TWI790193B - Methods and antibodies for modulation of immunoresponse - Google Patents
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本發明係關於免疫療法之領域。特定言之，本發明係關於藉由調節細胞上之CD11b表現以調控免疫反應之方法及抗體。 The present invention is in the field of immunotherapy. In particular, the present invention relates to methods and antibodies for regulating immune responses by modulating CD11b expression on cells.
普遍咸信表現免疫原性抗原之癌細胞可誘發抗腫瘤形成之有效免疫反應。另外，腫瘤微環境富含可觸發TLR傳訊以活化抗腫瘤反應之組分(Standiford TJ,Keshamouni VG(2012)Breaking the tolerance for tumor：Targeting negative regulators of TLR signaling.Oncoimmunology 1：340-345)。意謂，在疾病之初始階段，癌細胞可有機會經免疫系統識別及排斥，該免疫系統對發展中之腫瘤發揮宿主保護及腫瘤建模作用兩者。然而，癌細胞亦具有許多負調節機制以逃避免疫監視，諸如MHC分子之下調或抗原處理及呈現機械；增加抑制細胞介素之分泌；及表現抑制分子以誘發對癌細胞之免疫耐受性。因此，通常認為癌症病患具有較差之免疫力。因此，仍需要開發用於逆轉與免疫抑制相關之癌症之藥劑或療法。 It is generally believed that cancer cells expressing immunogenic antigens can induce effective immune responses against tumor formation. In addition, the tumor microenvironment is rich in components that can trigger TLR signaling to activate anti-tumor responses ( Standiford TJ, Keshamouni VG (2012) Breaking the tolerance for tumor: Targeting negative regulators of TLR signaling. Oncoimmunology 1: 340-345 ). This means that during the initial stages of disease, cancer cells may have the opportunity to be recognized and rejected by the immune system, which exerts both host-protective and tumor-modeling roles against the developing tumor. However, cancer cells also possess many negative regulatory mechanisms to evade immune surveillance, such as downregulation of MHC molecules or antigen processing and presentation machinery; increased secretion of inhibitory cytokines; and expression of inhibitory molecules to induce immune tolerance to cancer cells. Therefore, cancer patients are generally considered to have poor immunity. Therefore, there remains a need to develop agents or therapies for reversing cancers associated with immunosuppression.
整合素αM(CD11b、CR3A及ITGAM)係形成表現於許多免疫細胞(包括單核細胞、顆粒細胞、巨噬細胞、樹突狀細胞、自然殺手細胞及骨髓衍生之抑制細胞)之表面上之異二聚整合素αMβ2分子之蛋白質次單元。整合素αMβ2藉由通過其雜亂配體庫調節細胞黏附、遷移、 趨化作用及吞噬作用介導炎症。近期研究已指示藉由調控TLR4反應用於炎症之關鍵作用(Han C、Jin J、Xu S、Liu H、LiN等人，(2010)Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b.Nat Immunol 11：734-742)。血管之腔側內之各種內源性整合素αMβ2配體(諸如血纖維蛋白原)可觸發TLR4傳訊。與β2整合素偶合之ITAM之高結合性連接瞬時誘發TLR活化，但通過靶向MyD88及TRIF以進行Cbl-b介導之蛋白水解降解以迅速抑制TLR傳訊。因此，整合素αMβ2可充當選擇性抑制TLR傳訊通道之組分以阻斷TLR家族之效應之負調節劑(Wang L、Gordon RA、Huynh L、Su X、Park Min KH等人，(2010)Indirect inhibition of Toll-like receptor and type I interferon responses by ITAM-coupled receptors and integrins.Immunity 32：518-530)。 Integrin αM (CD11b, CR3A, and ITGAM) forms abnormalities expressed on the surface of many immune cells, including monocytes, granulocytes, macrophages, dendritic cells, natural killer cells, and myeloid-derived suppressor cells. Protein subunit of the dimeric integrin αMβ2 molecule. Integrin αMβ2 mediates inflammation by regulating cell adhesion, migration, chemotaxis and phagocytosis through its promiscuous ligand repertoire. Recent studies have indicated a key role for inflammation by regulating TLR4 responses (Han C, Jin J, Xu S, Liu H, LiN et al., (2010) Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b. Nat Immunol 11:734-742 ). Various endogenous integrin αMβ2 ligands, such as fibrinogen, within the luminal side of blood vessels can trigger TLR4 signaling. High-binding attachment of β2 integrin-coupled ITAMs transiently induces TLR activation but rapidly inhibits TLR signaling by targeting MyD88 and TRIF for Cbl-b-mediated proteolytic degradation. Thus, integrin αMβ2 may act as a negative regulator that selectively inhibits components of TLR signaling pathways to block the effects of the TLR family ( Wang L, Gordon RA, Huynh L, Su X, Park Min KH et al., (2010) Indirect inhibition of Toll-like receptors and type I interferon responses by ITAM-coupled receptors and integrins. Immunity 32:518-530 ).
PD-L1係共抑制蛋白中之一者，其以變化之濃度表現於許多類型之免疫細胞上且組成性地表現於單核細胞、巨噬細胞及樹突狀細胞、T細胞、B細胞、上皮細胞及血管內皮細胞上。一經正誘發(諸如IFN-γ及有絲***刺激)，則PD-L1將經進一步上調。PD-L1結合至其受體PD-1(其發現於經活化之T細胞上)，藉由於經活化之T細胞中誘發共抑制訊息(其促進T細胞凋亡及無反應性)以產生強效免疫抑制(Butte MJ、Keir ME、Phamduy TB、Sharpe AH、Freeman GJ(2007)Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses.Immunity 27：111-122；Francisco LM、Salinas VH、Brown KE、Vanguri VK、Freeman GJ等人，(2009)PD-L1 regulates the development,maintenance,and function of induced regulatory T cells.J Exp Med 206：3015-3029)。PD-L1/PD-1相互作用之完整性對避免過度免疫反應亦係重要的。PD-L1
與PD-1之間之相互作用之缺陷可導致免疫反應之失控傳播，從而導致諸如以下之病症：自體免疫疾病、超敏反應、移植排斥及移植物抗宿主疾病。
PD-L1 is one of the co-repressor proteins expressed in varying concentrations on many types of immune cells and constitutively expressed on monocytes, macrophages and dendritic cells, T cells, B cells, epithelial cells and vascular endothelial cells. Upon positive induction (such as IFN-γ and mitotic stimulation), PD-L1 will be further upregulated. PD-L1 binds to its receptor PD-1, which is found on activated T cells, to generate a strong Effective immunosuppression ( Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ (2007) Programmed death-1
US 8,008,449提供經分離之特異性結合至PD-1之單株抗體(特定言之人類單株抗體)。US 8,354,509係關於阻斷人類程式化死亡受體1(hPD-1)與其配體(hPD-L1或hPD-L2)之結合之抗體。US 8,900,587揭示阻斷hPD-1與hPD-L1或hPD-L2之結合之抗體及通過PD-1路徑增加(或減少下調)免疫細胞之活性之方法。US 9,067,999及US 9,073,994提供經由利用由PD-1、PD-L1或PD-L2誘發之免疫抑制訊息之抑制所引起之免疫增強作用以用於癌症或感染治療之組合物及使用其等之療法。然而，上文專利中提及之抗體對療法具有低反應率。US 20140099254A1提供誘發針對癌症或感染性疾病之免疫反應之方法，其包括向患有癌症或感染性疾病之個體投與選自由以下組成之群之兩種或更多種藥劑之組合：(i)白細胞重定向雙特異性抗體，其包括ADAM17、CD2、CD3、CD4、CD5、CD6、CD8、CD11a、CD11b、CD14、CD16、CD16b、CD25、CD28、CD30、CD32a、CD40、CD40L、CD44、CD45、CD56、CD57、CD64、CD69、CD74、CD89、CD90、CD137、CD177、CEACAM6、CEACAM8、HLA-DR α鏈、KIR及SLC44A2；(ii)干擾素；(iii)查核點抑制劑抗體，其包括CTLA4、PD1、PD-L1、LAG3、B7-H3、B7-H4.KIR及TIM3；及(iv)抗體-藥物結合物(ADC)。然而，此參考僅組合許多已知免疫相關成分，然而其對該等成分間之相互影響毫無提示。 US 8,008,449 provides isolated monoclonal antibodies (specifically human monoclonal antibodies) that specifically bind to PD-1. US 8,354,509 relates to an antibody that blocks the binding of human programmed death receptor 1 (hPD-1) to its ligand (hPD-L1 or hPD-L2). US 8,900,587 discloses an antibody that blocks the binding of hPD-1 to hPD-L1 or hPD-L2 and a method for increasing (or reducing down-regulation) the activity of immune cells through the PD-1 pathway. US 9,067,999 and US 9,073,994 provide compositions for the treatment of cancer or infection by utilizing immunoenhancing effects caused by suppression of immunosuppressive signals induced by PD-1, PD-L1 or PD-L2 and therapies using the same. However, the antibodies mentioned in the above patents have a low response rate to the therapy. US 20140099254A1 provides a method of inducing an immune response against cancer or infectious disease comprising administering to an individual suffering from cancer or infectious disease a combination of two or more agents selected from the group consisting of: (i) Leukocyte redirecting bispecific antibodies comprising ADAM17, CD2, CD3, CD4, CD5, CD6, CD8, CD11a, CD11b, CD14, CD16, CD16b, CD25, CD28, CD30, CD32a, CD40, CD40L, CD44, CD45, CD56, CD57, CD64, CD69, CD74, CD89, CD90, CD137, CD177, CEACAM6, CEACAM8, HLA-DR alpha chain, KIR, and SLC44A2; (ii) interferons; (iii) checkpoint inhibitor antibodies, including CTLA4 , PD1, PD-L1, LAG3, B7-H3, B7-H4, KIR, and TIM3; and (iv) antibody-drug conjugates (ADCs). However, this reference only combines many known immune-related components, yet it says nothing about the interplay between these components.
本發明意外發現PD-L1之表現可藉由使CD11b調節劑結合至免疫細胞及/或其他細胞上之CD11b來抑制。CD11b調節劑結合至CD11b將減少LPS致敏單核細胞上之PD-L1表現。在LPS誘發之免疫抑制單核細 胞或來自患有敗血性休克之病患之單核細胞中，當細胞受LPS激發時，CD11b調節劑結合至CD11b亦減少PD-L1表現。 The present invention unexpectedly found that the expression of PD-L1 can be inhibited by binding CD11b modulators to CD11b on immune cells and/or other cells. Binding of CD11b modulators to CD11b reduces PD-L1 expression on LPS-sensitized monocytes. In LPS-induced immunosuppressive monocytes Binding of CD11b modulators to CD11b also reduced PD-L1 expression in cells or monocytes from patients with septic shock when the cells were challenged with LPS.
本發明提供用於抑制免疫細胞中PD-L1表現之方法，其包括使該免疫細胞與結合至該細胞上CD11b之CD11b調節劑接觸，藉此調節該等免疫細胞之PD-L1表現。 The present invention provides a method for inhibiting the expression of PD-L1 in immune cells, which comprises contacting the immune cells with a CD11b modulator that binds to CD11b on the cells, thereby modulating the expression of PD-L1 in the immune cells.
本發明提供用於免疫細胞中逆轉免疫抑制或免疫衰竭或誘發預存免疫力之方法，其包括使該免疫細胞與結合至該細胞上CD11b之CD11b調節劑接觸。 The present invention provides a method for reversing immunosuppression or immune exhaustion or inducing pre-existing immunity in an immune cell comprising contacting the immune cell with a CD11b modulator that binds to CD11b on the cell.
本發明提供用於判定對CD11b調節劑具有反應性之個體之方法，該方法包括偵測生物樣品或個體中之PD-L1是否被抑制，方式為藉由使該生物樣品或該個體中之免疫細胞與CD11b調節劑接觸並偵測該CD11b調節劑對免疫細胞上PD-L1之抑制，其中該PD-L1抑制指示該個體對CD11b調節劑具有反應性。 The present invention provides a method for determining an individual responsive to a CD11b modulator, the method comprising detecting whether PD-L1 in a biological sample or in an individual is inhibited by immunizing the biological sample or in the individual The cells are contacted with a CD11b modulator and inhibition of PD-L1 on immune cells by the CD11b modulator is detected, wherein the PD-L1 inhibition indicates that the individual is responsive to the CD11b modulator.
在一些實施例中，本文描述之CD11b調節劑係抑制CD11b表現之RNAi劑、抗CD11b抗體或調控CD11b之小分子化合物。 In some embodiments, a CD11b modulator described herein is an RNAi agent that inhibits CD11b expression, an anti-CD11b antibody, or a small molecule compound that modulates CD11b.
在一個實施例中，該免疫細胞係T細胞或單核細胞或顆粒細胞或巨噬細胞或骨髓衍生之抑制細胞或自然殺手細胞。在一個實施例中，該CD11b結合增加IFN-γ、IL-12或CD8 T細胞。在另一實施例中，CD11b調節劑結合至細胞上之CD11b治療及/或預防與免疫抑制相關之疾病。在另一實施例中，該與免疫抑制或免疫衰竭相關之疾病係急性及/或慢性感染中之免疫細胞T細胞衰竭、敗血症、癌症中之免疫缺陷或老化中之免疫衰老。 In one embodiment, the immune cells are T cells or monocytes or granulocytes or macrophages or myeloid derived suppressor cells or natural killer cells. In one embodiment, the CD11b binding increases IFN-γ, IL-12 or CD8 T cells. In another embodiment, the CD11b modulator binds to CD11b on cells to treat and/or prevent diseases associated with immunosuppression. In another embodiment, the disease associated with immunosuppression or immune failure is immune cell T cell exhaustion in acute and/or chronic infection, sepsis, immunodeficiency in cancer or immunosenescence in aging.
在一個實施例中，預防及/或治療癌症之方法包括投與額外之活性劑或療法。在一些實施例中，該額外之活性劑係免疫查核點療法、放射療法或化學療法。 In one embodiment, the method of preventing and/or treating cancer comprises administering additional active agents or therapies. In some embodiments, the additional active agent is immune checkpoint therapy, radiation therapy or chemotherapy.
本發明亦提供抗CD11b抗體或其抗原結合部分，其包含以下中之 至少一者：由NYWIN(SEQ ID NO：1)或GFSLTSNSIS(SEQ ID NO：2)之胺基酸殘基或具有與SEQ ID NO：1或2具有至少85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之重鏈互補決定區1(H-CDR1)；由NIYPSDTYINHNQKFKD(SEQ ID NO：3)或AIWSGGGTDYNSDLKS(SEQ ID NO：4)之胺基酸殘基或具有與SEQ ID NO：3或4具有至少85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之重鏈CDR2(H-CDR2)；及由SAYANYFDY(SEQ ID NO：5)或RGGYPYYFDY(SEQ ID NO：6)之胺基酸殘基或具有與SEQ ID NO：5或6具有至少85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之重鏈CDR3(H-CDR3)；且包含以下中之至少一者：由RASQNIGTSIH(SEQ ID NO：7)或KSSQSLLYSENQENYLA(SEQ ID NO：8)之胺基酸殘基或具有與SEQ ID NO：7或8具有85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之輕鏈CDR1(L-CDR1)；由YASESIS(SEQ ID NO：9)或WASTRQS(SEQ ID NO：10)之胺基酸殘基或具有與SEQ ID NO：9或10中之任一者具有85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之輕鏈CDR2(L-CDR2)；及由QQSDSWPTLT(SEQ ID NO：11)或QQYYDTPLT(SEQ ID NO：12)之胺基酸殘基或具有與SEQ ID NO：11或12中之任一者具有85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之輕鏈CDR3(L-CDR3)；使得該經分離之抗體或其抗原結合部分結合至CD11b。 The present invention also provides an anti-CD11b antibody or an antigen-binding portion thereof comprising the following At least one: amino acid residues composed of NYWIN (SEQ ID NO: 1) or GFSLTSNSIS (SEQ ID NO: 2) or having at least 85%, 90%, 91%, 92% of SEQ ID NO: 1 or 2 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequence variants composed of heavy chain complementarity determining region 1 (H-CDR1); by NIYPSDTYINHNQKFKD (SEQ ID NO: 3) or the amino acid residue of AIWSGGGTDYNSDLKS (SEQ ID NO: 4) or having at least 85%, 90%, 91%, 92%, 93%, 94%, Heavy chain CDR2 (H-CDR2) consisting of variants of 95%, 96%, 97%, 98%, 99% identical amino acid sequences; and SAYANYFDY (SEQ ID NO: 5) or RGGYPYYFDY (SEQ ID NO: 6) amino acid residues or have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of SEQ ID NO: 5 or 6 Heavy chain CDR3 (H-CDR3) consisting of variants of amino acid sequences with 99% identity; and comprising at least one of the following: RASQNIGTSIH (SEQ ID NO: 7) or KSSQSLLYSENQENYLA (SEQ ID NO: 8) the amino acid residue or has 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 7 or 8 Light chain CDR1 (L-CDR1) consisting of variants of amino acid sequences with % identity; amino acid residues from YASESIS (SEQ ID NO: 9) or WASTRQS (SEQ ID NO: 10) or having the same amino acid residues as SEQ ID NO: 10 Amino acids with 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any of ID NO: 9 or 10 Light chain CDR2 (L-CDR2) that the variant composition of sequence; Any of variants consisting of amino acid sequences having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity Light chain CDR3 (L-CDR3); enables the isolated antibody, or antigen-binding portion thereof, to bind to CD11b.
在一些實施例中，本文描述之CDR包含一或更多種嵌入、取代及/或刪除。 In some embodiments, the CDRs described herein comprise one or more insertions, substitutions and/or deletions.
在另一實施例中，本發明提供抗CD11b抗體或其抗原結合部分，其包含(i)重鏈可變區，其包括包含SEQ ID NO：1之H-CDR1、包含SEQ ID NO：3之H-CDR2及包含SEQ ID NO：5之H-CDR3之重鏈可變區，及(ii)輕鏈可變區，其包括包含SEQ ID NO：7之L-CDR1、包含SEQ ID NO：9之L-CDR2及包含SEQ ID NO：11之L-CDR3；或(iii)重鏈可變區，其包括包含SEQ ID NO：2之H-CDR1、包含SEQ ID NO：4之H-CDR2及包含SEQ ID NO：6之H-CDR3之重鏈可變區，及(iv)輕鏈可變區，其包括包含SEQ ID NO：8之L-CDR1、包含SEQ ID NO：10之L-CDR2及包含SEQ ID NO：12之L-CDR3。在另一實施例中，H-CDR1具有由SEQ ID NO：1或2組成之胺基酸序列；H-CDR2具有由SEQ ID NO：3或4組成之胺基酸序列；H-CDR3具有由SEQ ID NO：5或6組成之胺基酸序列；L-CDR1具有由SEQ ID NO：7或8組成之胺基酸序列；L-CDR2具有由SEQ ID NO：9或10組成之胺基酸序列及L-CDR3具有由SEQ ID NO：11或12組成之胺基酸序列。 In another embodiment, the present invention provides an anti-CD11b antibody or an antigen-binding portion thereof comprising (i) a heavy chain variable region comprising H-CDR1 comprising SEQ ID NO:1, H-CDR1 comprising SEQ ID NO:3 H-CDR2 and heavy chain variable region comprising H-CDR3 of SEQ ID NO:5, and (ii) light chain variable region comprising L-CDR1 comprising SEQ ID NO:7, comprising SEQ ID NO:9 or (iii) a heavy chain variable region comprising H-CDR1 comprising SEQ ID NO: 2, H-CDR2 comprising SEQ ID NO: 4 and A heavy chain variable region comprising H-CDR3 of SEQ ID NO:6, and (iv) a light chain variable region comprising L-CDR1 comprising SEQ ID NO:8, L-CDR2 comprising SEQ ID NO:10 and L-CDR3 comprising SEQ ID NO:12. In another embodiment, H-CDR1 has an amino acid sequence consisting of SEQ ID NO: 1 or 2; H-CDR2 has an amino acid sequence consisting of SEQ ID NO: 3 or 4; H-CDR3 has an amino acid sequence consisting of The amino acid sequence consisting of SEQ ID NO: 5 or 6; L-CDR1 has the amino acid sequence consisting of SEQ ID NO: 7 or 8; L-CDR2 has the amino acid sequence consisting of SEQ ID NO: 9 or 10 Sequence and L-CDR3 has an amino acid sequence consisting of SEQ ID NO: 11 or 12.
另外，本發明提供人類化抗CD11b抗體或其抗原結合部分，其包含：(a)包含由SEQ ID NO：13組成之胺基酸序列之重鏈可變區，及(ii)包含由SEQ ID NO：23組成之胺基酸序列之輕鏈可變區；(c)包含由SEQ ID NO：14組成之胺基酸序列之重鏈可變區，及(ii)包含由SEQ ID NO：24組成之胺基酸序列之輕鏈可變區；(e)包含由SEQ ID NO：15組成之胺基酸序列之重鏈可變區，及(f)包含由SEQ ID NO：25組成之胺基酸序列之輕鏈可變區；(g)包含由SEQ ID NO：16組成之胺基酸序列之重鏈可變區，及(h)包含由SEQ ID NO：26組成之胺基酸序列之輕鏈可變區；(i)包含由SEQ ID NO：17組成之胺基酸序列之重鏈可變區，及(j)包含由SEQ ID NO：27組成之胺基酸序列之輕鏈可變區； (k)包含由SEQ ID NO：18組成之胺基酸序列之重鏈可變區，及(l)包含由SEQ ID NO：28組成之胺基酸序列之輕鏈可變區；(m)包含由SEQ ID NO：19組成之胺基酸序列之重鏈可變區，及(n)包含由SEQ ID NO：29組成之胺基酸序列之輕鏈可變區；(o)包含由SEQ ID NO：20組成之胺基酸序列之重鏈可變區，及(p)包含由SEQ ID NO：30組成之胺基酸序列之輕鏈可變區；(q)包含由SEQ ID NO：21組成之胺基酸序列之重鏈可變區，及(r)包含由SEQ ID NO：31組成之胺基酸序列之輕鏈可變區；或(s)包含由SEQ ID NO：22組成之胺基酸序列之重鏈可變區，及(t)包含由SEQ ID NO：32組成之胺基酸序列之輕鏈可變區。 In addition, the present invention provides a humanized anti-CD11b antibody or an antigen-binding portion thereof, comprising: (a) a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 13, and (ii) comprising an amino acid sequence consisting of SEQ ID NO: 13; The light chain variable region of the amino acid sequence consisting of NO: 23; (c) the heavy chain variable region comprising the amino acid sequence consisting of SEQ ID NO: 14, and (ii) comprising the amino acid sequence consisting of SEQ ID NO: 24 A light chain variable region consisting of an amino acid sequence; (e) a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 15, and (f) an amine comprising an amino acid sequence consisting of SEQ ID NO: 25 (g) a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 16, and (h) comprising an amino acid sequence consisting of SEQ ID NO: 26 (i) a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 17, and (j) comprising a light chain comprising an amino acid sequence consisting of SEQ ID NO: 27 variable region; (k) a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 18, and (l) a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 28; (m) A heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 19, and (n) a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 29; (o) comprising a sequence consisting of SEQ ID NO: ID NO: The heavy chain variable region of the amino acid sequence consisting of 20, and (p) the light chain variable region comprising the amino acid sequence consisting of SEQ ID NO: 30; (q) comprising the amino acid sequence consisting of SEQ ID NO: A heavy chain variable region comprising an amino acid sequence consisting of 21, and (r) a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 31; or (s) comprising an amino acid sequence consisting of SEQ ID NO: 22 and (t) a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO:32.
本發明亦提供包含抗CD11b抗體或其抗原結合部分之組合物。本發明亦提供包括向個體投與本發明之人類化抗CD11b抗體之方法。此等方法包括用於抑制免疫細胞中PD-L1表現；免疫細胞中逆轉免疫抑制或免疫衰竭或誘發預存免疫力；測定個體中之PD-L1；及治療或預防急性及/或慢性感染、敗血症、癌症中之免疫缺陷或老化中之免疫衰老之方法。本發明之抗CD11b抗體可用於上文提及之方法中。 The invention also provides compositions comprising anti-CD11b antibodies or antigen-binding portions thereof. The invention also provides methods comprising administering to a subject a humanized anti-CD11b antibody of the invention. Such methods include for inhibiting the expression of PD-L1 in immune cells; reversing immunosuppression or immune failure or inducing pre-existing immunity in immune cells; measuring PD-L1 in an individual; and treating or preventing acute and/or chronic infection, sepsis . A method for immunodeficiency in cancer or immunosenescence in aging. The anti-CD11b antibodies of the present invention can be used in the methods mentioned above.
圖1顯示CD11b與抗CD11b抗體之結合改變PD-L1之表面表現。人類單核細胞經LPS(100ng/ml)在同型對照IgG或抗CD11b抗體(ICRF44)之存在下刺激18hr。獲得該等細胞並使用流動式細胞測量術分析HLA-DR、PD-L1、CD80及CD86分子。表面分子表現呈現為MFI。值呈現為來自3組獨立實驗之平均值±SEM。 Figure 1 shows that binding of CD11b to anti-CD11b antibody alters the surface expression of PD-L1. Human monocytes were stimulated with LPS (100 ng/ml) for 18 hr in the presence of isotype control IgG or anti-CD11b antibody (ICRF44). The cells were obtained and analyzed for HLA-DR, PD-L1, CD80 and CD86 molecules using flow cytometry. Surface molecular representations are presented as MFI. Values are presented as mean ± SEM from 3 independent experiments.
圖2A及B分別顯示結合CD11b對細胞黏附血纖維蛋白原及減少PD-L1表現之效應。圖2A顯示ML-C19-A對K562/CD11b細胞黏附血纖維蛋白原之效應。25000個K562/CD11b細胞在10μM ML-C19-A或DMSO之存在下在37℃下黏附於塗覆血纖維蛋白原(20μg/ml)之孔之 底部，保持20min。結果藉由基於螢光素酶之CellTiter-Glo(Promega CO.)定量。各條柱表示來自代表性實驗之一式三份測定之平均值±SEM。圖2B顯示以CD11b拮抗劑結合CD11b會減少單核細胞上之PD-L1表現。人類單核細胞經LPS(100ng/ml)在DMSO對照或10μM之ML-C19-A之存在下刺激18hr。獲得該等細胞並使用流動式細胞測量術分析PD-L1分子。表面分子表現呈現為MFI。值呈現為來自10組獨立實驗之平均值±SEM。 Figures 2A and B show the effect of binding CD11b on cell adhesion to fibrinogen and reduction of PD-L1 expression, respectively. Figure 2A shows the effect of ML-C19-A on the adhesion of K562/CD11b cells to fibrinogen. 25,000 K562/CD11b cells adhered to wells coated with fibrinogen (20 μg/ml) in the presence of 10 μM ML-C19-A or DMSO at 37°C At the bottom, keep for 20min. Results were quantified by luciferase-based CellTiter-Glo (Promega CO.). Each bar represents the mean ± SEM of triplicate determinations from a representative experiment. Figure 2B shows that binding CD11b with a CD11b antagonist reduces PD-L1 expression on monocytes. Human monocytes were stimulated with LPS (100 ng/ml) for 18 hr in the presence of DMSO control or 10 μM of ML-C19-A. These cells were obtained and analyzed for PD-L1 molecules using flow cytometry. Surface molecular representations are presented as MFI. Values are presented as mean ± SEM from 10 independent experiments.
圖3顯示抗CD11b抗體單一療法對B16F10腫瘤之生長之效應。對C57BL/6小鼠在第0天皮下注射2 x 105個B16F10細胞。在第7天，對小鼠(n=5隻/組)腹腔內(ip)注射對照IgG(5mg/kg)或大鼠抗小鼠CD11b抗體。每三至四天重複注射。在第18天，處死小鼠。量測腫瘤體積且結果呈現為平均值±SEM。
Figure 3 shows the effect of anti-CD11b antibody monotherapy on the growth of B16F10 tumors. C57BL/6 mice were subcutaneously injected with 2 x 105 B16F10 cells on
圖4顯示抗CD11b抗體治療後之腫瘤浸潤性白細胞中之MDSC及CD8 T細胞群體。對C57BL/6小鼠在第0天皮下注射2 x 105個B16F10細胞。在第7天，對小鼠(n=5隻/組)腹腔內注射對照IgG(5mg/kg)或大鼠抗小鼠CD11b抗體。每三至四天重複注射。在第18天，處死小鼠。用膠原蛋白酶消化腫瘤及藉由流動式細胞測量術分析腫瘤浸潤性白細胞。
Figure 4 shows MDSC and CD8 T cell populations in tumor infiltrating leukocytes after anti-CD11b antibody treatment. C57BL/6 mice were subcutaneously injected with 2 x 105 B16F10 cells on
圖5顯示抗CD11b治療後之血液中之WBC及IAIE+/CD8 T細胞上之PD-L1表現。在第0天，經由尾靜脈向各小鼠內注射2x105個B16F10細胞。在第1天，對小鼠(n=3隻/組)腹腔內注射對照IgG(5mg/kg)或抗小鼠CD11b抗體(5mg/kg)。每三至四天重複注射。在第15天，處死小鼠。獲得WBC細胞並使用流動式細胞測量術分析PD-L1分子及IAIE+/CD8 T細胞。
Figure 5 shows the expression of PD-L1 on WBC and IAIE+/CD8 T cells in blood after anti-CD11b treatment. On
圖6顯示帶腫瘤之小鼠中之IFN-γ、IL-12及TNF-α之產生藉由使用抗CD11b抗體之治療逆轉。在第0天經由尾靜脈向各小鼠內注射2x105
個B16F10細胞。在第1天，對小鼠(n=3隻/組)腹腔內注射對照IgG(5mg/kg)或大鼠抗小鼠CD11b抗體(5mg/kg)。每三至四天重複注射。在第9天，處死小鼠。血漿細胞介素藉由BD CBA小鼠炎症套組定量。
Figure 6 shows that the production of IFN-γ, IL-12 and TNF-α in tumor-bearing mice was reversed by treatment with anti-CD11b antibody. On
圖7顯示抗CD11b抗體單一療法對LLC1腫瘤之生長之效應。對C57BL/6小鼠在第0天皮下注射1 x 106個LLC1細胞。在第7天，對小鼠(n=5隻/組)腹腔內注射對照IgG(5mg/kg)或大鼠抗小鼠CD11b抗體。每三至四天重複注射。量測腫瘤體積且結果呈現為平均值±SEM。
Figure 7 shows the effect of anti-CD11b antibody monotherapy on the growth of LLC1 tumors. C57BL/6 mice were subcutaneously injected with 1 x 106 LLC1 cells on
圖8顯示抗CD11b抗體單一療法在LLC1腫瘤模型中對存活率之效應。對C57BL/6小鼠在第0天皮下注射1 x 106個LLC1細胞。在第7天，對小鼠(n=5隻/組)腹腔內注射對照IgG(5mg/kg)或大鼠抗小鼠CD11b抗體。每三至四天重複注射。針對抗CD11b抗體對各組中經治療之小鼠之長期存活率之效應來分析小鼠。
Figure 8 shows the effect of anti-CD11b antibody monotherapy on survival in the LLC1 tumor model. C57BL/6 mice were subcutaneously injected with 1 x 106 LLC1 cells on
圖9顯示抗CD11b抗體及抗PD1組合療法對LLC1肺轉移模型之效應。在第0天經由尾靜脈向各小鼠內注射1x106個LLC1細胞。在第1天，對小鼠(n=3隻/組)腹腔內注射對照IgG(10mg/kg)、抗小鼠CD11b抗體(10mg/kg)、抗PD1抗體(10mg/kg)或抗CD11b(10mg/kg)+抗PD1(10mg/kg)。每三至四天重複注射。在第15天，處死小鼠且接種之腫瘤數量計數為在顯微鏡下存在於肺中之結節之總數量。
Figure 9 shows the effect of anti-CD11b antibody and anti-PD1 combination therapy on LLC1 lung metastasis model. On
圖10顯示在肺轉移模型中抗CD11b抗體及抗PD1組合療法對存活率之影響。在第0天經由尾靜脈向各小鼠內注射1x106個LLC1細胞。在第1天，對小鼠(n=4-5隻/組)腹腔內注射對照IgG(10mg/kg)、抗小鼠CD11b抗體(10mg/kg)、抗PD1抗體(10mg/kg)或抗CD11b(10mg/kg)+抗PD1(10mg/kg)。每三至四天重複注射。針對組合療法對各組中經治療之小鼠之長期存活率之效應來分析小鼠。
Figure 10 shows the effect of anti-CD11b antibody and anti-PD1 combination therapy on survival in a lung metastasis model. On
圖11顯示抗CD11b抗體及紫杉醇組合療法對B16F10腫瘤之生長之效應。對C57BL/6小鼠在第0天皮下注射2 x105個B16F10細胞。在第7
天，對小鼠(n=5隻/組)腹腔內注射對照IgG(5mg/kg)、抗小鼠CD11b抗體(5mg/kg)、紫杉醇(10mg/kg)+對照IgG(5mg/kg)或紫杉醇(10mg/kg)+抗CD11b抗體(5mg/kg)。每三至四天重複注射。量測腫瘤體積且結果呈現為平均值±SEM。
Figure 11 shows the effect of anti-CD11b antibody and paclitaxel combination therapy on the growth of B16F10 tumors. C57BL/6 mice were subcutaneously injected with 2 x 105 B16F10 cells on
圖12顯示在B16F10模型中抗CD11b抗體及紫杉醇組合療法對存活率之效應。對C57BL/6小鼠在第0天皮下注射2 x105個B16F10細胞。在第7天，對小鼠(n=5隻/組)腹腔內注射對照IgG(5mg/kg)、抗小鼠CD11b抗體(5mg/kg)、紫杉醇(10mg/kg)+對照IgG(5mg/kg)或紫杉醇(10mg/kg)+抗CD11b(5mg/kg)。每三至四天重複注射。針對組合療法對各組中經治療之小鼠之長期存活率之效應來分析小鼠。
Figure 12 shows the effect of anti-CD11b antibody and paclitaxel combination therapy on survival in the B16F10 model. C57BL/6 mice were subcutaneously injected with 2 x 105 B16F10 cells on
圖13顯示以抗CD11b抗體結合CD11b會減少經1μg/ml LPS激發之LPS誘發之免疫抑制單核細胞中之PD-L1表現。(A)人類單核細胞係分離自健康志願者且經100ng/ml LPS預處理2天以誘發免疫抑制。(B)LPS誘發之免疫抑制單核細胞在10μg/ml IgG1或抗CD11b抗體(ICRF44)之存在下經1μg/ml LPS激發18hr。清洗經處理之細胞並藉由流動式細胞測量術分析。表面PD-L1表現呈現為MFI。 Figure 13 shows that binding CD11b with an anti-CD11b antibody reduces PD-L1 expression in LPS-induced immunosuppressive monocytes challenged with 1 μg/ml LPS. (A) Human monocytic cell lines were isolated from healthy volunteers and pretreated with 100 ng/ml LPS for 2 days to induce immunosuppression. (B) LPS-induced immunosuppression Monocytes were challenged with 1 μg/ml LPS for 18 hr in the presence of 10 μg/ml IgG1 or anti-CD11b antibody (ICRF44). Treated cells were washed and analyzed by flow cytometry. Surface PD-L1 manifestations presented as MFI.
圖14顯示當經1μg/ml LPS激發時，以抗CD11b抗體結合CD11b減少來自患有敗血性休克之病患之人類單核細胞中之PD-L1表現。人類單核細胞係分離自患有敗血性休克之病患且在10μg/ml IgG1或抗CD11b抗體之存在下經1μg/ml LPS激發18hr。清洗經處理之細胞並藉由流動式細胞測量術分析。表面PD-L1表現呈現為MFI。 Figure 14 shows that binding of CD11b with an anti-CD11b antibody reduces PD-L1 expression in human monocytes from patients with septic shock when challenged with 1 μg/ml LPS. Human monocytic cell lines were isolated from patients with septic shock and challenged with 1 μg/ml LPS for 18 hr in the presence of 10 μg/ml IgG1 or anti-CD11b antibody. Treated cells were washed and analyzed by flow cytometry. Surface PD-L1 manifestations presented as MFI.
圖15顯示人類化CD11b抗體之輕鏈可變區之胺基酸序列。CDR以加下劃線之字母顯示。 Figure 15 shows the amino acid sequence of the light chain variable region of the humanized CD11b antibody. CDRs are shown in underlined letters.
圖16顯示人類化CD11b抗體之重鏈可變區之胺基酸序列。CDR以加下劃線之字母顯示。 Figure 16 shows the amino acid sequence of the heavy chain variable region of the humanized CD11b antibody. CDRs are shown in underlined letters.
圖17顯示人類化抗CD11b抗體之結合活性。將K562細胞或經人 類CD11b轉染之細胞(K562/CD11b)用10μg/ml人類化抗CD11b抗體培養30min。經結合之Ab藉由結合FITC之小鼠抗人類IgG偵測。該等細胞藉由流動式細胞測量術分析。虛線表示結合K562細胞之抗體。實線表示結合至K562/CD11b細胞之抗體。 Figure 17 shows the binding activity of humanized anti-CD11b antibodies. K562 cells or human CD11b-like transfected cells (K562/CD11b) were incubated with 10 μg/ml humanized anti-CD11b antibody for 30 min. Bound Ab was detected by FITC-conjugated mouse anti-human IgG. The cells were analyzed by flow cytometry. Dashed lines indicate antibodies binding to K562 cells. The solid line indicates antibody binding to K562/CD11b cells.
圖18顯示以抗CD11b抗體結合CD11b減少LPS致敏人類單核細胞中之PD-L1表現。致敏單核細胞在同型對照IgG、抗CD11b抗體(ICRF44)或人類化抗CD11b抗體之存在下培養18hr。收穫該等細胞並使用流動式細胞測量術分析單核細胞上之PD-L1表現。 Figure 18 shows that binding CD11b with anti-CD11b antibody reduces PD-L1 expression in LPS-sensitized human monocytes. Sensitized monocytes were cultured for 18 hr in the presence of isotype control IgG, anti-CD11b antibody (ICRF44) or humanized anti-CD11b antibody. The cells were harvested and analyzed for PD-L1 expression on monocytes using flow cytometry.
在描述本發明之組合物、方法及分離方法論前，應瞭解此發明不受其等之限制，因為此等組合物、方法及條件可變化。亦應瞭解本文使用之術語係僅出於描述特定之實施例之目的，且無意具有限制性。 Before the compositions, methods and isolation methodology of the present invention are described, it is to be understood that this invention is not limited thereto since such compositions, methods and conditions may 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.
本發明意外發現PD-L1之表現可藉由使調節劑結合至免疫細胞及/或其他細胞上之CD11b來抑制，藉此治療及/或預防與免疫抑制相關之疾病，諸如慢性感染、敗血症、癌症中之免疫缺陷及老化中之免疫衰老。 The present invention unexpectedly found that the expression of PD-L1 can be inhibited by binding the regulator to CD11b on immune cells and/or other cells, thereby treating and/or preventing diseases related to immunosuppression, such as chronic infection, sepsis, Immunodeficiency in cancer and immunosenescence in aging.
定義 definition
除非另有定義，否則本文使用之所有技術及科學術語具有本發明所屬領域中的一般技術者通常所瞭解之相同含義。與彼等文本描述者類似或等效之任何方法及材料可用於本發明之實務或測試中，因為將瞭解修飾及變化包含於本發明之精神及範圍內。 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described in the text can be used in the practice or testing of the invention, with modifications and variations being understood to be included within the spirit and scope of the invention.
除非另有說明，否則「一」或「一個」意謂一或多個。 Unless stated otherwise, "a" or "an" means one or more.
如本文使用，如下縮寫胺基酸殘基：丙胺酸(Ala；A)、天冬醯胺酸(Asn；N)、天冬胺酸(Asp；D)、精胺酸(Arg；R)、半胱胺酸(Cys；C)、麩胺酸(Glu；E)、麩醯胺酸(Gln；Q)、甘胺酸(Gly；G)、組胺酸 (His；H)、異白胺酸(Ile；I)、白胺酸(Leu；L)、離胺酸(Lys；K)、甲硫胺酸(Met；M)、***酸(Phe；F)、脯胺酸(Pro；P)、絲胺酸(Ser；S)、蘇胺酸(Thr；T)、色胺酸(Trp；W)、酪胺酸(Tyr；Y)及纈胺酸(Val；V)。 As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartate (Asp; D), arginine (Arg; R), Cysteine (Cys; C), Glutamine (Glu; E), Glutamine (Gln; Q), Glycine (Gly; G), Histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F ), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).
如本文使用，術語「CD11b」係指整合素αM(ITGAM)，其係異二聚整合素αMβ2之次單元。整合素αMβ2之第二次單元係常見整合素β2次單元(稱為CD18)。整合素αMβ2亦稱為巨噬細胞-1抗原(Mac-1)或互補受體3(CR3)，其係表現於白細胞之表面上，該等白細胞包括單核細胞、顆粒細胞、巨噬細胞及自然殺手細胞。 As used herein, the term "CD11b" refers to integrin αM (ITGAM), which is a subunit of heterodimeric integrin αMβ2. The second subunit of integrin αMβ2 is the common integrin β2 subunit (called CD18). Integrin αMβ2, also known as macrophage-1 antigen (Mac-1) or complementarity receptor 3 (CR3), is expressed on the surface of leukocytes, including monocytes, granulocytes, macrophages and natural killer cells.
如本文使用，術語「PD-L1」係指程式化死亡配體1(PD-L1)，其係分化簇274(CD274)或B7同源物1(B7-H1)。PD-L1係40kDa 1型跨膜蛋白，其在特定事件(諸如懷孕、自體免疫疾病、癌症、敗血症及其他感染性疾病(諸如結核分枝桿菌(mycobacterium tuberculosis)、巨細胞病毒(cytomegalovirus)及肝炎))期間對抑制免疫系統起主要作用。
As used herein, the term "PD-L1" refers to programmed death-ligand 1 (PD-L1), which is cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). PD-L1 is a
如本文使用，術語「單核細胞」，亦稱為單核白細胞，其屬於涉及一線防禦機制之白血球之一種類型且公認可分化為樹突狀細胞或巨噬細胞前驅物。單核細胞通常在血液系統中移動。回應於外部刺激訊息時，單核細胞分泌許多免疫調節細胞介素，移動至組織中之感染部位並分化為巨噬細胞。 As used herein, the term "monocyte", also known as mononuclear leukocyte, is a type of white blood cell involved in first-line defense mechanisms and is recognized to differentiate into dendritic cells or macrophage precursors. Monocytes normally move through the blood system. In response to external stimuli, monocytes secrete many immunoregulatory cytokines, move to the site of infection in tissues and differentiate into macrophages.
如本文使用，術語「調控」包括相較於對照組通常處於統計學顯著量或生理學顯著量之「增加」或「刺激」及「降低」或「減少」。 As used herein, the term "modulate" includes both "increase" or "stimulation" and "decrease" or "decrease" compared to a control group, usually by a statistically significant amount or a physiologically significant amount.
如本文使用，術語「個體」意謂針對治療或療法所選擇之人類或非人類動物。 As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy.
如本文使用，「一致性」係指兩個或更多個多肽或蛋白質序列間之關係(如藉由比較該等序列判定)。在此項技術中，「一致性」亦係 指多肽或蛋白質間之序列相關程度(如藉由此等序列串之間之匹配判定)。「一致性」藉由已知的生物資訊方法可容易地計算。兩個聚核苷酸或兩個多肽序列之「一致性百分率」係藉由使用GAP電腦程式(GCG Wisconsin Package之一部分，10.3版(Accelrys,San Diego,Calif.))使用其預設參數比較該等序列而測定。 As used herein, "identity" refers to the relationship between two or more polypeptide or protein sequences (as determined by comparing the sequences). In this technology, "consistency" is also Refers to the degree of sequence relatedness between polypeptides or proteins (as determined by the match between such sequence strings). "Identity" can be easily calculated by known bioinformatics methods. The "percent identity" of two polynucleotide or two polypeptide sequences was compared by using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. determined by the sequence.
如本文使用，術語「肽」、「多肽」及「蛋白質」各係指包含藉由肽鍵彼此相連之兩個或更多個胺基酸殘基之分子。此等術語包含(例如)天然及人造蛋白質、蛋白質序列之蛋白質片段及多肽類似物(諸如突變體、變體及融合蛋白)及轉譯後(或以其他方式共價或非共價)修飾之蛋白質。肽、多肽或蛋白質可為單體或聚合的。 As used herein, the terms "peptide," "polypeptide," and "protein" each refer to a molecule comprising two or more amino acid residues linked to each other by peptide bonds. These terms include, for example, natural and man-made proteins, protein fragments of protein sequences, and polypeptide analogs such as mutants, variants, and fusion proteins, and proteins that are post-translationally (or otherwise covalently or non-covalently) modified . A peptide, polypeptide or protein may be monomeric or polymeric.
如本文使用，術語「親和力」係指在分子(例如，抗體)之單一結合位點與其結合配偶體(例如，抗原)之間之非共價相互作用之總強度。除非另有指示，否則如本文使用，「結合親和力」係指反映結合對(例如，抗體與抗原)之成員間之1：1相互作用之固有結合親和力。分子X對其配偶體Y之親和力可通常藉由解離常數(Kd)表示。親和力可藉由此項技術中已知的常用方法量測，該等方法包括彼等本文描述者。下文描述用於量測結合親和力之特定闡述性及例示性實施例。 As used herein, the term "affinity" refers to the total strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary examples for measuring binding affinity are described below.
如本文使用，術語「抗體」係以最廣義使用且特定涵蓋單株抗體(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)、單價抗體、多價抗體及抗體片段，只要其等顯示所需之生物活性即可(例如，Fab及/或單臂抗體)。 As used herein, the term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies and antibody fragments, so long as they exhibit the desired biological activity (eg, Fab and/or one-armed antibodies).
如本文使用，術語「抗體片段」係指除完整抗體外之分子，其包含完整抗體之一部分，其結合該完整抗體所結合之抗原。抗體片段之實例包括(但不限於)Fv、Fab、Fab'、Fab'-SH、F(ab')2；雙功能抗體；線性抗體；單鏈抗體分子(例如，scFv)；及自抗體片段形成之多特異性抗體。 As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single chain antibody molecules (e.g., scFv); Formation of multispecific antibodies.
如本文使用，術語抗體之「抗原結合片段」係指抗體之保留特異性結合至抗原之能力之一或多個部分。已顯示抗體之抗原結合功能可藉由全長抗體之片段進行。包含於術語抗體之「抗原結合片段」內之結合片段之實例包括(i)Fab片段，由VL、VH、CL及CH1域組成之單價片段；(ii)F(ab')2片段，包含藉由二硫鍵在鉸鏈區連接之兩個Fab片段之二價片段；(iii)由VH及CH1域組成之Fd片段；(iv)由抗體之單臂之VL及VH域組成之Fv片段；(v)由VH域組成之dAb片段；及(vi)經分離之互補決定區(CDR)。此等抗體片段係使用習知程序獲得，諸如蛋白水解片段化程序，如描述於J.Goding，Antibodies：Principles and Practice，第98至118頁(N.Y.Academic Press 1983)中。該等片段以與完整抗體相同之方式針對效用進行篩選。 As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of antibodies can be performed by fragments of full length antibodies. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH , CL and CH1 domains; ( ii) F(ab') 2 Fragments, comprising bivalent fragments of two Fab fragments connected at the hinge region by a disulfide bond; (iii) Fd fragment consisting of VH and CH1 domains; (iv) VL and V of a single arm of an antibody Fv fragments composed of H domains; (v) dAb fragments composed of V H domains; and (vi) isolated complementarity determining regions (CDRs). Such antibody fragments are obtained using known procedures, such as the proteolytic fragmentation procedure as described in J. Goding, Antibodies: Principles and Practice, pp. 98-118 (NY Academic Press 1983). These fragments are screened for utility in the same manner as whole antibodies.
如本文使用，術語「互補決定區」(CDR)係指抗體內之其中此等蛋白質互補抗原之形狀之區域。本文使用首字母縮略詞CDR意謂「互補決定區」。 As used herein, the term "complementarity determining region" (CDR) refers to the region within an antibody where these proteins complement the shape of the antigen. The acronym CDR is used herein to mean "complementarity determining region".
抗體之「可變區」係指抗體輕鏈之可變區或抗體重鏈之可變區(單獨或組合)。重鏈及輕鏈之可變區各由四個藉由三個CDR(亦稱為高度可變區)連接之框架區(FR)組成。各鏈中之CDR係與有助於形成抗體之抗原結合部位之來自其他鏈之CDR藉由FR靠近地固定在一起。可用以識別CDR之邊界之例示性公約包括(例如)Kabat定義及Chothia定義。該Kabat定義係基於序列變異性(參見Kabat等人，1992,Sequences of Proteins of Immunological Interest，第5版，Public Health Service,NIH,Washington D.C.)，該Chothia定義係基於結構環區之位置(Chothia等人，1989,Nature 342：877-883)。CDR識別之其他方法包括「IMGT定義」(Lefranc,M.-P.等人，1999,Nucleic Acids Res.27：209-212)及「AbM定義」，其係Kabat與Chothia間之折中且係使用Oxford Molecular's AbM抗體建模軟體衍生，或CDR之「接觸定 義」基於所觀察到之抗原接觸，闡述於MacCallum等人，1996,J.Mol.Biol.262：732-745中。如本文使用，CDR可係指藉由Kabat編號系統定義之CDR。 A "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three CDRs (also called hypervariable regions). The CDRs in each chain are closely held together by FRs with CDRs from other chains that contribute to the antigen binding site of the antibody. Exemplary conventions that can be used to identify the boundaries of a CDR include, for example, the Kabat definition and the Chothia definition. The Kabat definition is based on sequence variability (see Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, NIH, Washington D.C.), the Chothia definition is based on the location of structural loop regions (Chothia et al. Man, 1989, Nature 342:877-883). Other methods of CDR identification include the "IMGT definition" (Lefranc, M.-P. et al., 1999, Nucleic Acids Res. 27:209-212) and the "AbM definition", which is a compromise between Kabat and Chothia and is Derived using Oxford Molecular's AbM antibody modeling software, or the "contact definition" of CDR "Sense" is described in MacCallum et al., 1996, J. Mol. Biol. 262:732-745, based on observed antigen exposure. As used herein, a CDR may refer to a CDR as defined by the Kabat numbering system.
如本文使用，術語「人類化抗體」或「人類化抗體片段」係一種特定類型的嵌合抗體，其包括免疫球蛋白胺基酸序列變體或其片段，其可結合至預定抗原，且其包含一或多個大體上具有人類免疫球蛋白之胺基酸序列之框架(FR)及一或多個大體上具有非人類免疫球蛋白之胺基酸序列之互補決定區(CDR)。通常稱為「輸入」序列之此非人類胺基酸序列通常取自「輸入」抗體域，特別是可變域。一般而言，人類化抗體包括非人類抗體之至少該CDRs或高度可變區(HVLs)嵌入人類重鏈或輕鏈可變域之FRs間。 As used herein, the term "humanized antibody" or "humanized antibody fragment" refers to a specific type of chimeric antibody, which includes immunoglobulin amino acid sequence variants or fragments thereof, which bind to a predetermined antigen, and which Comprising one or more frameworks (FR) having substantially the amino acid sequence of a human immunoglobulin and one or more complementarity determining regions (CDR) having substantially the amino acid sequence of a non-human immunoglobulin. This non-human amino acid sequence, often referred to as the "import" sequence, is usually taken from an "import" antibody domain, particularly a variable domain. In general, humanized antibodies include at least the CDRs or hypervariable regions (HVLs) of a non-human antibody inserted between the FRs of a human heavy or light chain variable domain.
如本文使用，「人類抗體」係具有胺基酸序列對應於人類或人類細胞產生或利用人類抗體庫或其他人類抗體編碼序列自非人類來源衍生之抗體之胺基酸序列之抗體。人類抗體之此定義特定排除包含非人類抗原結合殘基之人類化抗體。 As used herein, a "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
如本文使用，術語「嵌合抗體」係指含有來自一種抗體之一或多個區域及來自一或多種其他抗體之一或多個區域之抗體。 As used herein, the term "chimeric antibody" refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.
如本文使用，術語「重鏈」包括全長重鏈及其具有足夠可變區序列以賦予對抗原決定基之特異性之片段。全長重鏈包括可變區域(VH)及三個恆定區域(CH1、CH2及CH3)。該VH域係位於該多肽之胺基端，及該CH3域係位於羧基端。 As used herein, the term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer specificity for an epitope. A full-length heavy chain includes a variable region ( VH ) and three constant regions ( CH1 , CH2 and CH3 ). The VH domain is at the amino-terminus of the polypeptide, and the CH3 domain is at the carboxy-terminus.
如本文使用，術語「輕鏈」包括全長輕鏈及其具有足夠可變區序列以賦予對抗原決定基之特異性之片段。全長輕鏈包括可變區域(VL)及恆定區域(CL)。類似於重鏈，輕鏈之可變區域係位於多肽之胺基端。 As used herein, the term "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequence to confer specificity for an epitope. A full-length light chain includes a variable region ( VL ) and a constant region ( CL ). Like the heavy chain, the variable region of the light chain is located at the amino terminus of the polypeptide.
如本文使用，術語「醫藥上可接受之載劑」係指醫藥調配物中 之除活性成分外之對個體無毒性的成分。醫藥上可接受之載劑包括(但不限於)緩衝劑、賦形劑、穩定劑或防腐劑。 As used herein, the term "pharmaceutically acceptable carrier" refers to the ingredients other than the active ingredient that are not toxic to the individual. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
如本文使用，術語「個體」係指脊椎動物，較佳係哺乳動物，更佳係人類。哺乳動物包括(但不限於)人類、農場動物、競技類動物及寵物。 As used herein, the term "individual" refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, humans, farm animals, sport animals, and pets.
如本文使用，術語「有效量」係指足以產生有利或所需之臨床結果之量。有效量可以一或更多次投與進行投與。出於此發明之目的，有效量係足以診斷、減輕、緩解、穩定、逆轉、減緩或延遲疾病狀態之發展之量。 As used herein, the term "effective amount" refers to an amount sufficient to produce a beneficial or desired clinical result. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount is an amount sufficient to diagnose, alleviate, alleviate, stabilize, reverse, slow or delay the progression of a disease state.
如本文使用，術語「治療(treatment、treating、treat及類似用語)」通常係指獲得所需之藥理及/或生理效應。該效應就完全或部分預防疾病或其症狀而言可為預防性的及/或就部分或完全穩定或治癒疾病及/或歸因於該疾病之不利影響而言係治療性的。如本文使用之「治療」涵蓋哺乳動物(特定言之，人類)之疾病之任何治療，且包括：(a)預防可能易患該疾病或症狀但未診斷為已患有其之個體中出現該疾病或病症；(b)抑制該疾病症狀，即，阻止其發展；或(c)緩解該疾病症狀，即，引起該疾病或症狀之消退。 As used herein, the terms "treatment, treating, treat, and similar terms" generally refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms and/or therapeutic in terms of partial or complete stabilization or cure of the disease and/or adverse effects attributable to the disease. "Treatment" as used herein encompasses any treatment of a disease in a mammal (specifically, a human) and includes: (a) prevention of the disease or condition in an individual who may be susceptible to it but has not been diagnosed as having it a disease or condition; (b) inhibiting the symptoms of the disease, ie, arresting its development; or (c) relieving the symptoms of the disease, ie, causing regression of the disease or symptoms.
如本文使用之術語「預防」係指阻止病患或個體中出現疾病狀態或病症之預防性(preventative)或預防性(prophylactic)措施。預防亦可包括減少病患或個體中出現疾病狀態或病症之可能性及阻礙或阻止該疾病狀態或病症之發作。 The term "prevention" as used herein refers to preventative or prophylactic measures to prevent the occurrence of a disease state or disorder in a patient or individual. Prevention can also include reducing the likelihood of a disease state or disorder occurring in a patient or individual and hindering or arresting the onset of the disease state or disorder.
在提供值範圍之情況下，應瞭解介於該範圍之上限值及下限值之間之各介入值(至下限值之單位之十分之一，除非內文明確規定)及該規定範圍中之任何其他規定值或介入值係包含於本發明內。此等較小範圍之上限值及下限值可獨立地包括於該等較小範圍中，且亦係包含於本發明內，受制於該規定範圍中之任何經特定排除之臨限值。在 規定範圍包括該等臨限值中之一者或兩者之情況下，排除彼等經包括之臨限值中之一者或者兩者外之範圍亦包括於本發明中。 Where a range of values is provided, each intervening value between the upper and lower values of the range (to the tenth of the unit of the lower limit, unless expressly stated in the context) and the provision Any other stated or intervening value within the range is encompassed within the invention. The upper and lower limits of such smaller ranges may independently be included in such smaller ranges and are also encompassed within the invention, subject to any specifically excluded threshold in that stated range. exist Where the stated range includes one or both of these cutoff values, ranges excluding either or both of those included cutoff values are also included in the invention.
影響PD-L1表現之CD-11b調節劑之結合Combination of CD-11b modulators affecting PD-L1 expression
本發明意外發現通過使用與表現於免疫細胞之表面上之CD11b分子反應之CD11b調節劑之治療逆轉與敗血症、慢性感染及癌症中涉及之免疫抑制狀態相關之症狀。 The present invention has unexpectedly found that symptoms associated with immunosuppressive states involved in sepsis, chronic infection and cancer are reversed by treatment with CD11b modulators that react with CD11b molecules expressed on the surface of immune cells.
在一個態樣中，本發明提供用於抑制免疫細胞中PD-L1表現之方法，其包括使該免疫細胞與結合該細胞上CD11b之CD11b調節劑接觸，藉此抑制該免疫細胞之PD-L1表現。或者，本發明提供CD11b調節劑在製造用於抑制免疫細胞中PD-L1表現之製劑中之用途。本發明亦提供用於抑制免疫細胞中PD-L1表現之CD11b調節劑。 In one aspect, the invention provides a method for inhibiting PD-L1 expression in an immune cell comprising contacting the immune cell with a CD11b modulator that binds CD11b on the cell, thereby inhibiting PD-L1 of the immune cell Performance. Alternatively, the present invention provides the use of a CD11b modulator in the manufacture of a preparation for inhibiting the expression of PD-L1 in immune cells. The present invention also provides CD11b modulators for inhibiting the expression of PD-L1 in immune cells.
在另一態樣中，本發明提供用於免疫細胞中逆轉免疫抑制或免疫衰竭或誘發預存免疫力之方法，其包括使該等免疫細胞與結合該等細胞上CD11b之CD11b調節劑接觸。或者，本發明提供CD11b調節劑在製造用於免疫細胞中逆轉免疫抑制或免疫衰竭或誘發預存免疫力之製劑中之用途。本發明亦提供用於免疫細胞中逆轉免疫抑制或免疫衰竭或誘發預存免疫力之CD11b調節劑。 In another aspect, the invention provides a method for reversing immunosuppression or immune exhaustion or inducing pre-existing immunity in immune cells comprising contacting the immune cells with a CD11b modulator that binds CD11b on the cells. Alternatively, the present invention provides the use of a modulator of CD11b in the manufacture of a formulation for reversing immunosuppression or immune failure or inducing pre-existing immunity in immune cells. The present invention also provides modulators of CD11b for reversing immunosuppression or immune exhaustion or inducing pre-existing immunity in immune cells.
在另一態樣中，本發明提供用於判定對CD11b調節劑具有反應性之個體之方法，該方法包括偵測生物樣品或個體中之PD-L1是否被抑制，方式為藉由使該生物樣品或該個體中之免疫細胞與CD11b調節劑接觸並偵測該CD11b調節劑對免疫細胞上PD-L1之抑制，其中該PD-L1抑制指示該個體對該CD11b調節劑具有反應性。 In another aspect, the invention provides a method for determining an individual responsive to a CD11b modulator comprising detecting whether PD-L1 is inhibited in a biological sample or individual by making the biological The sample or immune cells in the individual are contacted with the CD11b modulator and inhibition of PD-L1 on the immune cells by the CD11b modulator is detected, wherein the PD-L1 inhibition indicates that the individual is responsive to the CD11b modulator.
在一個實施例中，本文描述之CD11b調節劑係抑制CD11b表現之RNAi劑、抗CD11b抗體或調控CD11b之小分子化合物。 In one embodiment, the CD11b modulator described herein is an RNAi agent that inhibits the expression of CD11b, an anti-CD11b antibody, or a small molecule compound that modulates CD11b.
在一些實施例中，抑制CD11b表現之RNAi劑係抑制CD11b表現之微小RNA(miRNA)或短小干擾RNA(siRNA)。在一些實施例中，該抗 CD11b抗體係單株、嵌合、人類化、人類或雙特異性抗CD11b抗體。 In some embodiments, the RNAi agent that inhibits CD11b expression is a microRNA (miRNA) or short interfering RNA (siRNA) that inhibits CD11b expression. In some embodiments, the anti CD11b antibody monoclonal, chimeric, humanized, human or bispecific anti-CD11b antibody.
在一些實施例中，調控CD11b之小分子化合物之實例包括(但不限於)描述於US 8,268,816、US 20120035154、WO002007039616、WO002006111371、WO002007054128、WO00199901258、J Immunol 2010,184，第3917至26頁及Cancer Discov,2012,2，第1091至99頁中之化合物。較佳地，該化合物係選自由下列各物組成之群：
在一個實施例中，該免疫細胞係單核細胞、顆粒細胞、巨噬細胞、骨髓衍生之抑制細胞或自然殺手細胞或T細胞。 In one embodiment, the immune cells are monocytes, granulosa cells, macrophages, myeloid-derived suppressor cells or natural killer cells or T cells.
在一個實施例中，CD11b結合增加IFN-γ、IL-12或CD8 T細胞。在另一實施例中，CD11b調節劑結合至細胞上之CD11b治療及/或預防與免疫抑制相關之疾病。 In one embodiment, CD11b binding increases IFN-γ, IL-12 or CD8 T cells. In another embodiment, the CD11b modulator binds to CD11b on cells to treat and/or prevent diseases associated with immunosuppression.
在另一實施例中，與免疫抑制或免疫衰竭相關之疾病係急性及/或慢性感染中之T細胞衰竭、敗血症、癌症中之免疫缺陷或老化中之免疫衰老。因此，本發明提供用於治療或預防個體之急性及/或慢性感染、敗血症、癌症中之免疫缺陷或老化中之免疫衰老之方法，其包括向個體投與有效量之CD11b調節劑。 In another embodiment, the disease associated with immunosuppression or immune failure is T cell exhaustion in acute and/or chronic infection, sepsis, immunodeficiency in cancer or immunosenescence in aging. Accordingly, the present invention provides methods for treating or preventing acute and/or chronic infection, sepsis, immunodeficiency in cancer, or immunosenescence in aging in a subject comprising administering to the subject an effective amount of a CD11b modulator.
在一個實施例中，本文描述之癌症係對免疫療法具有反應性之癌症。對免疫療法具有反應性之癌症之實例包括(但不限於)黑色素瘤、肺癌、肺鱗狀細胞癌、頭頸癌、乳癌、卵巢癌、子宮癌、***癌、胃癌、子宮頸癌、食道癌、膀胱癌、腎癌、腦癌、肝癌、結腸癌、骨癌、胰臟癌、皮膚癌、皮膚或眼內惡性黑色素瘤、卵巢癌、直腸癌、肛門區癌、胃癌、睾丸癌、輸卵管癌、子宮內膜癌、子宮頸癌、***癌、陰門癌、霍奇金氏病(Hodgkin’s Disease)、非霍奇金氏淋巴瘤(non-Hodgkin’s lymphoma)、食道癌、小腸癌、內分泌系統癌、甲狀腺癌、副甲狀腺癌、腎上腺癌、軟組織肉瘤、尿道癌、陰莖 癌、慢性或急性白血病(包括急性骨髓性白血病、慢性骨髓性白血病、急性淋巴母細胞白血病、慢性淋巴球性白血病)、兒童之實體腫瘤、淋巴球性淋巴瘤、腎盂癌、中樞神經系統(CNS)之贅瘤、原發性CNS淋巴瘤、腫瘤血管生成、髓軸腫瘤、腦幹神經膠瘤、垂體腺瘤、卡波西氏肉瘤(Kaposi’s sarcoma)、表皮樣癌、鱗狀細胞癌及T細胞淋巴瘤。 In one embodiment, the cancer described herein is a cancer responsive to immunotherapy. Examples of cancers responsive to immunotherapy include, but are not limited to, melanoma, lung cancer, lung squamous cell carcinoma, head and neck cancer, breast cancer, ovarian cancer, uterine cancer, prostate cancer, gastric cancer, cervical cancer, esophageal cancer, Bladder cancer, kidney cancer, brain cancer, liver cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, skin or intraocular malignant melanoma, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, fallopian tube cancer, Endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, esophagus, small intestine, endocrine system, thyroid carcinoma, parathyroid carcinoma, adrenal gland carcinoma, soft tissue sarcoma, urethral carcinoma, penile carcinoma Cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), solid tumors in children, lymphocytic lymphoma, renal pelvis cancer, central nervous system (CNS ), primary CNS lymphoma, tumor angiogenesis, medullary axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma and T cell lymphoma.
在一個實施例中，該癌症係轉移癌、難治性癌症(refractory cancer)、復發性癌症(relapsed cancer)或晚期癌症(advanced cancer)。 In one embodiment, the cancer is metastatic cancer, refractory cancer, relapsed cancer or advanced cancer.
在一個實施例中，預防及/或治療癌症之方法包括投與額外之活性劑或療法。在一些實施例中，該額外之活性劑係免疫查核點療法、放射療法或化學療法。 In one embodiment, the method of preventing and/or treating cancer comprises administering additional active agents or therapies. In some embodiments, the additional active agent is immune checkpoint therapy, radiation therapy or chemotherapy.
在一個實施例中，該CD11b調節劑及該免疫查核點療法、放射療法或化學療法係同時、循序或分別投與。在另一實施例中，該免疫查核點療法包括投與免疫查核點蛋白。較佳地，該免疫查核點蛋白係抗PD-1配體或抗CTLA-4抗體或抗PD-L1抗體，或其抗原結合片段或其任何組合。抗PD-1配體之實例包括(但不限於)抗PD-1抗體(諸如納武單抗(nivolumab)及派姆單抗(pembrolizumab))及抗CTLA-4抗體(諸如易普利姆瑪單抗(ipilimumab))。 In one embodiment, the CD11b modulator and the immune checkpoint therapy, radiation therapy or chemotherapy are administered simultaneously, sequentially or separately. In another embodiment, the immune checkpoint therapy comprises administering an immune checkpoint protein. Preferably, the immune checkpoint protein is an anti-PD-1 ligand or an anti-CTLA-4 antibody or an anti-PD-L1 antibody, or an antigen-binding fragment thereof, or any combination thereof. Examples of anti-PD-1 ligands include, but are not limited to, anti-PD-1 antibodies (such as nivolumab and pembrolizumab) and anti-CTLA-4 antibodies (such as ipilimumab monoclonal antibody (ipilimumab)).
在另一實施例中，該化學療法包括投與化學治療劑。該化學治療劑之實例包括(但不限於)烷化劑、抗代謝物、抗微管劑、拓樸異構酶抑制劑或細胞毒性抗生素。較佳地，該化學治療劑係順鉑、5-Fu、紫杉醇、多西他賽(docetaxel)、長春瑞濱(vinorelbine)、長春地辛(vindesine)、長春氟寧(vinflunine)、吉西他濱(gemcitabine)、胺甲喋呤(methotrexate)、吉非替尼(gefitinib)、拉帕替尼(lapatinib)或埃羅替尼(erlotinib)。 In another embodiment, the chemotherapy comprises administering a chemotherapeutic agent. Examples of such chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, antimicrotubule agents, topoisomerase inhibitors, or cytotoxic antibiotics. Preferably, the chemotherapeutic agent is cisplatin, 5-Fu, paclitaxel, docetaxel, vinorelbine, vindesine, vinflunine, gemcitabine ), methotrexate, gefitinib, lapatinib, or erlotinib.
本文描述之CD11b調節劑及其他藥劑可調配成調配物或組合物。 本發明之調配物或醫藥組合物可以許多方法投與，其取決於需要局部治療抑或全身治療且取決於待治療之區域。投與可為經口或非經腸。 The CD11b modulators and other agents described herein can be formulated into formulations or compositions. The formulations or pharmaceutical compositions of the invention can be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be oral or parenteral.
在某些實施例中，如本文描述之化合物及組合物係非經腸投與。非經腸投與包括靜脈內、動脈內、皮下、腹腔內或肌內注射或輸注。 In certain embodiments, compounds and compositions as described herein are administered parenterally. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion.
在某些實施例中，用於非經腸投與之調配物或組合物可包括無菌水溶液，其等亦可含有緩衝劑、稀釋劑及其他合適之添加劑諸如(但不限於)滲透增強劑、載劑化合物及其他醫藥上可接受之載劑或賦形劑。 In certain embodiments, formulations or compositions for parenteral administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, Carrier compounds and other pharmaceutically acceptable carriers or excipients.
在某些實施例中，用於經口投與之調配物或組合物可包括(但不限於)醫藥載劑、賦形劑、粉劑或顆粒、微粒、奈米顆粒、溶於水或非水性介質中之懸浮液或溶液、膠囊、凝膠膠囊、藥囊、錠劑或迷你型錠劑。可能需要增稠劑、調味劑、稀釋劑、乳化劑、分散助劑或黏合劑。 In certain embodiments, formulations or compositions for oral administration may include, but are not limited to, pharmaceutical carriers, excipients, powders or granules, microparticles, nanoparticles, water-soluble or non-aqueous Suspension or solution in medium, capsules, gel capsules, sachets, lozenges or mini-lozenges. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be required.
給藥係取決於待治療之疾病狀態之嚴重性及反應性，且療程持續數天至數月，或直至實現治癒或達成疾病狀態之減少。給藥亦取決於藥物效力及代謝。 Dosing is dependent on the severity and responsiveness of the disease state being treated, and the course of treatment is continued for days to months, or until cure is achieved or reduction of the disease state is achieved. Dosing also depends on drug potency and metabolism.
免疫細胞中之PD-L1表現之水平可充當用於逆轉免疫抑制及免疫衰竭及誘發預存免疫力之新穎治療目標。 The level of PD-L1 expression in immune cells may serve as a novel therapeutic target for reversing immunosuppression and immune failure and inducing pre-existing immunity.
本發明之抗CD11b抗體Anti-CD11b antibody of the present invention
本文提供新穎抗CD11b抗體及其等於治療及/或預防與免疫抑制及免疫衰竭相關之疾病(諸如癌症免疫療法、慢性感染中之T細胞衰竭、敗血症、癌症中之免疫缺陷及老化中之免疫衰老)中之使用方法。 Provided herein are novel anti-CD11b antibodies and their equivalents for the treatment and/or prevention of diseases associated with immunosuppression and immune failure, such as cancer immunotherapy, T cell exhaustion in chronic infection, sepsis, immunodeficiency in cancer, and immunosenescence in aging. ) in the method of use.
在一個態樣中，本發明提供抗CD11b抗體或其抗原結合部分，其包含以下中之至少一者：由NYWIN(SEQ ID NO：1)或GFSLTSNSIS (SEQ ID NO：2)之胺基酸殘基或具有與SEQ ID NO：1或2具有至少85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之重鏈互補決定區1(H-CDR1)；由NIYPSDTYINHNQKFKD(SEQ ID NO：3)或AIWSGGGTDYNSDLKS(SEQ ID NO：4)之胺基酸殘基或具有與SEQ ID NO：3或4具有至少85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之重鏈CDR2(H-CDR2)；及由SAYANYFDY(SEQ ID NO：5)或RGGYPYYFDY(SEQ ID NO：6)之胺基酸殘基或具有與SEQ ID NO：5或6具有至少85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之重鏈CDR3(H-CDR3)；且包含以下中之至少一者：由RASQNIGTSIH(SEQ ID NO：7)或KSSQSLLYSENQENYLA(SEQ ID NO：8)之胺基酸殘基或具有與SEQ ID NO：7或8具有85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之輕鏈CDR1(L-CDR1)；由YASESIS(SEQ ID NO：9)或WASTRQS(SEQ ID NO：10)之胺基酸殘基或具有與SEQ ID NO：9或10中之任一者具有85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之輕鏈CDR2(L-CDR2)；及由QQSDSWPTLT(SEQ ID NO：11)或QQYYDTPLT(SEQ ID NO：12)之該等胺基酸殘基或具有與SEQ ID NO：11或12中之任一者具有85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之變體組成之輕鏈CDR3(L-CDR3)；使得該經分離之抗體或其抗原結合部分結合至CD11b。 In one aspect, the invention provides an anti-CD11b antibody or antigen-binding portion thereof comprising at least one of the following: produced by NYWIN (SEQ ID NO: 1) or GFSLTSNSIS (SEQ ID NO:2) or have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid residues of SEQ ID NO: 1 or 2 %, 98%, 99% identical amino acid sequence variants consisting of heavy chain complementarity determining region 1 (H-CDR1); composed of NIYPSDTYINHNQKFKD (SEQ ID NO: 3) or AIWSGGGTDYNSDLKS (SEQ ID NO: 4) Amino acid residues or have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 3 or 4 The heavy chain CDR2 (H-CDR2) that the variant of amino acid sequence is composed; NO: 5 or 6 have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequence variant composition The heavy chain CDR3 (H-CDR3); and comprising at least one of the following: by RASQNIGTSIH (SEQ ID NO: 7) or KSSQSLLYSENQENYLA (SEQ ID NO: 8) amino acid residues or with SEQ ID NO: 7 or 8 Light chains composed of variants of amino acid sequences with 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity CDR1 (L-CDR1); consists of amino acid residues from YASESIS (SEQ ID NO: 9) or WASTRQS (SEQ ID NO: 10) or has 85% of any of SEQ ID NO: 9 or 10, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequence variants of light chain CDR2 (L-CDR2); And by these amino acid residues of QQSDSWPTLT (SEQ ID NO: 11) or QQYYDTPLT (SEQ ID NO: 12) or having any one in SEQ ID NO: 11 or 12 has 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequence variants consisting of light chain CDR3 (L-CDR3); so that the isolated The antibody or antigen-binding portion thereof binds to CD11b.
在一些實施例中，本文描述之CDR包含一或更多種嵌入、取代及/或刪除。 In some embodiments, the CDRs described herein comprise one or more insertions, substitutions and/or deletions.
在另一實施例中，本發明提供抗CD11b抗體或其抗原結合部分， 其包含(i)重鏈可變區，其包括包含SEQ ID NO：1之H-CDR1、包含SEQ ID NO：3之H-CDR2及包含SEQ ID NO：5之H-CDR3之重鏈可變區，及(ii)輕鏈可變區，其包括包含SEQ ID NO：7之L-CDR1、包含SEQ ID NO：9之L-CDR2及包含SEQ ID NO：11之L-CDR3；或(iii)重鏈可變區，其包括包含SEQ ID NO：2之H-CDR1、包含SEQ ID NO：4之H-CDR2及包含SEQ ID NO：6之H-CDR3之重鏈可變區，及(iv)輕鏈可變區，其包括包含SEQ ID NO：8之L-CDR1、包含SEQ ID NO：10之L-CDR2及包含SEQ ID NO：12之L-CDR3。在另一實施例中，H-CDR1具有由SEQ ID NO：1或2組成之胺基酸序列；H-CDR2具有由SEQ ID NO：3或4組成之胺基酸序列；H-CDR3具有由SEQ ID NO：5或6組成之胺基酸序列；L-CDR1具有由SEQ ID NO：7或8組成之胺基酸序列；L-CDR2具有由SEQ ID NO：9或10組成之胺基酸序列及L-CDR3具有由SEQ ID NO：11或12組成之胺基酸序列。 In another embodiment, the present invention provides an anti-CD11b antibody or antigen-binding portion thereof, It comprises (i) a heavy chain variable region comprising H-CDR1 comprising SEQ ID NO:1, H-CDR2 comprising SEQ ID NO:3 and H-CDR3 comprising SEQ ID NO:5. region, and (ii) a light chain variable region comprising L-CDR1 comprising SEQ ID NO:7, L-CDR2 comprising SEQ ID NO:9 and L-CDR3 comprising SEQ ID NO:11; or (iii ) heavy chain variable region comprising H-CDR1 comprising SEQ ID NO: 2, H-CDR2 comprising SEQ ID NO: 4 and H-CDR3 comprising SEQ ID NO: 6, and ( iv) Light chain variable region comprising L-CDR1 comprising SEQ ID NO:8, L-CDR2 comprising SEQ ID NO:10 and L-CDR3 comprising SEQ ID NO:12. In another embodiment, H-CDR1 has an amino acid sequence consisting of SEQ ID NO: 1 or 2; H-CDR2 has an amino acid sequence consisting of SEQ ID NO: 3 or 4; H-CDR3 has an amino acid sequence consisting of The amino acid sequence consisting of SEQ ID NO: 5 or 6; L-CDR1 has the amino acid sequence consisting of SEQ ID NO: 7 or 8; L-CDR2 has the amino acid sequence consisting of SEQ ID NO: 9 or 10 Sequence and L-CDR3 has an amino acid sequence consisting of SEQ ID NO: 11 or 12.
在一個態樣中，本發明提供重鏈可變區或其抗原結合部分，其包含具有由SEQ ID NO：1或2組成之胺基酸序列之H-CDR1、具有由SEQ ID NO：3或4組成之胺基酸序列之H-CDR2及具有由SEQ ID NO：5或6組成之胺基酸序列之H-CDR3之重鏈可變區。 In one aspect, the present invention provides a heavy chain variable region or an antigen-binding portion thereof comprising H-CDR1 having an amino acid sequence consisting of SEQ ID NO: 1 or 2, having an amino acid sequence consisting of SEQ ID NO: 3 or The heavy chain variable region of H-CDR2 having an amino acid sequence consisting of 4 and H-CDR3 having an amino acid sequence consisting of SEQ ID NO: 5 or 6.
在一個態樣中，本發明提供輕鏈可變區或其抗原結合部分，其包含具有由SEQ ID NO：7或8組成之胺基酸序列之L-CDR1，具有由SEQ ID NO：9或10組成之胺基酸序列之L-CDR2，及具有由SEQ ID NO：11或12組成之胺基酸序列之L-CDR3。 In one aspect, the present invention provides a light chain variable region or an antigen-binding portion thereof comprising L-CDR1 having an amino acid sequence consisting of SEQ ID NO: 7 or 8, having an amino acid sequence consisting of SEQ ID NO: 9 or L-CDR2 having an amino acid sequence consisting of 10, and L-CDR3 having an amino acid sequence consisting of SEQ ID NO: 11 or 12.
在一個實施例中，本發明提供人類化抗CD11b抗體或其抗原結合部分，其包含(i)包含與SEQ ID NO：13至22之胺基酸序列中之任一者具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%一致性之胺基酸序列之重鏈可變區，及(ii)包含與SEQ ID NO：23至32之胺基酸序列中之任一者具有至少90%、91%、92%、93%、 94%、95%、96%、97%、98%、99%一致性之胺基酸序列之輕鏈可變區。 In one embodiment, the invention provides a humanized anti-CD11b antibody or antigen-binding portion thereof comprising (i) comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequence of the heavy chain variable region, and (ii) comprising and SEQ ID NO: 23 Any one of the amino acid sequences to 32 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences of light chain variable regions.
在另一實施例中，本發明提供人類化抗CD11b抗體或其抗原結合部分，其包括包含由SEQ ID NO：13至22組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：23至32組成之胺基酸序列之輕鏈可變區。 In another embodiment, the present invention provides a humanized anti-CD11b antibody or an antigen-binding portion thereof comprising a heavy chain variable region comprising the amino acid sequence consisting of SEQ ID NO: 13 to 22, and comprising the amino acid sequence represented by SEQ ID NO: 13 to 22. NO: The light chain variable region of the amino acid sequence consisting of 23 to 32.
較佳地，本發明提供人類化抗CD11b抗體或其抗原結合部分，其包含：(a)包含由SEQ ID NO：13組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：23組成之胺基酸序列之輕鏈可變區；(b)包含由SEQ ID NO：14組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：24組成之胺基酸序列之輕鏈可變區；(c)包含由SEQ ID NO：15組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：25組成之胺基酸序列之輕鏈可變區；(d)包含由SEQ ID NO：16組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：26組成之胺基酸序列之輕鏈可變區；(e)包含由SEQ ID NO：17組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：27組成之胺基酸序列之輕鏈可變區；(f)包含由SEQ ID NO：18組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：28組成之胺基酸序列之輕鏈可變區；(g)包含由SEQ ID NO：19組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：29組成之胺基酸序列之輕鏈可變區；(h)包含由SEQ ID NO：20組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：30組成之胺基酸序列之輕鏈可變區；(i)包含由SEQ ID NO：21組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：31組成之胺基酸序列之輕鏈可變區；或 (j)包含由SEQ ID NO：22組成之胺基酸序列之重鏈可變區，及包含由SEQ ID NO：32組成之胺基酸序列之輕鏈可變區。 Preferably, the present invention provides a humanized anti-CD11b antibody or an antigen-binding portion thereof, which comprises: (a) a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 13, and comprising a sequence consisting of SEQ ID NO : the light chain variable region of an amino acid sequence consisting of 23; (b) the heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 14, and comprising an amine group consisting of SEQ ID NO: 24 The light chain variable region of the acid sequence; (c) the heavy chain variable region comprising the amino acid sequence consisting of SEQ ID NO: 15, and the light chain comprising the amino acid sequence consisting of SEQ ID NO: 25 can be Variable region; (d) a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 16, and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 26; (e) A heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 17, and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 27; (f) comprising a sequence consisting of SEQ ID NO: A heavy chain variable region comprising an amino acid sequence consisting of 18, and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 28; (g) comprising an amino acid consisting of SEQ ID NO: 19 The heavy chain variable region of the sequence, and the light chain variable region comprising the amino acid sequence consisting of SEQ ID NO: 29; (h) the heavy chain variable region comprising the amino acid sequence consisting of SEQ ID NO: 20 Region, and the light chain variable region comprising the amino acid sequence consisting of SEQ ID NO: 30; (i) the heavy chain variable region comprising the amino acid sequence consisting of SEQ ID NO: 21, and comprising the amino acid sequence consisting of SEQ ID NO: ID NO: the light chain variable region of an amino acid sequence consisting of 31; or (j) A heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO:22, and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO:32.
SEQ ID NO：13至32之胺基酸序列如下所列：本發明之人類化抗CD11b抗體之重鏈可變區：(SEQ ID NO：13至22) The amino acid sequences of SEQ ID NOs: 13 to 32 are listed below: The heavy chain variable region of the humanized anti-CD11b antibody of the present invention: (SEQ ID NOs: 13 to 22)
VH1
VH2
VH3
VH4
VH5
HC1
HC2
HC3
HC4
HC5
本發明之人類化抗CD11b抗體之輕鏈可變區：(SEQ ID NO：23至32)The light chain variable region of the humanized anti-CD11b antibody of the present invention: (SEQ ID NO: 23 to 32)
VL1
VL2
VL3
VL4
VL5
LC1
LC2
LC3
LC4
LC5
此項技術中熟知用於製備實際上針對任何靶抗原之單株抗體之技術。參見，例如，Kohler及Milstein，Nature 256：495(1975)，及Coligan等人(編)，Current Protocols In Immunology，第1卷，第2.5.1至2.6.7頁(John Wiley & Sons 1991)。單株抗體可藉由以下步驟來獲得：向小鼠或雞注射包含抗原之組合物，移除脾以獲得B淋巴細胞，融合B淋巴細胞與骨髓瘤細胞以產生融合瘤，選殖該等融合瘤，篩選針對該抗原產生抗體之陽性純系，培養該等針對該抗原產生抗體之純系，及自該等融合瘤培養物中分離該等抗體。 Techniques for preparing monoclonal antibodies to virtually any target antigen are well known in the art. See, eg, Kohler and Milstein, Nature 256:495 (1975), and Coligan et al. (eds.), Current Protocols In Immunology, Vol. 1, pp. 2.5.1 to 2.6.7 (John Wiley & Sons 1991). Monoclonal antibodies can be obtained by injecting a composition comprising an antigen into a mouse or chicken, removing the spleen to obtain B lymphocytes, fusing B lymphocytes with myeloma cells to generate fusion tumors, and breeding the fusions For tumors, positive clones that produce antibodies against the antigen are screened, the clones that produce antibodies against the antigen are cultivated, and the antibodies are isolated from the fusion tumor cultures.
各種技術(諸如嵌合或人類化抗體之產生)可涉及抗體選殖及構築之程序。用於受關注之抗體之抗原結合可變輕鏈及可變重鏈序列可藉由各種分子選殖程序獲得。嵌合抗體係其中人類抗體之可變區已經被(例如)小鼠抗體之可變區(包括小鼠抗體之互補決定區(CDR))置換之重組蛋白。嵌合抗體當向個體投與時顯示減小之免疫原性及增加之穩定性。此項技術中熟知用於構築嵌合抗體之方法。嵌合多株抗體可藉由將來自小鼠免疫球蛋白之重及輕可變鏈之小鼠CDR轉移至人類抗體之相應可變域內來人類化。該嵌合多株抗體中之小鼠框架區(FR)亦經人類FR序列置換。 Various techniques, such as the production of chimeric or humanized antibodies, may involve procedures of antibody cloning and construction. Antigen-binding variable light and variable heavy chain sequences for antibodies of interest can be obtained by various molecular breeding procedures. Chimeric antibody system A recombinant protein in which the variable regions of a human antibody have been replaced by, for example, the variable regions of a mouse antibody, including the complementarity determining regions (CDRs) of the mouse antibody. Chimeric antibodies exhibit reduced immunogenicity and increased stability when administered to an individual. Methods for constructing chimeric antibodies are well known in the art. Chimeric polyclonal antibodies can be humanized by transferring mouse CDRs from the heavy and light variable chains of mouse immunoglobulins into the corresponding variable domains of human antibodies. The mouse framework regions (FRs) in this chimeric polyclonal antibody were also replaced with human FR sequences.
例如，編碼特異性結合CD11b之人類化抗體之VL及/或VH之核酸可藉由活體外方法(諸如聚合酶鏈反應(PCR)、連接酶鏈反應(LCR)、基於轉錄之擴增系統(TAS)等)加以選殖或擴增。例如，編碼該蛋白質之聚核苷酸可藉由cDNA之聚合酶鏈反應使用基於該分子之DNA序列之引物來分離。熟習此項技術者熟知各種選殖及活體外擴增方法論。聚核苷酸亦可藉由用選自所需之聚核苷酸之序列之探針在嚴苛之雜合條件下篩選基因體或cDNA庫而分離。 For example, nucleic acids encoding the VL and/or VH of a humanized antibody that specifically binds CD11b can be synthesized by in vitro methods such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems ( TAS) etc.) to be colonized or amplified. For example, polynucleotides encoding the protein can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule. Various cloning and in vitro expansion methodologies are familiar to those skilled in the art. Polynucleotides can also be isolated by screening genomic or cDNA libraries under stringent hybridization conditions with probes selected from the sequence of the desired polynucleotide.
該等聚核苷酸包括重組DNA，其併入載體內；併入自主複製質體或病毒內或併入原核生物或真核生物之基因體DNA內，或其作為獨立於其他序列之個別分子(例如，cDNA)存在。本發明之核苷酸可為核醣核苷酸、去氧核醣核苷酸或任何一種核苷酸之經修飾之形式。該術語包括DNA之單股及雙股形式。 Such polynucleotides include recombinant DNA, incorporated into a vector; into a self-replicating plastid or virus, or into the genomic DNA of a prokaryote or eukaryote, or as a separate molecule independent of other sequences (eg, cDNA) exists. The nucleotides of the present invention may be ribonucleotides, deoxyribonucleotides or modified forms of either nucleotide. The term includes both single- and double-stranded forms of DNA.
編碼特異性結合CD11b之人類化抗體之VL及/或VH之DNA序列可藉由DNA轉移至合適之宿主細胞內而在活體外表現。該細胞可為原核細胞或真核細胞。該術語亦包括標的宿主細胞之任何子代。應瞭解所有子代可能不同於親代細胞，因為在複製期間可能發生突變。此項技術中已知穩定轉移之方法，其意謂外源DNA連續保持於宿主中。 The DNA sequence encoding the VL and/or VH of the humanized antibody specifically binding to CD11b can be expressed in vitro by transferring the DNA into a suitable host cell. The cells may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It should be understood that all progeny may differ from the parental cells as mutations may occur during replication. Methods of stable transfer are known in the art, which means that foreign DNA is continuously maintained in the host.
編碼特異性結合CD11b之人類化抗體之VL及/或VH之聚核苷酸序列可操作地連接至表現控制序列。可操作地連接至編碼序列之表現控制序列係經接合使得該編碼序列之表現在與該等表現控制序列相容之條件下達成。該等表現控制序列包括(但不限於)適當之啟動子、強化子、轉錄終止子、在編碼蛋白質之基因前之起始密碼子(例如ATG)、用於內含子之剪接訊息(維持該基因之正確閱讀框架以允許mRNA之適當轉譯)，及終止密碼子。 The polynucleotide sequences encoding the VL and/or VH of the humanized antibody that specifically binds CD11b are operably linked to expression control sequences. Expression control sequences operably linked to a coding sequence are ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. Such expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, initiation codons (such as ATG) before genes encoding proteins, splicing messages for introns (to maintain the the correct reading frame of the gene to allow proper translation of the mRNA), and a stop codon.
編碼特異性結合CD11b之人類化抗體之VL及/或VH之聚核苷酸序列可嵌入表現載體內。該表現載體之實例包括(但不限於)質體、病 毒，或其他可經操作以容許序列之嵌入或併入且可表現於原核生物或真核生物中之媒介體。宿主可包括微生物、酵母、昆蟲及哺乳類生物。此項技術中熟知於原核生物中表現具有真核或病毒序列之DNA序列之方法。此項技術中已知可於宿主中表現及複製之生物功能病毒及質體DNA載體。以重組DNA轉形宿主細胞可藉由熟習此項技術者熟知之習知技術進行。 A polynucleotide sequence encoding the VL and/or VH of a humanized antibody that specifically binds CD11b can be embedded in an expression vector. Examples of such expression vectors include (but are not limited to) plastids, virus Viruses, or other vectors that can be manipulated to allow intercalation or incorporation of sequences and can be expressed in prokaryotes or eukaryotes. Hosts can include microorganisms, yeast, insects and mammalian organisms. Methods for expressing DNA sequences with eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plastid DNA vectors capable of expression and replication in a host are known in the art. Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
經重組表現之多肽之分離及純化可藉由習知方式(包括製備型層析法及免疫分離)進行。 Isolation and purification of recombinantly expressed polypeptides can be performed by conventional means including preparative chromatography and immunoisolation.
人類化可通常遵循此項技術中已知的習知方法進行，藉由以嚙齒動物CDR或CDR序列代替人類抗體之相應序列。因此，此等「人類化」抗體係其中大體上小於已經來自非人類物種之相應序列取代之完整人類可變域之抗體。實際上，人類化抗體通常係其中一些CDR殘基及(可能)一些FR殘基經來自非人類(例如)嚙齒動物抗體之類似位置之殘基取代之人類抗體。 Humanization can generally be performed following conventional methods known in the art by substituting rodent CDRs or CDR sequences for the corresponding sequences of human antibodies. Thus, such "humanized" antibodies are substantially smaller than those in which fully human variable domains have been substituted with corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and (possibly) some FR residues are substituted by residues from analogous positions in non-human, eg, rodent antibodies.
選擇待用於製造人類化抗體中之人類可變域(輕及重兩者)對減小抗原性係非常重要的。嚙齒動物抗體之可變域之序列針對整個已知人類可變域序列庫進行篩選。然後最接近嚙齒動物之序列之人類序列被公認為係用於人類化抗體之人類框架(FR)。另一方法使用衍生自輕鏈或重鏈之特定子群之所有人類抗體之一致性序列之特定框架。相同框架可用於數種不同之人類化抗體。 The choice of human variable domains (both light and heavy) to be used in making humanized antibodies is very important to reduce antigenicity. The sequences of the variable domains of rodent antibodies were screened against the entire library of known human variable domain sequences. The human sequences closest to that of the rodent are then recognized as human frameworks (FRs) for humanized antibodies. Another method uses a specific framework derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies.
抗體結合部分包括(例如)Fab、Fab'、F(ab)2、F(ab')2、Fv、scFv及類似物。此等片段使用此項技術中熟知之方法產生自完整抗體，例如藉由使用酶(諸如木瓜酶(以產生Fab片段)或胃蛋白酶(以產生F(ab')2片段))之蛋白水解裂解。 Antibody binding moieties include, for example, Fab, Fab', F(ab) 2 , F(ab') 2 , Fv, scFv, and the like. These fragments are generated from intact antibodies using methods well known in the art, for example, by proteolytic cleavage using enzymes such as papain (to generate Fab fragments) or pepsin (to generate F(ab') 2 fragments) .
可對編碼本文描述之多肽之核酸作出修飾而不降低其生物活性。可作出一些修飾以促進靶分子選殖、表現或併入融合蛋白中。此 等修飾係熟習此項技術者熟知且包括(例如)終止密碼子，添加至胺基端處以提供起始、位置之甲硫胺酸，放置於任一末端上以產生便於定位之限制位置之額外之胺基酸或在純化步驟中有幫助之額外胺基酸。除重組方法外，本發明之抗體亦可使用此項技術中熟知的標準肽合成以完整或部分構築。 Modifications can be made to the nucleic acids encoding the polypeptides described herein without reducing their biological activity. Several modifications can be made to facilitate the colonization, expression or incorporation of the target molecule into the fusion protein. this Such modifications are well known to those skilled in the art and include, for example, a stop codon, a methionine added to the amino terminus to provide initiation, a positional methionine, an additional codon placed at either end to create a restriction position for ease of positioning. amino acids or additional amino acids that are helpful in purification steps. In addition to recombinant methods, antibodies of the invention can also be constructed in whole or in part using standard peptide synthesis well known in the art.
在另一態樣中，本發明提供包含本發明之抗CD11b抗體之組合物。在一些實施例中，此等組合物可投與給個體。在一些實施例中，本發明之抗CD11b抗體可提供於包含一或更多種其他組分(包括(但不限於)醫藥上可接受之載劑、佐劑、潤濕劑或乳化劑、pH緩衝劑、防腐劑及/或任何其他適用於組合物之預期用途之組分)之組合物中。此等組合物可採取溶液、懸浮液、乳液及類似物之形式。術語「醫藥上可接受之載劑」包括各種稀釋劑、賦形劑及/或媒介體。醫藥上可接受之載劑包括(但不限於)已知對遞送至人類及/或其他動物個體係安全之載劑，及/或由聯邦或州政府之監管機構批准之載劑，及/或列於美國藥典中及/或其他公認之藥典中之載劑，及/或接受來自一或多個公認之監管機構之特定或個別批准以用於人類及/或其他動物中之載劑。此等醫藥上可接受之載劑包括(但不限於)水、水溶液(諸如生理鹽水溶液、緩衝劑及類似物)、有機溶劑(諸如某些醇及油，其等包括彼等石油、動物、蔬菜或合成起源之油(諸如花生油、大豆油、礦物油、芝麻油))及類似物。 In another aspect, the invention provides compositions comprising an anti-CD11b antibody of the invention. In some embodiments, such compositions can be administered to an individual. In some embodiments, an anti-CD11b antibody of the invention may be provided in a formulation comprising one or more additional components including, but not limited to, a pharmaceutically acceptable carrier, adjuvant, wetting or emulsifying agent, pH buffers, preservatives and/or any other components suitable for the intended use of the composition). These compositions can take the form of solutions, suspensions, emulsions and the like. The term "pharmaceutically acceptable carrier" includes various diluents, excipients and/or vehicles. Pharmaceutically acceptable carriers include, but are not limited to, carriers known to be safe for delivery to humans and/or other animal systems, and/or carriers approved by regulatory agencies of the federal or state governments, and/or Carriers listed in the US Pharmacopoeia and/or other recognized pharmacopeias, and/or carriers that have received specific or individual approval from one or more recognized regulatory agencies for use in humans and/or other animals. Such pharmaceutically acceptable carriers include, but are not limited to, water, aqueous solutions (such as saline solutions, buffers, and the like), organic solvents (such as certain alcohols, and oils, including those of petroleum, animal, Oils of vegetable or synthetic origin (such as peanut oil, soybean oil, mineral oil, sesame oil) and the like.
在一個實施例中，本發明之人類化抗CD11b抗體可提供於包含一或更多種「化學治療劑」(其等係用於治療癌症之化學化合物，亦稱為抗腫瘤藥物)之組合物中。抗腫瘤藥物通常根據化學結構及藥物起源之差異而歸類為烷化劑、抗新陳代謝藥物、抗腫瘤抗生素、蒽環類抗生素、抗腫瘤草藥及激素。取決於週期或相特異性，抗腫瘤之化學治療藥物可歸類為(1)細胞週期非特異性藥劑(CCNSA)，諸如烷化 劑、抗腫瘤抗生素及鉑配位錯合物等，及(2)細胞週期特異性藥劑(CCSA)，諸如抗代謝藥物、長春花生物鹼等。 In one embodiment, the humanized anti-CD11b antibodies of the invention may be provided in a composition comprising one or more "chemotherapeutic agents" (which are chemical compounds used to treat cancer, also known as antineoplastic drugs) middle. Antineoplastic drugs are usually classified into alkylating agents, antimetabolic drugs, antineoplastic antibiotics, anthracycline antibiotics, antineoplastic herbal medicines and hormones according to differences in chemical structure and drug origin. Depending on the cycle or phase specificity, antineoplastic chemotherapeutic drugs can be classified as (1) cell cycle non-specific agents (CCNSAs), such as alkylating agents, antitumor antibiotics and platinum coordination complexes, and (2) cell cycle specific agents (CCSA), such as antimetabolites, vinca alkaloids, etc.
在一些實施例中，本發明之組合物包含「有效量」之本發明之抗CD11b抗體。「有效量」係達成所需目標結果所需要之量。有效達成所需目標結果之本發明之人類化抗CD11b抗體之量將取決於各種因素，其等包括(但不限於)預期個體之物種(例如，是否係人類抑或係一些其他動物物種)、預期個體之年齡及/或性別、經計劃之投與途徑、經計劃之給藥方案、任何進行中之疾病或病症之嚴重性及類似因素。有效量(其可為有效量之範圍)可藉由標準技術測定而無需任何過度之實驗，例如，使用在預期個體物種或任何合適之動物模型物種中之活體外分析及/或活體內分析。合適之分析包括(但不限於)彼等涉及來自劑量-反應曲線之外推法及/或衍生自活體外及/或活體內模型系統之其他資料者。在一些實施例中，該有效量可根據醫學或獸醫從業者基於特定情況之判斷而判定。 In some embodiments, compositions of the invention comprise an "effective amount" of an anti-CD11b antibody of the invention. An "effective amount" is that amount necessary to achieve the desired objective result. The amount of a humanized anti-CD11b antibody of the invention effective to achieve the desired target result will depend on various factors including, but not limited to, the species of the desired individual (e.g., whether it is human or some other animal species), the desired The age and/or sex of the subject, the planned route of administration, the planned dosing regimen, the severity of any ongoing disease or condition, and similar factors. An effective amount (which may be a range of effective amounts) can be determined by standard techniques without any undue experimentation, for example, using in vitro and/or in vivo assays in the intended individual species or any suitable animal model species. Suitable analyzes include, but are not limited to, those involving extrapolation from dose-response curves and/or other data derived from in vitro and/or in vivo model systems. In some embodiments, the effective amount can be determined according to the judgment of a medical or veterinary practitioner based on the particular situation.
在一個實施例中，人類化抗CD11b抗體之有效量在自每次投與時介於約0.01mg/kg至約40mg/kg體重之範圍內；較佳地，介於約0.01mg/kg至約30mg/kg、約0.01mg/kg至約20mg/kg、約0.01mg/kg至約10mg/kg、約1mg/kg至約40mg/kg、約1mg/kg至約30mg/kg、約1mg/kg至約20mg/kg、約1mg/kg至約10mg/kg、約2mg/kg至約40mg/kg、約2mg/kg至約30mg/kg、約2mg/kg至約20mg/kg、約2mg/kg至約10mg/kg、約5mg/kg至約40mg/kg、約5mg/kg至約30mg/kg、約5mg/kg至約20mg/kg或約5mg/kg至約10mg/kg或約1mg/kg至約5mg/kg之範圍內。 In one embodiment, the effective amount of humanized anti-CD11b antibody ranges from about 0.01 mg/kg to about 40 mg/kg body weight per administration; preferably, from about 0.01 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 1 mg/kg to about 40 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg kg to about 20 mg/kg, about 1 mg/kg to about 10 mg/kg, about 2 mg/kg to about 40 mg/kg, about 2 mg/kg to about 30 mg/kg, about 2 mg/kg to about 20 mg/kg, about 2 mg/kg kg to about 10 mg/kg, about 5 mg/kg to about 40 mg/kg, about 5 mg/kg to about 30 mg/kg, about 5 mg/kg to about 20 mg/kg, or about 5 mg/kg to about 10 mg/kg or about 1 mg/kg kg to about 5 mg/kg.
在一些實施例中，本發明提供方法，該等方法包括向個體投與本發明之人類化抗CD11b抗體。此等方法包括用於抑制免疫細胞中PD-L1表現、免疫細胞中逆轉免疫抑制或免疫衰竭或誘發預存免疫 力、偵測個體中之PD-L1及治療或預防急性及/或慢性感染、敗血症、癌症中之免疫缺陷或老化中之免疫衰老之方法。本發明之抗CD11b抗體可用於上文提及之方法中。 In some embodiments, the invention provides methods comprising administering to a subject a humanized anti-CD11b antibody of the invention. Such methods include methods for inhibiting PD-L1 expression in immune cells, reversing immunosuppression or immune exhaustion in immune cells, or inducing pre-existing immunity Methods of detecting PD-L1 in an individual and treating or preventing acute and/or chronic infection, sepsis, immunodeficiency in cancer or immunosenescence in aging. The anti-CD11b antibodies of the present invention can be used in the methods mentioned above.
本文描述之癌症係對免疫療法具有反應性之癌症且該等癌症之實例係如本文描述。預防及/或治療癌症之方法包含投與額外之活性劑或療法。該等額外之活性劑、其等實施例及投與係如本文描述。 Cancers described herein are cancers that are responsive to immunotherapy and examples of such cancers are as described herein. Methods of preventing and/or treating cancer comprise administering additional active agents or therapies. The additional active agents, their embodiments and administration are as described herein.
可投與(例如，在治療之方法之過程中)本發明之抗CD11b抗體或包含該抗CD11b抗體之組合物之個體包括任何及所有動物物種。在一些實施例中，該等個體係哺乳類物種。哺乳類個體包括(但不限於)人類、非人類靈長類動物、嚙齒動物、兔及雪貂。 Individuals to whom an anti-CD11b antibody or a composition comprising such an anti-CD11b antibody of the invention may be administered (eg, during a method of treatment) include any and all animal species. In some embodiments, the individuals are mammalian species. Mammalian subjects include, but are not limited to, humans, non-human primates, rodents, rabbits, and ferrets.
此項技術中已知各種遞送系統且可使用任何合適之遞送系統以向個體投與本發明之組合物。此等遞送系統包括(但不限於)皮內、肌內、腹腔內、靜脈內、皮下、鼻內、硬膜上及經口遞送系統。本發明之組合物可藉由任何便捷之途徑投與，例如藉由輸注或快速濃注，藉由通過上皮或黏膜皮膚內襯(例如，口腔黏膜、直腸黏膜及腸黏膜等)吸收且可連同其他生物活性劑一起投與。投與可為全身投與或局部投與。 A variety of delivery systems are known in the art and any suitable delivery system can be used to administer the compositions of the invention to a subject. Such delivery systems include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral delivery systems. The compositions of the present invention may be administered by any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucocutaneous lining (e.g., oral mucosa, rectal mucosa, and intestinal mucosa, etc.) and may be combined with Administer with other biologically active agents. Administration can be systemic or localized.
在一些此類實施例中，單一劑量之投與較佳。然而，在其他實施例中，額外之劑量可藉由達成所需效應之相同或不同之途徑投與。在一些實施例中，給藥方案可包含單一投與。在其他實施例中，給藥方案可包含多次投與。 In some such embodiments, administration of a single dose is preferred. However, in other embodiments, additional doses may be administered by the same or a different route to achieve the desired effect. In some embodiments, a dosing regimen may comprise a single administration. In other embodiments, the dosing regimen may comprise multiple administrations.
實例example
下文描述用於下列實例中之材料及方法： Materials and methods used in the following examples are described below:
材料及方法 Materials and methods
人類細胞分離及細胞培養 Human Cell Isolation and Cell Culture
來自健康志願者之白血球濃縮物係獲得自台灣血液服務基金會 (臺北，台灣)。獲得用於參與研究之書面知情同意書，其經馬偕紀念醫院人體試驗委員會(Institutional Review Board of the Mackay Memorial Hospital)批准。人類單核細胞如先前描述經分離。簡而言之，周邊血液單核細胞(PBMC)使用Ficoll-Paque Plus(GE Healthcare)梯度離心分離法進行分離。該等單核細胞藉由使用CD14 MACS微珠(Miltenyi Biotec)進行CD14選擇來進一步純化。使用流動式細胞測量術分析證實之單核細胞純度係約90%。 Leukocyte concentrates from healthy volunteers were obtained from the Taiwan Blood Service Foundation (Taipei, Taiwan). Written informed consent was obtained for study participation, which was approved by the Institutional Review Board of the Mackay Memorial Hospital. Human monocytes were isolated as previously described. Briefly, peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Paque Plus (GE Healthcare) gradient centrifugation. The monocytes were further purified by CD14 selection using CD14 MACS microbeads (Miltenyi Biotec). The purity of monocytes was confirmed to be approximately 90% using flow cytometry analysis.
動物及腫瘤細胞系。 Animal and tumor cell lines.
C57BL/6小鼠(6至8週齡)購買自國家實驗動物中心(臺北，台灣)。所有動物實驗均在無特定病原之條件下且根據經馬偕紀念醫院動物護理及使用委員會(臺北，台灣)批准之指導方針進行。在治療開始時及在治療期間每天量測各小鼠之體重。B16F10係鼠科黑色素瘤細胞及LLC1係鼠科Lewis氏肺癌。所有細胞均係衍生自C57BL/6小鼠。細胞在37℃下於5% CO2濕潤氣氛中維持於杜貝卡氏改良伊格培養基(DMEM)、10%熱滅活胎小牛血清、2mM L-麩醯胺酸、青黴素(100U/ml)及鏈黴素(100μg/ml)中。 C57BL/6 mice (6 to 8 weeks old) were purchased from the National Center for Experimental Animals (Taipei, Taiwan). All animal experiments were performed under specific pathogen-free conditions and according to guidelines approved by the Mackay Memorial Hospital Animal Care and Use Committee (Taipei, Taiwan). The body weight of each mouse was measured at the beginning of treatment and daily during treatment. B16F10 is murine melanoma cells and LLC1 is murine Lewis lung cancer. All cell lines were derived from C57BL/6 mice. Cells were maintained at 37°C in a humidified atmosphere of 5% CO 2 in Dubecca's modified Eagle's medium (DMEM), 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, penicillin (100 U/ml ) and streptomycin (100 μg/ml).
抗體及試劑 Antibodies and Reagents
針對人類單核細胞研究 For human monocyte research
來自大腸桿菌(O111：B4)之LPS係獲得自Sigma。對人類CD11b具有特異性之鼠科結合抗體(ICRF44)及用於對照抗體之小鼠IgG1均係購買自Biolegend。 LPS from E. coli (O111:B4) was obtained from Sigma. A murine binding antibody (ICRF44) specific for human CD11b and mouse IgG1 for a control antibody were purchased from Biolegend.
針對鼠科癌症模型 For murine cancer models
對鼠科CD11b(M1/70)具有特異性之大鼠結合抗體、大鼠對照IgG2b抗體(LTF-2)、亞美尼亞倉鼠抗鼠科PD1(J43)及亞美尼亞倉鼠對照IgG均係購買自BioXcell。紫杉醇係係獲得自馬偕紀念醫院之化學療法藥物。 Rat binding antibody specific to murine CD11b (M1/70), rat control IgG2b antibody (LTF-2), Armenian hamster anti-murine PD1 (J43) and Armenian hamster control IgG were purchased from BioXcell. Paclitaxel was a chemotherapy drug obtained from Mackay Memorial Hospital.
癌症治療之方案 cancer treatment plan
皮下腫瘤模型 Subcutaneous tumor model
對C57BL/6小鼠皮下接種2×105個B16F10細胞或1x106個LLC1細胞。在腫瘤接種後7天，開始治療。用不同抗體及化學藥物每週兩次腹腔內(ip)治療帶腫瘤小鼠。監測小鼠並每週兩次針對可觸知的腫瘤之形成進行評分且若腫瘤超過3000mm3之預定尺寸則處死該等小鼠。腫瘤體積用卡尺量測並用下式計算：A×B2×0.54，其中A係最大直徑及B係最小直徑。 C57BL/6 mice were subcutaneously inoculated with 2×10 5 B16F10 cells or 1×10 6 LLC1 cells. Seven days after tumor inoculation, treatment was initiated. Tumor-bearing mice were treated intraperitoneally (ip) twice a week with different antibodies and chemotherapeutics. Mice were monitored and scored twice weekly for the development of palpable tumors and sacrificed if tumors exceeded a predetermined size of 3000 mm 3 . Tumor volume was measured with a caliper and calculated with the following formula: A×B 2 ×0.54, where A is the largest diameter and B is the smallest diameter.
肺轉移模型 Lung Metastasis Model
在第0天經由尾靜脈向各小鼠注射2x105個B16F10細胞或LLC1細胞。在第1天，小鼠腹腔內注射各種抗體。每三至四天重複注射。在第15天，處死小鼠且將腫瘤接種之量計算為在顯微鏡下存在於肺中之結節之總數量。在其他實驗中，針對組合療法對各組中經治療之小鼠之長期存活率之效應分析小鼠。
On
流動式細胞測量分析 Flow Cytometry Analysis
針對人類單核細胞研究 For human monocyte research
單核細胞係經抗CD11b(ICRF44)或適當之同型對照抗體預先培養1小時。隨後向該等細胞中添加100ng/ml LPS並培養整夜。為分析LPS致敏單核細胞之表面表現型，將該等細胞在冰上於黑暗中用下列稀釋於含有1% BSA之磷酸鹽緩衝鹽水(PBS)中之mAb培養30分鐘：PD-L1-FITC、CD80-PE、CD86-PE、HLA-DR-PE及CD14-PerCP(BD Biosciences)。單核細胞、多形核白細胞(PMN)及淋巴細胞基於其等FSC/SSC性質進行閘控。使用FACS Calibur偵測螢光，且使用FCS Express，第3版(De Novo軟體)進行資料分析。 Monocyte cell lines were pre-incubated for 1 hour with anti-CD11b (ICRF44) or an appropriate isotype control antibody. Then 100 ng/ml LPS was added to the cells and incubated overnight. To analyze the surface phenotype of LPS-sensitized monocytes, the cells were incubated for 30 minutes on ice in the dark with the following mAbs diluted in phosphate-buffered saline (PBS) containing 1% BSA: PD-L1- FITC, CD80-PE, CD86-PE, HLA-DR-PE and CD14-PerCP (BD Biosciences). Monocytes, polymorphonuclear leukocytes (PMNs) and lymphocytes are gated based on their FSC/SSC properties. Fluorescence was detected using a FACS Calibur, and data analysis was performed using FCS Express, version 3 (De Novo software).
針對鼠科癌症研究 For murine cancer research
為獲得腫瘤浸潤性白細胞，藉由膠原蛋白酶IV(Sigma)消化腫瘤 組織。單一細胞懸浮液用下列抗體染色：CD45-PE、Ly-6G-FITC、Ly-6C-APC及CD8b.2-FITC。腫瘤浸潤性白細胞係經閘控自CD45+群體。使用FACS Calibur偵測螢光，且使用FCS Express，第3版(De Novo軟體)進行資料分析。 To obtain tumor-infiltrating leukocytes, tumors were digested by collagenase IV (Sigma) organize. Single cell suspensions were stained with the following antibodies: CD45-PE, Ly-6G-FITC, Ly-6C-APC and CD8b.2-FITC. Tumor-infiltrating leukocytes were gated from the CD45+ population. Fluorescence was detected using a FACS Calibur, and data analysis was performed using FCS Express, version 3 (De Novo software).
為分離來自各實驗之白血球(WBC)，全血細胞藉由RBC裂解緩衝液裂解。單一細胞懸浮液用下列抗體染色：PD-L1-APC、IAIE-APC及CD8b.2-FITC(Biolegend)。單核細胞、多形核白細胞(PMN)及淋巴細胞係基於其等FSC/SSC性質。使用FACS Calibur偵測螢光，且使用FCS Express，第3版(De Novo軟體)進行資料分析。 To isolate white blood cells (WBC) from each experiment, whole blood cells were lysed by RBC lysis buffer. Single cell suspensions were stained with the following antibodies: PD-L1-APC, IAIE-APC and CD8b.2-FITC (Biolegend). Monocytes, polymorphonuclear leukocytes (PMN) and lymphocyte lines are based on their FSC/SSC properties. Fluorescence was detected using a FACS Calibur, and data analysis was performed using FCS Express, version 3 (De Novo software).
細胞介素定量 Interleukin quantification
培養物上清液中之人類IL-6、IL-10、IL-12及TNF-α藉由商業酶聯免疫吸附法(ELISA；R&D系統)根據製造商使用說明進行偵測。血漿中之鼠科IL-12、IFN-γ及TNF-α藉由BD CBA小鼠炎症套組定量。 Human IL-6, IL-10, IL-12 and TNF-α in culture supernatants were detected by commercial enzyme-linked immunosorbent assay (ELISA; R&D Systems) according to the manufacturer's instructions. Murine IL-12, IFN-γ and TNF-α in plasma were quantified by BD CBA mouse inflammation kit.
實例1：結合CD11b將減少LPS致敏單核細胞上之PD-L1表現Example 1: Binding to CD11b reduces PD-L1 expression on LPS-sensitized monocytes
在此實例中，吾人研究整合素αMβ2(Mac-1)之阻斷是否可在功能上增加TLR反應。如圖1中所示，CD11b結合劑(諸如抗CD11b抗體(ICRF44))之投與可減少單核細胞上之LPS誘發之PD-L1表現。相比之下，抗CD11b抗體治療不改變LPS致敏單核細胞上之HLA-DR、CD80及CD86表現水平。以ML-C19-A(CD11b拮抗劑之小分子)(圖2A)結合CD11b亦證實LPS致敏單核細胞中之抑制PD-L1表現(圖2B)。此等結果一起表明CD11b在LPS致敏單核細胞上之PD-L1表現之誘發中起關鍵作用。 In this example, we investigated whether blockade of integrin αMβ2 (Mac-1) could functionally increase TLR responses. As shown in Figure 1, administration of a CD11b-binding agent, such as an anti-CD11b antibody (ICRF44), can reduce LPS-induced PD-L1 expression on monocytes. In contrast, anti-CD11b antibody treatment did not alter the expression levels of HLA-DR, CD80 and CD86 on LPS-sensitized monocytes. Binding of CD11b by ML-C19-A (a small molecule that is a CD11b antagonist) (FIG. 2A) also demonstrated inhibition of PD-L1 expression in LPS-sensitized monocytes (FIG. 2B). Together these results suggest that CD11b plays a critical role in the induction of PD-L1 expression on LPS-sensitized monocytes.
實例2：CD11b結合於抗腫瘤免疫力中之效應Example 2: Effect of CD11b Binding in Anti-tumor Immunity
為檢測CD11b結合於抗腫瘤免疫力中之效應，將抗小鼠CD11b(M1/70)抗體作為B16F10鼠科腫瘤模型中之單一療法進行測試。對C57BL/6小鼠在第0天皮下注射B16F10細胞。在第7天，對小鼠腹腔內
(ip)注射對照IgG(5mg/kg)或抗小鼠CD11b抗體(5mg/kg)。每三至四天重複注射。藉由針對各組監測腫瘤體積及長期存活率而測定效用。如圖3中所示，以抗小鼠CD11b抗體結合CD11b強效抑制B16F10腫瘤之皮下生長(在第18天，對照IgG相比於抗CD11b=1054±385.4mm3相比於502.7±268.2mm3)。吾人檢測免疫細胞群體於腫瘤中之比例。在腫瘤接種後之第18天，以抗CD11b抗體結合CD11b減少腫瘤浸潤性骨髓衍生之抑制細胞(MDSC)之局部聚集，MDSC抑制T細胞且導致經腫瘤浸潤之CD8 T細胞之增加(圖4)。同時，以抗CD11b抗體結合CD11b將免疫抑制腫瘤微環境轉變成免疫刺激狀態，其有利地有助於抗腫瘤效應。吾人進一步檢測免疫細胞群體在抗CD11b抗體治療後於周邊中之比例。在腫瘤注射後之第15天，抗CD11b治療導致CD11b陽性白血球中之PD-L1表現減少，同時IAIE陽性CD8 T細胞(經活化之T細胞)於CD8 T細胞中之百分率增加(圖5)。IFN-γ、IL-12及TNF-α之血漿濃度反映各種炎性或惡性疾病中之免疫刺激狀態。吾人量測經抗CD11b抗體治療之帶腫瘤之小鼠中之血漿IFN-γ、IL-12及TNF-α濃度。相較於對照IgG治療，經抗CD11b抗體治療之小鼠顯示高血漿IFN-γ、IL-12及TNF-α水平(圖6)。
To test the effect of CD11b binding in anti-tumor immunity, an anti-mouse CD11b (M1/70) antibody was tested as monotherapy in the B16F10 murine tumor model. C57BL/6 mice were subcutaneously injected with B16F10 cells on
CD11b結合亦證實不同之同基因LLC1腫瘤模型中之效用。使用5mg/kg抗CD11b抗體之治療強效抑制LLC1腫瘤之腫瘤生長(圖7)並延長動物存活(圖8)(中值存活天數對照IgG：31天；抗CD11b：42天)。 CD11b binding also demonstrated utility in different syngeneic LLC1 tumor models. Treatment with 5 mg/kg anti-CD11b antibody potently inhibited tumor growth of LLC1 tumors (Figure 7) and prolonged animal survival (Figure 8) (median survival days control IgG: 31 days; anti-CD11b: 42 days).
實例3：CD11b結合及免疫查核點療法於抗腫瘤免疫力中之協同效應Example 3: Synergistic effect of CD11b binding and immune checkpoint therapy in anti-tumor immunity
經組合之治療證實不同之同基因LLC1肺轉移模型中之效用。使用抗CD11b(10mg/kg)+抗PD-1(10mg/kg)抗體之治療強效減少LLC1腫瘤之腫瘤結節(圖9)(在第15天，對照IgG相比於抗CD11b相比於抗PD-1相比於抗CD11b+抗PD-1=200±13相比於167相比於164±11相
比於131±2)並延長動物存活(圖10)(中值存活天數對照IgG：24天；抗CD11b：24天；抗PD-1：22天；抗CD11b+抗PD-1：26天)。
Combination therapy demonstrated utility in different syngeneic LLC1 lung metastasis models. Treatment with anti-CD11b (10 mg/kg) + anti-PD-1 (10 mg/kg) antibody potently reduced tumor nodules of LLC1 tumors (Figure 9) (at
實例4：CD11b結合及化學療法於抗腫瘤免疫力中之協同效應Example 4: Synergistic effect of CD11b binding and chemotherapy in anti-tumor immunity
CD11b結合亦增強化學療法。在此實例中，在第0天植入B16F10細胞。在第7天，對小鼠腹腔內注射對照IgG(5mg/kg)、抗小鼠CD11b抗體(5mg/kg)、紫杉醇(10mg/kg)+對照IgG(5mg/kg)或紫杉醇(10mg/kg)+抗CD11b(5mg/kg)。每三至四天重複注射。如圖11中所示，使用紫杉醇加抗CD11b抗體之組合之治療有效控制腫瘤生長。組合治療之有效性亦證實於長期存活率中(圖12)(中值存活天數對照IgG：25天；抗CD11b：32天；紫杉醇+對照IgG：25天；紫杉醇+抗CD11b：32天)。
CD11b binding also enhances chemotherapy. In this example, B16F10 cells were implanted on
實例5：在來自患有敗血性休克之病患之LPS誘發之免疫抑制單核細胞或單核細胞中，以抗CD11b抗體結合CD11b在細胞經LPS挑戰時亦減少PD-L1表現。Example 5: In LPS-induced immunosuppressed monocytes or monocytes from patients with septic shock, binding of CD11b with an anti-CD11b antibody also reduced PD-L1 expression when the cells were challenged with LPS.
敗血症(一種由嚴重感染引起之全身性炎性反應症候群)仍係全球健康護理問題及威脅生命之疾病。越發明顯的係，敗血症啟動隨時間變化之雙相免疫反應。在敗血症之初始階段期間，全身性高度炎性免疫反應可全身產生炎性細胞介素(包括介白素(IL)-1、IL-6及腫瘤壞死因子(TNF)-α)，其可引起血液動力學不穩定性、多器官功能障礙、凝血異常及休克。隨高度炎性免疫反應而來者係幾乎同時產生抗炎性細胞介素(包括IL-10及腫瘤生長因子(TGF)-β)；免疫系統迅速進入免疫高度活性狀態(稱為免疫麻痺)，其表現為無法根除原發性感染及後期醫院內感染之發展。於患有敗血症之病患中觀察到之免疫麻痺之指示項包括淋巴細胞異常、單核細胞去活化伴減弱之人類白細胞抗原-DR(HLA-DR)表面表現、及在活體外刺激下之低TNF-α產生。單核細胞HLA-DR表現之持續減少指示患有敗血症之病患之醫院內感染及死亡 之高風險。最近，觀察到患有敗血性休克之病患之單核細胞中之高程式死亡配體-1(PD-L1)表現且其與繼發性醫院內感染及死亡率之高發生相關(Guignant C、Lepape A、Huang X、Kherouf H、Denis L等人，(2011)Programmed death-1 levels correlate with increased mortality,nosocomial infection and immune dysfunctions in septic shock patients.Crit Care 15：R99)。因此，單核細胞中之PD-L1表現之水平可充當免疫麻痺之新穎標誌。 Sepsis, a systemic inflammatory response syndrome caused by severe infection, remains a global healthcare problem and life-threatening disease. It is increasingly apparent that sepsis initiates a biphasic immune response that varies over time. During the initial stages of sepsis, a systemic hyperinflammatory immune response can systemically produce inflammatory interleukins (including interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α), which can cause Hemodynamic instability, multiple organ dysfunction, coagulation abnormalities, and shock. Those who come with the highly inflammatory immune response almost simultaneously produce anti-inflammatory cytokines (including IL-10 and tumor growth factor (TGF)-β); the immune system quickly enters a highly active state of immunity (called immune paralysis), It is manifested by the inability to eradicate the primary infection and the development of late nosocomial infections. Indices of immune paralysis observed in patients with sepsis include lymphocyte abnormalities, monocyte inactivation with attenuated surface expression of human leukocyte antigen-DR (HLA-DR), and low TNF-alpha production. Sustained reduction in monocyte HLA-DR expression indicates a high risk of nosocomial infection and death in patients with sepsis. Recently, high programmed death ligand-1 (PD-L1) expression in monocytes of patients with septic shock was observed and was associated with a high incidence of secondary nosocomial infections and mortality ( Guignant C , Lepape A, Huang X, Kherouf H, Denis L et al., (2011) Programmed death-1 levels correlate with increased mortality, nosocomial infection and immune dysfunctions in septic shock patients. Crit Care 15: R99). Therefore, the level of PD-L1 expression in monocytes may serve as a novel marker of immune paralysis.
已報告單核細胞對LPS歷時2天之先前曝露將使其等變為免疫抑制單核細胞(Wolk K，Docke WD，von Baehr V，Volk HD及Sabat R.(2000)Impaired antigen presentation by human monocytes during endotoxin tolerance.Blood 96：218)。臨床上，此等細胞與免疫麻痺及死亡率相關。吾人建立可複製之LPS誘發之免疫抑制單核細胞，其中人類單核細胞經100ng/ml LPS預先培養2天。相較於新鮮之經分離之人類單核細胞，LPS誘發之免疫抑制單核細胞在細胞表面上表現更高之PD-L1水平(圖13A)。為檢測CD11b調節劑在LPS誘發之免疫抑制單核細胞中之效應，使細胞在IgG1或抗CD11b抗體(ICRF44)之存在下曝露於1μg/ml LPS，歷時18hr。如圖13B中所示，以抗CD11b抗體(ICRF44)結合CD11b在細胞經LPS激發時減少LPS誘發之免疫抑制單核細胞中之PD-L1表現。此外，抗CD11b抗體(ICRF44)治療亦一經活體外LPS刺激即減少來自患有敗血性休克之病患之單核細胞中之PD-L1表現(圖14)。 Previous exposure of monocytes to LPS for 2 days has been reported to render them immunosuppressive monocytes ( Wolk K, Docke WD, von Baehr V, Volk HD and Sabat R. (2000) Impaired antigen presentation by human monocytes during endotoxin tolerance. Blood 96:218 ). Clinically, these cells are associated with immune paralysis and mortality. We established replicable LPS-induced immunosuppressive monocytes, in which human monocytes were pre-cultured with 100 ng/ml LPS for 2 days. LPS-induced immunosuppressive monocytes exhibited higher levels of PD-L1 on the cell surface compared to freshly isolated human monocytes (Fig. 13A). To test the effect of CD11b modulators in LPS-induced immunosuppressive monocytes, cells were exposed to 1 μg/ml LPS for 18 hr in the presence of IgG1 or anti-CD11b antibody (ICRF44). As shown in Figure 13B, binding of CD11b with an anti-CD11b antibody (ICRF44) reduced PD-L1 expression in LPS-induced immunosuppressive monocytes when the cells were challenged with LPS. Furthermore, anti-CD11b antibody (ICRF44) treatment also reduced PD-L1 expression in monocytes from patients with septic shock upon LPS stimulation in vitro ( FIG. 14 ).
實例6：結合人類CD11b之人類化抗體Example 6: Humanized antibodies that bind to human CD11b
針對人類抗體資料庫搜索鼠科抗人類CD11b抗體之可變域序列。選擇10組對鼠科抗人類CD11b具有高同源性之人類框架序列作為用於輕鏈及重鏈兩者之人類接受者。同時，分析N-醣化基序。因此應避免候選人類可變區中之潛在醣化位置。10個輕鏈之人類化可變域表示為 VL1、VL2、VL3、VL4、VL5、LC1、LC2、LC3、LC4及LC5(圖15)，而10個重鏈之人類化可變域表示為VH1、VH2、VH3、VH4、VH5、HC1、HC2、HC3、HC4及HC5(圖16)。此等輕鏈及重鏈肽序列可提供以高親和力結合至人類抗CD11b之人類化抗體或抗原結合部分。 The variable domain sequences of murine anti-human CD11b antibodies were searched against human antibody databases. Ten sets of human framework sequences with high homology to murine anti-human CD11b were selected as human recipients for both light and heavy chains. Simultaneously, N-glycosylation motifs were analyzed. Potential glycation positions in candidate class variable regions should therefore be avoided. The humanized variable domains of the 10 light chains are represented as VL1, VL2, VL3, VL4, VL5, LC1, LC2, LC3, LC4 and LC5 (Figure 15), while the humanized variable domains of the 10 heavy chains are represented as VH1, VH2, VH3, VH4, VH5, HC1, HC2 , HC3, HC4 and HC5 (Figure 16). These light and heavy chain peptide sequences can provide humanized antibodies or antigen binding portions that bind to human anti-CD11b with high affinity.
實例7：人類化CD11b抗體之功能活性Example 7: Functional activity of humanized CD11b antibodies
人類化抗CD11b抗體之特異性藉由流動式細胞測量術使用表現CD11b之K562細胞測定。如圖17中所示，此實例中之所有人類化抗CD11b抗體可結合至經CD11b轉染之K562細胞。相反，此等抗體不結合至K562細胞。綜合而言，此等結果證實人類化抗CD11b抗體可特異性結合至CD11b抗原決定基。 The specificity of humanized anti-CD11b antibodies was determined by flow cytometry using K562 cells expressing CD11b. As shown in Figure 17, all humanized anti-CD11b antibodies in this example could bind to K562 cells transfected with CD11b. In contrast, these antibodies did not bind to K562 cells. Taken together, these results demonstrate that humanized anti-CD11b antibodies can specifically bind to CD11b epitopes.
為檢測人類化抗CD11b抗體之功能活性，抗體係用於量測抗體抑制單核細胞之表面上之PD-L1表現之能力之LPS致敏單核細胞中。如圖18中所示，藉由LPS上調PD-L1可藉由人類化抗CD11b抗體顯著減少。 To test the functional activity of humanized anti-CD11b antibodies, the antibodies were used in LPS-sensitized monocytes to measure the ability of the antibodies to inhibit PD-L1 expression on the surface of monocytes. As shown in Figure 18, upregulation of PD-L1 by LPS could be significantly reduced by humanized anti-CD11b antibody.
總而言之，吾人描述一系列針對人類αM域之人類化抗CD11b抗體。人類化抗CD11b抗體之結合可減少LPS致敏單核細胞上之PD-L1表現。 In summary, we describe a series of humanized anti-CD11b antibodies directed against the human αM domain. Binding of a humanized anti-CD11b antibody reduces PD-L1 expression on LPS-sensitized monocytes.
<110> 台灣基督長老教會馬偕醫療財團法人馬偕紀念醫院 <110> Taiwan Presbyterian Church Mackay Medical Foundation Mackay Memorial Hospital
<120> 調控免疫反應之方法及抗體 <120> Methods and antibodies for modulating immune response
<130> L88340/CN24523 <130> L88340/CN24523
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<170> PatentIn version 3.5 <170> PatentIn version 3.5
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<213> 人造 <213> Artificial
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<223> H-CDR1 <223> H-CDR1
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<212> PRT <212> PRT
<213> 人造 <213> Artificial
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<400> 2
<210> 3 <210> 3
<211> 17 <211> 17
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> H-CDR2 <223> H-CDR2
<400> 3
<210> 4 <210> 4
<211> 16 <211> 16
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> H-CDR2 <223> H-CDR2
<400> 4
<210> 5 <210> 5
<211> 9 <211> 9
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> H-CDR3 <223> H-CDR3
<400> 5
<210> 6 <210> 6
<211> 10 <211> 10
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> H-CDR3 <223> H-CDR3
<400> 6
<210> 7 <210> 7
<211> 11 <211> 11
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> L-CDR1 <223> L-CDR1
<400> 7
<210> 8 <210> 8
<211> 17 <211> 17
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> L-CDR1 <223> L-CDR1
<400> 8
<210> 9 <210> 9
<211> 7 <211> 7
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> L-CDR2 <223> L-CDR2
<400> 9
<210> 10 <210> 10
<211> 7 <211> 7
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> L-CDR2 <223> L-CDR2
<400> 10
<210> 11 <210> 11
<211> 10 <211> 10
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> L-CDR3 <223> L-CDR3
<400> 11
<210> 12 <210> 12
<211> 9 <211> 9
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> L-CDR3 <223> L-CDR3
<400> 12
<210> 13 <210> 13
<211> 118 <211> 118
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之重鏈可變區-VH1 <223> Heavy chain variable region-VH1 of humanized anti-CD11b antibody
<400> 13
<210> 14 <210> 14
<211> 118 <211> 118
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之重鏈可變區-VH2 <223> Heavy chain variable region-VH2 of humanized anti-CD11b antibody
<400> 14
<210> 15 <210> 15
<211> 118 <211> 118
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之重鏈可變區-VH3 <223> Heavy chain variable region-VH3 of humanized anti-CD11b antibody
<400> 15
<210> 16 <210> 16
<211> 118 <211> 118
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之重鏈可變區-VH4 <223> Heavy chain variable region-VH4 of humanized anti-CD11b antibody
<400> 16
<210> 17 <210> 17
<211> 118 <211> 118
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之重鏈可變區-VH5 <223> Heavy chain variable region-VH5 of humanized anti-CD11b antibody
<400> 17
<210> 18 <210> 18
<211> 117 <211> 117
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之重鏈可變區-HC1 <223> Heavy Chain Variable Region of Humanized Anti-CD11b Antibody-HC1
<400> 18
<210> 19 <210> 19
<211> 117 <211> 117
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之重鏈可變區-HC2 <223> Heavy Chain Variable Region of Humanized Anti-CD11b Antibody-HC2
<400> 19
<210> 20 <210> 20
<211> 117 <211> 117
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之重鏈可變區-HC3 <223> Heavy Chain Variable Region of Humanized Anti-CD11b Antibody-HC3
<400> 20
<210> 21 <210> 21
<211> 117 <211> 117
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之重鏈可變區-HC4 <223> Heavy Chain Variable Region of Humanized Anti-CD11b Antibody-HC4
<400> 21
<210> 22 <210> 22
<211> 117 <211> 117
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之重鏈可變區-HC5 <223> Heavy Chain Variable Region of Humanized Anti-CD11b Antibody-HC5
<400> 22
<210> 23 <210> 23
<211> 108 <211> 108
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之輕鏈可變區-VL1 <223> Light chain variable region-VL1 of humanized anti-CD11b antibody
<400> 23
<210> 24 <210> 24
<211> 108 <211> 108
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之輕鏈可變區-VL2 <223> Light chain variable region-VL2 of humanized anti-CD11b antibody
<400> 24
<210> 25 <210> 25
<211> 108 <211> 108
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之輕鏈可變區-VL3 <223> Light chain variable region-VL3 of humanized anti-CD11b antibody
<400> 25
<210> 26 <210> 26
<211> 108 <211> 108
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之輕鏈可變區-VL4 <223> Light chain variable region-VL4 of humanized anti-CD11b antibody
<400> 26
<210> 27 <210> 27
<211> 108 <211> 108
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之輕鏈可變區-VL5 <223> Light chain variable region-VL5 of humanized anti-CD11b antibody
<400> 27
<210> 28 <210> 28
<211> 113 <211> 113
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之輕鏈可變區-LC1 <223> Light chain variable region of humanized anti-CD11b antibody-LC1
<400> 28
<210> 29 <210> 29
<211> 113 <211> 113
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之輕鏈可變區-LC2 <223> Light chain variable region of humanized anti-CD11b antibody-LC2
<400> 29
<210> 30 <210> 30
<211> 113 <211> 113
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之輕鏈可變區-LC3 <223> Light chain variable region of humanized anti-CD11b antibody-LC3
<400> 30
<210> 31 <210> 31
<211> 113 <211> 113
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之輕鏈可變區-LC4 <223> Light chain variable region of humanized anti-CD11b antibody-LC4
<400> 31
<210> 32 <210> 32
<211> 113 <211> 113
<212> PRT <212> PRT
<213> 人造 <213> Artificial
<220> <220>
<223> 人類化抗CD11b抗體之輕鏈可變區-LC5 <223> Light chain variable region of humanized anti-CD11b antibody-LC5
<400> 32
Claims (12)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201562174681P | 2015-06-12 | 2015-06-12 | |
| US62/174,681 | 2015-06-12 |
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|---|---|
| TW201713346A TW201713346A (en) | 2017-04-16 |
| TWI790193B true TWI790193B (en) | 2023-01-21 |
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| TW105118446A TW201704258A (en) | 2015-06-12 | 2016-06-13 | Methods and polypeptides for modulation of immunoresponse |
| TW105118447A TWI790193B (en) | 2015-06-12 | 2016-06-13 | Methods and antibodies for modulation of immunoresponse |
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| TW201704258A (en) | 2017-02-01 |
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