALB/c mice after repeated intraperitoneal administration of activatable antibody TY22402, parental antibody TY21580, or isotype control on days 1, 4, 7, and 11. Figure 48B shows the mean spleen weight of BALB/c mice after repeated intraperitoneal administration of activatable antibody TY22566, parental antibody TY21580, or isotype control on days 1, 4, 7, and 11. Figure 48C shows the mean spleen weight of BALB/c mice after repeated intraperitoneal administration of activatable antibody TY22401, parental antibody TY21580, or isotype control on days 1, 4, 7, and 11.

49顯示指定可活化抗體於50℃下7天後相較於對照條件之尺寸排阻層析法(SEC)譜。 Figure 49 shows size exclusion chromatography (SEC) spectra of designated activatable antibodies after 7 days at 50°C compared to control conditions.

50顯示指定可活化抗體於40℃下儲存7、14、21或28天後相較於對照條件之尺寸排阻層析法(SEC)譜。 Figure 50 shows size exclusion chromatography (SEC) profiles of designated activatable antibodies after storage at 40°C for 7, 14, 21 or 28 days compared to control conditions.

51顯示指定可活化抗體於六個凍熔循環後相較於對照條件之尺寸排阻層析法(SEC)譜。 Figure 51 shows size exclusion chromatography (SEC) spectra of designated activatable antibodies after six freeze-thaw cycles compared to control conditions.

52顯示指定可活化抗體於>115 mg/mL下儲存後之SEC主峰比率百分比。 Figure 52 shows the SEC main peak ratio percentage for a given activatable antibody after storage at >115 mg/mL.

53顯示穩定性資料之概述。 Figure 53 shows an overview of the stability data.

54描述人類及小鼠CTLA4之一部分與接觸殘基之多序列比對,該接觸殘基定位在人類CTLA4與CD80、CD86或伊匹單抗中之一者之間(基於兩個晶體結構)。接觸胺基酸係呈灰色陰影,關鍵接觸胺基酸係於框中,二聚體介面胺基酸係用點指示,及小鼠與人類CTLA4之間不同之胺基酸加底線並加粗。所示序列自頂部至底部由SEQ ID NO: 203至208表示。 Figure 54 depicts a multiple sequence alignment of portions of human and mouse CTLA4 with contact residues located between human CTLA4 and one of CD80, CD86, or ipilimumab (based on two crystal structures) . Contact amino acids are shaded gray, critical contact amino acids are boxed, dimer interface amino acids are indicated with dots, and amino acids that differ between mouse and human CTLA4 are underlined and bolded. The sequences shown are represented from top to bottom by SEQ ID NO: 203 to 208.

55A描述人類CTLA4與其配位體CD80之間之相互作用。 55B描述人類CTLA4與其配位體CD86之間之相互作用。 55C描述人類與小鼠CTLA4之間之結構比對。人類CTLA4為黑色,小鼠CTLA4為白色。 Figure 55A depicts the interaction between human CTLA4 and its ligand CD80. Figure 55B depicts the interaction between human CTLA4 and its ligand CD86. Figure 55C depicts a structural alignment between human and mouse CTLA4. Human CTLA4 is black and mouse CTLA4 is white.

56A 56E描述抗原決定基定位實驗之結果,其顯示TY21580 ( 56A)、伊匹單抗( 56B)、人類CD80 ( 56C)、人類CD86 ( 56D)及小鼠CD86 ( 56E)對人類CTLA4、小鼠CTLA4及CTLA4突變體之結合能力(藉由流動式細胞測量術)。 Figures 56A to 56E depict the results of epitope mapping experiments showing TY21580 ( Figure 56A ), ipilimumab ( Figure 56B ), human CD80 ( Figure 56C ), human CD86 ( Figure 56D ), and mouse CD86 ( Figure 56E ) Binding ability to human CTLA4, mouse CTLA4 and CTLA4 mutants (by flow cytometry).

57A 57D描述TY21580及伊匹單抗對人類CTLA4與CD80或CD86之間之受體-配位體結合阻斷之影響。 57A 57B顯示在TY21580、伊匹單抗或同型對照抗體之連續稀釋之存在下,人類CD80 ( 57A)或CD86 ( 57B)對板結合之人類重組CTLA4蛋白之結合曲線,如由ELISA所量測。 57C 57D顯示在TY21580、伊匹單抗或同型對照抗體之連續稀釋之存在下,人類重組CTLA4蛋白對板結合之人類CD80 ( 57C)或CD86 ( 57D)之結合曲線,如由ELISA所量測。 Figures 57A to 57D depict the effects of TY21580 and ipilimumab on blockade of receptor-ligand binding between human CTLA4 and CD80 or CD86. Figures 57A and 57B show binding curves for plate-bound human recombinant CTLA4 protein by human CD80 ( Figure 57A ) or CD86 ( Figure 57B ) in the presence of serial dilutions of TY21580, ipilimumab, or isotype control antibody, as determined by ELISA measured. Figures 57C to 57D show binding curves of human recombinant CTLA4 protein to plate-bound human CD80 ( Figure 57C ) or CD86 ( Figure 57D ) in the presence of serial dilutions of TY21580, ipilimumab, or isotype control antibody, as determined by ELISA measured.

58描述CD28路徑藉由抗CTLA4抗體之CTLA4阻斷介導之報告基因訊號活化。在經連續稀釋之抗CTLA4抗體之存在下,將Jurkat/CTLA4及aAPC/Raji細胞與作為同型對照之人類IgG1抗HEL抗體共培養。於過夜培育後用Bio-Glo螢光素酶受質量測發光信號及將相對螢光素酶單位(RLU)對空白對照標準化。將結果表示為平均RLU倍數± SEM。一式三份進行實驗。註釋:自分析排除TY21580之最高濃度(500 μg/mL)之數據點,同時擬合曲線,因為在此點處觀察到明顯鉤效應。 Figure 58 depicts reporter gene signaling activation of the CD28 pathway mediated by CTLA4 blockade with anti-CTLA4 antibodies. Jurkat/CTLA4 and aAPC/Raji cells were co-cultured with human IgG1 anti-HEL antibody as isotype control in the presence of serially diluted anti-CTLA4 antibodies. After overnight incubation, the luminescence signal was measured using a Bio-Glo luciferase absorber and the relative luciferase units (RLU) were normalized to the blank control. Results are expressed as mean RLU fold ± SEM. Experiments were performed in triplicate. Note: The data point at the highest concentration of TY21580 (500 μg/mL) was excluded from the analysis and curve fitting because a significant hook effect was observed at this point.

59描述藉由抗CTLA4抗體之ADCC報告基因訊號活化。在經連續稀釋之抗CTLA4抗體之存在下,將Jurkat/NFAT-Luc/CD16細胞及HEK293F/hCTLA4細胞與作為同型對照之人類IgG1抗HEL抗體共培養。於6小時培育後用ONE-Glo螢光素酶受質量測發光信號。將相對螢光素酶單位(RLU)對空白對照標準化,及將結果表示為平均RLU ± SEM。一式三份進行實驗。 Figure 59 depicts activation of ADCC reporter gene signaling by anti-CTLA4 antibodies. Jurkat/NFAT-Luc/CD16 cells and HEK293F/hCTLA4 cells were co-cultured with human IgG1 anti-HEL antibody as isotype control in the presence of serially diluted anti-CTLA4 antibodies. After 6 hours of incubation, the luminescence signal was measured using ONE-Glo luciferase substrate. Relative luciferase units (RLU) were normalized to blank control, and results are expressed as mean RLU ± SEM. Experiments were performed in triplicate.

60A 60B描述用抗CTLA4抗體處理之荷MC38腫瘤小鼠之腫瘤生長曲線。 60A描述用同型對照抗體(1 mg/kg BIW)、TY21580 (1 mg/kg或0.2 mg/kg BIW)或伊匹單抗(1 mg/kg或0.2 mg/kg BIW)處理之荷MC38腫瘤小鼠中隨時間之組平均腫瘤生長。數據點表示平均值;誤差線表示平均標準誤差(SEM)。 60B描述用同型對照抗體(組-1)、TY21580 (組-2及組-3)或伊匹單抗(組-4及組-5)處理之個別荷MC38腫瘤小鼠中隨時間之腫瘤生長。 Figures 60A and 60B depict tumor growth curves in MC38 tumor-bearing mice treated with anti-CTLA4 antibodies. Figure 60A depicts MC38-bearing tumors treated with isotype control antibody (1 mg/kg BIW), TY21580 (1 mg/kg or 0.2 mg/kg BIW), or ipilimumab (1 mg/kg or 0.2 mg/kg BIW) Group average tumor growth over time in mice. Data points represent means; error bars represent standard error of the mean (SEM). Figure 60B depicts tumors over time in individual MC38 tumor-bearing mice treated with isotype control antibody (Group-1), TY21580 (Group-2 and Group-3), or ipilimumab (Group-4 and Group-5) grow.

61A 61B描述TY21580及伊匹單抗對來自用TY21580或伊匹單抗處理之小鼠之皮下MC38腫瘤中之腫瘤內調節T (Treg)細胞水平的影響。 61A顯示自腫瘤單離之CD4+ T細胞中之T調節(Treg)細胞(CD4+ CD25+)之百分比。 61B描述自腫瘤單離之CD4+ T細胞亞群中之細胞毒性T淋巴細胞(CD8+ T細胞)與Treg細胞之比率(即,CD8+/Treg比率)。各數據點表示來自一種小鼠之資料。利用Prism 7 (GraphPad軟體)完成統計分析。使用多重T試驗計算 P-值。註釋: P>0.05;**:0.001< P<0.01,***: P<0.001。 Figures 61A and 61B depict the effects of TY21580 and ipilimumab on intratumoral regulatory T (Treg) cell levels in subcutaneous MC38 tumors from mice treated with TY21580 or ipilimumab. Figure 61A shows the percentage of T regulatory (Treg) cells (CD4+ CD25+) among CD4+ T cells isolated from tumors. Figure 61B depicts the ratio of cytotoxic T lymphocytes (CD8+ T cells) to Treg cells (i.e., CD8+/Treg ratio) in a subset of CD4+ T cells isolated from tumors. Each data point represents data from one species of mouse. Statistical analysis was completed using Prism 7 (GraphPad software). Calculate P -values using multiple T tests. Note: P >0.05; **: 0.001< P <0.01, ***: P <0.001.

62A 62B描述TY21580及伊匹單抗對來自用TY21580或伊匹單抗處理之小鼠之皮下CT26腫瘤中之腫瘤內調節T (Treg)細胞水平的影響。 62A顯示自腫瘤單離之CD4 +T細胞中之T調節(Treg)細胞(CD4 +CD25 +)之百分比。 62B描述自腫瘤單離之CD4 +T細胞亞群中之細胞毒性T淋巴細胞(CD8 +T細胞)與Treg細胞之比率(即,CD8 +/Treg比率)。各數據點表示來自一種小鼠之數據。利用Prism 7 (GraphPad軟體)完成統計分析。使用多重T試驗計算 P-值。註釋: P>0.05;**:0.001< P<0.01,***: P<0.001。 Figures 62A and 62B depict the effects of TY21580 and ipilimumab on intratumoral regulatory T (Treg) cell levels in subcutaneous CT26 tumors from mice treated with TY21580 or ipilimumab. Figure 62A shows the percentage of T regulatory (Treg) cells (CD4 + CD25 + ) among CD4 + T cells isolated from tumors. Figure 62B depicts the ratio of cytotoxic T lymphocytes (CD8 + T cells) to Treg cells (i.e., CD8 + /Treg ratio) in CD4 + T cell subsets isolated from tumors. Each data point represents data from one mouse species. Statistical analysis was completed using Prism 7 (GraphPad software). Calculate P -values using multiple T tests. Note: P >0.05; **: 0.001< P <0.01, ***: P <0.001.

63描述在用同型對照抗體或TY21580處理之荷CT26腫瘤小鼠中之FOXP3 +CD4 +Treg細胞上藉由平均螢光強度(MFI)量測之CTLA4表現水平。各數據點表示來自一種小鼠之數據。利用Prism 7 (GraphPad軟體)完成統計分析。使用多重T試驗計算 P-值。註釋: P>0.05;**:0.001< P<0.01,***: P<0.001。 Figure 63 depicts CTLA4 expression levels measured by mean fluorescence intensity (MFI) on FOXP3 + CD4 + Treg cells in CT26 tumor-bearing mice treated with isotype control antibody or TY21580. Each data point represents data from one mouse species. Statistical analysis was completed using Prism 7 (GraphPad software). Calculate P -values using multiple T tests. Note: P >0.05; **: 0.001< P <0.01, ***: P <0.001.

64A 64D描述用TY21580或同型對照抗體處理之荷小鼠H22肝癌小鼠之腫瘤生長曲線。 64A描述當腫瘤達到500 mm 3或800 mm 3時開始用TY21580處理,或當腫瘤達到500 mm 3時開始用同型對照抗體處理之組平均腫瘤生長。數據點表示來自8隻小鼠/組之腫瘤之平均值,誤差線表示平均標準誤差(SEM)。 64B 64D描述各小鼠之個別腫瘤生長。 64B描述用同型對照抗體處理之小鼠之腫瘤生長,其中當腫瘤達到500 mm 3時開始處理; 64C描述用TY21580處理之小鼠之腫瘤生長,其中當腫瘤達到500 mm 3時開始處理; 64D描述用TY21580處理之小鼠之腫瘤生長,當腫瘤達到800 mm 3時開始處理。 Figures 64A to 64D depict tumor growth curves in mice bearing H22 liver cancer treated with TY21580 or isotype control antibody. Figure 64A depicts mean tumor growth for groups that began treatment with TY21580 when tumors reached 500 mm3 or 800 mm3 , or with isotype control antibody when tumors reached 500 mm3 . Data points represent the mean of tumors from 8 mice/group, and error bars represent the standard error of the mean (SEM). Figures 64B to 64D depict individual tumor growth in each mouse. Figure 64B depicts tumor growth in mice treated with isotype control antibody, where treatment was initiated when tumors reached 500 mm; Figure 64C depicts tumor growth in mice treated with TY21580, where treatment was initiated when tumors reached 500 mm; Figure 64D depicts tumor growth in mice treated with TY21580, starting treatment when tumors reached 800 mm3 .

65A 65B描述含有可變長度之掩蔽肽之示例性可活化抗體相較於親本抗體TY21580之掩蔽效率。使用基於ELISA之方法測定掩蔽效率。 65A 65B表示為測試各種可活化抗CTLA4抗體使用相同實驗方法建立之兩個實驗。 Figures 65A and 65B depict the masking efficiency of exemplary activatable antibodies containing variable length masking peptides compared to the parent antibody TY21580. Masking efficiency was determined using an ELISA-based method. Figures 65A and 65B show two experiments set up using the same experimental method to test various activatable anti-CTLA4 antibodies.

66描述含有變化長度之裂解肽之示例性可活化抗體相較於親本抗體TY21580之掩蔽效率。使用基於ELISA之方法測定掩蔽效率。 Figure 66 depicts the masking efficiency of exemplary activatable antibodies containing cleavage peptides of varying lengths compared to the parent antibody TY21580. Masking efficiency was determined using an ELISA-based method.

         <![CDATA[<110> 英屬開曼群島商天演藥業有限公司(Adagene Inc.)]]>
          <![CDATA[<120> 抗CTLA4抗體及其製備及使用方法]]>
          <![CDATA[<130> TW 108119978]]>
          <![CDATA[<150> PCT/CN2019/074580]]>
          <![CDATA[<151> 2019-02-02]]>
          <![CDATA[<160> 230]]>
          <![CDATA[<170> FastSEQ for Windows Version 4.0]]>
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          Tyr Ser Ile Xaa Ser Gly Xaa Xaa Trp Xaa Trp Ile
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          <![CDATA[<223> Xaa = Ser或Tyr]]>
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          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 14]]>
          <![CDATA[<223> Xaa = Pro或Gln]]>
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          Ile Gly Xaa Ile Xaa His Ser Gly Ser Thr Tyr Tyr Ser Xaa Ser Leu
           1               5                  10                  15      
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                      20  
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          <![CDATA[<221]]>> 變異體        ]]&gt;
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          <br/>&lt;![CDATA[&lt;222&gt; 8, 10]]&gt;
          <br/>&lt;![CDATA[&lt;223&gt; Xaa = Gly或Ser]]&gt;
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          <br/><![CDATA[Ile Gly Xaa Ile Ser Pro Ser Xaa Gly Xaa Thr Xaa Tyr Ala Gln Lys
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          <![CDATA[<223> Xaa = Gly或Ser]]>
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          <![CDATA[<222> 10]]>
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           1               5                  10                  15      
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          <br/>&lt;![CDATA[&lt;222&gt; 3]]&gt;
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          <br/>&lt;![CDATA[&lt;400&gt; 7]]&gt;
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          <![CDATA[<210> 8]]>
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          <![CDATA[<223> Xaa = Asp或Leu]]>
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          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5]]>
          <![CDATA[<223> Xaa = Phe或Tyr]]>
          <![CDATA[<220> ]]>
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          <![CDATA[<222> 10]]>
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          <![CDATA[<400> 8]]>
          Ala Arg Xaa Gly Xaa Gly Tyr Phe Asp Xaa
           1               5                  10  
          <![CDATA[<210> ]]>9
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          <![CDATA[<222> 3]]>
          <![CDATA[<223> Xaa = Leu或Arg]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 4]]>
          <![CDATA[<223> Xaa = Ile或Pro]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5]]>
          <![CDATA[<223> Xaa = Ala或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6]]>
          <![CDATA[<223> Xaa = Ser或Thr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 8]]>
          <![CDATA[<223> Xaa = Thr或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 9]]>
          <![CDATA[<223> Xaa = Ala或Tyr]]>
          <![CDATA[<400> 9]]>
          Ala Arg Xaa Xaa Xaa Xaa Ala Xaa Xaa Phe Asp Tyr
           1               5                  10          
          <![CDATA[<210> 10]]>
          <![CDATA[<211> 17]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 4]]>
          <![CDATA[<223> Xaa = Ile或Val]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5]]>
          <![CDATA[<223> Xaa = Ala或His]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6]]>
          <![CDATA[<223> Xaa = Pro或Ser]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 13]]>
          <![CDATA[<223> Xaa = Asp或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 17]]>
          <![CDATA[<223> Xaa = Phe或Val]]>
          <![CDATA[<400> 10]]>
          Ala Arg Asp Xaa Xaa Xaa Gly Ser Ser Gly Tyr Tyr Xaa Gly Phe Asp
           1               5                  10                  15      
          Xaa
          <![CDATA[<210> 11]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5、7]]>
          <![CDATA[<223> Xaa = Gly或Ser]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6]]>
          <![CDATA[<223> Xaa = Ile或Val]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 9]]>
          <![CDATA[<223> Xaa = Ser或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 11]]>
          <![CDATA[<223> Xaa = Ala或Asn]]>
          <![CDATA[<400> 11]]>
          Arg Ala Ser Gln Xaa Xaa Xaa Ser Xaa Leu Xaa
           1               5                  10      
          <![CDATA[<210> 12]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5]]>
          <![CDATA[<223> Xaa = Ser或Thr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 7]]>
          <![CDATA[<223> Xaa = Phe、Arg或Ser]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 8]]>
          <![CDATA[<223> Xaa = Gly或Ser]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 10]]>
          <![CDATA[<223> Xaa = Phe或Tyr]]>
          <![CDATA[<400> 12]]>
          Arg Ala Ser Gln Xaa Val Xaa Xaa Arg Xaa Leu Ala
           1               5                  10          
          <![CDATA[<210> 13]]>
          <![CDATA[<211> 15]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 4]]>
          <![CDATA[<223> Xaa = Glu或Gln]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 9]]>
          <![CDATA[<223> Xaa = Asp、Phe、His或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 11]]>
          <![CDATA[<223> Xaa = Phe、Ile或Lys]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體]]>        
          <![CDATA[<222> 15]]>
          <![CDATA[<223> Xaa = Ala、Asp或His]]>
          <![CDATA[<400> 13]]>
          Arg Ala Ser Xaa Ser Val Asp Phe Xaa Gly Xaa Ser Phe Leu Xaa
           1               5                  10                  15  
          <![CDATA[<210> 14]]>
          <![CDATA[<211> 9]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1]]>
          <![CDATA[<223> Xaa = Ala或A]]>sp
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 4]]>
          <![CDATA[<223> Xaa = Asn、Ser或Thr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5]]>
          <![CDATA[<223> Xaa = Leu或Arg]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6]]>
          <![CDATA[<223> Xaa = Ala、Glu或Gln]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 7]]>
          <![CDATA[<223> Xaa = Ser或Thr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 9]]>
          <![CDATA[<223> Xaa = Ile或Val]]>
          <![CDATA[<400> 14]]>
          Xaa Ala Ser Xaa Xaa Xaa Xaa Gly Xaa
           1               5                  
          <![CDATA[<210> 15]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 3]]>
          <![CDATA[<223> Xaa = Glu、Gln或Val]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 4]]>
          <![CDATA[<223> Xaa = His或Gln]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5]]>
          <![CDATA[<223> Xaa = Ala、Gly、His、Arg或Ser]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6]]>
          <![CDATA[<223> Xaa = Asp、Leu、Ser或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 7]]>
          <![CDATA[<223> Xaa = Glu、Gly、Pro、Gln或Ser]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 8]]>
          <![CDATA[<223> Xaa = Leu、Thr、Val或Trp]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 10]]>
          <![CDATA[<223> Xaa = Phe、Leu、Pro、Trp或Tyr]]>
          <![CDATA[<400> 15]]>
          Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Thr
           1               5                  10      
          <![CDATA[<210> 16]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5]]>
          <![CDATA[<223> Xaa = Se]]>r或Tyr
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6]]>
          <![CDATA[<223> Xaa = Asp或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 7]]>
          <![CDATA[<223> Xaa = Gln或Tyr]]>
          <![CDATA[<400> 16]]>
          Tyr Cys Gln Gln Xaa Xaa Xaa Trp Pro Pro Trp Thr
           1               5                  10          
          <![CDATA[<210> 17]]>
          <![CDATA[<211> 13]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 4]]>
          <![CDATA[<223> Xaa = His或Gln]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6]]>
          <![CDATA[<223> Xaa = Thr或Val]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 11]]>
          <![CDATA[<223> Xaa = Glu或Val]]>
          <![CDATA[<400> 17]]>
          Tyr Cys Gln Xaa Tyr Xaa Ser Ser Pro Pro Xaa Tyr Thr
           1               5                  10              
          <![CDATA[<210> 18]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 18]]>
          Phe Thr Phe Ser Asp Tyr Ala Ile His Trp Val
           1               5                  10      
          <![CDATA[<210> 19]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 19]]>
          Tyr Ser Ile Thr Ser Gly Tyr Tyr Trp Ala Trp Ile
           1               5                  10          
          <![CDATA[<210> 20]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 20]]>
          Phe Thr Phe Ser Asp Tyr Gly Ile His Trp Val
           1               5                  10      
          <![CDATA[<210> 21]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 21]]>
          Tyr Ser Ile Ser Ser Gly Tyr His Trp Asp Trp Ile
           1               5                  10          
          <![CDATA[<210> 22]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 22]]>
          Phe Thr Phe Ser Asp Tyr Trp Ile His Trp Val
           1               5                  10      
          <![CDATA[<210> 23]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 23]]>
          Tyr Ser Ile Ser Ser Gly Tyr His Trp Ser Trp Ile
           1               5                  10          
          <![CDATA[<210> 24]]>
          <![CDATA[<211> 13]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 24]]>
          Phe Ser Leu Ser Thr Gly Gly Val Ala Val Ser Trp Ile
           1               5                  10              
          <![CDATA[<210> 25]]>
          <![CDATA[<211> 13]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 25]]>
          Phe Ser Leu Ser Thr Gly Gly Val Ala Val Gly Trp Ile
           1               5                  10              
          <![CDATA[<210> 26]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 26]]>
          Phe Thr Phe Ser Gly Tyr Ala Ile His Trp Val
           1               5                  10      
          <![CDATA[<210> 27]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 2]]>7
          Tyr Thr Phe Ser Gly Tyr Gly Ile His Trp Val
           1               5                  10      
          <![CDATA[<210> 28]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 28]]>
          Tyr Thr Phe Ser Gly Tyr Ala Ile His Trp Val
           1               5                  10      
          <![CDATA[<210> 29]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 29]]>
          Tyr Ser Ile Thr Ser Gly His Tyr Trp Ser Trp Ile
           1               5                  10          
          <![CDATA[<210> 30]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 30]]>
          Ile Gly Ile Ile Ser Pro Ser Ser Gly Ser Thr Asn Tyr Ala Gln Lys
           1               5                  10                  15      
          Phe Gln Gly Arg Val
                      20      
          <![CDATA[<210> 31]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 31]]>
          Val Ser Ser Ile Ser Gly Ser Gly Ser Thr Thr Tyr Tyr Ala Asp Ser
           1               5                  10                  15      
          Val Lys Gly Arg Phe
                      20      
          <![CDATA[<210> 32]]>
          <![CDATA[<211> 20]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 32]]>
          Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu
           1               5                  10                  15      
          Lys Ser Arg Val
                      20  
          <![CDATA[<210> 33]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 33]]>
          Val Ser Gly Ile Ser Gly Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
           1               5                  10                  15      
          Val Lys Gly Arg Phe
                      20      
          <![CDATA[<210> 34]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 34]]>
          Ile Gly Trp Ile Ser Pro Ser Gly Gly Gly Thr Lys Tyr Ala Gln Lys
           1               5                  10                  15      
          Phe Gln Gly Arg Val
                      20      
          <![CDATA[<210> 35]]>
          <![CDATA[<211> 20]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 35]]>
          Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser Leu
           1               5                  10                  15      
          Lys Ser Arg Leu
                      20  
          <![CDATA[<210> 36]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 36]]>
          Val Ser Ala Ile Ser Gly Tyr Gly Ser Thr Thr Tyr Tyr Ala Asp Ser
           1               5                  10                  15      
          Val Lys Gly Arg Phe
                      20      
          <![CDATA[<210> 37]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 37]]>
          Ile Gly Ile Ile Ser Pro Ser Gly Gly Gly Thr Lys Tyr Ala Gln Lys
           1               5                  10                  15      
          Phe Gln Gly Arg Val
                      20      
          <![CDATA[<210> 38]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<40]]>0> 38]]&gt;
          <br/><![CDATA[Ile Gly Ile Ile Ser Pro Ser Gly Gly Ser Thr Lys Tyr Ala Gln Lys
           1               5                  10                  15      
          Phe Gln Gly Arg Val
                      20      
          <![CDATA[<210> 39]]>
          <![CDATA[<211> 20]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 39]]>
          Ile Gly Asp Ile Ser His Ser Gly Ser Thr Tyr Tyr Ser Gln Ser Leu
           1               5                  10                  15      
          Lys Ser Arg Val
                      20  
          <![CDATA[<210> 40]]>
          <![CDATA[<211> 17]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 40]]>
          Ala Arg Asp Ile His Ser Gly Ser Ser Gly Tyr Tyr Tyr Gly Phe Asp
           1               5                  10                  15      
          Val
          <![CDATA[<210> 41]]>
          <![CDATA[<211> 10]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 41]]>
          Ala Arg Asp Gly Phe Gly Tyr Phe Asp Tyr
           1               5                  10  
          <![CDATA[<210> 42]]>
          <![CDATA[<211> 17]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 42]]>
          Ala Arg Asp Val Ala Pro Gly Ser Ser Gly Tyr Tyr Asp Gly Phe Asp
           1               5                  10                  15      
          Phe
          <![CDATA[<210> 43]]>
          <![CDATA[<211> 13]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 43]]>
          Ala Arg His Ser Tyr Tyr Gly Ser Gly Asn Phe Asp Tyr
           1               5                  10              
          <![CDATA[<210> 44]]>
          <![CDATA[<211> 9]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<40]]>0> 44]]&gt;
          <br/><![CDATA[Ala Arg Gly Ala Tyr Glu Phe Asp Tyr
           1               5                  
          <![CDATA[<210> 45]]>
          <![CDATA[<211> 9]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 45]]>
          Ala Arg Ser Tyr Val Tyr Phe Asp Tyr
           1               5                  
          <![CDATA[<210> 46]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人]]>工序列
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 46]]>
          Ala Arg Arg Ile Ala Thr Ala Thr Tyr Phe Asp Tyr
           1               5                  10          
          <![CDATA[<210> 47]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 47]]>
          Ala Arg Leu Pro Tyr Ser Ala Tyr Ala Phe Asp Tyr
           1               5                  10          
          <![CDATA[<210> 48]]>
          <![CDATA[<211> 7]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 48]]>
          Ala Arg His Pro Phe Ala Tyr
           1               5          
          <![CDATA[<210> 49]]>
          <![CDATA[<211> 9]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 49]]>
          Ala Arg Arg Ile Asp Ala Phe Asp Ile
           1               5                  
          <![CDATA[<210> 50]]>
          <![CDATA[<211> 8]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 50]]>
          Ala Arg Leu Tyr Asp Val Ala Tyr
           1               5              
          <![CDATA[<210> 51]]>
          <![CDATA[<211> 10]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 51]]>
          Ala Arg Leu Gly Tyr Gly Tyr Phe Asp Val
           1               5                  10  
          <![CDATA[<210> 52]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 52]]>
          Ala Arg Gly Ser Arg Thr Gly Tyr Phe Asp Tyr
           1               5                  10      
          <![CDATA[<210> 53]]>
          <![CDATA[<211> 15]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 53]]>
          Arg Ala Ser Glu Ser Val Asp Phe Phe Gly Ile Ser Phe Leu Ala
           1               5                  10                  15  
          <![CDATA[<210> 54]]>
          <![CDATA[<211> 10]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 54]]>
          Ser Ala Ser Ser Ser Val Ser Tyr Val Tyr
           1               5                  10  
          <![CDATA[<210> 55]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 55]]>
          Arg Ala Ser Gln Gly Ile Gly Ser Ser Leu Ala
           1               5                  10      
          <![CDATA[<210> 56]]>
          <![CDATA[<211> 15]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 56]]>
          Arg Ala Ser Glu Ser Val Asp Phe Phe Gly Lys Ser Phe Leu His
           1               5                  10                  15  
          <![CDATA[<210> 57]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 57]]>
          Arg Ala Ser Gln Ser Val Ser Ser Arg Phe Leu Ala
           1               5                  10          
          <![CDATA[<210> 58]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 58]]>
          Arg Ala Ser Gln Ser Val Arg Gly Arg Phe Leu Ala
           1               5                  10          
          <![CDATA[<210> 59]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 59]]>
          Arg Ala Ser Gln Thr Val Phe Ser Arg Tyr Leu Ala
           1               5                  10          
          <![CDATA[<210> 60]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 60]]>
          Arg Ala Ser Gln Gly Val Ser Ser Tyr Leu Ala
           1               5                  10      
          <![CDATA[<210> 61]]>
          <![CDATA[<211> 15]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 61]]>
          Arg Ala Ser Gln Ser Val Asp Phe Tyr Gly Ile Ser Phe Leu Asp
           1               5                  10                  15  
          <![CDATA[<210> 62]]>
          <![CDATA[<211> 15]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 62]]>
          Arg Ala Ser Gln Ser Val Asp Phe Asp Gly Phe Ser Phe Leu His
           1               5                  10                  15  
          <![CDATA[<210> 63]]>
          <![CDATA[<211> 15]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 63]]>
          Arg Ala Ser Gln Ser Val Asp Phe His Gly Lys Ser Phe Leu His
           1               5                  10                  15  
          <![CDATA[<210> 64]]>
          <![CDATA[<211> 15]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223]]>> 合成構築體]]&gt;
          <br/>
          <br/>&lt;![CDATA[&lt;400&gt; 64]]&gt;
          <br/><![CDATA[Arg Ala Ser Gln Ser Val Asp Phe Tyr Gly Ile Ser Phe Leu His
           1               5                  10                  15  
          <![CDATA[<210> 65]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 65]]>
          Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn
           1               5                  10      
          <![CDATA[<210> 66]]>
          <![CDATA[<211> 9]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 66]]>
          Asp Ala Ser Asn Arg Ala Thr Gly Ile
           1               5                  
          <![CDATA[<210> 67]]>
          <![CDATA[<211> 9]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 67]]>
          Asp Ala Ser Ser Leu Glu Ser Gly Val
           1               5                  
          <![CDATA[<210> 68]]>
          <![CDATA[<211> 9]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 68]]>
          Asp Ala Ser Asn Leu Glu Thr Gly Val
           1               5                  
          <![CDATA[<210> 69]]>
          <![CDATA[<211> 9]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 69]]>
          Ala Ala Ser Thr Leu Gln Ser Gly Val
           1               5                  
          <![CDATA[<210> 70]]>
          <![CDATA[<211> 13]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 70]]>
          Tyr Cys Gln His Tyr Thr Ser Ser Pro Pro Val Tyr Thr
           1               5                  10              
          <![CDATA[<210> 71]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 71]]>
          Tyr Cys Val Gln Gly Leu Gln Thr Pro Trp Thr
           1               5                  10      
          <![CDATA[<210> 72]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 72]]>
          Tyr Cys Gln Gln Tyr Asp Gln Trp Pro Pro Trp Thr
           1               5                  10          
          <![CDATA[<210> 73]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 73]]>
          Tyr Cys Gln Gln Ser Tyr Ser Trp Pro Pro Thr
           1               5                  10      
          <![CDATA[<210> 74]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 74]]>
          Tyr Cys Gln Gln Ser Tyr Pro Thr Pro Leu Thr
           1               5                  10      
          <![CDATA[<210> 75]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 75]]>
          Tyr Cys Gln Gln Ser Ser Ser Trp Pro Pro Thr
           1               5                  10      
          <![CDATA[<210> 76]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 76]]>
          Tyr Cys Gln Gln Ser Tyr Tyr Trp Pro Pro Trp Thr
           1               5                  10          
          <![CDATA[<210> 77]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 77]]>
          Tyr Cys Gln His His Tyr Gly Thr Pro Leu Thr
           1               5                  10      
          <![CDATA[<210> 78]]>
          <![CDATA[<211> 13]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 78]]>
          Tyr Cys Gln Gln Tyr Val Ser Ser Pro Pro Glu Tyr Thr
           1               5                  10              
          <![CDATA[<210> 79]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 79]]>
          Tyr Cys Gln Gln Arg Asp Ser Trp Pro Tyr Thr
           1               5                  10      
          <![CDATA[<210> 80]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> ]]>合成構築體
          <![CDATA[<400> 80]]>
          Tyr Cys Glu Gln Ser Leu Glu Val Pro Phe Thr
           1               5                  10      
          <![CDATA[<210> 81]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 81]]>
          Tyr Cys Val Gln Ala Leu Gln Leu Pro Leu Thr
           1               5                  10      
          <![CDATA[<210> 82]]>
          <![CDATA[<211> 124]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 82]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
                      20                  25                  30          
          Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35                  40                  45              
          Gly Ile Ile Ser Pro Ser Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe
              50                  55                  60                  
          Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65                  70                  75                  80  
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85                  90                  95      
          Ala Arg Asp Ile His Ser Gly Ser Ser Gly Tyr Tyr Tyr Gly Phe Asp
                      100                 105                 110         
          Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115                 120                 
          <![CDATA[<210> 83]]>
          <![CDATA[<211> 118]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 83]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly
                      20                  25                  30          
          Tyr Tyr Trp Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
                  35                  40                  45              
          Val Ser Ser Ile Ser Gly Ser Gly Ser Thr Thr Tyr Tyr Ala Asp Ser
              50                  55                  60                  
          Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
          65                  70                  75                  80  
          Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
                          85                  90                  95      
          Cys Ala Arg Asp Gly Phe Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
                      100                 105                 110         
          Leu Val Thr Val Ser Ser
                  115             
          <![CDATA[<210> 84]]>
          <![CDATA[<211> 123]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 84]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
                      20                  25                  30          
          Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35                  40                  45              
          Gly Glu Ile Tyr His Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Lys
              50                  55                  60                  
          Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
          65                  70                  75                  80  
          Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
                          85                  90                  95      
          Arg Asp Val Ala Pro Gly Ser Ser Gly Tyr Tyr Asp Gly Phe Asp Phe
                      100                 105                 110         
          Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115                 120             
          <![CDATA[<210> 85]]>
          <![CDATA[<211> 121]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 85]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly
                      20                  25                  30          
          Tyr His Trp Asp Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
                  35                  40                  45              
          Val Ser Gly Ile Ser Gly Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
              50                  55                  60                  
          Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
          65                  70                  75                  80  
          Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
                          85                  90                  95      
          Cys Ala Arg His Ser Tyr Tyr Gly Ser Gly Asn Phe Asp Tyr Trp Gly
                      100                 105                 110         
          Gln Gly Thr Leu Val Thr Val Ser Ser
                  115                 120     
          <![CDATA[<210> 86]]>
          <![CDATA[<211> 116]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 86]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
                      20                  25                  30          
          Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35                  40                  45              
          Gly Trp Ile Ser Pro Ser Gly Gly Gly Thr Lys Tyr Ala Gln Lys Phe
              50                  55                  60                  
          Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65                  70                  75                  80  
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85                  90                  95      
          Ala Arg Gly Ala Tyr Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
                      100                 105                 110         
          Thr Val Ser Ser
                  115     
          <![CDATA[<210> 87]]>
          <![CDATA[<211> 116]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 87]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly
                      20                  25                  30          
          Tyr His Trp Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
                  35                  40                  45              
          Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser Leu
              50                  55                  60                  
          Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65                  70                  75                  80  
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85                  90                  95      
          Ala Arg Ser Tyr Val Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
                      100                 105                 110         
          Thr Val Ser Ser
                  115     
          <![CDATA[<210> 88]]>
          <![CDATA[<211> 120]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 88]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly
                      20                  25                  30          
          Gly Val Ala Val Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu
                  35                  40                  45              
          Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Tyr Tyr Ser Pro Ser
              50                  55                  60                  
          Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
          65                  70                  75                  80  
          Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
                          85                  90                  95      
          Cys Ala Arg Arg Ile Ala Thr Ala Thr Tyr Phe Asp Tyr Trp Gly Gln
                      100                 105                 110         
          Gly Thr Leu Val Thr Val Ser Ser
                  115                 120 
          <![CDATA[<210> 89]]>
          <![CDATA[<211> 121]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 89]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly
                      20                  25                  30          
          Gly Val Ala Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu
                  35                  40                  45              
          Trp Val Ser Ala Ile Ser Gly Tyr Gly Ser Thr Thr Tyr Tyr Ala Asp
              50                  55                  60                  
          Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
          65                  70                  75                  80  
          Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
                          85                  90                  95      
          Tyr Cys Ala Arg Leu Pro Tyr Ser Ala Tyr Ala Phe Asp Tyr Trp Gly
                      100                 105                 110         
          Gln Gly Thr Leu Val Thr Val Ser Ser
                  115                 120     
          <![CDATA[<210> 90]]>
          <![CDATA[<211> 114]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 90]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr
                      20                  25                  30          
          Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35                  40                  45              
          Gly Ile Ile Ser Pro Ser Gly Gly Gly Thr Lys Tyr Ala Gln Lys Phe
              50                  55                  60                  
          Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65                  70                  75                  80  
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85                  90                  95      
          Ala Arg His Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
                      100                 105                 110         
          Ser Ser
          <![CDATA[<210> 91]]>
          <![CDATA[<211> 115]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 91]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Gly Tyr
                      20                  25                  30          
          Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35                  40                  45              
          Gly Glu Ile Tyr His Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Lys
              50                  55                  60                  
          Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
          65                  70                  75                  80  
          Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
                          85                  90                  95      
          Arg Arg Ile Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu Val Thr
                      100                 105                 110         
          Val Ser Ser
                  115 
          <![CDATA[<210> 92]]>
          <![CDATA[<211> 115]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 92]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Gly Tyr
                      20                  25                  30          
          Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35                  40                  45              
          Gly Ile Ile Ser Pro Ser Gly Gly Gly Thr Lys Tyr Ala Gln Lys Phe
              50                  55                  60                  
          Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65                  70                  75                  80  
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85                  90                  95      
          Ala Arg Leu Tyr Asp Val Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
                      100                 105                 110         
          Val Ser Ser
                  115 
          <![CDATA[<210> 93]]>
          <![CDATA[<211> 117]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 93]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
                      20                  25                  30          
          Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35                  40                  45              
          Gly Ile Ile Ser Pro Ser Gly Gly Ser Thr Lys Tyr Ala Gln Lys Phe
              50                  55                  60                  
          Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65                  70                  75                  80  
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85                  90                  95      
          Ala Arg Leu Gly Tyr Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu
                      100                 105                 110         
          Val Thr Val Ser Ser
                  115         
          <![CDATA[<210> 94]]>
          <![CDATA[<211> 118]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 94]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly
                      20                  25                  30          
          His Tyr Trp Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
                  35                  40                  45              
          Ile Gly Asp Ile Ser His Ser Gly Ser Thr Tyr Tyr Ser Gln Ser Leu
              50                  55                  60                  
          Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65                  70                  75                  80  
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85                  90                  95      
          Ala Arg Gly Ser Arg Thr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
                      100                 105                 110         
          Leu Val Thr Val Ser Ser
                  115             
          <![CDATA[<210>]]> 95
          <![CDATA[<211> 114]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 95]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Phe Phe
                      20                  25                  30          
          Gly Ile Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35                  40                  45              
          Lys Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser
              50                  55                  60                  
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65                  70                  75                  80  
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Thr
                          85                  90                  95      
          Ser Ser Pro Pro Val Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
                      100                 105                 110         
          Lys Arg
          <![CDATA[<210> 96]]>
          <![CDATA[<211> 107]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 96]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Val
                      20                  25                  30          
          Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
                  35                  40                  45              
          Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
              50                  55                  60                  
          Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
          65                  70                  75                  80  
          Asp Phe Ala Thr Tyr Tyr Cys Val Gln Gly Leu Gln Thr Pro Trp Thr
                          85                  90                  95      
          Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105         
          <![CDATA[<210> 97]]>
          <![CDATA[<211> 109]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 97]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Ser
                      20                  25                  30          
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35                  40                  45              
          Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly
              50                  55                  60                  
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65                  70                  75                  80  
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Gln Trp Pro Pro
                          85                  90                  95      
          Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105                 
          <![CDATA[<210> 98]]>
          <![CDATA[<211> 112]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 98]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Phe Phe
                      20                  25                  30          
          Gly Lys Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35                  40                  45              
          Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser
              50                  55                  60                  
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65                  70                  75                  80  
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
                          85                  90                  95      
          Ser Trp Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105                 110         
          <![CDATA[<210> 99]]>
          <![CDATA[<211> 109]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 99]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Arg
                      20                  25                  30          
          Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
                  35                  40                  45              
          Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser
              50                  55                  60                  
          Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
          65                  70                  75                  80  
          Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Pro Thr Pro
                          85                  90                  95      
          Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105                 
          <![CDATA[<210> 100]]>
          <![CDATA[<211> 109]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 100]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Arg Gly Arg
                      20                  25                  30          
          Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
                  35                  40                  45              
          Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser
              50                  55                  60                  
          Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
          65                  70                  75                  80  
          Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Ser Trp Pro
                          85                  90                  95      
          Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105                 
          <![CDATA[<210> 101]]>
          <![CDATA[<211> 110]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 101]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Val Phe Ser Arg
                      20                  25                  30          
          Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
                  35                  40                  45              
          Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser
              50                  55                  60                  
          Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
          65                  70                  75                  80  
          Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Tyr Trp Pro
                          85                  90                  95      
          Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105                 110 
          <![CDATA[<210> 102]]>
          <![CDATA[<211> 108]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 102]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Ser Ser Tyr
                      20                  25                  30          
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35                  40                  45              
          Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
              50                  55                  60                  
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65                  70                  75                  80  
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Leu
                          85                  90                  95      
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105             
          <![CDATA[<210> 103]]>
          <![CDATA[<211> 114]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> ]]>人工序列
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 103]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Phe Tyr
                      20                  25                  30          
          Gly Ile Ser Phe Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35                  40                  45              
          Lys Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser
              50                  55                  60                  
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65                  70                  75                  80  
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Val
                          85                  90                  95      
          Ser Ser Pro Pro Glu Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
                      100                 105                 110         
          Lys Arg
          <![CDATA[<210> 104]]>
          <![CDATA[<211> 112]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 104]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Phe Asp
                      20                  25                  30          
          Gly Phe Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35                  40                  45              
          Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser
              50                  55                  60                  
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65                  70                  75                  80  
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Asp
                          85                  90                  95      
          Ser Trp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105                 110         
          <![CDATA[<210> 105]]>
          <![CDATA[<211> 112]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 105]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Phe His
                      20                  25                  30          
          Gly Lys Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35                  40                  45              
          Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser
              50                  55                  60                  
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65                  70                  75                  80  
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glu Gln Ser Leu
                          85                  90                  95      
          Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105                 110         
          <![CDATA[<210> 106]]>
          <![CDATA[<211> 112]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 106]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Phe Tyr
                      20                  25                  30          
          Gly Ile Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35                  40                  45              
          Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser
              50                  55                  60                  
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65                  70                  75                  80  
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Ala Leu
                          85                  90                  95      
          Gln Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105                 110         
          <![CDATA[<210> 107]]>
          <![CDATA[<211> 108]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 107]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
                      20                  25                  30          
          Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35                  40                  45              
          Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
              50                  55                  60                  
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65                  70                  75                  80  
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Leu
                          85                  90                  95      
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100                 105             
          <![CDATA[<210> 108]]>
          <![CDATA[<211> 372]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 108]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggatt caccttctcc gactacgcta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gactcgagtg gatcggtatc atctccccat ctagcggttc tactaactac 180
          gcccagaagt tccagggtcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagagacatt 300
          cactctggtt cttctggtta ctactacggt ttcgacgtct ggggtcaagg aacactagtc 360
          accgtctcct cg                                                     372
          <![CDATA[<210> 109]]>
          <![CDATA[<211> 354]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 109]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggata ctctatcacc tctggttact actgggcctg gattcgtcag 120
          gccccgggta agggcctcga gtgggtgtct tccatctctg gttccggttc tactacctac 180
          tacgccgact ctgtcaaggg ccgtttcact ataagtcgcg acaattcgaa aaacacactg 240
          tacctacaac tgaacagctt aagagctgag gacactgccg tctattattg cgccagagat 300
          ggtttcggct acttcgacta ctggggtcaa ggaacactag tcaccgtctc ctcg       354
          <![CDATA[<210> 110]]>
          <![CDATA[<211> 369]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 110]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggatt caccttctcc gactacggta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggtgaa atctaccact ctggttctac ctactactct 180
          ccatctctga agtctcgtgt gactataagt cgcgacaatt cgaaaaacac actgtaccta 240
          caactgaaca gcttaagagc tgaggacact gccgtctatt attgcgccag agacgttgcc 300
          cctggttctt ctggttacta cgacggtttc gacttctggg gtcaaggaac actagtcacc 360
          gtctcctcg                                                         369
          <![CDATA[<210> 111]]>
          <![CDATA[<211> 363]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 111]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggata ctctatctcc tctggttacc actgggactg gattcgtcag 120
          gccccgggta agggcctcga gtgggtgtct ggtatctctg gttacggtgg ttctacctac 180
          tacgccgact ctgtcaaggg ccgtttcact ataagtcgcg acaattcgaa aaacacactg 240
          tacctacaac tgaacagctt aagagctgag gacactgccg tctattattg cgccagacac 300
          agttattacg gttccggtaa tttcgactac tggggtcaag gaacactagt caccgtctcc 360
          tcg                                                               363
          <![CDATA[<210> 112]]>
          <![CDATA[<211> 348]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> ]]>人工序列
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 112]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggatt caccttctcc gactactgga ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggttgg atctccccat ctggcggtgg tactaagtac 180
          gcccagaagt tccagggtcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagaggggct 300
          tacgaatttg actactgggg tcaaggaaca ctagtcaccg tctcctcg              348
          <![CDATA[<210> 113]]>
          <![CDATA[<211> 348]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 113]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggata ctctatctcc tctggttacc actggagctg gattcgtcag 120
          gccccgggta agggcctcga gtggctggcc cggatcgact gggacgatga caagtactac 180
          tctacctctc tgaagtctcg tctgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagatcgtac 300
          gtgtacttcg actactgggg tcaaggaaca ctagtcaccg tctcctcg              348
          <![CDATA[<210> 114]]>
          <![CDATA[<211> 360]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 114]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggatt ctctctgtct accggcggtg tggctgtgag ctggattcgt 120
          caggccccgg gtaagggcct cgagtggatc ggtgaaatct accactctgg ttctacctac 180
          tactctccat ctctgaagtc tcgtgtgact ataagtcgcg acaattcgaa aaacacactg 240
          tacctacaac tgaacagctt aagagctgag gacactgccg tctattattg cgcccgtcgt 300
          atcgccaccg ctacttactt cgactactgg ggtcaaggaa cactagtcac cgtctcctcg 360
          <![CDATA[<210> 115]]>
          <![CDATA[<211> 363]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 115]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggatt ctctctgtct accggcggtg tggctgtggg ctggattcgt 120
          caggccccgg gtaagggcct cgagtgggtg tctgctatct ctggttacgg ttctactacc 180
          tactacgccg actctgtcaa gggccgtttc actataagtc gcgacaattc gaaaaacaca 240
          ctgtacctac aactgaacag cttaagagct gaggacactg ccgtctatta ttgcgccaga 300
          ttgccatact ccgcctacgc tttcgactac tggggtcaag gaacactagt caccgtctcc 360
          tcg                                                               363
          <![CDATA[<210> 116]]>
          <![CDATA[<211> 342]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 116]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggatt caccttctcc ggctacgcta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggtatc atctccccat ctggcggtgg tactaagtac 180
          gcccagaagt tccagggtcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagacaccca 300
          ttcgcctact ggggtcaagg aacactagtc accgtctcct cg                    342
          <![CDATA[<210> 117]]>
          <![CDATA[<211> 345]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 117]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggata caccttctcc ggctacggta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggtgaa atctaccact ctggttctac ctactactct 180
          ccatctctga agtctcgtgt gactataagt cgcgacaatt cgaaaaacac actgtaccta 240
          caactgaaca gcttaagagc tgaggacact gccgtctatt attgcgccag aagaattgac 300
          gccttcgaca tctggggtca aggaacacta gtcaccgtct cctcg                 345
          <![CDATA[<210> 118]]>
          <![CDATA[<211> 345]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 118]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggata caccttctcc ggctacgcta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggtatc atctccccat ctggcggtgg tactaagtac 180
          gcccagaagt tccagggtcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagactctat 300
          gacgttgcct actggggtca aggaacacta gtcaccgtct cctcg                 345
          <![CDATA[<210> 119]]>
          <![CDATA[<211> 351]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 119]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggatt caccttctcc gactacgcta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggtatc atctccccat ctggcggttc tactaagtac 180
          gcccagaagt tccagggtcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagactcggt 300
          tacgggtact tcgacgtctg gggtcaagga acactagtca ccgtctcctc g          351
          <![CDATA[<210> 120]]>
          <![CDATA[<211> 354]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 120]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
          tcctgtgcag cttccggata ctctatcacc tctggtcact actggagctg gattcgtcag 120
          gccccgggta agggcctcga gtggatcggt gacatctccc actctggttc tacctactac 180
          tctcaatctc tgaagtctcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc gcgtggtagt 300
          aggaccggct acttcgacta ttggggtcaa ggaacactag tcaccgtctc ctcg       354
          <![CDATA[<210> 121]]>
          <![CDATA[<211> 342]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 121]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctga gtctgtggac ttcttcggta tctctttcct ggcctggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctctaa ccgtgccacc 180
          ggtatcccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgccagc actacacctc ttcgccacca 300
          gtgtacacct tcggacaggg taccaaggtg gagatcaaac ga                    342
          <![CDATA[<210> 122]]>
          <![CDATA[<211> 321]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 122]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgct ctgcctcttc tagcgtgagc tacgtgtact ggtatcaaca gaaaccagga 120
          aaagctccga agcttctgat ctacgacgcc tcttctctgg aatctggtgt gccatctcgc 180
          ttctctggat ccggttccgg gacggatttc actctgacca tcagcagtct gcagccggaa 240
          gacttcgcaa cttattactg cgtgcagggt cttcagaccc cttggacctt cggacagggt 300
          accaaggtgg agatcaaacg a                                           321
          <![CDATA[<210> 123]]>
          <![CDATA[<211> 327]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 123]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gggtattggc tcttccctgg cttggtatca acagaaacca 120
          ggaaaagctc cgaagcttct gatctacgac gcctctaacc gtgccaccgg tatcccatct 180
          cgcttctctg gatccggttc cgggacggat ttcactctga ccatcagcag tctgcagccg 240
          gaagacttcg caacttatta ctgccagcag tacgaccaat ggccaccttg gaccttcgga 300
          cagggtacca aggtggagat caaacga                                     327
          <![CDATA[<210> 124]]>
          <![CDATA[<211> 336]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 124]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctga gtctgtggac ttcttcggta agtctttcct gcactggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctctaa cctggaaacc 180
          ggtgtgccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgccagc agtcctactc ctggcctccg 300
          accttcggac agggtaccaa ggtggagatc aaacga                           336
          <![CDATA[<210> 125]]>
          <![CDATA[<211> 327]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 12]]>5
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtgagc agccgtttcc tggcctggta tcaacagaaa 120
          ccaggaaaag ctccgaagct tctgatctac gacgcctcta accgtgccac cggtatccca 180
          tctcgcttct ctggatccgg ttccgggacg gatttcactc tgaccatcag cagtctgcag 240
          ccggaagact tcgcaactta ttactgccag cagtcctacc ccacccctct taccttcgga 300
          cagggtacca aggtggagat caaacga                                     327
          <![CDATA[<210> 126]]>
          <![CDATA[<211> 327]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 126]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtgcgc ggccgtttcc tggcctggta tcaacagaaa 120
          ccaggaaaag ctccgaagct tctgatctac gacgcctcta accgtgccac cggtatccca 180
          tctcgcttct ctggatccgg ttccgggacg gatttcactc tgaccatcag cagtctgcag 240
          ccggaagact tcgcaactta ttactgccag cagtcctcct cctggcctcc gaccttcgga 300
          cagggtacca aggtggagat caaacga                                     327
          <![CDATA[<210> 127]]>
          <![CDATA[<211> 330]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 127]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gaccgtgttc tctcgttacc tggcttggta tcaacagaaa 120
          ccaggaaaag ctccgaagct tctgatctac gacgcctcta accgtgccac cggtatccca 180
          tctcgcttct ctggatccgg ttccgggacg gatttcactc tgaccatcag cagtctgcag 240
          ccggaagact tcgcaactta ttactgccag cagtcctact actggccacc ttggaccttc 300
          ggacagggta ccaaggtgga gatcaaacga                                  330
          <![CDATA[<210> 128]]>
          <![CDATA[<211> 324]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 128]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gggtgtgtct tcttacctgg cctggtatca acagaaacca 120
          ggaaaagctc cgaagcttct gatctacgcc gcctctacct tgcagtctgg tgtgccatct 180
          cgcttctctg gatccggttc cgggacggat ttcactctga ccatcagcag tctgcagccg 240
          gaagacttcg caacttacta ctgccagcac cactacggca ccccactgac cttcggtcag 300
          ggtaccaagg tggagatcaa acga                                        324
          <![CDATA[<210> 129]]>
          <![CDATA[<211> 342]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 129]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtggac ttctacggta tctctttcct ggactggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctctaa ccgtgccacc 180
          ggtatcccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgccagc agtacgtctc ttcgccacca 300
          gagtacacct tcggacaggg taccaaggtg gagatcaaac ga                    342
          <![CDATA[<210> 130]]>
          <![CDATA[<211> 336]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 130]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtggac ttcgacggtt tctctttcct gcactggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctcttc tctggaatct 180
          ggtgtgccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgccagc agcgtgactc ctggccttac 300
          accttcggac agggtaccaa ggtggagatc aaacga                           336
          <![CDATA[<210> 131]]>
          <![CDATA[<211> 336]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 131]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtggac ttccacggta agtctttcct gcactggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctcttc tctggaatct 180
          ggtgtgccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgcgagc aatccctgga agtcccattc 300
          accttcggac agggtaccaa ggtggagatc aaacga                           336
          <![CDATA[<210> 132]]>
          <![CDATA[<211> 336]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 132]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtggac ttctacggta tctctttcct gcactggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctcttc tctggaatct 180
          ggtgtgccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgcgtgc aggctcttca gttgcctctt 300
          accttcggac agggtaccaa ggtggagatc aaacga                           336
          <![CDATA[<210> 133]]>
          <![CDATA[<211> 324]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 133]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctatctct tcttacctga actggtatca acagaaacca 120
          ggaaaagctc cgaagcttct gatctacgac gcctctaacc tggaaaccgg tgtgccatct 180
          cgcttctctg gatccggttc cgggacggat ttcactctga ccatcagcag tctgcagccg 240
          gaagacttcg caacttacta ctgccagcac cactacggca ccccactgac cttcggtcag 300
          ggtaccaagg tggagatcaa acga                                        324
          <![CDATA[<210> 134]]>
          <![CDATA[<211> 5]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213]]>> 人工序列]]&gt;
          <br/>
          <br/>&lt;![CDATA[&lt;220&gt; ]]&gt;
          <br/>&lt;![CDATA[&lt;223&gt; 合成構築體]]&gt;
          <br/>
          <br/>&lt;![CDATA[&lt;220&gt; ]]&gt;
          <br/>&lt;![CDATA[&lt;221&gt; 變異體        ]]&gt;
          <br/>&lt;![CDATA[&lt;222&gt; 1]]&gt;
          <br/>&lt;![CDATA[&lt;223&gt; 在至少2個且至多10個的]]&gt;<![CDATA[重複序列中存在
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 3]]>
          <![CDATA[<223> 在至少3個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1, 3]]>
          <![CDATA[<223> Xaa = Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、Lys、Leu、]]>
          Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp或Tyr
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5]]>
          <![CDATA[<223> 在至少1個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His或Pro
          <![CDATA[<400> 134]]>
          Xaa Cys Xaa Cys Xaa
           1               5  
          <![CDATA[<210> 135]]>
          <![CDATA[<211> 5]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1]]>
          <![CDATA[<223> 在至少2個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 3]]>
          <![CDATA[<223> 在至少3個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5]]>
          <![CDATA[<223> 在至少1個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1, 3, 5]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His或Pro
          <![CDATA[<400> 135]]>
          Xaa Cys Xaa Cys Xaa
           1               5  
          <![CDATA[<210> 136]]>
          <![CDATA[<211> 15]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> misc_feature   ]]>
          <![CDATA[<222> 1, 2, 3]]>
          <![CDATA[<223> 在至少2個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> misc_feature   ]]>
          <![CDATA[<222> 7, 8, 9]]>
          <![CDATA[<223> 在至少3個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> misc_feature   ]]>
          <![CDATA[<222> 13, 14, 15]]>
          <![CDATA[<223> 在至少1個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> misc_feature   ]]>
          <![CDATA[<222> 1, 2, 7, 8, 13]]>
          <![CDATA[<223> n = A、T、C或G]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> misc_feature   ]]>
          <![CDATA[<222> 3, 9]]>
          <![CDATA[<223> n = T或G]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> misc_feature   ]]>
          <![CDATA[<222> 6, 12]]>
          <![CDATA[<223> n = T或C]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> misc_feature   ]]>
          <![CDATA[<222> 14]]>
          <![CDATA[<223> n = A、T或C]]>
          <![CDATA[<400> 136]]>
          nnntgnnnnt gnnnc                                                  15
          <![CDATA[<210> 137]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1, 2, 3,]]> 4, 5, 6, 15, 16
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His或Pro
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 8, 9, 10, 11, 12, 13]]>
          <![CDATA[<223> Xaa = Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、Lys、Leu、]]>
          Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp或Tyr
          <![CDATA[<400> 137]]>
          Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
           1               5                  10                  15      
          <![CDATA[<210> 138]]>
          <![CDATA[<211> 18]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1, 2, 3, 4, 5, 6, 17, 18]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、P]]>he、Val、
          His或Pro
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 8, 9, 10, 11, 12, 13, 14, 15]]>
          <![CDATA[<223> Xaa = Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、Lys、Leu、]]>
          Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp或Tyr
          <![CDATA[<400> 138]]>
          Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
           1               5                  10                  15      
          Xaa Xaa
          <![CDATA[<210> 139]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His或Pro
          <![CDATA[<400> 139]]>
          Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
           1               5                  10                  15      
          <![CDATA[<210> 140]]>
          <![CDATA[<211> 18]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr]]>、Ser、Thr、Asn、Ile、Leu、Phe、Val、
          His或Pro
          <![CDATA[<400> 140]]>
          Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
           1               5                  10                  15      
          Xaa Xaa
          <![CDATA[<210> 141]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 141]]>
          Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro Tyr Pro Cys Ser Ala
           1               5                  10                  15      
          <![CDATA[<210> 142]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 142]]>
          Ile Val His His Ser Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser
           1               5                  10                  15      
          <![CDATA[<210> 143]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 143]]>
          Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr Pro Tyr Cys Asn Ser
           1               5                  10                  15      
          <![CDATA[<210> 144]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 144]]>
          Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1               5                  10                  15      
          <![CDATA[<210> 145]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 145]]>
          Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr Pro Tyr Cys Gln Ser
           1               5                  10                  15      
          <![CDATA[<210> 146]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 146]]>
          Pro Gln Pro Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1               5                  10                  15      
          <![CDATA[<210> 147]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 147]]>
          Pro Asn Pro Ala Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1               5                  10                  15      
          <![CDATA[<210> 148]]>
          <![CDATA[<211> 5]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 148]]>
          Glu Val Gly Ser Tyr
           1               5  
          <![CDATA[<210> 149]]>
          <![CDATA[<211> 5]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 149]]>
          Ser Gly Arg Ser Ala
           1               5  
          <![CDATA[<210> 150]]>
          <![CDATA[<21]]>1> 6]]&gt;
          <br/>&lt;![CDATA[&lt;212&gt; PRT]]&gt;
          <br/>&lt;![CDATA[&lt;213&gt; 人工序列]]&gt;
          <br/>
          <br/>&lt;![CDATA[&lt;220&gt; ]]&gt;
          <br/>&lt;![CDATA[&lt;223&gt; 合成構築體]]&gt;
          <br/>
          <br/>&lt;![CDATA[&lt;400&gt; 150]]&gt;
          <br/><![CDATA[Pro Leu Gly Leu Ala Gly
           1               5      
          <![CDATA[<210> 151]]>
          <![CDATA[<211> 7]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 151]]>
          Glu Asn Leu Tyr Phe Gln Gly
           1               5          
          <![CDATA[<210> 152]]>
          <![CDATA[<211> 26]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 20, 21]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His或Pro
          <![CDATA[<400> 152]]>
          Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
           1               5                  10                  15      
          Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala
                      20                  25      
          <![CDATA[<210> 153]]>
          <![CDATA[<211> 26]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6, 7, 8, 9, 10, 11, 20, 21]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His或Pro
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 13, 14, 15, 16, 17, 18]]>
          <![CDATA[<223> Xaa = Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、Lys、Leu、]]>
          Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp或Tyr
          <![CDATA[<400> 153]]>
          Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
           1               5                  10                  15      
          Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala
                      20                  25      
          <![CDATA[<210> 154]]>
          <![CDATA[<211> 28]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His或Pro
          <![CDATA[<400> 154]]>
          Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
           1               5                  10                  15      
          Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala
                      20                  25              
          <![CDATA[<210> 155]]>
          <![CDATA[<211> 28]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6, 7, 8, 9, 10, 11, 22, 23]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His,或Pro
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 13, 14, 15, 16, 17, 18, 19, 20]]>
          <![CDATA[<223> Xaa = Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、Lys、Leu、]]>
          Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp或Tyr
          <![CDATA[<400> 155]]>
          Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
           1               5                  10                  15      
          Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala
                      20                  25              
          <![CDATA[<210> 156]]>
          <![CDATA[<211> 5]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 156]]>
          Gly Gly Gly Gly Ser
           1               5  
          <![CDATA[<210> 157]]>
          <![CDATA[<211> 4]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 157]]>
          Ser Gly Gly Ser
           1              
          <![CDATA[<210> 158]]>
          <![CDATA[<211> 4]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 158]]>
          Gly Gly Ser Gly
           1              
          <![CDATA[<210> 159]]>
          <![CDATA[<211> 5]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 159]]>
          Gly Gly Ser Gly Gly
           1               5  
          <![CDATA[<210> 160]]>
          <![CDATA[<211> 5]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 160]]>
          Gly Ser Gly Ser Gly
           1               5  
          <![CDATA[<210> 161]]>
          <![CDATA[<211> 5]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 161]]>
          Gly Ser Gly Gly Gly
           1               5  
          <![CDATA[<210> 162]]>
          <![CDATA[<211> 5]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 162]]>
          Gly Gly Gly Ser Gly
           1               5  
          <![CDATA[<210> 163]]>
          <![CDATA[<211> 5]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 163]]>
          Gly Ser Ser Ser Gly
           1               5  
          <![CDATA[<210> 164]]>
          <![CDATA[<211> 42]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 6]]>
          <![CDATA[<223> Xaa = Ala、Asp、Ile、Asn、Pro或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 7]]>
          <![CDATA[<223> Xaa =  Ala、Phe、Asn、Ser或Val]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 8]]>
          <![CDATA[<223> Xaa = Ala、His、Leu、Pro、Ser、Val或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 9]]>
          <![CDATA[<223> Xaa =  Ala、His、Ser或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 10]]>
          <![CDATA[<223> Xaa = Ala、Asp、Pro、Ser、Val或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 11]]>
          <![CDATA[<223> Xaa = Ala、Asp、Leu、Ser或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 13]]>
          <![CDATA[<223> Xaa = Asp、Pro或Val]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 14]]>
          <![CDATA[<223> Xaa = Ala、Asp、Hi]]>s、Pro、Ser或Thr
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 15]]>
          <![CDATA[<223> Xaa = Ala、Asp、Phe、His、Pro或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 16]]>
          <![CDATA[<223> Xaa =  Leu、Pro或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 17]]>
          <![CDATA[<223> Xaa = Phe、Pro或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 18]]>
          <![CDATA[<223> Xaa = Ala、Pro、Ser或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 20]]>
          <![CDATA[<223> Xaa = Ala、Asp、Asn、Ser、Thr或Tyr]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 21]]>
          <![CDATA[<223> Xaa = Ala、Ser或Tyr]]>
          <![CDATA[<400> 164]]>
          Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
           1               5                  10                  15      
          Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20                  25                  30          
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35                  40          
          <![CDATA[<210> 165]]>
          <![CDATA[<211> 42]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 165]]>
          Glu Val Gly Ser Tyr Asp Ala Leu His Tyr Ala Cys Pro Pro Asp Tyr
           1               5                  10                  15      
          Tyr Ala Cys Tyr Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20                  25                  30          
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35                  40          
          <![CDATA[<210> 166]]>
          <![CDATA[<211> 42]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 166]]>
          Glu Val Gly Ser Tyr Asn Ser Tyr His Ala Tyr Cys Pro His Pro Leu
           1               5                  10                  15      
          Tyr Pro Cys Thr Ala Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20                  25                  30          
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35                  40          
          <![CDATA[<210> 167]]>
          <![CDATA[<211> 42]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 167]]>
          Glu Val Gly Ser Tyr Ala Ser Ser Ala Val Leu Cys Val Thr Ala Tyr
           1               5                  10                  15      
          Phe Ser Cys Asn Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20                  25                  30          
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35                  40          
          <![CDATA[<210> 168]]>
          <![CDATA[<211> 41]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 168]]>
          Glu Val Gly Ser Tyr Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro
           1               5                  10                  15      
          Tyr Pro Cys Ser Ala Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20                  25                  30          
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35                  40      
          <![CDATA[<210> 169]]>
          <![CDATA[<211> 42]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 169]]>
          Glu Val Gly Ser Tyr Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro
           1               5                  10                  15      
          Tyr Pro Cys Ser Ala Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20                  25                  30          
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35                  40          
          <![CDATA[<210> 170]]>
          <![CDATA[<211> 41]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 1]]>70
          Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr
           1               5                  10                  15      
          Pro Tyr Cys Asp Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20                  25                  30          
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35                  40      
          <![CDATA[<210> 171]]>
          <![CDATA[<211> 42]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 171]]>
          Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr
           1               5                  10                  15      
          Pro Tyr Cys Asp Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20                  25                  30          
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35                  40          
          <![CDATA[<210> 172]]>
          <![CDATA[<211> 41]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 172]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1               5                  10                  15      
          Pro Tyr Cys Asn Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20                  25                  30          
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35                  40      
          <![CDATA[<210> 173]]>
          <![CDATA[<211> 42]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 173]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1               5                  10                  15      
          Pro Tyr Cys Asn Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20                  25                  30          
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35                  40          
          <![CDATA[<210> 174]]>
          <![CDATA[<211> 41]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 174]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1               5                  10                  15      
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20                  25                  30          
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35                  40      
          <![CDATA[<210> 175]]>
          <![CDATA[<211> 42]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 175]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1               5                  10                  15      
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20                  25                  30          
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35                  40          
          <![CDATA[<210> 176]]>
          <![CDATA[<211> 41]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 176]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1               5                  10                  15      
          Pro Tyr Cys Gln Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20                  25                  30          
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35                  40      
          <![CDATA[<210> 177]]>
          <![CDATA[<211> 41]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 177]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1               5                  10                  15      
          Pro Tyr Cys Asn Ser Ala Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20                  25                  30          
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35                  40      
          <![CDATA[<210> 178]]>
          <![CDATA[<211> 41]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 178]]>
          Glu Val Gly Ser Tyr Pro Gln Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1               5                  10                  15      
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20                  25                  30          
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35                  40      
          <![CDATA[<210> 179]]>
          <![CDATA[<211> 41]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 179]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ala Ser Asp Cys Val Pro Tyr Tyr
           1               5                  10                  15      
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20                  25                  30          
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35                  40      
          <![CDATA[<210> 180]]>
          <![CDATA[<211> 20]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 180]]>
          Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro Leu Gly Leu Ala Gly
           1               5                  10                  15      
          Ser Gly Gly Ser
                      20  
          <![CDATA[<210> 181]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1]]>
          <![CDATA[<223> 在至少2個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1, 10, 11]]>
          <![CDATA[<223> Xaa = Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、Lys、Leu、]]>
          Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp或Tyr
          <![CDATA[<400> 181]]>
          Xaa Cys Pro Asp His Pro Tyr Pro Cys Xaa Xaa
           1               5                  10      
          <![CDATA[<210> 182]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1]]>
          <![CDATA[<223> 在至少2個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1, 10, 11]]>
          <![CDATA[<223> Xaa = Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、Lys、Leu、]]>
          Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp或Tyr
          <![CDATA[<400> 182]]>
          Xaa Cys Asp Ala Phe Tyr Pro Tyr Cys Xaa Xaa
           1               5                  10      
          <![CDATA[<210> 183]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1]]>
          <![CDATA[<223> 在至少2個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1, 10, 11]]>
          <![CDATA[<223> Xaa = Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、Lys、Leu、]]>
          Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp或Tyr
          <![CDATA[<400> 183]]>
          Xaa Cys Asp Ser His Tyr Pro Tyr Cys Xaa Xaa
           1               5                  10      
          <![CDATA[<210> 184]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1]]>
          <![CDATA[<223> 在至少2個且至多10個的重複序列中存在]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 1, 10, 11]]>
          <![CDATA[<223> Xaa = Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、Lys、Leu、]]>
          Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp或Tyr
          <![CDATA[<400> 184]]>
          Xaa Cys Val Pro Tyr Tyr Tyr Ala Cys Xaa Xaa
           1               5                  10      
          <![CDATA[<210> 185]]>
          <![CDATA[<400> 185]]>
          000     
          <![CDATA[<210> 186]]>
          <![CDATA[<400> 186]]>
          000    
          <![CDATA[<210> 187]]>
          <![CDATA[<400> 187]]>
          000      
          <![CDATA[<210> 188]]>
          <![CDATA[<400> 188]]>
          000     
          <![CDATA[<210> 189]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 189]]>
          Glu Val Gly Ser Tyr Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro
           1               5                  10                  15      
          Tyr Pro Cys Ser Ala
                      20      
          <![CDATA[<210> 190]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 190]]>
          Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr
           1               5                  10                  15      
          Pro Tyr Cys Asp Ser
                      20      
          <![CDATA[<210> 191]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 191]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1               5                  10                  15      
          Pro Tyr Cys Asn Ser
                      20      
          <![CDATA[<210> 192]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 192]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1               5                  10                  15      
          Tyr Ala Cys Ala Tyr
                      20      
          <![CDATA[<210> 193]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 193]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1               5                  10                  15      
          Pro Tyr Cys Gln Ser
                      20      
          <![CDATA[<210> 194]]>
          <![CDATA[<400> 194]]>
          000  
          <![CDATA[<210> 195]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 195]]>
          Glu Val Gly Ser Tyr Pro Gln Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1               5                  10                  15      
          Tyr Ala Cys Ala Tyr
                      20      
          <![CDATA[<210> 196]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 196]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ala Ser Asp Cys Val Pro Tyr Tyr
           1               5                  10                  15      
          Tyr Ala Cys Ala Tyr
                      20      
          <![CDATA[<210> 197]]>
          <![CDATA[<211> 41]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 197]]>
          Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr
           1               5                  10                  15      
          Pro Tyr Cys Asp Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro
                      20                  25                  30          
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35                  40      
          <![CDATA[<210> 198]]>
          <![CDATA[<211> 36]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 198]]>
          Glu Val Gly His Ser Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser
           1               5                  10                  15      
          Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly
                      20                  25                  30          
          Ser Gly Gly Ser
                  35      
          <![CDATA[<210> 199]]>
          <![CDATA[<211> 32]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 199]]>
          Glu Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser Ser Gly Arg Ser
           1               5                  10                  15      
          Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly Ser Gly Gly Ser
                      20                  25                  30          
          <![CDATA[<210> 200]]>
          <![CDATA[<211> 41]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 200]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1               5                  10                  15      
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro
                      20                  25                  30          
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35                  40      
          <![CDATA[<210> 201]]>
          <![CDATA[<211> 36]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 201]]>
          Glu Val Gly Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1               5                  10                  15      
          Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly
                      20                  25                  30          
          Ser Gly Gly Ser
                  35      
          <![CDATA[<210> 202]]>
          <![CDATA[<211> 32]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 202]]>
          Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Ser Gly Arg Ser
           1               5                  10                  15      
          Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly Ser Gly Gly Ser
                      20                  25                  30          
          <![CDATA[<210> 203]]>
          <![CDATA[<211> 118]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 203]]>
          Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
           1               5                  10                  15      
          Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
                      20                  25                  30          
          Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
                  35                  40                  45              
          Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
              50                  55                  60                  
          Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
          65                  70                  75                  80  
          Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
                          85                  90                  95      
          Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Ala Gln Ile
                      100                 105                 110         
          Tyr Val Ile Asp Pro Glu
                  115             
          <![CDATA[<210> 204]]>
          <![CDATA[<211> 118]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 204]]>
          Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
           1               5                  10                  15      
          Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
                      20                  25                  30          
          Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
                  35                  40                  45              
          Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
              50                  55                  60                  
          Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
          65                  70                  75                  80  
          Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
                          85                  90                  95      
          Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
                      100                 105                 110         
          Tyr Val Ile Asp Pro Glu
                  115             
          <![CDATA[<210> 205]]>
          <![CDATA[<211> 115]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 205]]>
          Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg
           1               5                  10                  15      
          Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
                      20                  25                  30          
          Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
                  35                  40                  45              
          Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Ile Cys
              50                  55                  60                  
          Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu
          65                  70                  75                  80  
          Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr
                          85                  90                  95      
          Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val
                      100                 105                 110         
          Ile Asp Pro
                  115 
          <![CDATA[<210> 206]]>
          <![CDATA[<211> 114]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 206]]>
          Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
           1               5                  10                  15      
          Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
                      20                  25                  30          
          Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
                  35                  40                  45              
          Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Asp Ser Ile
              50                  55                  60                  
          Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly
          65                  70                  75                  80  
          Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met
                          85                  90                  95      
          Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr
                      100                 105                 110         
          Val Ile
          <![CDATA[<210> 207]]>
          <![CDATA[<211> 120]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 智人]]>
          <![CDATA[<400> 207]]>
          Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg
           1               5                  10                  15      
          Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
                      20                  25                  30          
          Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
                  35                  40                  45              
          Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp
              50                  55                  60                  
          Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr
          65                  70                  75                  80  
          Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val
                          85                  90                  95      
          Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
                      100                 105                 110         
          Gln Ile Tyr Val Ile Asp Pro Glu
                  115                 120 
          <![CDATA[<210> 208]]>
          <![CDATA[<211> 120]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 小家鼠]]>
          <![CDATA[<400> 208]]>
          Glu Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser His
           1               5                  10                  15      
          Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp
                      20                  25                  30          
          Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln Met Thr Glu
                  35                  40                  45              
          Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp
              50                  55                  60                  
          Tyr Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr
          65                  70                  75                  80  
          Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu Cys Lys Val
                          85                  90                  95      
          Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr
                      100                 105                 110         
          Gln Ile Tyr Val Ile Asp Pro Glu
                  115                 120 
          <![CDATA[<210> 209]]>
          <![CDATA[<211> 31]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 209]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1               5                  10                  15      
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Pro Leu Gly Leu Ala
                      20                  25                  30      
          <![CDATA[<210> 210]]>
          <![CDATA[<211> 22]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 210]]>
          Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Ser Gly Arg Ser
           1               5                  10                  15      
          Ala Pro Leu Gly Leu Ala
                      20          
          <![CDATA[<210> 211]]>
          <![CDATA[<211> 17]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 211]]>
          Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Ser Gly Arg Ser
           1               5                  10                  15      
          Ala
          <![CDATA[<210> 212]]>
          <![CDATA[<211> 17]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 212]]>
          Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Pro Leu Gly Leu
           1               5                  10                  15      
          Ala
          <![CDATA[<210> 213]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 213]]>
          Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1               5                  10          
          <![CDATA[<210> 214]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 214]]>
          Glu Val Gly Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1               5                  10                  15      
          <![CDATA[<210> 215]]>
          <![CDATA[<211> 12]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 215]]>
          Glu Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser
           1               5                  10          
          <![CDATA[<210> 216]]>
          <![CDATA[<211> 16]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 216]]>
          Glu Val Gly His Ser Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser
           1               5                  10                  15      
          <![CDATA[<210> 217]]>
          <![CDATA[<211> 20]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 3]]>
          <![CDATA[<223> Present in repeats of at least one and up to 11]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 5, 6, 7, 8, 9, 10, 11, 12]]>
          <![CDATA[<223> Xaa = Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、L]]>ys、Leu、
          Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp或Tyr
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 3, 14, 15]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His或Pro
          <![CDATA[<400> 217]]>
          Glu Val Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser
           1               5                  10                  15      
          Gly Arg Ser Ala
                      20  
          <![CDATA[<210> 218]]>
          <![CDATA[<211> 17]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 4, 5, 6, 7, 8, 9, 11, 12]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His或Pro
          <![CDATA[<400> 218]]>
          Glu Asp Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser
           1               5                  10                  15      
          Ala
          <![CDATA[<210> 219]]>
          <![CDATA[<211> 17]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<220> ]]>
          <![CDATA[<221> 變異體        ]]>
          <![CDATA[<222> 4, 5, 6, 7, 8, 9, 11, 12]]>
          <![CDATA[<223> Xaa = Asp、Ala、Tyr、Ser、Thr、Asn、Ile、Leu、Phe、Val、]]>
          His或Pro
          <![CDATA[<400> 219]]>
          Glu Asp Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Pro Leu Gly Leu
           1               5                  10                  15      
          Ala
          <![CDATA[<210> 220]]>
          <![CDATA[<211> 21]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 220]]>
          Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu Asn Leu Tyr Phe Gln
           1               5                  10                  15      
          Gly Ser Gly Gly Ser
                      20      
          <![CDATA[<210> 221]]>
          <![CDATA[<211> 20]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 221]]>
          Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly
           1               5                  10                  15      
          Ser Gly Gly Ser
                      20  
          <![CDATA[<210> 222]]>
          <![CDATA[<211> 10]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 222]]>
          Ser Gly Arg Ser Ala Pro Leu Gly Leu Ala
           1               5                  10  
          <![CDATA[<210> 223]]>
          <![CDATA[<211> 13]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 223]]>
          Phe Ser Leu Ser Thr Gly Gly Val Gly Val Gly Trp Ile
           1               5                  10              
          <![CDATA[<210> 224]]>
          <![CDATA[<211> 20]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 224]]>
          Leu Ala Leu Ile Asp Trp Ala Asp Asp Lys Tyr Tyr Ser Pro Ser Leu
           1               5                  10                  15      
          Lys Ser Arg Leu
                      20  
          <![CDATA[<210> 225]]>
          <![CDATA[<211> 15]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 225]]>
          Ala Arg Gly Gly Ser Asp Thr Val Ile Gly Asp Trp Phe Ala Tyr
           1               5                  10                  15  
          <![CDATA[<210> 226]]>
          <![CDATA[<211> 11]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 226]]>
          Arg Ala Ser Gln Ser Ile Gly Ser Tyr Leu Ala
           1               5                  10      
          <![CDATA[<210> 227]]>
          <![CDATA[<211> 9]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 227]]>
          Asp Ala Ser Asn Leu Glu Thr Gly Val
           1               5                  
          <![CDATA[<210> 228]]>
          <![CDATA[<211> 10]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 228]]>
          Tyr Cys Gln Gln Gly Tyr Tyr Leu Trp Thr
           1               5                  10  
          <![CDATA[<210> 229]]>
          <![CDATA[<211> 123]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 229]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1               5                  10                  15      
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly
                      20                  25                  30          
          Gly Val Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu
                  35                  40                  45              
          Trp Leu Ala Leu Ile Asp Trp Ala Asp Asp Lys Tyr Tyr Ser Pro Ser
              50                  55                  60                  
          Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
          65                  70                  75                  80  
          Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
                          85                  90                  95      
          Cys Ala Arg Gly Gly Ser Asp Thr Val Ile Gly Asp Trp Phe Ala Tyr
                      100                 105                 110         
          Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115                 120             
          <![CDATA[<210> 230]]>
          <![CDATA[<211> 106]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> 人工序列]]>
          <![CDATA[<220> ]]>
          <![CDATA[<223> 合成構築體]]>
          <![CDATA[<400> 230]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1               5                  10                  15      
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Tyr
                      20                  25                  30          
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35                  40                  45              
          Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
              50                  55                  60                  
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65                  70                  75                  80  
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Tyr Leu Trp Thr
                          85                  90                  95      
          Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
                      100                 105     
          <![CDATA[ <110> British Cayman Islands Adagene Pharmaceutical Co., Ltd. (Adagene Inc.)]]>
           <![CDATA[ <120> Anti-CTLA4 antibody and preparation and use methods]]>
           <![CDATA[ <130>TW 108119978]]>
           <![CDATA[ <150> PCT/CN2019/074580]]>
           <![CDATA[ <151> 2019-02-02]]>
           <![CDATA[ <160> 230]]>
           <![CDATA[ <170> FastSEQ for Windows Version 4.0]]>
           <![CDATA[ <210> 1]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1]]>
           <![CDATA[ <223> Xaa = Phe or T]]>yr
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Xaa = Asp or Gly]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 7]]>
           <![CDATA[ <223> Xaa = Ala, Gly or Trp]]>
           <![CDATA[ <400> 1]]>
          Xaa Thr Phe Ser Xaa Tyr Xaa Ile His Trp Val
           1 5 10
           <![CDATA[ <210> 2]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 4]]>
           <![CDATA[ <223> Xaa = Ser or Thr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 7, 8]]>
           <![CDATA[ <223> Xaa = His or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 10]]>
           <![CDATA[ <223> Xaa = Ala, Asp or Ser]]>
           <![CDATA[ <400> 2]]>
          Tyr Ser Ile Xaa Ser Gly Xaa Xaa Trp Xaa Trp Ile
           1 5 10
           <![CDATA[ <210> 3]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 11]]>
           <![CDATA[ <223> Xaa = Gly or Ser]]>
           <![CDATA[ <400> 3]]>
          Phe Ser Leu Ser Thr Gly Gly Val Ala Val Xaa Trp Ile
           1 5 10
           <![CDATA[ <210> 4]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 3]]>
           <![CDATA[ <223> Xaa = Asp or Glu]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Xaa = Ser or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 14]]>
           <![CDATA[ <223> Xaa = Pro or Gln]]>
           <![CDATA[ <400> 4]]>
          Ile Gly Xaa Ile Xaa His Ser Gly Ser Thr Tyr Tyr Ser Xaa Ser Leu
           1 5 10 15
          Lys Ser Arg Val
                      20
           <![CDATA[ <210> 5]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221]]>> Variant ]]&gt;
           <br/> &lt;![CDATA[ &lt;222&gt;3]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Xaa = Ile or Trp]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;221&gt; Variant ]]&gt;
           <br/> &lt;![CDATA[ &lt;222&gt; 8, 10]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Xaa = Gly or Ser]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;221&gt; Variant ]]&gt;
           <br/> &lt;![CDATA[ &lt;222&gt;12]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Xaa = Lys or Asn]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;400&gt;5]]&gt;
           <br/> <![CDATA[Ile Gly Xaa Ile Ser Pro Ser Xaa Gly Xaa Thr Xaa Tyr Ala Gln Lys
           1 5 10 15
          Phe Gln Gly Arg Val
                      20
           <![CDATA[ <210> 6]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 3]]>
           <![CDATA[ <223> Xaa = Ala, Gly or Ser]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 7]]>
           <![CDATA[ <223> Xaa = Ser or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 9]]>
           <![CDATA[ <223> Xaa = Gly or Ser]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 10]]>
           <![CDATA[ <223> Xaa = Ser or Thr]]>
           <![CDATA[ <400> 6]]>
          Val Ser Xaa Ile Ser Gly Xaa Gly Xaa Xaa Thr Tyr Tyr Ala Asp Ser
           1 5 10 15
          Val Lys Gly Arg Phe
                      20
           <![CDATA[ <210> 7]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221]]>> Variant ]]&gt;
           <br/> &lt;![CDATA[ &lt;222&gt;3]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Xaa = Gly, Arg or Ser]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;221&gt; Variant ]]&gt;
           <br/> &lt;![CDATA[ &lt;222&gt;4]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Xaa = Ala, Ile or Tyr]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;221&gt; Variant ]]&gt;
           <br/> &lt;![CDATA[ &lt;222&gt;5]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Xaa = Asp, Val or Tyr]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;221&gt; Variant ]]&gt;
           <br/> &lt;![CDATA[ &lt;222&gt;6]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Xaa = Ala, Glu or Tyr]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;221&gt; Variant ]]&gt;
           <br/> &lt;![CDATA[ &lt;222&gt;9]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Xaa = Ile or Tyr]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;400&gt;7]]&gt;
           <br/> <![CDATA[Ala Arg Xaa Xaa Xaa Xaa Phe Asp Xaa
           1 5
           <![CDATA[ <210> 8]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 3]]>
           <![CDATA[ <223> Xaa = Asp or Leu]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Xaa = Phe or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 10]]>
           <![CDATA[ <223> Xaa = Val or Tyr]]>
           <![CDATA[ <400> 8]]>
          Ala Arg Xaa Gly Xaa Gly Tyr Phe Asp Xaa
           1 5 10
           <![CDATA[ <210> ]]>9
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 3]]>
           <![CDATA[ <223> Xaa = Leu or Arg]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 4]]>
           <![CDATA[ <223> Xaa = Ile or Pro]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Xaa = Ala or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6]]>
           <![CDATA[ <223> Xaa = Ser or Thr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 8]]>
           <![CDATA[ <223> Xaa = Thr or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 9]]>
           <![CDATA[ <223> Xaa = Ala or Tyr]]>
           <![CDATA[ <400> 9]]>
          Ala Arg Xaa Xaa Xaa Xaa Ala Xaa Xaa Phe Asp Tyr
           1 5 10
           <![CDATA[ <210> 10]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 4]]>
           <![CDATA[ <223> Xaa = Ile or Val]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Xaa = Ala or His]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6]]>
           <![CDATA[ <223> Xaa = Pro or Ser]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 13]]>
           <![CDATA[ <223> Xaa = Asp or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 17]]>
           <![CDATA[ <223> Xaa = Phe or Val]]>
           <![CDATA[ <400> 10]]>
          Ala Arg Asp Xaa Xaa Xaa Gly Ser Ser Gly Tyr Tyr Xaa Gly Phe Asp
           1 5 10 15
          Xaa
           <![CDATA[ <210> 11]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5, 7]]>
           <![CDATA[ <223> Xaa = Gly or Ser]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6]]>
           <![CDATA[ <223> Xaa = Ile or Val]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 9]]>
           <![CDATA[ <223> Xaa = Ser or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 11]]>
           <![CDATA[ <223> Xaa = Ala or Asn]]>
           <![CDATA[ <400> 11]]>
          Arg Ala Ser Gln Xaa Xaa Xaa Ser Xaa Leu Xaa
           1 5 10
           <![CDATA[ <210> 12]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Xaa = Ser or Thr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 7]]>
           <![CDATA[ <223> Xaa = Phe, Arg or Ser]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 8]]>
           <![CDATA[ <223> Xaa = Gly or Ser]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 10]]>
           <![CDATA[ <223> Xaa = Phe or Tyr]]>
           <![CDATA[ <400> 12]]>
          Arg Ala Ser Gln Xaa Val Xaa Xaa Arg Xaa Leu Ala
           1 5 10
           <![CDATA[ <210> 13]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 4]]>
           <![CDATA[ <223> Xaa = Glu or Gln]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 9]]>
           <![CDATA[ <223> Xaa = Asp, Phe, His or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 11]]>
           <![CDATA[ <223> Xaa = Phe, Ile or Lys]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant]]>
           <![CDATA[ <222> 15]]>
           <![CDATA[ <223> Xaa = Ala, Asp or His]]>
           <![CDATA[ <400> 13]]>
          Arg Ala Ser Xaa Ser Val Asp Phe Xaa Gly Xaa Ser Phe Leu Xaa
           1 5 10 15
           <![CDATA[ <210> 14]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1]]>
           <![CDATA[ <223> Xaa = Ala or A]]>sp
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 4]]>
           <![CDATA[ <223> Xaa = Asn, Ser or Thr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Xaa = Leu or Arg]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6]]>
           <![CDATA[ <223> Xaa = Ala, Glu or Gln]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 7]]>
           <![CDATA[ <223> Xaa = Ser or Thr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 9]]>
           <![CDATA[ <223> Xaa = Ile or Val]]>
           <![CDATA[ <400> 14]]>
          Xaa Ala Ser Xaa Xaa Xaa Xaa Gly Xaa
           1 5
           <![CDATA[ <210> 15]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 3]]>
           <![CDATA[ <223> Xaa = Glu, Gln or Val]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 4]]>
           <![CDATA[ <223> Xaa = His or Gln]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Xaa = Ala, Gly, His, Arg or Ser]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6]]>
           <![CDATA[ <223> Xaa = Asp, Leu, Ser or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 7]]>
           <![CDATA[ <223> Xaa = Glu, Gly, Pro, Gln or Ser]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 8]]>
           <![CDATA[ <223> Xaa = Leu, Thr, Val or Trp]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 10]]>
           <![CDATA[ <223> Xaa = Phe, Leu, Pro, Trp or Tyr]]>
           <![CDATA[ <400> 15]]>
          Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Thr
           1 5 10
           <![CDATA[ <210> 16]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Xaa = Se]]>r or Tyr
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6]]>
           <![CDATA[ <223> Xaa = Asp or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 7]]>
           <![CDATA[ <223> Xaa = Gln or Tyr]]>
           <![CDATA[ <400> 16]]>
          Tyr Cys Gln Gln Xaa Xaa Xaa Trp Pro Pro Trp Thr
           1 5 10
           <![CDATA[ <210> 17]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 4]]>
           <![CDATA[ <223> Xaa = His or Gln]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6]]>
           <![CDATA[ <223> Xaa = Thr or Val]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 11]]>
           <![CDATA[ <223> Xaa = Glu or Val]]>
           <![CDATA[ <400> 17]]>
          Tyr Cys Gln Xaa Tyr Xaa Ser Ser Pro Pro Xaa Tyr Thr
           1 5 10
           <![CDATA[ <210> 18]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 18]]>
          Phe Thr Phe Ser Asp Tyr Ala Ile His Trp Val
           1 5 10
           <![CDATA[ <210> 19]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 19]]>
          Tyr Ser Ile Thr Ser Gly Tyr Tyr Trp Ala Trp Ile
           1 5 10
           <![CDATA[ <210> 20]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 20]]>
          Phe Thr Phe Ser Asp Tyr Gly Ile His Trp Val
           1 5 10
           <![CDATA[ <210> 21]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 21]]>
          Tyr Ser Ile Ser Ser Gly Tyr His Trp Asp Trp Ile
           1 5 10
           <![CDATA[ <210> 22]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 22]]>
          Phe Thr Phe Ser Asp Tyr Trp Ile His Trp Val
           1 5 10
           <![CDATA[ <210> 23]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 23]]>
          Tyr Ser Ile Ser Ser Gly Tyr His Trp Ser Trp Ile
           1 5 10
           <![CDATA[ <210> 24]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 24]]>
          Phe Ser Leu Ser Thr Gly Gly Val Ala Val Ser Trp Ile
           1 5 10
           <![CDATA[ <210> 25]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 25]]>
          Phe Ser Leu Ser Thr Gly Gly Val Ala Val Gly Trp Ile
           1 5 10
           <![CDATA[ <210> 26]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 26]]>
          Phe Thr Phe Ser Gly Tyr Ala Ile His Trp Val
           1 5 10
           <![CDATA[ <210> 27]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 2]]>7
          Tyr Thr Phe Ser Gly Tyr Gly Ile His Trp Val
           1 5 10
           <![CDATA[ <210> 28]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 28]]>
          Tyr Thr Phe Ser Gly Tyr Ala Ile His Trp Val
           1 5 10
           <![CDATA[ <210> 29]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 29]]>
          Tyr Ser Ile Thr Ser Gly His Tyr Trp Ser Trp Ile
           1 5 10
           <![CDATA[ <210> 30]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 30]]>
          Ile Gly Ile Ile Ser Pro Ser Ser Gly Ser Thr Asn Tyr Ala Gln Lys
           1 5 10 15
          Phe Gln Gly Arg Val
                      20
           <![CDATA[ <210> 31]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 31]]>
          Val Ser Ser Ile Ser Gly Ser Gly Ser Thr Thr Tyr Tyr Tyr Ala Asp Ser
           1 5 10 15
          Val Lys Gly Arg Phe
                      20
           <![CDATA[ <210> 32]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 32]]>
          Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu
           1 5 10 15
          Lys Ser Arg Val
                      20
           <![CDATA[ <210> 33]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 33]]>
          Val Ser Gly Ile Ser Gly Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
           1 5 10 15
          Val Lys Gly Arg Phe
                      20
           <![CDATA[ <210> 34]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 34]]>
          Ile Gly Trp Ile Ser Pro Ser Gly Gly Gly Thr Lys Tyr Ala Gln Lys
           1 5 10 15
          Phe Gln Gly Arg Val
                      20
           <![CDATA[ <210> 35]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 35]]>
          Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser Leu
           1 5 10 15
          Lys Ser Arg Leu
                      20
           <![CDATA[ <210> 36]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 36]]>
          Val Ser Ala Ile Ser Gly Tyr Gly Ser Thr Thr Tyr Tyr Ala Asp Ser
           1 5 10 15
          Val Lys Gly Arg Phe
                      20
           <![CDATA[ <210> 37]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 37]]>
          Ile Gly Ile Ile Ser Pro Ser Gly Gly Gly Thr Lys Tyr Ala Gln Lys
           1 5 10 15
          Phe Gln Gly Arg Val
                      20
           <![CDATA[ <210> 38]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <40]]>0> 38]]&gt;
           <br/> <![CDATA[Ile Gly Ile Ile Ser Pro Ser Gly Gly Ser Thr Lys Tyr Ala Gln Lys
           1 5 10 15
          Phe Gln Gly Arg Val
                      20
           <![CDATA[ <210> 39]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 39]]>
          Ile Gly Asp Ile Ser His Ser Gly Ser Thr Tyr Tyr Ser Gln Ser Leu
           1 5 10 15
          Lys Ser Arg Val
                      20
           <![CDATA[ <210> 40]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 40]]>
          Ala Arg Asp Ile His Ser Gly Ser Ser Ser Gly Tyr Tyr Tyr Gly Phe Asp
           1 5 10 15
          Val
           <![CDATA[ <210> 41]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 41]]>
          Ala Arg Asp Gly Phe Gly Tyr Phe Asp Tyr
           1 5 10
           <![CDATA[ <210> 42]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 42]]>
          Ala Arg Asp Val Ala Pro Gly Ser Ser Gly Tyr Tyr Asp Gly Phe Asp
           1 5 10 15
          Phe
           <![CDATA[ <210> 43]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 43]]>
          Ala Arg His Ser Tyr Tyr Gly Ser Gly Asn Phe Asp Tyr
           1 5 10
           <![CDATA[ <210> 44]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <40]]>0> 44]]&gt;
           <br/> <![CDATA[Ala Arg Gly Ala Tyr Glu Phe Asp Tyr
           1 5
           <![CDATA[ <210> 45]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 45]]>
          Ala Arg Ser Tyr Val Tyr Phe Asp Tyr
           1 5
           <![CDATA[ <210> 46]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Person]]>Work sequence
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 46]]>
          Ala Arg Arg Ile Ala Thr Ala Thr Tyr Phe Asp Tyr
           1 5 10
           <![CDATA[ <210> 47]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 47]]>
          Ala Arg Leu Pro Tyr Ser Ala Tyr Ala Phe Asp Tyr
           1 5 10
           <![CDATA[ <210> 48]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 48]]>
          Ala Arg His Pro Phe Ala Tyr
           1 5
           <![CDATA[ <210> 49]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 49]]>
          Ala Arg Arg Ile Asp Ala Phe Asp Ile
           1 5
           <![CDATA[ <210> 50]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 50]]>
          Ala Arg Leu Tyr Asp Val Ala Tyr
           1 5
           <![CDATA[ <210> 51]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 51]]>
          Ala Arg Leu Gly Tyr Gly Tyr Phe Asp Val
           1 5 10
           <![CDATA[ <210> 52]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 52]]>
          Ala Arg Gly Ser Arg Thr Gly Tyr Phe Asp Tyr
           1 5 10
           <![CDATA[ <210> 53]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 53]]>
          Arg Ala Ser Glu Ser Val Asp Phe Phe Gly Ile Ser Phe Leu Ala
           1 5 10 15
           <![CDATA[ <210> 54]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 54]]>
          Ser Ala Ser Ser Ser Val Ser Tyr Val Tyr
           1 5 10
           <![CDATA[ <210> 55]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 55]]>
          Arg Ala Ser Gln Gly Ile Gly Ser Ser Leu Ala
           1 5 10
           <![CDATA[ <210> 56]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 56]]>
          Arg Ala Ser Glu Ser Val Asp Phe Phe Gly Lys Ser Phe Leu His
           1 5 10 15
           <![CDATA[ <210> 57]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 57]]>
          Arg Ala Ser Gln Ser Val Ser Ser Arg Phe Leu Ala
           1 5 10
           <![CDATA[ <210> 58]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 58]]>
          Arg Ala Ser Gln Ser Val Arg Gly Arg Phe Leu Ala
           1 5 10
           <![CDATA[ <210> 59]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 59]]>
          Arg Ala Ser Gln Thr Val Phe Ser Arg Tyr Leu Ala
           1 5 10
           <![CDATA[ <210> 60]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 60]]>
          Arg Ala Ser Gln Gly Val Ser Ser Tyr Leu Ala
           1 5 10
           <![CDATA[ <210> 61]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 61]]>
          Arg Ala Ser Gln Ser Val Asp Phe Tyr Gly Ile Ser Phe Leu Asp
           1 5 10 15
           <![CDATA[ <210> 62]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 62]]>
          Arg Ala Ser Gln Ser Val Asp Phe Asp Gly Phe Ser Phe Leu His
           1 5 10 15
           <![CDATA[ <210> 63]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 63]]>
          Arg Ala Ser Gln Ser Val Asp Phe His Gly Lys Ser Phe Leu His
           1 5 10 15
           <![CDATA[ <210> 64]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223]]>> Synthetic construct]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;400&gt;64]]&gt;
           <br/> <![CDATA[Arg Ala Ser Gln Ser Val Asp Phe Tyr Gly Ile Ser Phe Leu His
           1 5 10 15
           <![CDATA[ <210> 65]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 65]]>
          Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn
           1 5 10
           <![CDATA[ <210> 66]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 66]]>
          Asp Ala Ser Asn Arg Ala Thr Gly Ile
           1 5
           <![CDATA[ <210> 67]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 67]]>
          Asp Ala Ser Ser Leu Glu Ser Gly Val
           1 5
           <![CDATA[ <210> 68]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 68]]>
          Asp Ala Ser Asn Leu Glu Thr Gly Val
           1 5
           <![CDATA[ <210> 69]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 69]]>
          Ala Ala Ser Thr Leu Gln Ser Gly Val
           1 5
           <![CDATA[ <210> 70]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 70]]>
          Tyr Cys Gln His Tyr Thr Ser Ser Pro Pro Val Tyr Thr
           1 5 10
           <![CDATA[ <210> 71]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 71]]>
          Tyr Cys Val Gln Gly Leu Gln Thr Pro Trp Thr
           1 5 10
           <![CDATA[ <210> 72]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 72]]>
          Tyr Cys Gln Gln Tyr Asp Gln Trp Pro Pro Trp Thr
           1 5 10
           <![CDATA[ <210> 73]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 73]]>
          Tyr Cys Gln Gln Ser Tyr Ser Trp Pro Pro Thr
           1 5 10
           <![CDATA[ <210> 74]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 74]]>
          Tyr Cys Gln Gln Ser Tyr Pro Thr Pro Leu Thr
           1 5 10
           <![CDATA[ <210> 75]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 75]]>
          Tyr Cys Gln Gln Ser Ser Ser Trp Pro Pro Thr
           1 5 10
           <![CDATA[ <210> 76]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 76]]>
          Tyr Cys Gln Gln Ser Tyr Tyr Trp Pro Pro Trp Thr
           1 5 10
           <![CDATA[ <210> 77]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 77]]>
          Tyr Cys Gln His His Tyr Gly Thr Pro Leu Thr
           1 5 10
           <![CDATA[ <210> 78]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 78]]>
          Tyr Cys Gln Gln Tyr Val Ser Ser Pro Pro Glu Tyr Thr
           1 5 10
           <![CDATA[ <210> 79]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 79]]>
          Tyr Cys Gln Gln Arg Asp Ser Trp Pro Tyr Thr
           1 5 10
           <![CDATA[ <210> 80]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> ]]>Synthetic construct
           <![CDATA[ <400> 80]]>
          Tyr Cys Glu Gln Ser Leu Glu Val Pro Phe Thr
           1 5 10
           <![CDATA[ <210> 81]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 81]]>
          Tyr Cys Val Gln Ala Leu Gln Leu Pro Leu Thr
           1 5 10
           <![CDATA[ <210> 82]]>
           <![CDATA[ <211> 124]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 82]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
                      20 25 30
          Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35 40 45
          Gly Ile Ile Ser Pro Ser Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Asp Ile His Ser Gly Ser Ser Ser Gly Tyr Tyr Tyr Gly Phe Asp
                      100 105 110
          Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 83]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 83]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly
                      20 25 30
          Tyr Tyr Trp Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
                  35 40 45
          Val Ser Ser Ile Ser Gly Ser Gly Ser Thr Thr Tyr Tyr Tyr Ala Asp Ser
              50 55 60
          Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
          65 70 75 80
          Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
                          85 90 95
          Cys Ala Arg Asp Gly Phe Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
                      100 105 110
          Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 84]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 84]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
                      20 25 30
          Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35 40 45
          Gly Glu Ile Tyr His Ser Gly Ser Thr Tyr Tyr Tyr Ser Pro Ser Leu Lys
              50 55 60
          Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
          65 70 75 80
          Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
                          85 90 95
          Arg Asp Val Ala Pro Gly Ser Ser Gly Tyr Tyr Asp Gly Phe Asp Phe
                      100 105 110
          Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 85]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 85]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly
                      20 25 30
          Tyr His Trp Asp Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
                  35 40 45
          Val Ser Gly Ile Ser Gly Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
              50 55 60
          Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
          65 70 75 80
          Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
                          85 90 95
          Cys Ala Arg His Ser Tyr Tyr Gly Ser Gly Asn Phe Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 86]]>
           <![CDATA[ <211> 116]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 86]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
                      20 25 30
          Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35 40 45
          Gly Trp Ile Ser Pro Ser Gly Gly Gly Thr Lys Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Gly Ala Tyr Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
                      100 105 110
          Thr Val Ser Ser
                  115
           <![CDATA[ <210> 87]]>
           <![CDATA[ <211> 116]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 87]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly
                      20 25 30
          Tyr His Trp Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
                  35 40 45
          Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser Leu
              50 55 60
          Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Ser Tyr Val Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
                      100 105 110
          Thr Val Ser Ser
                  115
           <![CDATA[ <210> 88]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 88]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly
                      20 25 30
          Gly Val Ala Val Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu
                  35 40 45
          Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Tyr Tyr Ser Pro Ser
              50 55 60
          Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
          65 70 75 80
          Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
                          85 90 95
          Cys Ala Arg Arg Ile Ala Thr Ala Thr Tyr Phe Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 89]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 89]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly
                      20 25 30
          Gly Val Ala Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu
                  35 40 45
          Trp Val Ser Ala Ile Ser Gly Tyr Gly Ser Thr Thr Tyr Tyr Ala Asp
              50 55 60
          Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Ala Arg Leu Pro Tyr Ser Ala Tyr Ala Phe Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 90]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 90]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr
                      20 25 30
          Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35 40 45
          Gly Ile Ile Ser Pro Ser Gly Gly Gly Thr Lys Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg His Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
                      100 105 110
          Ser Ser
           <![CDATA[ <210> 91]]>
           <![CDATA[ <211> 115]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 91]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Gly Tyr
                      20 25 30
          Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35 40 45
          Gly Glu Ile Tyr His Ser Gly Ser Thr Tyr Tyr Tyr Ser Pro Ser Leu Lys
              50 55 60
          Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
          65 70 75 80
          Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
                          85 90 95
          Arg Arg Ile Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu Val Thr
                      100 105 110
          Val Ser Ser
                  115
           <![CDATA[ <210> 92]]>
           <![CDATA[ <211> 115]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 92]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Gly Tyr
                      20 25 30
          Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35 40 45
          Gly Ile Ile Ser Pro Ser Gly Gly Gly Thr Lys Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Leu Tyr Asp Val Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
                      100 105 110
          Val Ser Ser
                  115
           <![CDATA[ <210> 93]]>
           <![CDATA[ <211> 117]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 93]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
                      20 25 30
          Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35 40 45
          Gly Ile Ile Ser Pro Ser Gly Gly Ser Thr Lys Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Leu Gly Tyr Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu
                      100 105 110
          Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 94]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 94]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly
                      20 25 30
          His Tyr Trp Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
                  35 40 45
          Ile Gly Asp Ile Ser His Ser Gly Ser Thr Tyr Tyr Ser Gln Ser Leu
              50 55 60
          Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Gly Ser Arg Thr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
                      100 105 110
          Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210>]]> 95
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 95]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Phe Phe
                      20 25 30
          Gly Ile Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35 40 45
          Lys Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65 70 75 80
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Thr
                          85 90 95
          Ser Ser Pro Pro Val Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
                      100 105 110
          Lys Arg
           <![CDATA[ <210> 96]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 96]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Val
                      20 25 30
          Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
                  35 40 45
          Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
              50 55 60
          Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
          65 70 75 80
          Asp Phe Ala Thr Tyr Tyr Cys Val Gln Gly Leu Gln Thr Pro Trp Thr
                          85 90 95
          Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 97]]>
           <![CDATA[ <211> 109]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 97]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Ser
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Gln Trp Pro Pro
                          85 90 95
          Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 98]]>
           <![CDATA[ <211> 112]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 98]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Phe Phe
                      20 25 30
          Gly Lys Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35 40 45
          Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65 70 75 80
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
                          85 90 95
          Ser Trp Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105 110
           <![CDATA[ <210> 99]]>
           <![CDATA[ <211> 109]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 99]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Arg
                      20 25 30
          Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
                  35 40 45
          Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser
              50 55 60
          Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
          65 70 75 80
          Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Pro Thr Pro
                          85 90 95
          Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 100]]>
           <![CDATA[ <211> 109]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 100]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Arg Gly Arg
                      20 25 30
          Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
                  35 40 45
          Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser
              50 55 60
          Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
          65 70 75 80
          Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Ser Trp Pro
                          85 90 95
          Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 101]]>
           <![CDATA[ <211> 110]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 101]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Val Phe Ser Arg
                      20 25 30
          Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
                  35 40 45
          Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser
              50 55 60
          Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
          65 70 75 80
          Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Tyr Trp Pro
                          85 90 95
          Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105 110
           <![CDATA[ <210> 102]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 102]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Ser Ser Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Leu
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 103]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> ]]>Artificial sequence
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 103]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Phe Tyr
                      20 25 30
          Gly Ile Ser Phe Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35 40 45
          Lys Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65 70 75 80
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Val
                          85 90 95
          Ser Ser Pro Pro Glu Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
                      100 105 110
          Lys Arg
           <![CDATA[ <210> 104]]>
           <![CDATA[ <211> 112]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 104]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Phe Asp
                      20 25 30
          Gly Phe Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35 40 45
          Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65 70 75 80
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Asp
                          85 90 95
          Ser Trp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105 110
           <![CDATA[ <210> 105]]>
           <![CDATA[ <211> 112]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 105]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Phe His
                      20 25 30
          Gly Lys Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35 40 45
          Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65 70 75 80
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glu Gln Ser Leu
                          85 90 95
          Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105 110
           <![CDATA[ <210> 106]]>
           <![CDATA[ <211> 112]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 106]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Phe Tyr
                      20 25 30
          Gly Ile Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
                  35 40 45
          Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65 70 75 80
          Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Ala Leu
                          85 90 95
          Gln Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105 110
           <![CDATA[ <210> 107]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 107]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
                      20 25 30
          Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Leu
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 108]]>
           <![CDATA[ <211> 372]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 108]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggatt caccttctcc gactacgcta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gactcgagtg gatcggtatc atctccccat ctagcggttc tactaactac 180
          gcccagaagt tccagggtcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagagacatt 300
          cactctggtt cttctggtta ctactacggt ttcgacgtct ggggtcaagg aacactagtc 360
          accgtctcct cg 372
           <![CDATA[ <210> 109]]>
           <![CDATA[ <211> 354]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 109]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggata ctctatcacc tctggttat actgggcctg gattcgtcag 120
          gccccgggta agggcctcga gtgggtgtct tccatctctg gttccggttc tactacctac 180
          tacgccgact ctgtcaaggg ccgtttcact ataagtcgcg acaattcgaa aaacacactg 240
          tacctacaac tgaacagctt aagagctgag gacactgccg tctattattg cgccagagat 300
          ggtttcggct acttcgacta ctggggtcaa ggaacactag tcaccgtctc ctcg 354
           <![CDATA[ <210> 110]]>
           <![CDATA[ <211> 369]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 110]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggatt caccttctcc gactacggta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggtgaa atctaccact ctggttctac ctactactct 180
          ccatctctga agtctcgtgt gactataagt cgcgacaatt cgaaaaacac actgtaccta 240
          caactgaaca gcttaagagc tgaggacact gccgtctatt attgcgccag agacgttgcc 300
          cctggttctt ctggttacta cgacggtttc gacttctggg gtcaaggaac actagtcacc 360
          gtctcctcg 369
           <![CDATA[ <210> 111]]>
           <![CDATA[ <211> 363]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 111]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggata ctctatctcc tctggttacc actgggactg gattcgtcag 120
          gccccgggta agggcctcga gtgggtgtct ggtatctctg gttacggtgg ttctacctac 180
          tacgccgact ctgtcaaggg ccgtttcact ataagtcgcg acaattcgaa aaacacactg 240
          tacctacaac tgaacagctt aagagctgag gacactgccg tctattattg cgccagacac 300
          agttattacg gttccggtaa tttcgactac tggggtcaag gaacactagt caccgtctcc 360
          tcg 363
           <![CDATA[ <210> 112]]>
           <![CDATA[ <211> 348]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> ]]>Artificial sequence
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 112]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggatt caccttctcc gactactgga ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggttgg atctccccat ctggcggtgg tactaagtac 180
          gcccagaagt tccagggtcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagaggggct 300
          tacgaatttg actactgggg tcaaggaaca ctagtcaccg tctcctcg 348
           <![CDATA[ <210> 113]]>
           <![CDATA[ <211> 348]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 113]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggata ctctatctcc tctggttacc actggagctg gattcgtcag 120
          gccccgggta agggcctcga gtggctggcc cggatcgact gggacgatga caagtactac 180
          tctacctctc tgaagtctcg tctgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagatcgtac 300
          gtgtacttcg actactgggg tcaaggaaca ctagtcaccg tctcctcg 348
           <![CDATA[ <210> 114]]>
           <![CDATA[ <211> 360]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 114]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggatt ctctctgtct accggcggtg tggctgtgag ctggattcgt 120
          caggccccgg gtaagggcct cgagtggatc ggtgaaatct accactctgg ttctacctac 180
          tactctccat ctctgaagtc tcgtgtgact ataagtcgcg acaattcgaa aaacacactg 240
          tacctacaac tgaacagctt aagagctgag gacactgccg tctattattg cgcccgtcgt 300
          atcgccaccg ctacttactt cgactactgg ggtcaaggaa cactagtcac cgtctcctcg 360
           <![CDATA[ <210> 115]]>
           <![CDATA[ <211> 363]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 115]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggatt ctctctgtct accggcggtg tggctgtggg ctggattcgt 120
          caggccccgg gtaagggcct cgagtgggtg tctgctatct ctggttacgg ttctactacc 180
          tactacgccg actctgtcaa gggccgtttc actataagtc gcgacaattc gaaaaacaca 240
          ctgtacctac aactgaacag cttaagagct gaggacactg ccgtctatta ttgcgccaga 300
          ttgccatact ccgcctacgc tttcgactac tggggtcaag gaacactagt caccgtctcc 360
          tcg 363
           <![CDATA[ <210> 116]]>
           <![CDATA[ <211> 342]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 116]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggatt caccttctcc ggctacgcta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggtatc atctccccat ctggcggtgg tactaagtac 180
          gcccagaagt tccagggtcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagacaccca 300
          ttcgcctact ggggtcaagg aacactagtc accgtctcct cg 342
           <![CDATA[ <210> 117]]>
           <![CDATA[ <211> 345]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 117]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggata caccttctcc ggctacggta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggtgaa atctaccact ctggttctac ctactactct 180
          ccatctctga agtctcgtgt gactataagt cgcgacaatt cgaaaaacac actgtaccta 240
          caactgaaca gcttaagagc tgaggacact gccgtctatt attgcgccag aagaattgac 300
          gccttcgaca tctggggtca aggaacacta gtcaccgtct cctcg 345
           <![CDATA[ <210> 118]]>
           <![CDATA[ <211> 345]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 118]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggata caccttctcc ggctacgcta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggtatc atctccccat ctggcggtgg tactaagtac 180
          gcccagaagt tccagggtcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagactctat 300
          gacgttgcct actggggtca aggaacacta gtcaccgtct cctcg 345
           <![CDATA[ <210> 119]]>
           <![CDATA[ <211> 351]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 119]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggatt caccttctcc gactacgcta ttcactgggt gcgtcaggcc 120
          ccgggtaagg gcctcgagtg gatcggtatc atctccccat ctggcggttc tactaagtac 180
          gcccagaagt tccagggtcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc cagactcggt 300
          tacgggtact tcgacgtctg gggtcaagga acactagtca ccgtctcctc g 351
           <![CDATA[ <210> 120]]>
           <![CDATA[ <211> 354]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 120]]>
          gaggttcagc tggtggagtc tggcggtggc ctggtgcagc caggggggctc actccgtttg 60
          tcctgtgcag cttccggata ctctatcacc tctggtcact actggagctg gattcgtcag 120
          gccccgggta agggcctcga gtggatcggt gacatctccc actctggttc tacctactac 180
          tctcaatctc tgaagtctcg tgtgactata agtcgcgaca attcgaaaaa cacactgtac 240
          ctacaactga acagcttaag agctgaggac actgccgtct attattgcgc gcgtggtagt 300
          aggaccggct acttcgacta ttggggtcaa ggaacactag tcaccgtctc ctcg 354
           <![CDATA[ <210> 121]]>
           <![CDATA[ <211> 342]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 121]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctga gtctgtggac ttcttcggta tctctttcct ggcctggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctctaa ccgtgccacc 180
          ggtatcccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgccagc actacacctc ttcgccacca 300
          gtgtacacct tcggacaggg taccaaggtg gagatcaaac ga 342
           <![CDATA[ <210> 122]]>
           <![CDATA[ <211> 321]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 122]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgct ctgcctcttc tagcgtgagc tacgtgtact ggtatcaaca gaaaccagga 120
          aaagctccga agcttctgat ctacgacgcc tcttctctgg aatctggtgt gccatctcgc 180
          ttctctggat ccggttccgg gacggatttc actctgacca tcagcagtct gcagccggaa 240
          gacttcgcaa cttattactg cgtgcagggt cttcagaccc cttggacctt cggacagggt 300
          accaaggtgg agatcaaacg a 321
           <![CDATA[ <210> 123]]>
           <![CDATA[ <211> 327]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 123]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gggtattggc tcttccctgg cttggtatca acagaaacca 120
          ggaaaagctc cgaagcttct gatctacgac gcctctaacc gtgccaccgg tatcccatct 180
          cgcttctctg gatccggttc cgggacggat ttcactctga ccatcagcag tctgcagccg 240
          gaagacttcg caacttatta ctgccagcag tacgaccaat ggccaccttg gaccttcgga 300
          cagggtacca aggtggagat caaacga 327
           <![CDATA[ <210> 124]]>
           <![CDATA[ <211> 336]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 124]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctga gtctgtggac ttcttcggta agtctttcct gcactggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctctaa cctggaaacc 180
          ggtgtgccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgccagc agtcctactc ctggcctccg 300
          accttcggac agggtaccaa ggtggagatc aaacga 336
           <![CDATA[ <210> 125]]>
           <![CDATA[ <211> 327]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 12]]>5
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtgagc agccgtttcc tggcctggta tcaacagaaa 120
          ccaggaaaag ctccgaagct tctgatctac gacgcctcta accgtgccac cggtatccca 180
          tctcgcttct ctggatccgg ttccgggacg gatttcactc tgaccatcag cagtctgcag 240
          ccggaagact tcgcaactta ttactgccag cagtcctacc ccacccctct taccttcgga 300
          cagggtacca aggtggagat caaacga 327
           <![CDATA[ <210> 126]]>
           <![CDATA[ <211> 327]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 126]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtgcgc ggccgtttcc tggcctggta tcaacagaaa 120
          ccaggaaaag ctccgaagct tctgatctac gacgcctcta accgtgccac cggtatccca 180
          tctcgcttct ctggatccgg ttccgggacg gatttcactc tgaccatcag cagtctgcag 240
          ccggaagact tcgcaactta ttactgccag cagtcctcct cctggcctcc gaccttcgga 300
          cagggtacca aggtggagat caaacga 327
           <![CDATA[ <210> 127]]>
           <![CDATA[ <211> 330]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 127]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gaccgtgttc tctcgttacc tggcttggta tcaacagaaa 120
          ccaggaaaag ctccgaagct tctgatctac gacgcctcta accgtgccac cggtatccca 180
          tctcgcttct ctggatccgg ttccgggacg gatttcactc tgaccatcag cagtctgcag 240
          ccggaagact tcgcaactta ttactgccag cagtcctact actggccacc ttggaccttc 300
          ggacagggta ccaaggtgga gatcaaacga 330
           <![CDATA[ <210> 128]]>
           <![CDATA[ <211> 324]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 128]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gggtgtgtct tcttacctgg cctggtatca acagaaacca 120
          ggaaaagctc cgaagcttct gatctacgcc gcctctacct tgcagtctgg tgtgccatct 180
          cgcttctctg gatccggttc cgggacggat ttcactctga ccatcagcag tctgcagccg 240
          gaagacttcg caacttacta ctgccagcac cactacggca ccccactgac cttcggtcag 300
          ggtaccaagg tggagatcaa acga 324
           <![CDATA[ <210> 129]]>
           <![CDATA[ <211> 342]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 129]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtggac ttctacggta tctctttcct ggactggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctctaa ccgtgccacc 180
          ggtatcccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgccagc agtacgtctc ttcgccacca 300
          gagtacacct tcggacaggg taccaaggtg gagatcaaac ga 342
           <![CDATA[ <210> 130]]>
           <![CDATA[ <211> 336]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 130]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtggac ttcgacggtt tctctttcct gcactggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctcttc tctggaatct 180
          ggtgtgccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgccagc agcgtgactc ctggccttac 300
          accttcggac agggtaccaa ggtggagatc aaacga 336
           <![CDATA[ <210> 131]]>
           <![CDATA[ <211> 336]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 131]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtggac ttccacggta agtctttcct gcactggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctcttc tctggaatct 180
          ggtgtgccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgcgagc aatccctgga agtcccattc 300
          accttcggac agggtaccaa ggtggagatc aaacga 336
           <![CDATA[ <210> 132]]>
           <![CDATA[ <211> 336]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 132]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctgtggac ttctacggta tctctttcct gcactggtat 120
          caacagaaac caggaaaagc tccgaagctt ctgatctacg acgcctcttc tctggaatct 180
          ggtgtgccat ctcgcttctc tggatccggt tccgggacgg atttcactct gaccatcagc 240
          agtctgcagc cggaagactt cgcaacttat tactgcgtgc aggctcttca gttgcctctt 300
          accttcggac agggtaccaa ggtggagatc aaacga 336
           <![CDATA[ <210> 133]]>
           <![CDATA[ <211> 324]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 133]]>
          gatatccagt tgacccagtc cccgagttcc ctgtccgcct ctgtgggcga tcgggtcacc 60
          atcacctgcc gtgcctctca gtctatctct tcttacctga actggtatca acagaaacca 120
          ggaaaagctc cgaagcttct gatctacgac gcctctaacc tggaaaccgg tgtgccatct 180
          cgcttctctg gatccggttc cgggacggat ttcactctga ccatcagcag tctgcagccg 240
          gaagacttcg caacttacta ctgccagcac cactacggca ccccactgac cttcggtcag 300
          ggtaccaagg tggagatcaa acga 324
           <![CDATA[ <210> 134]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213]]>> Artificial sequence]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Synthetic construct]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;221&gt; Variant ]]&gt;
           <br/> &lt;![CDATA[ &lt;222&gt;1]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; in at least 2 and at most 10]]&gt; <![CDATA[Exists in repeated sequences
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 3]]>
           <![CDATA[ <223> Exists in at least 3 and at most 10 repeat sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1, 3]]>
           <![CDATA[ <223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ]]>
          Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Exists in at least 1 and at most 10 repeating sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His or Pro
           <![CDATA[ <400> 134]]>
          Xaa Cys Xaa Cys Xaa
           1 5
           <![CDATA[ <210> 135]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1]]>
           <![CDATA[ <223> Exists in at least 2 and at most 10 repeat sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 3]]>
           <![CDATA[ <223> Exists in at least 3 and at most 10 repeat sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5]]>
           <![CDATA[ <223> Exists in at least 1 and at most 10 repeating sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1, 3, 5]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His or Pro
           <![CDATA[ <400> 135]]>
          Xaa Cys Xaa Cys Xaa
           1 5
           <![CDATA[ <210> 136]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> misc_feature ]]>
           <![CDATA[ <222> 1, 2, 3]]>
           <![CDATA[ <223> Exists in at least 2 and at most 10 repeat sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> misc_feature ]]>
           <![CDATA[ <222> 7, 8, 9]]>
           <![CDATA[ <223> Exists in at least 3 and at most 10 repeat sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> misc_feature ]]>
           <![CDATA[ <222> 13, 14, 15]]>
           <![CDATA[ <223> Exists in at least 1 and at most 10 repeating sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> misc_feature ]]>
           <![CDATA[ <222> 1, 2, 7, 8, 13]]>
           <![CDATA[ <223> n = A, T, C or G]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> misc_feature ]]>
           <![CDATA[ <222> 3, 9]]>
           <![CDATA[ <223> n = T or G]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> misc_feature ]]>
           <![CDATA[ <222> 6, 12]]>
           <![CDATA[ <223> n = T or C]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> misc_feature ]]>
           <![CDATA[ <222> 14]]>
           <![CDATA[ <223> n = A, T or C]]>
           <![CDATA[ <400> 136]]>
          nnntgnnnnt gnnnc 15
           <![CDATA[ <210> 137]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1, 2, 3,]]> 4, 5, 6, 15, 16
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His or Pro
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 8, 9, 10, 11, 12, 13]]>
           <![CDATA[ <223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ]]>
          Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
           <![CDATA[ <400> 137]]>
          Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
           1 5 10 15
           <![CDATA[ <210> 138]]>
           <![CDATA[ <211> 18]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1, 2, 3, 4, 5, 6, 17, 18]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, P]]>he, Val,
          His or Pro
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 8, 9, 10, 11, 12, 13, 14, 15]]>
           <![CDATA[ <223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ]]>
          Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
           <![CDATA[ <400> 138]]>
          Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
           1 5 10 15
          Xaa Xaa
           <![CDATA[ <210> 139]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His or Pro
           <![CDATA[ <400> 139]]>
          Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
           1 5 10 15
           <![CDATA[ <210> 140]]>
           <![CDATA[ <211> 18]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr]]>, Ser, Thr, Asn, Ile, Leu, Phe, Val,
          His or Pro
           <![CDATA[ <400> 140]]>
          Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
           1 5 10 15
          Xaa Xaa
           <![CDATA[ <210> 141]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 141]]>
          Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro Tyr Pro Cys Ser Ala
           1 5 10 15
           <![CDATA[ <210> 142]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 142]]>
          Ile Val His His Ser Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser
           1 5 10 15
           <![CDATA[ <210> 143]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 143]]>
          Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr Pro Tyr Cys Asn Ser
           1 5 10 15
           <![CDATA[ <210> 144]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 144]]>
          Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1 5 10 15
           <![CDATA[ <210> 145]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 145]]>
          Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr Pro Tyr Cys Gln Ser
           1 5 10 15
           <![CDATA[ <210> 146]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 146]]>
          Pro Gln Pro Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1 5 10 15
           <![CDATA[ <210> 147]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 147]]>
          Pro Asn Pro Ala Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1 5 10 15
           <![CDATA[ <210> 148]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 148]]>
          Glu Val Gly Ser Tyr
           1 5
           <![CDATA[ <210> 149]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 149]]>
          Ser Gly Arg Ser Ala
           1 5
           <![CDATA[ <210> 150]]>
           <![CDATA[ <21]]>1> 6]]&gt;
           <br/> &lt;![CDATA[ &lt;212&gt;PRT]]&gt;
           <br/> &lt;![CDATA[ &lt;213&gt; Artificial sequence]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Synthetic construct]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;400&gt;150]]&gt;
           <br/> <![CDATA[Pro Leu Gly Leu Ala Gly
           1 5
           <![CDATA[ <210> 151]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 151]]>
          Glu Asn Leu Tyr Phe Gln Gly
           1 5
           <![CDATA[ <210> 152]]>
           <![CDATA[ <211> 26]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 20, 21]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His or Pro
           <![CDATA[ <400> 152]]>
          Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
           1 5 10 15
          Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala
                      20 25
           <![CDATA[ <210> 153]]>
           <![CDATA[ <211> 26]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6, 7, 8, 9, 10, 11, 20, 21]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His or Pro
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 13, 14, 15, 16, 17, 18]]>
           <![CDATA[ <223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ]]>
          Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
           <![CDATA[ <400> 153]]>
          Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
           1 5 10 15
          Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala
                      20 25
           <![CDATA[ <210> 154]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His or Pro
           <![CDATA[ <400> 154]]>
          Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
           1 5 10 15
          Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala
                      20 25
           <![CDATA[ <210> 155]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6, 7, 8, 9, 10, 11, 22, 23]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His, or Pro
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 13, 14, 15, 16, 17, 18, 19, 20]]>
           <![CDATA[ <223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ]]>
          Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
           <![CDATA[ <400> 155]]>
          Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
           1 5 10 15
          Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala
                      20 25
           <![CDATA[ <210> 156]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 156]]>
          Gly Gly Gly Gly Ser
           1 5
           <![CDATA[ <210> 157]]>
           <![CDATA[ <211> 4]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 157]]>
          Ser Gly Gly Ser
           1              
           <![CDATA[ <210> 158]]>
           <![CDATA[ <211> 4]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 158]]>
          Gly Gly Ser Gly
           1              
           <![CDATA[ <210> 159]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 159]]>
          Gly Gly Ser Gly Gly
           1 5
           <![CDATA[ <210> 160]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 160]]>
          Gly Ser Gly Ser Gly
           1 5
           <![CDATA[ <210> 161]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 161]]>
          Gly Ser Gly Gly Gly
           1 5
           <![CDATA[ <210> 162]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 162]]>
          Gly Gly Gly Ser Gly
           1 5
           <![CDATA[ <210> 163]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 163]]>
          Gly Ser Ser Ser Gly
           1 5
           <![CDATA[ <210> 164]]>
           <![CDATA[ <211> 42]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 6]]>
           <![CDATA[ <223> Xaa = Ala, Asp, Ile, Asn, Pro or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 7]]>
           <![CDATA[ <223> Xaa = Ala, Phe, Asn, Ser or Val]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 8]]>
           <![CDATA[ <223> Xaa = Ala, His, Leu, Pro, Ser, Val or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 9]]>
           <![CDATA[ <223> Xaa = Ala, His, Ser or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 10]]>
           <![CDATA[ <223> Xaa = Ala, Asp, Pro, Ser, Val or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 11]]>
           <![CDATA[ <223> Xaa = Ala, Asp, Leu, Ser or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 13]]>
           <![CDATA[ <223> Xaa = Asp, Pro or Val]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 14]]>
           <![CDATA[ <223> Xaa = Ala, Asp, Hi]]>s, Pro, Ser or Thr
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 15]]>
           <![CDATA[ <223> Xaa = Ala, Asp, Phe, His, Pro or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 16]]>
           <![CDATA[ <223> Xaa = Leu, Pro or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 17]]>
           <![CDATA[ <223> Xaa = Phe, Pro or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 18]]>
           <![CDATA[ <223> Xaa = Ala, Pro, Ser or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 20]]>
           <![CDATA[ <223> Xaa = Ala, Asp, Asn, Ser, Thr or Tyr]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 21]]>
           <![CDATA[ <223> Xaa = Ala, Ser or Tyr]]>
           <![CDATA[ <400> 164]]>
          Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
           1 5 10 15
          Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20 25 30
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 165]]>
           <![CDATA[ <211> 42]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 165]]>
          Glu Val Gly Ser Tyr Asp Ala Leu His Tyr Ala Cys Pro Pro Asp Tyr
           1 5 10 15
          Tyr Ala Cys Tyr Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20 25 30
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 166]]>
           <![CDATA[ <211> 42]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 166]]>
          Glu Val Gly Ser Tyr Asn Ser Tyr His Ala Tyr Cys Pro His Pro Leu
           1 5 10 15
          Tyr Pro Cys Thr Ala Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20 25 30
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 167]]>
           <![CDATA[ <211> 42]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 167]]>
          Glu Val Gly Ser Tyr Ala Ser Ser Ala Val Leu Cys Val Thr Ala Tyr
           1 5 10 15
          Phe Ser Cys Asn Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20 25 30
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 168]]>
           <![CDATA[ <211> 41]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 168]]>
          Glu Val Gly Ser Tyr Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro
           1 5 10 15
          Tyr Pro Cys Ser Ala Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20 25 30
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 169]]>
           <![CDATA[ <211> 42]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 169]]>
          Glu Val Gly Ser Tyr Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro
           1 5 10 15
          Tyr Pro Cys Ser Ala Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20 25 30
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 170]]>
           <![CDATA[ <211> 41]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 1]]>70
          Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr
           1 5 10 15
          Pro Tyr Cys Asp Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20 25 30
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 171]]>
           <![CDATA[ <211> 42]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 171]]>
          Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr
           1 5 10 15
          Pro Tyr Cys Asp Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20 25 30
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 172]]>
           <![CDATA[ <211> 41]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 172]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1 5 10 15
          Pro Tyr Cys Asn Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20 25 30
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 173]]>
           <![CDATA[ <211> 42]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 173]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1 5 10 15
          Pro Tyr Cys Asn Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20 25 30
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 174]]>
           <![CDATA[ <211> 41]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 174]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1 5 10 15
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20 25 30
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 175]]>
           <![CDATA[ <211> 42]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 175]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1 5 10 15
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu
                      20 25 30
          Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 176]]>
           <![CDATA[ <211> 41]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 176]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1 5 10 15
          Pro Tyr Cys Gln Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20 25 30
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 177]]>
           <![CDATA[ <211> 41]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 177]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1 5 10 15
          Pro Tyr Cys Asn Ser Ala Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20 25 30
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 178]]>
           <![CDATA[ <211> 41]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 178]]>
          Glu Val Gly Ser Tyr Pro Gln Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1 5 10 15
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20 25 30
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 179]]>
           <![CDATA[ <211> 41]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 179]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ala Ser Asp Cys Val Pro Tyr Tyr
           1 5 10 15
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro
                      20 25 30
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 180]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 180]]>
          Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro Leu Gly Leu Ala Gly
           1 5 10 15
          Ser Gly Gly Ser
                      20
           <![CDATA[ <210> 181]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1]]>
           <![CDATA[ <223> Exists in at least 2 and at most 10 repeat sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1, 10, 11]]>
           <![CDATA[ <223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ]]>
          Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
           <![CDATA[ <400> 181]]>
          Xaa Cys Pro Asp His Pro Tyr Pro Cys Xaa Xaa
           1 5 10
           <![CDATA[ <210> 182]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1]]>
           <![CDATA[ <223> Exists in at least 2 and at most 10 repeat sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1, 10, 11]]>
           <![CDATA[ <223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ]]>
          Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
           <![CDATA[ <400> 182]]>
          Xaa Cys Asp Ala Phe Tyr Pro Tyr Cys Xaa Xaa
           1 5 10
           <![CDATA[ <210> 183]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1]]>
           <![CDATA[ <223> Exists in at least 2 and at most 10 repeat sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1, 10, 11]]>
           <![CDATA[ <223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ]]>
          Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
           <![CDATA[ <400> 183]]>
          Xaa Cys Asp Ser His Tyr Pro Tyr Cys Xaa Xaa
           1 5 10
           <![CDATA[ <210> 184]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1]]>
           <![CDATA[ <223> Exists in at least 2 and at most 10 repeat sequences]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 1, 10, 11]]>
           <![CDATA[ <223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ]]>
          Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
           <![CDATA[ <400> 184]]>
          Xaa Cys Val Pro Tyr Tyr Tyr Ala Cys Xaa Xaa
           1 5 10
           <![CDATA[ <210> 185]]>
           <![CDATA[ <400> 185]]>
          000
           <![CDATA[ <210> 186]]>
           <![CDATA[ <400> 186]]>
          000
           <![CDATA[ <210> 187]]>
           <![CDATA[ <400> 187]]>
          000
           <![CDATA[ <210> 188]]>
           <![CDATA[ <400> 188]]>
          000
           <![CDATA[ <210> 189]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 189]]>
          Glu Val Gly Ser Tyr Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro
           1 5 10 15
          Tyr Pro Cys Ser Ala
                      20
           <![CDATA[ <210> 190]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 190]]>
          Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr
           1 5 10 15
          Pro Tyr Cys Asp Ser
                      20
           <![CDATA[ <210> 191]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 191]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1 5 10 15
          Pro Tyr Cys Asn Ser
                      20
           <![CDATA[ <210> 192]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 192]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1 5 10 15
          Tyr Ala Cys Ala Tyr
                      20
           <![CDATA[ <210> 193]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 193]]>
          Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr
           1 5 10 15
          Pro Tyr Cys Gln Ser
                      20
           <![CDATA[ <210> 194]]>
           <![CDATA[ <400> 194]]>
          000
           <![CDATA[ <210> 195]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 195]]>
          Glu Val Gly Ser Tyr Pro Gln Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1 5 10 15
          Tyr Ala Cys Ala Tyr
                      20
           <![CDATA[ <210> 196]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 196]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ala Ser Asp Cys Val Pro Tyr Tyr
           1 5 10 15
          Tyr Ala Cys Ala Tyr
                      20
           <![CDATA[ <210> 197]]>
           <![CDATA[ <211> 41]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 197]]>
          Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr
           1 5 10 15
          Pro Tyr Cys Asp Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro
                      20 25 30
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 198]]>
           <![CDATA[ <211> 36]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 198]]>
          Glu Val Gly His Ser Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser
           1 5 10 15
          Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly
                      20 25 30
          Ser Gly Gly Ser
                  35
           <![CDATA[ <210> 199]]>
           <![CDATA[ <211> 32]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 199]]>
          Glu Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser Ser Gly Arg Ser
           1 5 10 15
          Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly Ser Gly Gly Ser
                      20 25 30
           <![CDATA[ <210> 200]]>
           <![CDATA[ <211> 41]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 200]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1 5 10 15
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro
                      20 25 30
          Leu Gly Leu Ala Gly Ser Gly Gly Ser
                  35 40
           <![CDATA[ <210> 201]]>
           <![CDATA[ <211> 36]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 201]]>
          Glu Val Gly Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1 5 10 15
          Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly
                      20 25 30
          Ser Gly Gly Ser
                  35
           <![CDATA[ <210> 202]]>
           <![CDATA[ <211> 32]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 202]]>
          Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Ser Gly Arg Ser
           1 5 10 15
          Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly Ser Gly Gly Ser
                      20 25 30
           <![CDATA[ <210> 203]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 203]]>
          Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
           1 5 10 15
          Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
                      20 25 30
          Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
                  35 40 45
          Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
              50 55 60
          Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
          65 70 75 80
          Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
                          85 90 95
          Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Ala Gln Ile
                      100 105 110
          Tyr Val Ile Asp Pro Glu
                  115
           <![CDATA[ <210> 204]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 204]]>
          Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
           1 5 10 15
          Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
                      20 25 30
          Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
                  35 40 45
          Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
              50 55 60
          Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
          65 70 75 80
          Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
                          85 90 95
          Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
                      100 105 110
          Tyr Val Ile Asp Pro Glu
                  115
           <![CDATA[ <210> 205]]>
           <![CDATA[ <211> 115]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 205]]>
          Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg
           1 5 10 15
          Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
                      20 25 30
          Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
                  35 40 45
          Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Ile Cys
              50 55 60
          Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu
          65 70 75 80
          Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr
                          85 90 95
          Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val
                      100 105 110
          Ile Asp Pro
                  115
           <![CDATA[ <210> 206]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 206]]>
          Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
           1 5 10 15
          Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
                      20 25 30
          Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
                  35 40 45
          Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Asp Ser Ile
              50 55 60
          Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly
          65 70 75 80
          Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met
                          85 90 95
          Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr
                      100 105 110
          Val Ile
           <![CDATA[ <210> 207]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 207]]>
          Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg
           1 5 10 15
          Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
                      20 25 30
          Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
                  35 40 45
          Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp
              50 55 60
          Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr
          65 70 75 80
          Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val
                          85 90 95
          Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
                      100 105 110
          Gln Ile Tyr Val Ile Asp Pro Glu
                  115 120
           <![CDATA[ <210> 208]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Mus musculus]]>
           <![CDATA[ <400> 208]]>
          Glu Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser His
           1 5 10 15
          Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp
                      20 25 30
          Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asn Asp Gln Met Thr Glu
                  35 40 45
          Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp
              50 55 60
          Tyr Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr
          65 70 75 80
          Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu Cys Lys Val
                          85 90 95
          Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr
                      100 105 110
          Gln Ile Tyr Val Ile Asp Pro Glu
                  115 120
           <![CDATA[ <210> 209]]>
           <![CDATA[ <211> 31]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 209]]>
          Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr
           1 5 10 15
          Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Pro Leu Gly Leu Ala
                      20 25 30
           <![CDATA[ <210> 210]]>
           <![CDATA[ <211> 22]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 210]]>
          Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Ser Gly Arg Ser
           1 5 10 15
          Ala Pro Leu Gly Leu Ala
                      20
           <![CDATA[ <210> 211]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 211]]>
          Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Ser Gly Arg Ser
           1 5 10 15
          Ala
           <![CDATA[ <210> 212]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 212]]>
          Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Pro Leu Gly Leu
           1 5 10 15
          Ala
           <![CDATA[ <210> 213]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 213]]>
          Glu Asp Cys Val Pro Tyr Tyr Tyr Tyr Ala Cys Ala Tyr
           1 5 10
           <![CDATA[ <210> 214]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 214]]>
          Glu Val Gly Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr
           1 5 10 15
           <![CDATA[ <210> 215]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 215]]>
          Glu Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser
           1 5 10
           <![CDATA[ <210> 216]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 216]]>
          Glu Val Gly His Ser Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser
           1 5 10 15
           <![CDATA[ <210> 217]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 3]]>
           <![CDATA[ <223> Present in repeats of at least one and up to 11]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 5, 6, 7, 8, 9, 10, 11, 12]]>
           <![CDATA[ <223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, L]]>ys, Leu,
          Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 3, 14, 15]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His or Pro
           <![CDATA[ <400> 217]]>
          Glu Val Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser
           1 5 10 15
          Gly Arg Ser Ala
                      20
           <![CDATA[ <210> 218]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 4, 5, 6, 7, 8, 9, 11, 12]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His or Pro
           <![CDATA[ <400> 218]]>
          Glu Asp Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser
           1 5 10 15
          Ala
           <![CDATA[ <210> 219]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <221> Variant ]]>
           <![CDATA[ <222> 4, 5, 6, 7, 8, 9, 11, 12]]>
           <![CDATA[ <223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val,]]>
          His or Pro
           <![CDATA[ <400> 219]]>
          Glu Asp Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Pro Leu Gly Leu
           1 5 10 15
          Ala
           <![CDATA[ <210> 220]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 220]]>
          Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu Asn Leu Tyr Phe Gln
           1 5 10 15
          Gly Ser Gly Gly Ser
                      20
           <![CDATA[ <210> 221]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 221]]>
          Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly
           1 5 10 15
          Ser Gly Gly Ser
                      20
           <![CDATA[ <210> 222]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 222]]>
          Ser Gly Arg Ser Ala Pro Leu Gly Leu Ala
           1 5 10
           <![CDATA[ <210> 223]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 223]]>
          Phe Ser Leu Ser Thr Gly Gly Val Gly Val Gly Trp Ile
           1 5 10
           <![CDATA[ <210> 224]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 224]]>
          Leu Ala Leu Ile Asp Trp Ala Asp Asp Lys Tyr Tyr Ser Pro Ser Leu
           1 5 10 15
          Lys Ser Arg Leu
                      20
           <![CDATA[ <210> 225]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 225]]>
          Ala Arg Gly Gly Ser Asp Thr Val Ile Gly Asp Trp Phe Ala Tyr
           1 5 10 15
           <![CDATA[ <210> 226]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 226]]>
          Arg Ala Ser Gln Ser Ile Gly Ser Tyr Leu Ala
           1 5 10
           <![CDATA[ <210> 227]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 227]]>
          Asp Ala Ser Asn Leu Glu Thr Gly Val
           1 5
           <![CDATA[ <210> 228]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 228]]>
          Tyr Cys Gln Gln Gly Tyr Tyr Leu Trp Thr
           1 5 10
           <![CDATA[ <210> 229]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 229]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
           1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly
                      20 25 30
          Gly Val Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu
                  35 40 45
          Trp Leu Ala Leu Ile Asp Trp Ala Asp Asp Lys Tyr Tyr Ser Pro Ser
              50 55 60
          Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
          65 70 75 80
          Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
                          85 90 95
          Cys Ala Arg Gly Gly Ser Asp Thr Val Ile Gly Asp Trp Phe Ala Tyr
                      100 105 110
          Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 230]]>
           <![CDATA[ <211> 106]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequence]]>
           <![CDATA[ <220> ]]>
           <![CDATA[ <223> Synthetic construct]]>
           <![CDATA[ <400> 230]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
           1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Tyr Leu Trp Thr
                          85 90 95
          Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
                      100 105
          
      

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0001

Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0002

Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0003

Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0004

Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0005

Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0006

Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0007

Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0008

Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0009

Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0010

Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0011

Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0012

Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0013

Figure 12_A0101_SEQ_0014
Figure 12_A0101_SEQ_0014

Figure 12_A0101_SEQ_0015
Figure 12_A0101_SEQ_0015

Figure 12_A0101_SEQ_0016
Figure 12_A0101_SEQ_0016

Figure 12_A0101_SEQ_0017
Figure 12_A0101_SEQ_0017

Figure 12_A0101_SEQ_0018
Figure 12_A0101_SEQ_0018

Figure 12_A0101_SEQ_0019
Figure 12_A0101_SEQ_0019

Figure 12_A0101_SEQ_0020
Figure 12_A0101_SEQ_0020

Figure 12_A0101_SEQ_0021
Figure 12_A0101_SEQ_0021

Figure 12_A0101_SEQ_0022
Figure 12_A0101_SEQ_0022

Figure 12_A0101_SEQ_0023
Figure 12_A0101_SEQ_0023

Figure 12_A0101_SEQ_0024
Figure 12_A0101_SEQ_0024

Figure 12_A0101_SEQ_0025
Figure 12_A0101_SEQ_0025

Figure 12_A0101_SEQ_0026
Figure 12_A0101_SEQ_0026

Figure 12_A0101_SEQ_0027
Figure 12_A0101_SEQ_0027

Figure 12_A0101_SEQ_0028
Figure 12_A0101_SEQ_0028

Figure 12_A0101_SEQ_0029
Figure 12_A0101_SEQ_0029

Figure 12_A0101_SEQ_0030
Figure 12_A0101_SEQ_0030

Figure 12_A0101_SEQ_0031
Figure 12_A0101_SEQ_0031

Figure 12_A0101_SEQ_0032
Figure 12_A0101_SEQ_0032

Figure 12_A0101_SEQ_0033
Figure 12_A0101_SEQ_0033

Figure 12_A0101_SEQ_0034
Figure 12_A0101_SEQ_0034

Figure 12_A0101_SEQ_0035
Figure 12_A0101_SEQ_0035

Figure 12_A0101_SEQ_0036
Figure 12_A0101_SEQ_0036

Figure 12_A0101_SEQ_0037
Figure 12_A0101_SEQ_0037

Figure 12_A0101_SEQ_0038
Figure 12_A0101_SEQ_0038

Figure 12_A0101_SEQ_0039
Figure 12_A0101_SEQ_0039

Figure 12_A0101_SEQ_0040
Figure 12_A0101_SEQ_0040

Figure 12_A0101_SEQ_0041
Figure 12_A0101_SEQ_0041

Figure 12_A0101_SEQ_0042
Figure 12_A0101_SEQ_0042

Figure 12_A0101_SEQ_0043
Figure 12_A0101_SEQ_0043

Figure 12_A0101_SEQ_0044
Figure 12_A0101_SEQ_0044

Figure 12_A0101_SEQ_0045
Figure 12_A0101_SEQ_0045

Figure 12_A0101_SEQ_0046
Figure 12_A0101_SEQ_0046

Figure 12_A0101_SEQ_0047
Figure 12_A0101_SEQ_0047

Figure 12_A0101_SEQ_0048
Figure 12_A0101_SEQ_0048

Figure 12_A0101_SEQ_0049
Figure 12_A0101_SEQ_0049

Figure 12_A0101_SEQ_0050
Figure 12_A0101_SEQ_0050

Figure 12_A0101_SEQ_0051
Figure 12_A0101_SEQ_0051

Figure 12_A0101_SEQ_0052
Figure 12_A0101_SEQ_0052

Figure 12_A0101_SEQ_0053
Figure 12_A0101_SEQ_0053

Figure 12_A0101_SEQ_0054
Figure 12_A0101_SEQ_0054

Figure 12_A0101_SEQ_0055
Figure 12_A0101_SEQ_0055

Figure 12_A0101_SEQ_0056
Figure 12_A0101_SEQ_0056

Figure 12_A0101_SEQ_0057
Figure 12_A0101_SEQ_0057

Figure 12_A0101_SEQ_0058
Figure 12_A0101_SEQ_0058

Figure 12_A0101_SEQ_0059
Figure 12_A0101_SEQ_0059

Figure 12_A0101_SEQ_0060
Figure 12_A0101_SEQ_0060

Figure 12_A0101_SEQ_0061
Figure 12_A0101_SEQ_0061

Figure 12_A0101_SEQ_0062
Figure 12_A0101_SEQ_0062

Figure 12_A0101_SEQ_0063
Figure 12_A0101_SEQ_0063

Figure 12_A0101_SEQ_0064
Figure 12_A0101_SEQ_0064

Figure 12_A0101_SEQ_0065
Figure 12_A0101_SEQ_0065

Figure 12_A0101_SEQ_0066
Figure 12_A0101_SEQ_0066

Figure 12_A0101_SEQ_0067
Figure 12_A0101_SEQ_0067

Figure 12_A0101_SEQ_0068
Figure 12_A0101_SEQ_0068

Figure 12_A0101_SEQ_0069
Figure 12_A0101_SEQ_0069

Figure 12_A0101_SEQ_0070
Figure 12_A0101_SEQ_0070

Figure 12_A0101_SEQ_0071
Figure 12_A0101_SEQ_0071

Figure 12_A0101_SEQ_0072
Figure 12_A0101_SEQ_0072

Figure 12_A0101_SEQ_0073
Figure 12_A0101_SEQ_0073

Figure 12_A0101_SEQ_0074
Figure 12_A0101_SEQ_0074

Figure 12_A0101_SEQ_0075
Figure 12_A0101_SEQ_0075

Figure 12_A0101_SEQ_0076
Figure 12_A0101_SEQ_0076

Figure 12_A0101_SEQ_0077
Figure 12_A0101_SEQ_0077

Figure 12_A0101_SEQ_0078
Figure 12_A0101_SEQ_0078

Figure 12_A0101_SEQ_0079
Figure 12_A0101_SEQ_0079

Figure 12_A0101_SEQ_0080
Figure 12_A0101_SEQ_0080

Figure 12_A0101_SEQ_0081
Figure 12_A0101_SEQ_0081

Figure 12_A0101_SEQ_0082
Figure 12_A0101_SEQ_0082

Figure 12_A0101_SEQ_0083
Figure 12_A0101_SEQ_0083

Figure 12_A0101_SEQ_0084
Figure 12_A0101_SEQ_0084

Figure 12_A0101_SEQ_0085
Figure 12_A0101_SEQ_0085

Figure 12_A0101_SEQ_0086
Figure 12_A0101_SEQ_0086

Claims (24)

一種可活化抗體,其包含: 第一多肽,該第一多肽自N端至C端包含掩蔽部分(MM)、可裂解部分(CM)及靶結合部分(TBM), 其中該MM包含選自由SEQ ID NO: 189-193、195-196及213-216所組成之群之胺基酸序列; 其中該CM包含至少第一裂解位點; 其中: a)該TBM包含抗體輕鏈可變區(VL),且該可活化抗體另包含第二多肽,該第二多肽包含抗體重鏈可變區(VH); b)該TBM包含抗體重鏈可變區(VH),且該可活化抗體另包含第二多肽,該第二多肽包含抗體輕鏈可變區(VL); c)該TBM自N端至C端包含抗體輕鏈可變區(VL)及抗體重鏈可變區(VH);或 d)該TBM自N端至C端包含抗體重鏈可變區(VH)及抗體輕鏈可變區(VL); 其中該VH包含HVR-H1、HVR-H2及HVR-H3,該HVR-H1包含YSISSGYHWSWI (SEQ ID NO: 23)之胺基酸序列,該HVR-H2包含LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35)之胺基酸序列,且該HVR-H3包含ARSYVYFDY (SEQ ID NO: 45)之胺基酸序列; 其中該VL包含HVR-L1、HVR-L2及HVR-L3,該HVR-L1包含RASQSVRGRFLA (SEQ ID NO: 58)之胺基酸序列,該HVR-L2包含DASNRATGI (SEQ ID NO: 66)之胺基酸序列,且該HVR-L3包含YCQQSSSWPPT (SEQ ID NO: 75)之胺基酸序列;及 其中當該CM裂解時,該可活化抗體經由該VH及VL結合至人類CTLA4。 An activatable antibody comprising: a first polypeptide comprising a masking moiety (MM), a cleavable moiety (CM) and a target binding moiety (TBM) from the N-terminus to the C-terminus, Wherein the MM includes an amino acid sequence selected from the group consisting of SEQ ID NO: 189-193, 195-196 and 213-216; wherein the CM contains at least a first cleavage site; in: a) The TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide, the second polypeptide comprising an antibody heavy chain variable region (VH); b) the TBM comprises an antibody heavy chain variable region (VH), and the activatable antibody further comprises a second polypeptide, the second polypeptide comprising an antibody light chain variable region (VL); c) The TBM contains the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH) from the N-terminus to the C-terminus; or d) The TBM contains the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) from the N-terminus to the C-terminus; Wherein the VH includes HVR-H1, HVR-H2 and HVR-H3, the HVR-H1 includes the amino acid sequence of YSISSGYHWSWI (SEQ ID NO: 23), and the HVR-H2 includes the amine of LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35) The amino acid sequence, and the HVR-H3 includes the amino acid sequence of ARSYVYFDY (SEQ ID NO: 45); Wherein the VL includes HVR-L1, HVR-L2 and HVR-L3, the HVR-L1 includes the amino acid sequence of RASQSVRGRFLA (SEQ ID NO: 58), and the HVR-L2 includes the amine of DASNRATGI (SEQ ID NO: 66) The amino acid sequence of HVR-L3 includes the amino acid sequence of YCQQSSSWPPT (SEQ ID NO: 75); and Wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 via the VH and VL. 如請求項1之可活化抗體,其中該TBM包含抗體輕鏈可變區(VL),且該可活化抗體另包含第二多肽,該第二多肽包含抗體重鏈可變區(VH)。The activatable antibody of claim 1, wherein the TBM includes an antibody light chain variable region (VL), and the activatable antibody further includes a second polypeptide, and the second polypeptide includes an antibody heavy chain variable region (VH). . 如請求項1或2之可活化抗體,其中該第一裂解位點為選自由以下組成之群之蛋白酶之蛋白酶裂解位點:尿激酶型纖維蛋白溶酶原活化因子(uPA)、基質金屬蛋白酶-1 (MMP-1)、MMP-2、MMP-3、MMP-8、MMP-9、MMP-14、菸草蝕紋病毒(Tobacco Etch Virus)(TEV)蛋白酶、胞漿素(plasmin)、凝血酶(Thrombin)、X因子、PSA、PSMA、組織蛋白酶(Cathepsin) D、組織蛋白酶K、組織蛋白酶S、ADAM10、ADAM12、ADAMTS、卡斯蛋白酶(Caspase)-1、卡斯蛋白酶-2、卡斯蛋白酶-3、卡斯蛋白酶-4、卡斯蛋白酶-5、卡斯蛋白酶-6、卡斯蛋白酶-7、卡斯蛋白酶-8、卡斯蛋白酶-9、卡斯蛋白酶-10、卡斯蛋白酶-11、卡斯蛋白酶-12、卡斯蛋白酶-13、卡斯蛋白酶-14、及TACE。The activatable antibody of claim 1 or 2, wherein the first cleavage site is a protease cleavage site of a protease selected from the group consisting of: urokinase plasminogen activator (uPA), matrix metalloproteinase -1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, coagulation Enzyme (Thrombin), factor Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase- 11. Caspsin-12, Caspsin-13, Caspsin-14, and TACE. 如請求項1或2之可活化抗體,其中該CM另包含至該第一裂解位點C端的第一連接子(L 1)。 The activatable antibody of claim 1 or 2, wherein the CM further includes a first linker (L 1 ) to the C-terminus of the first cleavage site. 如請求項4之可活化抗體,其中該L 1包含選自由SEQ ID NO: 156至163組成之群之胺基酸序列。 The activatable antibody of claim 4, wherein L 1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 156 to 163. 如請求項1或2之可活化抗體,其中該CM另包含第二裂解位點。The activatable antibody of claim 1 or 2, wherein the CM further includes a second cleavage site. 如請求項6之可活化抗體,其中該CM另包含至該第一裂解位點C端的第一連接子(L 1),且該第二裂解位點為該L 1之C端。 The activatable antibody of claim 6, wherein the CM further includes a first linker (L 1 ) to the C-terminus of the first cleavage site, and the second cleavage site is the C-terminus of L 1 . 如請求項6之可活化抗體,其中該第二裂解位點為選自由以下組成之群之蛋白酶之蛋白酶裂解位點:尿激酶型纖維蛋白溶酶原活化因子(uPA)、基質金屬蛋白酶-1 (MMP-1)、MMP-2、MMP-3、MMP-8、MMP-9、MMP-14、菸草蝕紋病毒(TEV)蛋白酶、胞漿素、凝血酶、X因子、PSA、PSMA、組織蛋白酶D、組織蛋白酶K、組織蛋白酶S、ADAM10、ADAM12、ADAMTS、卡斯蛋白酶-1、卡斯蛋白酶-2、卡斯蛋白酶-3、卡斯蛋白酶-4、卡斯蛋白酶-5、卡斯蛋白酶-6、卡斯蛋白酶-7、卡斯蛋白酶-8、卡斯蛋白酶-9、卡斯蛋白酶-10、卡斯蛋白酶-11、卡斯蛋白酶-12、卡斯蛋白酶-13、卡斯蛋白酶-14、及TACE。The activatable antibody of claim 6, wherein the second cleavage site is a protease cleavage site selected from the group consisting of urokinase plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, cytoplasmin, thrombin, factor X, PSA, PSMA, tissue Protease D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-1 -6, Caspsin-7, Caspsin-8, Caspsin-9, Caspsin-10, Caspsin-11, Caspsin-12, Caspsin-13, Caspsin-14 , and TACE. 如請求項6之可活化抗體,其中該第一裂解位點及第二裂解位點係不同。The activatable antibody of claim 6, wherein the first cleavage site and the second cleavage site are different. 如請求項6之可活化抗體,其中該CM另包含至該第二裂解位點C端之第二連接子(L 2)。 The activatable antibody of claim 6, wherein the CM further includes a second linker (L 2 ) to the C-terminus of the second cleavage site. 如請求項10之可活化抗體,其中該L 2包含選自由SEQ ID NO: 156至163組成之群之胺基酸序列。 The activatable antibody of claim 10, wherein the L 2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 156 to 163. 如請求項1或2之可活化抗體,其中該CM另包含至該第一裂解位點N端之第三連接子(L 3)。 The activatable antibody of claim 1 or 2, wherein the CM further includes a third linker (L 3 ) to the N-terminus of the first cleavage site. 如請求項1或2之可活化抗體,其中該CM包含至少一個第一蛋白酶裂解位點,且用選自由以下組成之群之一或多種蛋白酶裂解:尿激酶型纖維蛋白溶酶原活化因子(uPA)、基質金屬蛋白酶-1 (MMP-1)、MMP-2、MMP-3、MMP-8、MMP-9、MMP-14、菸草蝕紋病毒(TEV)蛋白酶、胞漿素、凝血酶、X因子、PSA、PSMA、組織蛋白酶D、組織蛋白酶K、組織蛋白酶S、ADAM10、ADAM12、ADAMTS、卡斯蛋白酶-1、卡斯蛋白酶-2、卡斯蛋白酶-3、卡斯蛋白酶-4、卡斯蛋白酶-5、卡斯蛋白酶-6、卡斯蛋白酶-7、卡斯蛋白酶-8、卡斯蛋白酶-9、卡斯蛋白酶-10、卡斯蛋白酶-11、卡斯蛋白酶-12、卡斯蛋白酶-13、卡斯蛋白酶-14、及TACE。The activatable antibody of claim 1 or 2, wherein the CM comprises at least one first protease cleavage site and is cleaved with one or more proteases selected from the group consisting of: urokinase-type plasminogen activating factor ( uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, cytoplasmin, thrombin, Factor Caspsin-5, Caspsin-6, Caspsin-7, Caspsin-8, Caspsin-9, Caspsin-10, Caspsin-11, Caspsin-12, Caspsin-12 -13, Caspsin-14, and TACE. 如請求項1或2之可活化抗體,其中該可活化抗體包含選自由SEQ ID NO: 165至179及198至202組成之群之胺基酸序列。The activatable antibody of claim 1 or 2, wherein the activatable antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 165 to 179 and 198 to 202. 如請求項1之可活化抗體,其中該VH包含SEQ ID NO: 87之胺基酸序列,且該VL包含SEQ ID NO: 100之胺基酸序列。The activatable antibody of claim 1, wherein the VH includes the amino acid sequence of SEQ ID NO: 87, and the VL includes the amino acid sequence of SEQ ID NO: 100. 如請求項1、2及15中任一項之可活化抗體,其中該MM包含PNPSSDCVPYYYACAY (SEQ ID NO: 144)之胺基酸序列。The activatable antibody of any one of claims 1, 2 and 15, wherein the MM includes the amino acid sequence of PNPSSDCVPYYYACAY (SEQ ID NO: 144). 如請求項1、2及15中任一項之可活化抗體,其中該可活化抗體包含EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192)之胺基酸序列。The activatable antibody of any one of claims 1, 2 and 15, wherein the activatable antibody comprises the amino acid sequence of EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192). 一種可活化抗體,其包含: (a) 第一多肽,該第一多肽自N端至C端包含掩蔽部分(MM)、可裂解部分(CM)及抗體輕鏈可變區(VL);及 (b) 第二多肽,該第二多肽包含抗體重鏈可變區(VH); 其中該MM包含PNPSSDCVPYYYACAY (SEQ ID NO: 144)之胺基酸序列; 其中該CM包含至少第一裂解位點; 其中該VH包含SEQ ID NO: 87之胺基酸序列; 其中該VL包含SEQ ID NO: 100之胺基酸序列;及 其中當該CM裂解時,該可活化抗體經由該VH及VL結合至人類CTLA4。 An activatable antibody comprising: (a) a first polypeptide comprising a masking moiety (MM), a cleavable moiety (CM) and an antibody light chain variable region (VL) from the N-terminus to the C-terminus; and (b) a second polypeptide comprising an antibody heavy chain variable region (VH); Wherein the MM contains the amino acid sequence of PNPSSDCVPYYYACAY (SEQ ID NO: 144); wherein the CM contains at least a first cleavage site; Wherein the VH includes the amino acid sequence of SEQ ID NO: 87; wherein the VL includes the amino acid sequence of SEQ ID NO: 100; and Wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 via the VH and VL. 如請求項18之可活化抗體,其中該第一多肽包含EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200)之胺基酸序列。The activatable antibody of claim 18, wherein the first polypeptide comprises the amino acid sequence of EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200). 如請求項1、2、15、18及19中任一項之可活化抗體,其中該可活化抗體包含人類IgG1 Fc區,該人類IgG1 Fc區包含增加抗體依賴性細胞毒性(ADCC)活性之一或多個突變。The activatable antibody of any one of claims 1, 2, 15, 18 and 19, wherein the activatable antibody includes a human IgG1 Fc region, and the human IgG1 Fc region includes one of the activities of increasing antibody-dependent cellular cytotoxicity (ADCC) or multiple mutations. 一種醫藥組合物,其包含如請求項1至20中任一項之可活化抗體及醫藥上可接受之載劑。A pharmaceutical composition comprising the activatable antibody according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier. 一種如請求項1至20中任一項之可活化抗體或如請求項21之醫藥組合物之用途,其係用於製備於有需要之個體治療癌症或延遲癌症進展之藥劑。A use of the activatable antibody according to any one of claims 1 to 20 or the pharmaceutical composition according to claim 21, which is used to prepare a medicament for treating cancer or delaying the progression of cancer in an individual in need. 一種如請求項1至20中任一項之可活化抗體或如請求項21之醫藥組合物之用途,其係用於製備減少有需要個體中實體腫瘤大小之藥劑,其中該實體腫瘤具有約400至1000 mm 3之大小。 Use of an activatable antibody according to any one of claims 1 to 20 or a pharmaceutical composition according to claim 21 for the preparation of a medicament for reducing the size of a solid tumor in an individual in need thereof, wherein the solid tumor has about 400 to 1000 mm3 size. 如請求項23之用途,其中該藥劑進一步包含至少一種另外治療劑或與至少一種另外治療劑組合使用,其中該至少一種另外治療劑係選自由病毒基因療法、免疫檢查點抑制劑、靶療法、放射療法、疫苗接種療法及化療組成之群。The use of claim 23, wherein the medicament further comprises or is used in combination with at least one additional therapeutic agent, wherein the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, target therapy, A group consisting of radiation therapy, vaccination therapy and chemotherapy.
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