TWI742008B - Immune modulation and treatment of solid tumors with antibodies that specifically bind cd38 - Google Patents
Immune modulation and treatment of solid tumors with antibodies that specifically bind cd38 Download PDFInfo
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本申請案主張2016年6月24日申請之美國申請案第15/191808號、2016年6月24日申請之國際申請案第US16/39165號、2016年5月4日申請之美國臨時申請案第62/331,489號、2015年12月4日申請之美國臨時申請案第62/263,307號、及2015年11月4日申請之美國臨時申請案第62/250,566號、以及2015年11月2日申請之美國臨時申請案第62/249,546號之權益,其全部內容以引用的方式併入本文中。 This application claims U.S. Application No. 15/191808 filed on June 24, 2016, International Application No. US16/39165 filed on June 24, 2016, and U.S. Provisional Application filed on May 4, 2016 No. 62/331,489, U.S. Provisional Application No. 62/263,307 filed on December 4, 2015, and U.S. Provisional Application No. 62/250,566 filed on November 4, 2015, and November 2, 2015 The rights and interests of the US Provisional Application No. 62/249,546, the entire contents of which are incorporated herein by reference.
本發明係關於用特異性結合CD38之抗體免疫調節及治療固態腫瘤之方法。 The present invention relates to methods for immunomodulating and treating solid tumors with antibodies that specifically bind to CD38.
免疫系統係受共刺激及共抑制配位體及受體之網路嚴密控制。這些分子為T細胞活化提供二級訊號,且提供正訊號及負訊號之平衡網路,以最大化針對感染及腫瘤之免疫反應,同時限制對自身的免疫性(Wang等人,(Epub Mar.7,2011)J Exp Med 208(3):577-92;Lepenies等人,(2008)Endocr Metab Immune Disord Drug Targets 8:279-288)。 The immune system is tightly controlled by a network of co-stimulatory and co-inhibitory ligands and receptors. These molecules provide secondary signals for T cell activation, and provide a balanced network of positive and negative signals to maximize the immune response to infections and tumors, while limiting immunity to self (Wang et al., (Epub Mar. 7, 2011) J Exp Med 208(3): 577-92; Lepenies et al. (2008) Endocr Metab Immune Disord Drug Targets 8: 279-288).
治療固態腫瘤之免疫檢查點療法(其靶向T細胞中之共抑制途徑以促進抗瘤免疫反應)在核准抗CTLA-4及抗PD-1抗體 YERVOY®(伊匹單抗(ipilimumab))、KEYTRUDA®(派立珠單抗(pembrolizumab))、及OPDIVO®(尼沃魯單抗(nivolumab))之情況下導致了癌症病患之臨床照護的進步。儘管抗PD-1/PD-L1抗體展示了促進患有多種固態腫瘤之病患之臨床反應,但是反應率仍然相當低,其在經預治療的病患中係15%至20%(Swaika等人,(2015)Mol Immunol doi:10.1016/j.molimm.2015.02.009)。 The immune checkpoint therapy for the treatment of solid tumors (which targets the co-suppressive pathway in T cells to promote anti-tumor immune responses) is approved for anti-CTLA-4 and anti-PD-1 antibodies YERVOY ® (ipilimumab), KEYTRUDA ® (pembrolizumab) and OPDIVO ® (nivolumab) have led to the advancement of clinical care for cancer patients. Although anti-PD-1/PD-L1 antibodies have been shown to promote clinical response in patients with a variety of solid tumors, the response rate is still quite low, which is 15% to 20% in pre-treated patients (Swaika et al. Human, (2015) Mol Immunol doi: 10.1016/j.molimm.2015.02.009).
儘管自然殺手細胞(NK)、樹突細胞(DC)、及效應T細胞能夠驅動強力的抗瘤反應,但腫瘤細胞時常誘導免疫抑制性微環境,其有利於免疫抑制性免疫細胞群之發展,諸如骨髓衍生抑制細胞(MDSC)、調節T細胞(Treg)、或調節B細胞(Breg),其等導致癌症病患及實驗腫瘤模型中之腫瘤免疫耐受性及免疫療法方案之失敗。 Although natural killer cells (NK), dendritic cells (DC), and effector T cells can drive a strong anti-tumor response, tumor cells often induce an immunosuppressive microenvironment, which is conducive to the development of immunosuppressive immune cell populations. Such as bone marrow-derived suppressor cells (MDSC), regulatory T cells (Treg), or regulatory B cells (Breg), which lead to the failure of tumor immune tolerance and immunotherapy regimens in cancer patients and experimental tumor models.
因此,仍需要發展新的癌症免疫療法,其等誘導針對腫瘤之適應性免疫反應或靶向免疫抑制性免疫細胞。 Therefore, there is still a need to develop new cancer immunotherapies, which induce adaptive immune responses against tumors or target immunosuppressive immune cells.
本發明提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體。 The present invention provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need.
本發明亦提供一種用於治療患有調節T細胞(Treg)媒介之疾病的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體。 The present invention also provides a method for treating a disease mediated by regulatory T cells (Treg), which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to a patient in need of the disease.
本發明亦提供一種用於治療患有骨髓衍生抑制細胞(MDSC)媒介之疾病的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體。 The present invention also provides a method for treating a disease mediated by bone marrow-derived suppressor cells (MDSC), which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need.
本發明亦提供一種用於治療患有調節B細胞(Breg)媒介之疾病的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體。 The present invention also provides a method for the treatment of a disease mediated by regulatory B cells (Breg), which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to a patient in need of the disease.
本發明亦提供一種抑制調節T細胞(Treg)之活性的方法,其包含使該Treg接觸特異性結合CD38之抗體。 The present invention also provides a method for inhibiting the activity of regulatory T cells (Treg), which comprises contacting the Treg with an antibody that specifically binds to CD38.
本發明亦提供一種抑制骨髓衍生抑制細胞(MDSC)之活性的方法,其包含使該MDSC接觸特異性結合CD38之抗體。 The present invention also provides a method for inhibiting the activity of bone marrow-derived suppressor cells (MDSC), which comprises contacting the MDSC with an antibody that specifically binds to CD38.
本發明亦提供一種抑制調節B細胞(Breg)之活性的方法,其包含使該Breg接觸特異性結合CD38之抗體。 The present invention also provides a method for inhibiting the activity of regulating B cells (Breg), which comprises contacting the Breg with an antibody that specifically binds to CD38.
本發明亦提供一種增強病患之免疫反應的方法,其包含向該病患投予特異性結合CD38之抗體。 The present invention also provides a method for enhancing the immune response of a patient, which comprises administering an antibody that specifically binds to CD38 to the patient.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含藉由向該病患投予特異性結合CD38之抗體來減少該病患中Treg細胞之數目。 The present invention also provides a method for treating a patient suffering from solid tumors, which comprises reducing the number of Treg cells in the patient by administering an antibody that specifically binds to CD38 to the patient.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含藉由向該病患投予特異性結合CD38之抗體來減少該病患中骨髓衍生抑制細胞(MDSC)之數目。 The present invention also provides a method for treating a patient suffering from solid tumors, which comprises reducing the number of bone marrow-derived suppressor cells (MDSC) in the patient by administering to the patient an antibody that specifically binds to CD38.
本發明亦提供一種抑制免疫抑制細胞之活性的方法,其包含使該免疫抑制細胞接觸特異性結合CD38之抗體。 The present invention also provides a method for inhibiting the activity of immunosuppressive cells, which comprises contacting the immunosuppressive cells with an antibody that specifically binds to CD38.
本發明亦提供一種治療患有病毒感染之病患的方法,其包含向有彼之需要之該病患投予特異性結合CD38之抗體。 The present invention also provides a method for treating a patient suffering from a viral infection, which comprises administering an antibody that specifically binds to CD38 to the patient in need.
圖1顯示,病患中淋巴球數目之中位數係對以8mg/kg(上方線)或16mg/kg(下方線)劑量的DARZALEXTM(達拉單抗)治療反應隨時間推移而增加,並在治療結束後淋巴球數目返回至基期。研究:SIRIUS。X軸指示時間,其係以治療週期及在各治療週期內之給藥天數表示(C1D1:第1週期第1天;C1D4:第1週期第4天等等)。SCR:基期;EOT:治療結束;WK:週;POST-WK:在治療後之指定週數;post-PD FU:進展後之追蹤(follow-up)。以灰色陰影強調的範圍指示反應者之各訪視之數據點的四分位數間距(IQR)係25至27%。 Figure 1 shows that the median number of lymphocytes in the patient increases with time in response to DARZALEX TM (dalazumab) at a dose of 8 mg/kg (upper line) or 16 mg/kg (lower line). And after the end of treatment, the number of lymphocytes returned to the baseline. Research: SIRIUS. The X-axis indicates the time, which is represented by the treatment cycle and the number of days of administration in each treatment cycle (C1D1: the first day of the first cycle; C1D4: the fourth day of the first cycle, etc.). SCR: base period; EOT: end of treatment; WK: weeks; POST-WK: designated weeks after treatment; post-PD FU: follow-up. The range highlighted in gray indicates that the interquartile range (IQR) of the data points for each visit of the respondent is 25 to 27%.
圖2顯示針對各個別病患(淺灰色線)經DARZALEXTM(達拉單抗)治療的病患之周邊血液中CD3+ T細胞之絕對計數自基期的變 化百分比(%)。研究:SIRIUS(MMY2002)。X軸指示時間,其係以治療週期及在各治療週期內之給藥天數表示(C1D1:第1週期第1天;C1D4:第1週期第4天等等)。WK:週;POST-WK:在治療後之指定週數;POST-PD FU:進展後之追蹤。黑色線顯示所有病患之中位數變化%。 Figure 2 shows the percentage change (%) of the absolute counts of CD3 + T cells in the peripheral blood of patients treated with DARZALEX TM (dalazumab) for each individual patient (light gray line) from the baseline. Research: SIRIUS (MMY2002). The X-axis indicates the time, which is represented by the treatment cycle and the number of days of administration in each treatment cycle (C1D1: the first day of the first cycle; C1D4: the fourth day of the first cycle, etc.). WK: week; POST-WK: specified number of weeks after treatment; POST-PD FU: follow-up after progress. The black line shows the% change in the median for all patients.
圖3顯示針對各個別病患(淺灰色線)經DARZALEXTM(達拉單抗)治療的病患之周邊血液中CD4+ T細胞之絕對計數自基期的變化百分比(%)。研究:SIRIUS。X軸指示時間,其係以治療週期及在各治療週期內之給藥天數表示(C1D1:第1週期第1天;C1D4:第1週期第4天等等)。WK:週;POST-TMT:治療後。黑色線顯示所有病患之中位數變化%。 Figure 3 shows the percentage change (%) of the absolute count of CD4 + T cells in the peripheral blood of patients treated with DARZALEX TM (dalazumab) for each individual patient (light gray line) from the baseline. Research: SIRIUS. The X-axis indicates the time, which is represented by the treatment cycle and the number of days of administration in each treatment cycle (C1D1: the first day of the first cycle; C1D4: the fourth day of the first cycle, etc.). WK: Week; POST-TMT: After treatment. The black line shows the% change in the median for all patients.
圖4顯示針對各個別病患(淺灰色線)經DARZALEXTM(達拉單抗)治療的病患之周邊血液中CD8+ T細胞之絕對計數自基期的變化百分比(%)。研究:SIRIUS。X軸指示時間,其係以治療週期及在各治療週期內之給藥天數表示(C1D1:第1週期,第1天;C1D4:第1週期第4天等等)。WK:週;Pre-PD FU:進展前之追蹤;Post-PD FU:進展後之追蹤。黑色線顯示所有病患之中位數變化%。
Figure 4 shows the percentage change (%) of the absolute counts of CD8 + T cells in the peripheral blood of patients treated with DARZALEX TM (dalazumab) for each individual patient (light gray line) from the baseline. Research: SIRIUS. The X axis indicates the time, which is represented by the treatment cycle and the number of days of administration in each treatment cycle (C1D1:
圖5顯示骨髓抽出物中CD45+CD3+細胞之數目(測量為淋巴球之百分比)在以劑量8mg/kg或16mg/kg的DARZALEXTM(達拉單抗)治療期間隨時間推移而增加。該圖表包括如所指示的反應者及無反應者。研究:SIRIUS。X軸指示時間,其係以治療週期及在各治療週期內之給藥天數表示(C2D22:第2週期第22天;等等)。SCR:基期;Post-PD FU1:進展後之追蹤。以灰色陰影強調的範圍指示針對以8mg/kg給藥的無反應者、以16mg/kg給藥的反應者、或以16mg/kg給藥的無反應者,各訪視之數據點的四分位數間距(IQR)分別係25至27%。NR:無反應者;R:反應者。 Figure 5 shows that the number of CD45 + CD3 + cells in bone marrow aspirates (measured as a percentage of lymphocytes) increased over time during treatment with DARZALEX™ (dalazumab) at a dose of 8 mg/kg or 16 mg/kg. The chart includes responders and non-responders as indicated. Research: SIRIUS. The X axis indicates the time, which is expressed in terms of the treatment cycle and the number of days of administration in each treatment cycle (C2D22: 22nd day of the second cycle; etc.). SCR: base period; Post-PD FU1: follow-up after progress. The range highlighted with gray shading indicates a quarter of the data points of each visit for non-responders who were dosed at 8 mg/kg, responders who were dosed at 16 mg/kg, or non-responders who were dosed at 16 mg/kg. The digit spacing (IQR) is 25 to 27%, respectively. NR: non-responders; R: responders.
圖6顯示骨髓抽出物中CD45+CD3+ CD8+細胞之數目(呈淋巴球之百分比測量)在以劑量8mg/kg或16mg/kg的DARZALEXTM (達拉單抗)治療期間隨時間推移而增加。該圖表包括如所指示的反應者及無反應者。研究:SIRIUS。X軸指示時間,其係以治療週期及在各治療週期內之給藥天數表示(C2D22:第2週期第22天等等)。SCR:基期;Post-PD FU1:進展後之追蹤。以灰色陰影強調的範圍指示針對以8mg/kg給藥的無反應者、以16mg/kg給藥的反應者、或以16mg/kg給藥的無反應者,各訪視之數據點的四分位數間距(IQR)分別係25至27%。NR:無反應者;R:反應者。 Figure 6 shows that the number of CD45 + CD3 + CD8 + cells in bone marrow aspirate (measured as a percentage of lymphocytes) increased over time during treatment with DARZALEX TM (dalazumab) at a dose of 8 mg/kg or 16 mg/kg . The chart includes responders and non-responders as indicated. Research: SIRIUS. The X axis indicates the time, which is represented by the treatment cycle and the number of days of administration in each treatment cycle (C2D22: 22nd day of the second cycle, etc.). SCR: base period; Post-PD FU1: follow-up after progress. The range highlighted with gray shading indicates a quarter of the data points of each visit for non-responders who were dosed at 8 mg/kg, responders who were dosed at 16 mg/kg, or non-responders who were dosed at 16 mg/kg. The digit spacing (IQR) is 25 to 27%, respectively. NR: non-responders; R: responders.
圖7A顯示在DARZALEXTM(達拉單抗)治療期間,以所有治療病患之中位數值所表示的周邊血液中CD8+/Treg及CD8+/CD4+細胞之比率增加。時間點:C1D1:第1週期第1天;C3D1:第3週期第1天;C4D1:第4週期第1天。研究:SIRIUS。SRC:基期。 Figure 7A shows that during DARZALEX TM (darumab) treatment, the ratio of CD8 + /Treg and CD8 + /CD4 + cells in the peripheral blood represented by the median value of all treated patients increased. Time point: C1D1: the first day of the first cycle; C3D1: the first day of the third cycle; C4D1: the first day of the fourth cycle. Research: SIRIUS. SRC: Base period.
圖7B顯示在DARZALEXTM(達拉單抗)治療期間,以所有經治療病患之中位數值所表示的骨髓抽出物中CD8+/Treg細胞之比率隨時間推移而增加。時間點:C1D1:第1週期第1天;C3D1:第3週期第1天;C4D1:第4週期第1天。研究:SIRIUS。 Fig. 7B shows that the ratio of CD8 + /Treg cells in the bone marrow aspirate expressed as the median value of all treated patients during DARZALEX TM (dalazumab) treatment increased over time. Time point: C1D1: the first day of the first cycle; C3D1: the first day of the third cycle; C4D1: the first day of the fourth cycle. Research: SIRIUS.
圖8A顯示當相較於無反應者時,反應者之CD8+ T細胞純系性增加,如使用特定純系細胞之豐度變化(CIA)%所測量。研究:GEN501 17病患子集。 Figure 8A shows that when compared to non-responders, the CD8 + T cell lineage of responders increases, as measured using the change in abundance (CIA)% of specific lineage cells. Study: A subset of GEN501 17 patients.
圖8B顯示DARZALEXTM(達拉單抗)治療之前之於之後,個別病患中CD8+ T細胞純系性之倍數變化。反應者係經星號指示。純系性係測量為特定純系細胞之豐度變化(CIA)倍數。研究:GEN501 17病患子集。 Figure 8B shows the fold change of CD8 + T cell homologousness in individual patients before and after DARZALEX TM (dalazumab) treatment. Respondents are indicated by asterisks. Homogenous lineage is measured as a multiple of the change in abundance (CIA) of a specific inline cell line. Study: A subset of GEN501 17 patients.
圖8C顯示當相較於無反應者(B組)時,反應者(A組)在TCR貯庫中總擴增較大,其係使用CIA(豐度變化)測量。P=0.037。研究:GEN501 17病患子集。 Figure 8C shows that when compared to non-responders (group B), responders (group A) have a greater total amplification in the TCR reservoir, which is measured using CIA (change in abundance). P=0.037. Study: A subset of GEN501 17 patients.
圖8D顯示反應者及無反應者中各擴增T細胞殖株之絕對豐度變化(CIA)之和。反應者(A組)與無反應者(B組)之間的P=0.035。研究:GEN501 17病患子集。 Figure 8D shows the sum of the absolute abundance change (CIA) of each expanded T cell clone in responders and non-responders. The P=0.035 between responders (group A) and non-responders (group B). Study: A subset of GEN501 17 patients.
圖8E顯示反應者(A組)及無反應者(B組)中之單個細胞殖株的最大CIA。研究:GEN501 17病患子集。 Figure 8E shows the maximum CIA of single cell clones in responders (group A) and non-responders (group B). Study: A subset of GEN501 17 patients.
圖8F顯示當相較於無反應者(B組)時,反應者(A組)之單一殖株之最大擴增較大,其係使用最大CIA%測量。P=0.0477。研究:GEN501 17病患子集。 Figure 8F shows that the maximum amplification of a single clone of the responder (group A) is greater when compared to the non-responder (group B), which is measured using the maximum CIA%. P=0.0477. Study: A subset of GEN501 17 patients.
圖9A顯示在基期、或在治療2週、4週、或8週、或復發後,無反應者(NR,黑色方形)及對DARZALEXTM(達拉單抗)具有至少最小反應之病患(MR,白色方形)的周邊血液中之CD8+初始細胞(nave cell)的百分比(%)。研究:GEN501 17病患子集。**p=1.82×10-4。 Figure 9A shows that in the baseline period, or after 2 weeks, 4 weeks, or 8 weeks of treatment, or after recurrence, non-responders (NR, black squares) and patients with at least the least response to DARZALEX TM (dalazumab) ( MR, white square) CD8 + initial cells in the peripheral blood (na ve cell) percentage (%). Study: A subset of GEN501 17 patients. **p=1.82×10 -4 .
圖9B顯示在基期、或在治療2週、4週、或8週、或復發後,無反應者(NR,黑色方形)及對DARZALEXTM(達拉單抗)具有至少最小反應之病患(MR,白色方形)的周邊血液中之CD8+中央記憶細胞(Tem)之百分比。研究:GEN501 17病患子集。*p=4.88×10-2。 Figure 9B shows that in the baseline period, or after 2 weeks, 4 weeks, or 8 weeks of treatment, or after recurrence, non-responders (NR, black squares) and patients with at least the least response to DARZALEX TM (dalazumab) ( MR, white square ) the percentage of CD8 + central memory cells (Tem) in the peripheral blood. Study: A subset of GEN501 17 patients. *p=4.88×10 -2 .
圖9C顯示在基期、或在治療第1、4、或8週、或復發後,周邊血液中HLA I類限制性CD8+ T細胞之增加百分比。研究:GEN501 17病患子集。 Figure 9C shows the percentage increase of HLA class I restricted CD8 + T cells in the peripheral blood during the baseline period, or after 1, 4, or 8 weeks of treatment, or after recurrence. Study: A subset of GEN501 17 patients.
圖9D顯示在基期或治療中,CD38在周邊血液之CD8+初始T細胞及CD8+中央記憶細胞(Tem)中以低水平表現。研究:GEN501 17病患子集。MFI:平均螢光強度。 Figure 9D shows that CD38 is expressed at low levels in CD8 + naive T cells and CD8 + central memory cells (Tem) in the peripheral blood during the baseline or treatment. Study: A subset of GEN501 17 patients. MFI: Average fluorescence intensity.
圖10A顯示FACS分析之分佈圖,其顯示在基期,多發性骨髓瘤病患中Treg(CD3+ CD3+CD4+CD25+CD127dim)之頻率(上分佈圖,P4細胞群)及CD38+ Treg在Treg群內之頻率(下分佈圖,P5細胞群)。研究:GEN501 17病患子集。 Figure 10A shows the distribution diagram of FACS analysis, which shows that in the base stage, the frequency of Treg (CD3 + CD3 + CD4 + CD25 + CD127 dim ) in patients with multiple myeloma (upper distribution diagram, P4 cell population) and CD38 + Treg Frequency within the Treg population (distribution below, P5 cell population). Study: A subset of GEN501 17 patients.
圖10B顯示FACS分析之分佈圖,其顯示在DARZALEXTM(達拉單抗)治療之後,多發性骨髓瘤病患中Treg(CD3+ CD3+CD4+CD25+CD127dim)之頻率(上分佈圖,P4細胞群)及CD38+ Treg在Treg群內之頻率(下分佈圖,P5細胞群)。 DARZALEXTM(達拉單抗)治療使CD38+ Treg損耗。研究:GEN501 17病患子集。 Figure 10B shows the distribution diagram of FACS analysis, which shows the frequency of Treg (CD3 + CD3 + CD4 + CD25 + CD127 dim ) in patients with multiple myeloma after DARZALEX TM (dalazumab) treatment (upper distribution diagram, P4 cell population) and the frequency of CD38 + Treg in the Treg population (distribution diagram below, P5 cell population). DARZALEX (TM ) treatment depletes CD38 + Treg. Study: A subset of GEN501 17 patients.
圖10C顯示在基期、或在1週、4週、8週、復發之後、或在6個月治療結束(EOT)時,經DARZALEXTM(達拉單抗)治療的病患中CD38高CD3+CD4+CD25+CD127dim Treg之頻率。CD38高Treg之頻率隨著DARZALEXTM(達拉單抗)治療減少,並在EOT時返回至基期。Y軸:CD3+ T細胞之CD38高CD3+CD4+CD25+CD127dim Treg的%。研究:GEN501 17病患子集。 Figure 10C shows that in the base period, or at 1 week, 4 weeks, 8 weeks, after recurrence, or at the end of treatment (EOT) for 6 months, CD38 was high in patients treated with DARZALEX TM (dalazumab). CD4 + CD25 + CD127 dim Treg frequency. The frequency of CD38 high Treg decreased with DARZALEX TM (dalazumab) treatment, and returned to the baseline at EOT. Y axis:% of CD3 + T cell CD38 high CD3 + CD4 + CD25 + CD127 dim Treg. Study: A subset of GEN501 17 patients.
圖10D顯示在基期、在治療1週、4週、及8週,反應者及無反應者中之CD8+/Treg細胞比率。在治療第8週時,反應者之於無反應者之CD8+/Treg細胞比率顯著地較高(p=0.00955)。研究:GEN501 17病患子集。 Figure 10D shows the ratio of CD8 + /Treg cells among responders and non-responders in the baseline period, at 1 week, 4 weeks, and 8 weeks of treatment. At the 8th week of treatment, the ratio of CD8 + /Treg cells among responders to non-responders was significantly higher (p=0.00955). Study: A subset of GEN501 17 patients.
圖10E顯示當相較於CD38- Treg或陰性對照時,在CD38+ Treg存在下更有效地抑制效應細胞的增生。誤差條代表標準誤差。星號標示顯著變化。樣本係自多個健康供體獲得。透過羧基螢光素琥珀醯亞胺酯(CFSE)之稀釋評估細胞增生。 Figure 10E shows that when compared to CD38 - Treg or negative control, the proliferation of effector cells is more effectively inhibited in the presence of CD38 + Treg. Error bars represent standard errors. Asterisks indicate significant changes. The samples were obtained from multiple healthy donors. Cell proliferation was assessed by dilution of carboxyfluorescein succinimidyl ester (CFSE).
圖11顯示骨髓衍生抑制細胞(MDSC)存在於多發性骨髓瘤病患中(上圖表,加框的細胞),且約一半的細胞表現CD38(中間圖表,加框的細胞)。CD38高MDSC群於經一次DARZALEXTM(達拉單抗)輸液治療的病患中損耗(下圖表,加框的細胞)。研究:GEN501 17病患子集。 Figure 11 shows that bone marrow-derived suppressor cells (MDSC) are present in patients with multiple myeloma (top chart, boxed cells), and about half of the cells express CD38 (middle chart, boxed cells). The CD38 high MDSC population was depleted in patients treated with a DARZALEX TM (dalazumab) infusion (bottom chart, boxed cells). Study: A subset of GEN501 17 patients.
圖12顯示當相較於基期時,在經DARZALEXTM(達拉單抗)治療1週、4週、或8週之後,病患中之CD38高MDSC(CD11b+HLADR-CD14-CD33+CD15+)之數目減少,並在治療結束(EOT)後返回至接近基期。復發病患仍展示CD38高MDSC減少。黑色方形:無反應者;白色方形:對DARZALEXTM(達拉單抗)治療具有至少最小反應的病患。垂直線指示各組中之中位數值。病患2、4、15、16、及17展示出高的最初CD38高MDSC群。研究:GEN501 17病患子集。
Figure 12 shows that when compared to the base period, after undergoing DARZALEX TM (Dara mAb) for 1 week, 4 weeks or 8 weeks, the patients in the high CD38 MDSC (CD11b + HLADR - CD14 - CD33 + CD15 + ) Decreased, and returned to near the base period after the end of treatment (EOT). Relapsed patients still showed a reduction in CD38 high MDSC. Black squares: non-responders; white squares: patients who have at least the least response to DARZALEX TM (dalazumab) treatment. The vertical line indicates the median value in each group.
圖13顯示具最高CD38高MDSC的病患(病患2、4、15、16、及17)具有最高的無進展存活期(PFS)。這些病患對DARZALEXTM(達拉單抗)治療具有部分反應(PR)或最小反應(MR)。SD:疾病穩定;PD:疾病進展。X軸顯示各個別編號病患之PFS。
Figure 13 shows that patients with the highest CD38 high MDSC (
圖14顯示MDSC對DARZALEXTM(達拉單抗)誘導之ADCC敏感。道迪細胞(Daudi cell)在測定中用作目標細胞之陽性對照。測量細胞裂解%。 Figure 14 shows that MDSC is sensitive to ADCC induced by DARZALEX(TM). Daudi cells are used as a positive control for target cells in the assay. Measure% cell lysis.
圖15A顯示在治療之第1週、第4週、及第8週時,CD38+ Breg在經DARZALEXTM(達拉單抗)治療的病患中損耗。 Figure 15A shows that CD38 + Breg was depleted in patients treated with DARZALEX TM (dalazumab) at the first week, the fourth week, and the eighth week of treatment.
圖15B顯示CD38+ Breg在刺激後分泌IL-10。 Figure 15B shows that CD38 + Breg secretes IL-10 after stimulation.
圖16A顯示在基期及在治療期間的指定時間,透過具有VGPR的經DARZALEXTM(達拉單抗)治療的病患之PBMC中CMV、EBV、及流感病毒特異性(CEF)IFN-γ的產生所測量之抗病毒反應。OD:光學密度。白色條:陰性對照;黑色條:添加CEF;短劃條:僅同種異體PBMC。星號指示統計學顯著的變化。Pre 4,8,10=治療之第4、8、或10週。
Figure 16A shows the production of CMV, EBV, and influenza virus-specific (CEF) IFN-γ in the PBMC of patients treated with DARZALEX TM (dalazumab) with VGPR at the baseline and at designated times during treatment Antiviral response measured. OD: Optical density. White bar: negative control; black bar: CEF added; dashed bar: only allogeneic PBMC. Asterisks indicate statistically significant changes.
圖16B顯示在基期及在治療期間的指定時間,透過具有CR的經DARZALEXTM(達拉單抗)治療的病患之PBMC中CMV、EBV、及流感病毒特異性(CEF)IFN-γ的產生所測量之抗病毒反應。OD:光學密度。白色條:陰性對照;黑色條:添加CEF;短劃條:僅同種異體PBMC。星號指示統計學顯著的變化。Pre 4,8,10=治療之第4、8、或10週。
Figure 16B shows the production of CMV, EBV, and influenza virus-specific (CEF) IFN-γ in the PBMC of patients treated with DARZALEX TM (dalazumab) with CR at the baseline and at the designated time during treatment Antiviral response measured. OD: Optical density. White bar: negative control; black bar: CEF added; dashed bar: only allogeneic PBMC. Asterisks indicate statistically significant changes.
圖16C顯示在基期及在治療期間的指定時間,透過具有PD的經DARZALEXTM(達拉單抗)治療的病患之PBMC中CMV、EBV、及流感病毒特異性(CEF)IFN-γ的產生所測量之抗病毒反應。OD:光學密度。白色條:陰性對照;黑色條:添加CEF;短劃條:僅同種異體PBMC。Ns:不顯著。Pre 4,8=治療之第4或8週。
Figure 16C shows the production of CMV, EBV, and influenza virus-specific (CEF) IFN-γ in the PBMC of patients treated with DARZALEX TM (dalazumab) with PD at the baseline and at the designated time during the treatment period Antiviral response measured. OD: Optical density. White bar: negative control; black bar: CEF added; dashed bar: only allogeneic PBMC. Ns: Not significant.
圖16D顯示在基期及在治療期間的指定時間,透過具有MR的經DARZALEXTM(達拉單抗)治療的病患之PBMC中CMV、EBV、及流感病毒特異性(CEF)IFN-γ的產生所測量之抗病毒反應。OD:光學密度。白色條:陰性對照;黑色條:添加CEF;短劃條:僅同種異體PBMC。Ns:不顯著。Pre 4,8=治療之第4或8週。
Figure 16D shows the production of CMV, EBV, and influenza virus-specific (CEF) IFN-γ in the PBMC of patients treated with DARZALEX TM (dalazumab) at the base period and at the designated time during the treatment period. Antiviral response measured. OD: Optical density. White bar: negative control; black bar: CEF added; dashed bar: only allogeneic PBMC. Ns: Not significant.
圖16E顯示在基期及在治療期間所指定的時間,具有VGPR的經DARZALEXTM(達拉單抗)治療的病患之PBMC中增生病毒反應性T細胞之百分比(%)。白色條:陰性對照;黑色條:添加CEF。星號指示統計學顯著的變化。Pre 4,8,10=治療之第4、8、或10週。
Figure 16E shows the percentage (%) of proliferating virus-reactive T cells in the PBMC of patients treated with DARZALEX TM (dalazumab) with VGPR during the base period and at the designated time during the treatment. White bar: negative control; black bar: CEF added. Asterisks indicate statistically significant changes.
圖16F顯示在基期及在治療期間所指定的時間,具有CR的經DARZALEXTM(達拉單抗)治療的病患之PBMC中增生病毒反應性T細胞之百分比(%)。白色條:陰性對照;黑色條:添加CEF。
星號指示統計學顯著的變化。Pre 4,8,10=治療之第4、8、或10週。
Figure 16F shows the percentage (%) of proliferating virus-reactive T cells in the PBMC of patients treated with DARZALEX™ (dalazumab) with CR at the base period and at the designated time during the treatment. White bar: negative control; black bar: CEF added. Asterisks indicate statistically significant changes.
圖17A顯示FACS分析之分佈圖,其顯示CD38在健康供體之自然殺手細胞(NK)、單核球、B細胞、及T細胞上的表現水平。 Figure 17A shows a distribution diagram of FACS analysis, which shows the expression level of CD38 on natural killer cells (NK), monocytes, B cells, and T cells from healthy donors.
圖17B顯示FACS分析之分佈圖,其顯示CD38在多發性骨髓瘤病患之漿細胞、自然殺手細胞(NK)、單核球、B細胞、及T細胞上的表現水平。 Figure 17B shows the distribution diagram of FACS analysis, which shows the expression level of CD38 on plasma cells, natural killer cells (NK), monocytes, B cells, and T cells in patients with multiple myeloma.
圖17C顯示復發性及難治性多發性骨髓瘤病患之CD38+ Treg、Breg、NK、B細胞、及T細胞中CD38之平均螢光強度(MFI)的比較。當相較於CD38+Treg、Bregs、及NK細胞時,CD38以較低水平表現於B細胞及T細胞中。 Figure 17C shows a comparison of the mean fluorescence intensity (MFI) of CD38 in CD38+ Treg, Breg, NK, B cells, and T cells in patients with relapsed and refractory multiple myeloma. When compared to CD38+Treg, Bregs, and NK cells, CD38 is expressed in B cells and T cells at a lower level.
圖18顯示PD-L1蛋白隨時間推移在反應者(R)之PBMC樣本中下調且在無反應者(NR)中上調。SD:疾病穩定。C1D1:第1週期第1天;C3D1:第3週期第1天。Y軸顯示log2蛋白濃度值。 Figure 18 shows that PD-L1 protein is down-regulated in the PBMC samples of responders (R) and up-regulated in non-responders (NR) over time. SD: Stable disease. C1D1: the first day of the first cycle; C3D1: the first day of the third cycle. The Y axis shows the value of log2 protein concentration.
於本說明書及隨附的申請專利範圍中,除非內文另有明確說明,否則單數形式的「一(a/an)」及「該(the)」皆包括複數指稱。因此,例如對於「一細胞(a cell)」之指稱包括兩或更多個細胞之組合、及類似者。 In the scope of this specification and the accompanying patent application, unless the content clearly indicates otherwise, the singular forms of "一 (a/an)" and "the (the)" include plural references. Therefore, for example, the reference to "a cell" includes a combination of two or more cells, and the like.
「CD38」係指人類CD38蛋白(同義字:ADP核糖基環化酶1、cADPr水解酶1、環ADP核糖水解酶1)。人類CD38具有GenBank登錄號NP_001766及SEQ ID NO:1所示之胺基酸序列。熟知的是,CD38係一種II型單次跨膜蛋白,其具有:胺基酸殘基1至21,其等代表胞質域;胺基酸殘基22至42,其等代表跨膜域;及殘基43至300,其等代表CD38之胞外域。
"CD38" refers to human CD38 protein (synonyms:
SEQ ID NO:1 SEQ ID NO: 1
本文中所用之「抗體(antibody)」係以廣義的方式意指並包括免疫球蛋白(immunoglobulin)分子,其包括單株抗體(包括鼠類、人類、人化(humanized)、及嵌合單株抗體)、抗體片段、雙特異性或多特異性抗體、二聚體、四聚體、或多聚體抗體、單鏈抗體、域抗體、以及任何其他包含具有所需特異性之抗原結合部位(antigen binding site)的免疫球蛋白之修飾組態。 As used herein, "antibody" refers to and includes immunoglobulin molecules in a broad sense, including monoclonal antibodies (including murine, human, humanized, and chimeric monoclonal antibodies). Antibodies), antibody fragments, bispecific or multispecific antibodies, dimers, tetramers, or multimer antibodies, single chain antibodies, domain antibodies, and any other antigen binding sites that contain the desired specificity ( The modified configuration of the immunoglobulin of the antigen binding site).
免疫球蛋白可分派為五大類,即IgA、IgD、IgE、IgG及、IgM,取決於重鏈恆定域(constant domain)胺基酸序列。IgA及IgG被進一步細分為同型IgA1、IgA2、IgG1、IgG2、IgG3、及IgG4。任何脊椎動物物種的抗體輕鏈可分派為兩種截然不同類型(即kappa(κ)及lambda(λ))之一者,視其等恆定域的胺基酸序列而定。 Immunoglobulins can be classified into five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the constant domain of the heavy chain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be assigned to one of two distinct types (ie, kappa (κ) and lambda (λ)), depending on the amino acid sequence of their constant domains.
「抗體片段(Antibody fragment)」係指免疫球蛋白分子的一個部分,其保留重鏈及/或輕鏈抗原結合部位,諸如重鏈互補決定區(HCDR)1、2、及3、輕鏈互補決定區(LCDR)1、2、及3、重鏈可變區(VH)、或輕鏈可變區(VL)。抗體片段包括Fab片段,即由VL、VH、CL、及CH1域所組成之單價片段;F(ab)2片段,即二價片段,包含在絞鏈區域(hinge region)藉由二硫橋連結的兩個Fab片段;Fd片段,由VH及CH1域所組成;Fv片段,由抗體單臂的VL及VH域所組成;域抗體(dAb)片段(Ward等人,Nature 341:544-6,1989),其係由VH域所組成。VH及VL域可經工程改造並經由合成連接子連接在一起以形成各種類型的單鏈抗體設計,其中VH/VL域會進行分子內配對,或者在VH及VL域係由分開之單鏈抗體建構體所表現之情況下則會進行分子間配對,以形成單價抗原結合部位,諸如單鏈Fv(scFv)或雙價抗體(diabody);例如描述於PCT國際公開第WO1998/44001號、第WO1988/01649號、第WO1994/13804號、及第WO1992/01047號中者。這些抗體片段係使用所屬技術領域中具有通常知識者所知悉的熟知技術而獲得,且該些片段係經篩選具有如全長抗體相同方式之效用。 "Antibody fragment" refers to a part of an immunoglobulin molecule that retains heavy and/or light chain antigen binding sites, such as heavy chain complementarity determining regions (HCDR) 1, 2, and 3, and light chain complementation Determining regions (LCDR) 1, 2, and 3, heavy chain variable region (VH), or light chain variable region (VL). Antibody fragments include Fab fragments, which are monovalent fragments composed of VL, VH, CL, and CH1 domains; F(ab) 2 fragments, which are bivalent fragments, are contained in the hinge region connected by a disulfide bridge Two Fab fragments; Fd fragment, composed of VH and CH1 domains; Fv fragment, composed of VL and VH domains of one arm of an antibody; domain antibody (dAb) fragments (Ward et al., Nature 341:544-6, 1989), which is composed of the VH domain. The VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs, where the VH/VL domains are paired intramolecularly, or the VH and VL domains are separated by single chain antibodies In the case of constructs, intermolecular pairing will be performed to form a monovalent antigen binding site, such as a single chain Fv (scFv) or a diabody; for example, it is described in PCT International Publication No. WO1998/44001, No. WO1988 /01649, WO1994/13804, and WO1992/01047. These antibody fragments are obtained using well-known techniques known to those with ordinary knowledge in the art, and these fragments are screened to have the same utility as full-length antibodies.
「經單離之抗體(isolated antibody)」係指實質上不含其他具有不同抗原特異性之抗體的抗體或抗體片段(例如,特異性結合CD38的經單離之抗體實質上不含特異性結合人類CD38以外之抗原的抗體)。然而,特異性結合CD38的經單離之抗體可能會與其他抗原有交叉反應,諸如人類CD38的異種同源物,諸如食蟹獼猴(Macaca fascicularis,cynomolgus)CD38。在雙特異性抗體之情況下,雙特異性抗體特異性結合兩種感興趣的抗原,且實質上不含特異性結合非為兩種感興趣抗原之抗原的抗體。此外,經單離之抗體可實質上不含其他細胞材料和/或化學物。「經單離之抗體」涵蓋經單離至較高純度的抗體,諸如係80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%純的抗體。 "Isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to CD38 is substantially free of specific binding Antibodies to antigens other than human CD38). However, isolated antibodies that specifically bind to CD38 may cross-react with other antigens, such as heterologous human CD38, such as cynomolgus ( Macaca fascicularis , cynomolgus) CD38. In the case of bispecific antibodies, the bispecific antibody specifically binds to two antigens of interest, and is substantially free of antibodies that specifically bind to antigens that are not two antigens of interest. In addition, the isolated antibody may be substantially free of other cellular materials and/or chemicals. "Isolated antibody" covers antibodies that have been isolated to a higher purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure antibody.
「特異性結合(specific binding,specific bind)」或「結合(bind)」係指抗體以大於其他抗原的親和力結合至一抗原或該抗原內之表位。通常,抗體以約1×10-8M或更小(例如約1×10-9M或更小、約1×10-10M或更小、約1×10-11M或更小、或約1×10-12M或更小)的平衡解離常數(KD),結合至抗原或抗原內之表位,通常係以其結合至非特異性抗原(例如,BSA、酪蛋白)之KD小至少一百倍的KD結合。解離常數可使用標準程序來測量。然而,特異性結合至抗原或抗原內之表位的抗體可能對於其他相關抗原具有交叉反應性,例如對於來自其他物種(諸如人類或猴)的相同抗原(同源物(homolog)),該猴例如食蟹獼猴(Macaca fascicularis,cynomolgus,cyno)、黑猩猩(Pan troglodytes,chimpanzee,chimp)、或狨(Callithrix jacchus,common marmoset,marmoset)。儘管單特異性抗體特異性結合一種抗原或一種表位,但雙特異性抗體特異性結合兩種不同抗原或兩種不同表位。 "Specific binding (specific binding)" or "bind" means that an antibody binds to an antigen or an epitope within the antigen with greater affinity than other antigens. Generally, the antibody is about 1×10 -8 M or less (e.g., about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, or The equilibrium dissociation constant (K D ) of about 1×10 -12 M or less), which binds to an antigen or an epitope within an antigen, usually by the K of its binding to a non-specific antigen (eg, BSA, casein) K D combination with D at least one hundred times smaller. The dissociation constant can be measured using standard procedures. However, antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity with other related antigens, for example, for the same antigen (homolog) from another species (such as human or monkey), the monkey For example, crab-eating macaques ( Macaca fascicularis , cynomolgus, cyno), chimpanzees ( Pan troglodytes , chimpanzee, chimp), or marmosets ( Callithrix jacchus , common marmoset, marmoset). Although monospecific antibodies specifically bind to one antigen or one epitope, bispecific antibodies specifically bind to two different antigens or two different epitopes.
抗體可變區係由被三個「抗原結合部位(antigen binding site)」中斷的「架構(framework)」區所組成。該等抗原結合部位係使用各種用語定義:互補決定區(CDR)(三個在VH(HCDR1,HCDR2,HCDR3)且三個在VL(LCDR1,LCDR2,LCDR3))係基於序列變異性(Wu及Kabat(1970)J Exp Med 132:211-50;Kabat等人Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991);「高度變異區(Hypervariable region)」、「HVR」、或「HV」(三個在VH(H1,H2,H3)且三個在VL(L1,L2,L3))係指如Chothia及Lesk所定義般在結構上係高度變異(hypervariable)之抗體可變域中的區域(Chothia及Lesk(1987)Mol Biol 196:901-17)。其他用語包括「IMGT-CDR」(Lefranc等人,(2003)Dev Comparat Immunol 27:55-77)及「特異性決定殘基用途(Specificity Determining Residue Usage)」(SDRU)(Almagro(2004)Mol Recognit 17:132-43)。國際免疫遺傳學(International ImMunoGeneTics,IMGT)數據庫(http://www_imgt_org)提供了標準化編號及抗原結合部位的定義。CDR、HV、及IMGT描 繪之間的對應性係描述於Lefranc等人,(2003)Dev Comparat Immunol 27:55-77中。 The antibody variable region is composed of "framework" regions interrupted by three "antigen binding sites". These antigen binding sites are defined using various terms: complementarity determining regions (CDR) (three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3)) are based on sequence variability (Wu and Kabat (1970) J Exp Med 132: 211-50; Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991); "Hypervariable region (Hypervariable region) )”, “HVR”, or “HV” (three in VH (H1, H2, H3) and three in VL (L1, L2, L3)) refer to the height of the structure as defined by Chothia and Lesk Hypervariable regions in the variable domains of antibodies (Chothia and Lesk (1987) Mol Biol 196:901-17). Other terms include "IMGT-CDR" (Lefranc et al. (2003) Dev Comparat Immunol 27:55-77) and "Specificity Determining Residue Usage" (SDRU) (Almagro (2004) Mol Recognit 17:132-43). The International ImMunoGeneTics (IMGT) database (http://www_imgt_org) provides standardized numbers and definitions of antigen binding sites. The correspondence between CDR, HV, and IMGT descriptions is described in Lefranc et al. (2003) Dev Comparat Immunol 27:55-77.
本文中所用之「Chothia殘基(Chothia residue)」為根據Al-Lazikani所編號的抗體VL及VH殘基(Al-Lazikani等人,(1997)J Mol Biol 273:927-48)。 As used herein, "Chothia residues" are antibody VL and VH residues numbered according to Al-Lazikani (Al-Lazikani et al. (1997) J Mol Biol 273:927-48).
「架構(framwork)」或「架構序列(framwork sequence)」為可變區中被定義為抗原結合部位以外的其餘序列。因為抗原結合部位可用如上所述之各種用語來定義,架構之確切胺基酸序列取決於如何定義抗原結合部位。 "Framwork" or "framwork sequence" refers to the remaining sequences in the variable region that are defined as the antigen binding site. Because the antigen binding site can be defined in various terms as described above, the exact amino acid sequence of the framework depends on how the antigen binding site is defined.
「人化抗體(humanized antibody)」係指抗原結合部位係衍生自非人類物種且可變區架構係衍生自人類免疫球蛋白序列的抗體。人化抗體可在架構區中包括取代,所以該架構可能不是所表現人類免疫球蛋白或生殖系基因序列的確切複本。 "Humanized antibody" refers to an antibody whose antigen binding site is derived from a non-human species and the variable region framework is derived from human immunoglobulin sequences. Humanized antibodies can include substitutions in the framework region, so the framework may not be an exact copy of the human immunoglobulin or germline gene sequence represented.
「人類抗體(human antibody)」係指具有重鏈及輕鏈可變區的抗體,其中架構及抗原結合部位兩者皆衍生自人源序列。若該抗體含有恆定區,則該恆定區亦衍生自人源序列。 "Human antibody" refers to an antibody with heavy and light chain variable regions, in which both the framework and the antigen binding site are derived from human sequences. If the antibody contains a constant region, the constant region is also derived from human sequences.
人類抗體包含「衍生自(derived from)」人源序列的重或輕鏈可變區,其中該抗體的可變區係獲自使用人類生殖系免疫球蛋白或重排(rearranged)免疫球蛋白基因的系統。該等系統包括經呈現在噬菌體上的人類免疫球蛋白基因庫(gene library)、及基因轉殖非人類動物(諸如帶有人類免疫球蛋白基因座的小鼠)。「人類抗體」在與人類生殖系免疫球蛋白或重排免疫球蛋白基因比較時可能含有胺基酸差異,此係因於例如天然發生之體細胞突變、或在架構或抗原結合部位中刻意引入取代、或兩者。通常,「人類抗體」係在胺基酸序列上與由人類生殖系免疫球蛋白或重排免疫球蛋白基因所編碼的胺基酸序列具有至少約80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%同一性。在一些情況下,「人類抗體」可能含有自人類架構序列分析導出的共有架構序列,例如Knappik等人,(2000)J Mol Biol 296:57-86中所述,或合併至經呈現在噬菌體上的人類免疫球 蛋白基因庫中的合成HCDR3,例如Shi等人,(2010)J Mol Biol 397:385-96及國際專利公開第WO2009/085462號中所述。 A human antibody contains a heavy or light chain variable region "derived from" a human sequence, wherein the variable region of the antibody is obtained from the use of human germline immunoglobulin or rearranged immunoglobulin genes system. These systems include human immunoglobulin gene libraries displayed on phage, and gene transfer of non-human animals (such as mice with human immunoglobulin loci). "Human antibodies" may contain amino acid differences when compared with human germline immunoglobulin or rearranged immunoglobulin genes, due to, for example, naturally occurring somatic mutations or deliberate introduction in the framework or antigen binding site Replace, or both. Generally, "human antibodies" have at least about 80%, 81%, 82%, 83%, and the amino acid sequence encoded by the human germline immunoglobulin or rearranged immunoglobulin gene in the amino acid sequence. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % Identity. In some cases, "human antibodies" may contain consensus framework sequences derived from human framework sequence analysis, such as those described in Knappik et al., (2000) J Mol Biol 296:57-86, or incorporated into phage display Synthetic HCDR3 in the human immunoglobulin gene library, for example, Shi et al., (2010) J Mol Biol 397:385-96 and International Patent Publication No. WO2009/085462.
衍生自人類免疫球蛋白序列的人類抗體可使用諸如合併有合成CDR及/或合成架構之噬菌體呈現(phage display)的系統來產生,或者可在體外進行突變誘發以改良抗體性質,從而得到在體內人類抗體生殖系貯庫(repertoire)內不會天然存在的抗體。 Human antibodies derived from human immunoglobulin sequences can be produced using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or can be mutagenized in vitro to improve antibody properties, thereby obtaining in vivo Human antibodies are not naturally occurring antibodies in the repertoire of human antibodies.
抗原結合部位衍生自非人類物種的抗體不包括在人類抗體的定義中。 Antibodies whose antigen binding sites are derived from non-human species are not included in the definition of human antibodies.
「重組抗體(recombinant antibody)」包括所有藉由重組手段製備、表現、創建、或單離之抗體。諸如自基因轉殖或染色體轉殖有人類免疫球蛋白基因的動物(例如小鼠或大鼠)或由其製備的融合瘤(於以下進一步描述)單離之抗體;自經轉形(transform)以表現抗體之宿主細胞單離之抗體;自重組、組合抗體庫單離之抗體;及藉由任何涉及將人類免疫球蛋白基因序列剪接到其他DNA序列的其他手段製備、表現、創建、或單離之抗體;或在體外使用Fab臂交換所產生的抗體諸如雙特異性抗體。 "Recombinant antibody" includes all antibodies that are prepared, expressed, created, or isolated by recombinant means. Such as antibodies that are isolated from animals (such as mice or rats) or fusion tumors (described further below) prepared from gene transfer or chromosomal transfer of human immunoglobulin genes; self-transformation (transform) Antibodies isolated from host cells expressing antibodies; antibodies isolated from recombinant or combinatorial antibody libraries; and prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences Isolated antibodies; or antibodies such as bispecific antibodies produced in vitro using Fab arm exchange.
「單株抗體(monoclonal antibody)」係指由單一分子組成之抗體分子的製劑。單株抗體組成物對特定表位呈現出單一結合特異性及親和力,或者在雙特異性單株抗體的情況下,對兩個不同的表位有雙結合特異性。「單株抗體」因此係指在各重鏈及各輕鏈中具有單一胺基酸組成之抗體群,除了可能熟知的變更,諸如自抗體重鏈移除C端離胺酸。在抗體群內,單株抗體可具有異質性醣基化。單株抗體可係單特異性或多特異性,或單價、二價、或多價。雙特異性抗體係包括在用語單株抗體中。 "Monoclonal antibody" refers to a preparation of antibody molecules composed of a single molecule. The monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope, or in the case of a bispecific monoclonal antibody, it has dual binding specificities for two different epitopes. A "monoclonal antibody" therefore refers to a group of antibodies with a single amino acid composition in each heavy chain and each light chain, except for possible well-known changes, such as the removal of the C-terminal lysine from the heavy chain of the antibody. Within the antibody population, monoclonal antibodies can have heterogeneous glycosylation. Monoclonal antibodies can be monospecific or multispecific, or monovalent, bivalent, or multivalent. The bispecific antibody system is included in the term monoclonal antibody.
「表位(epitope)」意指與抗體特異性結合的抗原部分。表位經常係由分子部分(諸如胺基酸或多醣側鏈)之化學活性(諸如極性、非極性、或疏水性)表面分組(grouping)所組成,並且可具有特定三維結構特性,以及特定電荷特性。表位可由形成構形空間單元之鄰接(contiguous)及/或非鄰接(noncontiguous)胺基酸所構成。針對非 鄰接表位,來自抗原線性序列之相異部分的胺基酸會透過蛋白質分子的摺疊而在3維空間中緊密靠近。 "Epitope" means the part of an antigen that specifically binds to an antibody. Epitopes are often composed of chemically active (such as polar, non-polar, or hydrophobic) surface groupings of molecular parts (such as amino acids or polysaccharide side chains), and can have specific three-dimensional structural characteristics and specific charges characteristic. Epitopes can be composed of contiguous and/or noncontiguous amino acids that form structural space units. Against non Adjacent to epitopes, amino acids from different parts of the linear sequence of the antigen will close together in a 3-dimensional space through the folding of protein molecules.
「變異體(variant)」係指藉由一或多個修改(例如取代、插入、或刪除)而不同於參考多肽或參考多核苷酸的多肽或多核苷酸。 "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications (such as substitutions, insertions, or deletions).
「組合(in combination with)」意指二或更多種治療劑一起以混合物形式投予至對象,同時以單劑投予或以任何順序以單劑依序投予。一般而言,各藥劑將以一劑量及/或按照針對該藥劑所判定之時間表投予。 "In combination with" means that two or more therapeutic agents are administered to a subject together as a mixture, simultaneously administered in a single dose or sequentially administered in a single dose in any order. In general, each agent will be administered in a dose and/or according to a schedule determined for that agent.
「治療(treat或treatment)」係指治療性處理,其中目的係在於減緩(減輕)非所欲的生理變化或疾病,諸如腫瘤或腫瘤細胞的發展或蔓延,或在治療過程中提供有益或所欲的臨床結果。有益或所欲的臨床結果包括症狀的減輕、疾病程度的減小、疾病狀態的穩定化(即,不惡化)、疾病進程的延緩或減慢、不發生轉移、疾病狀態的改善或緩和、及緩解(無論部分或完全),無論是可偵測或不可偵測的。「治療」亦可意指相較於未接受治療之對象的預期存活期,延長存活期。那些需要治療的對象包括那些已經患有非所欲的生理變化或疾病的對象,以及那些易患有該生理變化或疾病的對象。 "Treat (treat or treatment)" refers to therapeutic treatment, in which the purpose is to slow down (relieve) undesirable physiological changes or diseases, such as the development or spread of tumors or tumor cells, or to provide benefits or benefits in the course of treatment. Desired clinical results. Beneficial or desired clinical results include alleviation of symptoms, reduction of disease severity, stabilization of disease state (i.e., no deterioration), delay or slowdown of disease progression, no metastasis, improvement or alleviation of disease state, and Mitigation (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging the survival period compared to the expected survival period of an untreated subject. Those in need of treatment include those who have suffered undesired physiological changes or diseases, and those who are susceptible to such physiological changes or diseases.
「治療有效量(therapeutically effective amount)」係指達到所欲治療成果所需之劑量及時間的有效量。治療有效量可根據不同因素而異,諸如對象之疾病狀態、年齡、性別、及體重、以及治療劑或治療劑的組合在對象中引發所欲反應的能力。有效治療劑或治療劑組合的例示性指標包括例如病患幸福感的改善、腫瘤負荷的減少、腫瘤生長的減緩或停止、及/或癌細胞未轉移至身體的其他位置。 "Therapeutically effective amount" refers to the effective amount of the dose and time required to achieve the desired therapeutic effect. The therapeutically effective amount may vary according to different factors, such as the disease state, age, sex, and weight of the subject, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the subject. Exemplary indicators of an effective therapeutic agent or therapeutic agent combination include, for example, improvement in patient well-being, reduction in tumor burden, slowing or cessation of tumor growth, and/or cancer cells not metastasizing to other locations in the body.
「抑制生長(inhibit growth)」(例如,提及腫瘤細胞時)係指當相較於在治療劑或治療藥品之組合不存在的情況下相同腫瘤細胞或腫瘤組織之生長的降低或延緩時,當與治療劑或治療劑或藥品之組合接觸時,在體外或體內的腫瘤細胞生長或腫瘤組織有可測量的降低或延緩。在體外或體內的腫瘤細胞或腫瘤組織之生長的抑制可 係至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、99%、或100%。 "Inhibit growth" (for example, when referring to tumor cells) refers to when the growth of the same tumor cell or tumor tissue is reduced or delayed compared to the absence of a therapeutic agent or a combination of therapeutic drugs, When contacted with a therapeutic agent or a combination of therapeutic agents or drugs, there is a measurable decrease or delay in tumor cell growth or tumor tissue in vitro or in vivo. Inhibition of tumor cell or tumor tissue growth in vitro or in vivo can be It is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
「調節T細胞(regulatory T cell)」、或「Tregs」、或「Treg」係指調節(一或多種)其他T細胞及/或其他免疫細胞之活性(經常藉由抑制其等活性)之T淋巴球。Treg可係CD3+CD4+CD25+CD127dim T細胞。應瞭解,Treg可不完全限於此表型,且可表現Foxp3。 "Regulatory T cell", or "Tregs", or "Treg" refers to T cells that regulate the activity of (one or more) other T cells and/or other immune cells (often by inhibiting their activities) Lymphocytes. Treg can be CD3 + CD4 + CD25 + CD127 dim T cells. It should be understood that Treg may not be completely limited to this phenotype, and may express Foxp3.
「效應T細胞(effector T cell)」、或「Teffs_」、或「Teff」係指執行免疫反應之功能的T淋巴球,諸如殺滅腫瘤細胞及/或活化抗瘤免疫反應,其可導致腫瘤細胞自身體清除。Teff可係CD3+與CD4+或CD8+。Teff可分泌、含有、或表現諸如IFN-γ、顆粒酶B、及ICOS之標誌。應瞭解,Teffs可不完全限於這些表型。 "Effector T cell", or "Teffs_", or "Teff" refers to T lymphocytes that perform immune response functions, such as killing tumor cells and/or activating anti-tumor immune responses, which can cause tumors Cells are cleared from the body. Teff can be CD3 + and CD4 + or CD8 + . Teff can secrete, contain, or exhibit markers such as IFN-γ, granzyme B, and ICOS. It should be understood that Teffs may not be completely limited to these phenotypes.
「Treg之功能(Function of Tregs)」或「Treg功能(Treg function)」係指Treg之抑制性功能,其係關於宿主免疫反應之調節及/或自體免疫之預防。Treg之功能可係抑制由CD8+ T細胞、自然殺手(NK)細胞、MØ細胞、B細胞、或樹突細胞(DC)所引發之抗瘤反應,或抑制效應T細胞之增生。 "Function of Tregs" or "Treg function" refers to the inhibitory function of Treg, which relates to the regulation of host immune response and/or the prevention of autoimmunity. The function of Treg can be to inhibit the anti-tumor response triggered by CD8 + T cells, natural killer (NK) cells, MØ cells, B cells, or dendritic cells (DC), or to inhibit the proliferation of effector T cells.
「抑制Treg之功能(Inhibit function of Tregs)」或「抑制Treg功能(inhibit Treg function)」係指降低在體外或在動物或人類對象體內的Treg之功能的水平,其可藉由所屬技術領域中已知的習知技術判定。Treg之功能的水平可降低例如至少約1%、5%、10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、99%、或100%。「抑制Treg之功能」包括減少Treg之數目,例如藉由經由抗體效應功能(諸如抗體依賴性細胞毒性(ADCC))來殺滅Treg。 "Inhibit function of Tregs" or "inhibit Treg function" refers to reducing the level of Treg function in vitro or in an animal or human subject. Known conventional technology determination. The level of Treg function can be reduced, for example, by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%. "Inhibiting the function of Treg" includes reducing the number of Tregs, for example, by killing Tregs through antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC).
「骨髓衍生抑制細胞(myeloid-derived suppressor cell)」、或「MDSCs」、或「MDSC」係指為造血譜系且表現巨噬細胞/單核球標誌CD11b及顆粒球標誌Gr-1/Ly-6G之特化細胞群。MDSC之表型可係例如CD11b+HLA-DR-CD14-CD33+CD15+。MDSC表現成熟抗原呈遞細胞標誌MHC II類及F480之低或不可偵測的表現。MDSC係骨髓譜系之未成熟細胞,且可進一步分化成若干細胞型,包 括巨噬細胞、嗜中性球、樹突細胞、單核球、或顆粒球。MDSC可天然地發現於人類及動物之正常成人骨髓中或正常造血之部位(諸如脾)中。 "Myeloid-derived suppressor cells", or "MDSCs", or "MDSC" refer to the hematopoietic lineage and express the macrophage/monocyte marker CD11b and the particle marker Gr-1/Ly-6G The specialized cell population. The phenotype of MDSC can be, for example, CD11b + HLA-DR - CD14 - CD33 + CD15 + . MDSC shows low or undetectable performance of mature antigen-presenting cell markers MHC class II and F480. MDSC is an immature cell of the bone marrow lineage, and can be further differentiated into several cell types, including macrophages, neutrophils, dendritic cells, monocytes, or granulocytes. MDSC can be naturally found in normal adult bone marrow of humans and animals or in normal hematopoietic sites (such as the spleen).
「抑制MDSC之功能(Inhibit function of MDSCs)」或「抑制MDSC功能(inhibit MDSC function)」係指降低在體外或在動物或人類對象體內的MDSC之功能的水平,其可藉由所屬技術領域中已知的習知技術判定。MDSC之功能的水平可降低例如至少約1%、5%、10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、99%、或100%。「抑制MDSC之功能」包括減少MDSC之數目,例如藉由經由抗體效應功能(諸如ADCC)來殺滅MDSC。MDSC可藉由各種機制(諸如活性氧化物過氧化亞硝酸鹽的產生、因高水平精胺酸酶所致的精胺酸酶代謝的增加、及氧化亞氮合成酶的增加)抑制T細胞反應諸如增生、純系擴增、或細胞介素產生。MDSC可對IFN-γ及若干細胞介素(諸如IL-4及IL-13)反應。IFN-γ可活化MDSC,其誘導一氧化氮合成酶2(NOS2)之活性。可替代地,Th2細胞介素諸如介白素-4(IL-4)及IL-13可活化MDSC,其可引起誘導精胺酸酶-1(ARG1)之活性。藉由NOS2或ARG1代謝L-精胺酸可引起抑制T細胞的增生,且兩種酶的活性一起可透過產生活性氮氧化物而導致T細胞的細胞凋亡。 "Inhibit function of MDSCs" or "inhibit MDSC function" refers to reducing the level of MDSC function in vitro or in an animal or human subject. Known conventional technology determination. The functional level of MDSC can be reduced, for example, by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%. "The function of inhibiting MDSC" includes reducing the number of MDSCs, for example by killing MDSCs through antibody effector functions (such as ADCC). MDSC can inhibit T cell response by various mechanisms (such as the production of active oxide peroxynitrite, the increase in sperminase metabolism due to high levels of sperminase, and the increase in nitrous oxide synthase) Such as hyperplasia, clone expansion, or cytokine production. MDSC can respond to IFN-γ and several cytokines such as IL-4 and IL-13. IFN-γ can activate MDSC, which induces the activity of nitric oxide synthase 2 (NOS2). Alternatively, Th2 cytokines such as interleukin-4 (IL-4) and IL-13 can activate MDSC, which can induce the activity of arginase-1 (ARG1). Metabolism of L-arginine by NOS2 or ARG1 can inhibit the proliferation of T cells, and the activities of the two enzymes together can lead to apoptosis of T cells through the production of active nitrogen oxides.
「Treg相關疾病(Treg related disease)」係指關係到T調節細胞(Treg)之疾病或病症。Treg相關疾病可由Treg功能(例如,抑制抗瘤反應或抑制效應T細胞增生)造成。Treg媒介之疾病可係癌症。「Treg相關疾病」及「Treg媒介之疾病(Treg mediated disease)」在本文中可互換使用。 "Treg related disease (Treg related disease)" refers to a disease or disorder related to T regulatory cells (Treg). Treg-related diseases can be caused by Treg functions (e.g., suppression of anti-tumor response or suppression of effector T cell proliferation). Treg-mediated diseases can be cancer. "Treg-related disease" and "Treg mediated disease" are used interchangeably in this article.
「增強效應T細胞之反應(Enhance response of effector T cells)」或「增強T細胞之反應(enhance T cell responses)」係指在體外或在動物或人類對象體內增強或刺激效應T細胞以具有持續的或放大的生物功能,或更新或再活化耗盡的或非活性的T細胞。例示性T細胞反應係增生、γ-干擾素自CD 8+ T細胞分泌、抗原反應性、或 純系擴增。測量此增強之方式對於所屬技術領域中具有通常知識者係已知的。 "Enhance response of effector T cells" or "enhance T cell responses" refer to the enhancement or stimulation of effector T cells in vitro or in animal or human subjects to have sustained Or amplify biological functions, or renew or reactivate depleted or inactive T cells. Exemplary T cell response line proliferation, gamma interferon secretion from CD 8 + T cells, antigen reactivity, or pure line expansion. The way to measure this enhancement is known to those of ordinary knowledge in the art.
「MDSC相關疾病(MDSC related disease)」係指關係到骨髓衍生抑制細胞(MDSC)之疾病或病症。MDSC相關疾病可由MDSC功能(例如,抑制抗瘤反應或效應T細胞增生)造成。MDSC媒介之疾病可係癌症。「MDSC相關疾病」及「MDSC媒介之疾病(Treg mediated disease)」在本文中可互換使用。 "MDSC related disease" refers to a disease or disorder related to bone marrow-derived suppressor cells (MDSC). MDSC-related diseases can be caused by MDSC functions (for example, inhibition of anti-tumor response or effector T cell proliferation). MDSC-mediated diseases can be cancer. "MDSC-related disease" and "Treg mediated disease" are used interchangeably in this article.
「調節B細胞(Regulatory B cell)」、或「Breg」、或「Bregs」係指抑制免疫反應之B淋巴球。Breg可係CD19+CD24+CD38+細胞,且可藉由抑制由Breg所分泌之IL-10所媒介之T細胞增生來抑制免疫反應。應瞭解,存在其他Breg子集,且描述於例如Ding等人,(2015)Human Immunology 76:615-621中。 "Regulatory B cell", or "Breg", or "Bregs" refers to B lymphocytes that suppress immune response. Breg can be CD19 + CD24 + CD38 + cells, and can suppress the immune response by inhibiting the proliferation of T cells mediated by IL-10 secreted by Breg. It should be understood that other Breg subsets exist and are described in, for example, Ding et al., (2015) Human Immunology 76:615-621.
「Breg相關疾病(Breg related disease)」係指關係到調節B細胞之疾病或病症。Breg相關疾病可由例如Breg媒介之抗瘤反應的抑制或效應T細胞增生造成。Breg媒介之疾病可係癌症。「Breg相關疾病」及「Breg媒介之疾病(Breg mediated disease)」在本文中可互換使用。 "Breg related disease" refers to a disease or disorder related to regulatory B cells. Breg-related diseases can be caused by, for example, Breg-mediated anti-tumor response suppression or effector T cell proliferation. Breg-mediated diseases can be cancer. "Breg related disease" and "Breg mediated disease" are used interchangeably in this article.
「病患(Patient)」包括任何人類或非人類動物(nonhuman animal)。「非人類動物」包括所有脊椎動物,例如,哺乳動物及非哺乳動物,諸如非人類靈長類、綿羊、狗、貓、馬、牛、雞、兩棲類、爬蟲類、等等。「病患」及「對象(subject)」在本文中可互換使用。 "Patient" includes any human or nonhuman animal. "Non-human animals" include all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and so on. "Patient" and "subject" are used interchangeably in this article.
本發明提供一種用特異性結合CD38之抗體治療患有固態腫瘤之病患的方法,不管腫瘤細胞是否表現CD38。本發明進一步提供用於治療患有調節T細胞(Treg)、骨髓衍生抑制細胞(MDSC)、或調節B細胞(Breg)媒介之疾病之病患的方法。本發明進一步提供用於調節Treg、MDSC、或Breg活性以治療固態腫瘤的方法,該等固態腫瘤係CD38陽性及/或與這些免疫抑制性細胞之高水平相關聯。 The present invention provides a method for treating patients with solid tumors with antibodies that specifically bind CD38, regardless of whether the tumor cells express CD38. The present invention further provides methods for treating patients suffering from diseases mediated by regulatory T cells (Treg), bone marrow-derived suppressor cells (MDSC), or regulatory B cells (Breg). The present invention further provides methods for regulating the activity of Treg, MDSC, or Breg to treat solid tumors that are CD38 positive and/or are associated with high levels of these immunosuppressive cells.
本發明係至少部分地基於以下發現,即抗CD38抗體DARZALEXTM(達拉單抗(daratumumab))在病患中具有免疫調節活 性,其減少免疫抑制性Treg、MDSC、及Breg之數目,增加CD8+ T細胞之數目及CD8+對Treg之比率,促進CD8+中央記憶細胞形成,及增加T細胞純系性。 The present invention is based at least in part on the discovery that the anti-CD38 antibody DARZALEX TM (daratumumab) has immunomodulatory activity in patients, which reduces the number of immunosuppressive Treg, MDSC, and Breg, and increases CD8 + The number of T cells and the ratio of CD8 + to Treg, promote the formation of CD8 + central memory cells, and increase the homologousness of T cells.
臨床上正評估DARZALEXTM(達拉單抗)及其他抗CD38抗體治療血基質惡性腫瘤及漿細胞病症之功效,包括多發性骨髓瘤,其係藉由該抗體藉由抗體效應功能(諸如ADCC、CDC、ACDP、及細胞凋亡)消除CD38-陽性細胞之能力,但是其等在促進適應性免疫反應中之免疫調節活性尚未被認識。其他免疫調節抗體(抗PD1、抗CTLA4)透過靶向免疫系統之抑制抗瘤反應的組分起作用。例如,抗PD1抗體已被展示增加T細胞增生,刺激抗原特異性記憶反應,且部分減輕在體外Treg媒介之效應T細胞的抑制(例如參見,美國專利第8,779,105號)。兩種抗PD-1抗體目前被核准用於黑色素瘤的治療,OPDIVO®(尼沃魯單抗(nivolumab))及KEYTRUDA®(派立珠單抗(pembrolizumab)),且這些抗體處於各種固態腫瘤之臨床開發中,諸如非小細胞肺癌、前列腺癌、頭部及頸部癌、胃腸癌、胃癌、前列腺癌、輸卵管癌、卵巢癌、胰腺癌、乳癌及腦癌、腎癌、膀胱癌、尿道癌、食管癌、及結腸直腸癌。抗CTLA-4抗體YERVOY®(伊匹單抗(ipilimumab))已被核准用於黑色素瘤的治療。YERVOY®(伊匹單抗)及另一個抗CTLA-4抗體(曲美木單抗(tremelimumab))亦正開發用於前列腺癌、非小細胞肺癌、卵巢癌、胃腸癌、胃癌、結腸直腸癌、腎癌、食管癌、及泌尿生殖癌。 Clinically, the efficacy of DARZALEX TM (darumab) and other anti-CD38 antibodies in the treatment of blood stromal malignancies and plasma cell disorders, including multiple myeloma, is being evaluated by the antibody through antibody effector functions (such as ADCC, CDC, ACDP, and apoptosis) have the ability to eliminate CD38-positive cells, but their immunomodulatory activity in promoting adaptive immune responses has not yet been recognized. Other immunomodulatory antibodies (anti-PD1, anti-CTLA4) act by targeting components of the immune system that inhibit the anti-tumor response. For example, anti-PD1 antibodies have been shown to increase T cell proliferation, stimulate antigen-specific memory responses, and partially alleviate Treg-mediated effector T cell suppression in vitro (see, for example, U.S. Patent No. 8,779,105). Two anti-PD-1 antibodies are currently approved for the treatment of melanoma, OPDIVO® (nivolumab) and KEYTRUDA® (pembrolizumab), and these antibodies are in various solid tumors In clinical development, such as non-small cell lung cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, prostate cancer, fallopian tube cancer, ovarian cancer, pancreatic cancer, breast cancer and brain cancer, kidney cancer, bladder cancer, urethra Cancer, esophageal cancer, and colorectal cancer. The anti-CTLA-4 antibody YERVOY® (ipilimumab) has been approved for the treatment of melanoma. YERVOY® (ipilimumab) and another anti-CTLA-4 antibody (tremelimumab) are also being developed for prostate cancer, non-small cell lung cancer, ovarian cancer, gastrointestinal cancer, gastric cancer, colorectal cancer , Kidney cancer, esophageal cancer, and urogenital cancer.
不希望受任何特定理論束縛,基於用本文所述之DARZALEXTM(達拉單抗)觀察到之免疫調節效應,DARZALEXTM(達拉單抗)及其他抗CD38抗體可能在固態腫瘤之治療中係有效的。因在經DARZALEXTM(達拉單抗)治療之病患中觀察到免疫反應之一般活化,患有CD38-陰性固態腫瘤之病患可能同樣會對抗CD38抗體療法反應。 Without wishing to be bound by any particular theory, based on the immunomodulatory effects observed with DARZALEX TM (darumab) described herein, DARZALEX TM (darumab) and other anti-CD38 antibodies may be involved in the treatment of solid tumors. Effective. Due to the general activation of the immune response observed in patients treated with DARZALEX TM (darumab), patients with CD38-negative solid tumors may also respond to anti-CD38 antibody therapy.
本發明提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體達一段足以治療該固態腫瘤的時間。 The present invention provides a method for treating a patient suffering from a solid tumor, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need thereof for a period of time sufficient to treat the solid tumor.
本發明亦提供一種治療患有調節T細胞(Treg)媒介之疾病之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體達一段足以治療該Treg媒介之疾病的時間。 The present invention also provides a method for treating a patient suffering from a disease mediated by regulatory T cells (Treg), which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need thereof for a period of time sufficient for treatment The time of the disease of the Treg vector.
本發明亦提供一種治療患有骨髓衍生抑制細胞(MDSC)媒介之疾病之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體達一段足以治療該MDSC媒介之疾病的時間。 The present invention also provides a method for treating a patient suffering from a disease mediated by bone marrow-derived suppressor cells (MDSC), which comprises administering to the patient in need a therapeutically effective amount of an antibody that specifically binds CD38 for a period of time sufficient The time to treat the disease of the MDSC vector.
本發明亦提供一種治療患有調節B細胞(Breg)媒介之疾病之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體達一段足以治療該Breg媒介之疾病的時間。 The present invention also provides a method for treating a patient suffering from a disease mediated by regulatory B cells (Breg), which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient who needs it for a period of time sufficient for treatment The time of the disease of the Breg vector.
本發明亦提供一種抑制調節T細胞(Treg)之活性的方法,其包含使該調節T細胞接觸特異性結合CD38之抗體。 The present invention also provides a method for inhibiting the activity of regulatory T cells (Treg), which comprises contacting the regulatory T cells with an antibody that specifically binds to CD38.
本發明亦提供一種抑制骨髓衍生抑制細胞(MDSC)之活性的方法,其包含使該MDSC接觸特異性結合CD38之抗體。 The present invention also provides a method for inhibiting the activity of bone marrow-derived suppressor cells (MDSC), which comprises contacting the MDSC with an antibody that specifically binds to CD38.
本發明亦提供一種抑制調節B細胞(Breg)之活性的方法,其包含使該Breg接觸特異性結合CD38之抗體。 The present invention also provides a method for inhibiting the activity of regulating B cells (Breg), which comprises contacting the Breg with an antibody that specifically binds to CD38.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含藉由向該病患投予特異性結合CD38之抗體來減少該病患中調節T細胞(Treg)之數目。 The present invention also provides a method for treating a patient suffering from solid tumors, which comprises reducing the number of regulatory T cells (Treg) in the patient by administering an antibody that specifically binds to CD38 to the patient.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含藉由向該病患投予特異性結合CD38之抗體來減少該病患中骨髓衍生抑制細胞(MDSC)之數目。 The present invention also provides a method for treating a patient suffering from solid tumors, which comprises reducing the number of bone marrow-derived suppressor cells (MDSC) in the patient by administering to the patient an antibody that specifically binds to CD38.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含藉由向該病患投予特異性結合CD38之抗體來減少該病患中調節B細胞(Breg)之數目。 The present invention also provides a method for treating a patient suffering from solid tumors, which comprises reducing the number of regulatory B cells (Breg) in the patient by administering to the patient an antibody that specifically binds to CD38.
本發明亦提供一種增強病患之免疫反應的方法,其包含向有彼之需要之該病患投予特異性結合CD38之抗體達一段足以增強該免疫反應的時間。 The present invention also provides a method for enhancing the immune response of a patient, which comprises administering an antibody that specifically binds to CD38 to the patient who needs it for a period of time sufficient to enhance the immune response.
在一些實施例中,該病患患有病毒感染。 In some embodiments, the patient has a viral infection.
本發明亦提供一種治療患有病毒感染之病患的方法,其包含向有彼之需要之該病患投予特異性結合CD38之抗體達一段足以治療該病毒感染的時間。 The present invention also provides a method for treating a patient suffering from a viral infection, which comprises administering an antibody that specifically binds to CD38 to the patient in need thereof for a period of time sufficient to treat the viral infection.
在一些實施例中,該免疫反應係效應T細胞(Teff)反應。 In some embodiments, the immune response is an effector T cell (Teff) response.
在一些實施例中,該Teff反應係由CD4+ T細胞或CD8+ T細胞媒介。 In some embodiments, the Teff response is mediated by CD4 + T cells or CD8 + T cells.
在一些實施例中,該Teff反應係由CD4+ T細胞媒介。 In some embodiments, the Teff response is mediated by CD4 + T cells.
在一些實施例中,該Teff反應係由CD8+ T細胞媒介。 In some embodiments, the Teff response is mediated by CD8 + T cells.
在一些實施例中,該Teff反應係CD8+ T細胞之數目的增加、CD8+ T細胞增生的增加、T細胞純系擴增的增加、CD8+記憶細胞形成的增加、抗原依賴性抗體產生的增加、或細胞介素、趨化介素、或介白素產生的增加。 In some embodiments, the Teff response is an increase in the number of CD8 + T cells, an increase in CD8 + T cell proliferation, an increase in T cell lineage expansion, an increase in CD8 + memory cell formation, and an increase in antigen-dependent antibody production. , Or increased production of cytokines, chemokines, or interleukins.
T細胞之增生可例如藉由使用氚化胸苷測量DNA合成之速率,或測量體外干擾素-γ(IFN-γ)之產生,或使用已知方法測量病患樣本之細胞群中T細胞之絕對數目或百分比來評估。 The proliferation of T cells can be achieved, for example, by measuring the rate of DNA synthesis using tritiated thymidine, or measuring the production of interferon-γ (IFN-γ) in vitro, or measuring the number of T cells in the cell population of patient samples using known methods. Evaluate in absolute numbers or percentages.
純系擴增可藉由例如使用已知方法定序T細胞池之TCR來評估。 Homogenous expansion can be assessed by, for example, sequencing the TCR of the T cell pool using known methods.
記憶細胞之形成可藉由使用例如FACS測量初始(nave)T細胞(CD45RO-/CD62L+)對記憶T細胞(CD45RO+/CD62L高)之比率來評估。 The formation of memory cells can be measured by using, for example, FACS to measure the initial (na VE) T cells (CD45RO - / CD62L +) ratio of memory T cells (CD45RO + / CD62L high) of the assessed.
細胞介素、趨化介素、或介白素之產生(諸如干擾素-γ(IFN-γ)、腫瘤壞死因子-α(TNF-α)、IL-1、IL-2、IL-3、IL-4、IL-6、IL-8、IL-10、IL-12、IL-13、IL-16、IL-18、及IL-23、MIP-1α、MIP-1β、RANTES、CCL4的產生)可使用標準方法諸如ELISA或ELLISPOT測定評估。 The production of cytokines, chemokines, or interleukins (such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-1, IL-2, IL-3, IL- 4. Production of IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, and IL-23, MIP-1α, MIP-1β, RANTES, CCL4) Use standard methods such as ELISA or ELLISPOT assay for evaluation.
抗原特異性抗體之產生可使用標準方法(諸如ELISA或放射免疫測定(RIA))從衍生自病患之樣本評估。 The production of antigen-specific antibodies can be assessed from patient-derived samples using standard methods such as ELISA or radioimmunoassay (RIA).
「增加(increase或increasing)」各種Teff反應之含義係易於理解的。在測試樣本中或在對象中當相較於對照時,舉例而 言,例如在經抗CD38抗體治療的病患中當相較於治療之前的相同病患時,或在對抗CD38抗體治療作出反應之病患或病患之群組中當相較於對相同治療未作出反應之病患或病患之群組中,增加可係增加至少約5%、至少約10%、25%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%、110%、120%、130%、140%、150%、200%、250%、300%、350%、400%、或更多。通常,增加係統計學顯著的。 The meaning of "increase or increasing" various Teff reactions is easy to understand. In the test sample or in the subject when compared to the control, for example and In other words, for example, in patients treated with anti-CD38 antibodies when compared to the same patients before treatment, or in patients or groups of patients who have responded to anti-CD38 antibody treatment, when compared to the same treatment In patients or groups of patients who do not respond, the increase can be an increase of at least about 5%, at least about 10%, 25%, 50%, 51%, 52%, 53%, 54%, 55%, 56 %, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 200 %, 250%, 300%, 350%, 400%, or more. Generally, the increase is statistically significant.
類似地,「減少(reduce或reducing)」或「降低(decreasing或decrease)」Treg、MDSC、及/或Breg之數目之含義係易於理解的。在測試樣本中或在對象中當相較於對照時,舉例而言,例如在經抗CD38抗體治療的病患中當相較於治療之前的相同病患時,或在對抗CD38抗體治療作出反應之病患或病患之群組中當相較於對相同治療未作出反應之病患或病患之群組中,降低可係至少約10%、25%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%、110%、120%、130%、140%、150%、200%、250%、300%、350%、400%、或更多。一般而言,降低係統計學顯著的。 Similarly, the meaning of "reduce (reducing)" or "decreasing (decrease)" the number of Treg, MDSC, and/or Breg is easy to understand. In a test sample or in a subject when compared to a control, for example, in a patient treated with an anti-CD38 antibody when compared to the same patient before treatment, or in response to an anti-CD38 antibody treatment When compared with those who did not respond to the same treatment or the group of patients, the reduction can be at least about 10%, 25%, 50%, 51%, 52% , 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69 %, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120% , 130%, 140%, 150%, 200%, 250%, 300%, 350%, 400%, or more. Generally speaking, the reduction is statistically significant.
在一些實施例中,特異性結合CD38之抗體抑制免疫抑制細胞之功能。 In some embodiments, antibodies that specifically bind to CD38 inhibit the function of immunosuppressive cells.
在一些實施例中,免疫抑制細胞係調節T細胞(Treg)、骨髓衍生抑制細胞(MDSC)、或調節B細胞(Breg)。 In some embodiments, the immunosuppressive cell line is regulatory T cells (Treg), bone marrow-derived suppressor cells (MDSC), or regulatory B cells (Breg).
在一些實施例中,Treg係CD3+CD4+CD25+CD127dim T細胞。 In some embodiments, Tregs are CD3 + CD4 + CD25 + CD127 dim T cells.
在一些實施例中,CD3+CD4+CD25+CD127dim細胞表現Foxp3。 In some embodiments, CD3 + CD4 + CD25 + CD127 dim cells express Foxp3.
在一些實施例中,CD3+CD4+CD25+CD127dim T細胞表現CD38。 In some embodiments, CD3 + CD4 + CD25 + CD127 dim T cells express CD38.
Treg功能,諸如其等抑制Teff細胞之能力,可使用已知方法評估,諸如評估混合淋巴球反應(MLR)中Treg抑制Teff增生之能力。 Treg function, such as its ability to inhibit Teff cells, can be evaluated using known methods, such as evaluating the ability of Treg to inhibit Teff proliferation in mixed lymphocyte response (MLR).
Treg功能可藉由例如透過直接殺滅Treg或Treg之亞群(諸如CD38+ Treg)減少Treg當相較於Teff時的相對數目(例如增加CD8+/Treg細胞之比率)來抑制。 Treg function can be inhibited by, for example, reducing the relative number of Tregs when compared to Teff (e.g. increasing the ratio of CD8+ /Treg cells) by directly killing Tregs or subpopulations of Tregs (such as CD38 + Treg).
在一些實施例中,Treg功能係藉由殺滅Treg細胞來抑制。 In some embodiments, Treg function is inhibited by killing Treg cells.
在一些實施例中,Treg殺滅係藉由抗體誘導之抗體依賴性細胞毒性(ADCC)、抗體依賴性細胞吞噬作用(ADCP)、補體依賴性細胞毒性(CDC)、或特異性結合CD38之抗體所誘導的細胞凋亡來媒介。 In some embodiments, Treg killing is by antibody-induced antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or an antibody that specifically binds CD38 The induced apoptosis is mediated.
在一些實施例中,Treg殺滅係藉由ADCC媒介。 In some embodiments, Treg killing is through the ADCC medium.
在一些實施例中,CD38+ Treg係被殺滅的。 In some embodiments, CD38 + Treg is killed.
在一些實施例中,1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、或60%的Treg係被殺滅的。 In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% , 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32 %, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 60% of Treg were killed.
由於CD38僅在Treg及MDSC之一部分中表現,所以預期治療患有固態腫瘤之病患將不會導致Treg及MDSC之全身性損耗,因此可能提供改善的安全概況。 Since CD38 is only expressed in part of Treg and MDSC, it is expected that treatment of patients with solid tumors will not lead to systemic depletion of Treg and MDSC, and therefore may provide an improved safety profile.
在一些實施例中,MDSC係CD11b+HLA-DR-CD14- CD33+CD15+細胞。 In some embodiments, the MDSC is CD11b + HLA-DR - CD14 - CD33 + CD15 + cells.
在一些實施例中,CD11b+HLA-DR-CD14-CD33+CD15+ MDSC表現CD38。 In some embodiments, CD11b + HLA-DR - CD14 - CD33 + CD15 + MDSC exhibits CD38.
MDSC功能可例如藉由透過直接殺滅該等細胞減少MDSC之數目來抑制。 MDSC function can be inhibited, for example, by reducing the number of MDSCs by directly killing the cells.
在一些實施例中,MDSC功能係藉由殺滅CD38+ MDSC來抑制。 In some embodiments, MDSC function is inhibited by killing CD38 + MDSC.
在一些實施例中,MDSC殺滅係藉由抗體誘導之抗體依賴性細胞毒性(ADCC)、抗體依賴性細胞吞噬作用(ADCP)、補體依賴性細胞毒性(CDC)、或特異性結合CD38之抗體所誘導的細胞凋亡來媒介。 In some embodiments, MDSC killing is by antibody-induced antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or antibodies that specifically bind CD38 The induced apoptosis is mediated.
在一些實施例中,MDSC殺滅係藉由ADCC媒介。 In some embodiments, MDSC is killed by the ADCC medium.
在一些實施例中,1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、或60%的MDSC係被殺滅的。 In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% , 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32 %, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 60% of MDSCs were killed.
在一些實施例中,Breg係CD19+CD24+CD38+細胞。 In some embodiments, Breg are CD19 + CD24 + CD38 + cells.
Breg功能可例如藉由透過直接殺滅Breg減少Breg之數目來抑制。 The Breg function can be inhibited, for example, by reducing the number of Bregs by directly killing Bregs.
在一些實施例中,Breg功能係藉由殺滅CD38+ Breg來抑制。 In some embodiments, Breg function is inhibited by killing CD38 + Breg.
在一些實施例中,Breg殺滅係藉由抗體誘導之抗體依賴性細胞毒性(ADCC)、抗體依賴性細胞吞噬作用(ADCP)、補體依賴性細胞毒性(CDC)、或特異性結合CD38之抗體所誘導的細胞凋亡來媒介。 In some embodiments, Breg killing is by antibody-induced antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or an antibody that specifically binds to CD38 The induced apoptosis is mediated.
在一些實施例中,Breg殺滅係藉由ADCC媒介。 In some embodiments, Breg killing is through the ADCC medium.
在一些實施例中,1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、或60%的Breg係被殺滅的。 In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% , 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32 %, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 60% of Breg are killed.
Treg在維持周邊身耐受性中起關鍵作用。天然發生之CD4+CD25hi Treg係產生於胸腺中,並表現Foxp3,其係用於建立及維持Treg譜系鑑別及抑制功能所需的轉錄因子。Treg可聚積在疾病部位(例如在腫瘤內),在該處其等抑制腫瘤抗原特異性T細胞之效應功能,導致抗瘤反應不足。腫瘤浸潤Foxp3+ Treg之增加的密度與各種固態腫瘤(包括胰腺癌、卵巢癌、及肝細胞癌)中之不良預後相關聯。Treg之損耗導致鼠類模型中之抗瘤免疫及腫瘤排斥增強,但亦可導致自體免疫疾病之發展。 Treg plays a key role in maintaining tolerance of the peripheral body. The naturally occurring CD4 + CD25 hi Treg line is produced in the thymus and expresses Foxp3, which is a transcription factor required to establish and maintain Treg lineage identification and suppression functions. Tregs can accumulate in diseased sites (for example, in tumors), where they inhibit the effector function of tumor antigen-specific T cells, resulting in insufficient anti-tumor response. The increased density of tumor infiltrating Foxp3 + Treg is associated with poor prognosis in various solid tumors (including pancreatic cancer, ovarian cancer, and hepatocellular carcinoma). The depletion of Treg leads to enhanced anti-tumor immunity and tumor rejection in murine models, but it can also lead to the development of autoimmune diseases.
骨髓衍生抑制細胞(MDSC)係在不同分化階段之早期骨髓前驅細胞、未成熟顆粒球、巨噬細胞、及樹突細胞之異質性族群。其等大量聚積於癌症病患中,且其等具有強力的免疫抑制性功能,抑制自然殺手細胞(NK)及自然殺手T細胞(NKT)兩者之細胞毒性活性、及由CD8+ T細胞媒介之適應性免疫反應。儘管NK細胞抑制之機制目前未被很好地理解,但MDSC媒介之T細胞抑制係由多個途徑負責,包括精胺酸酶1/ARG1之產生及一氧化氮合成酶2(NOS2)之上調。ARG1及NOS2代謝L-精胺酸,且係一起或分開地阻斷T細胞CD3ζ鏈之轉譯,抑制T細胞增生,及促進T細胞凋亡。此外,MDSC分泌免疫抑制性細胞介素並誘導調節T細胞發展。
Bone marrow-derived suppressor cells (MDSC) are a heterogeneous group of early bone marrow precursor cells, immature granular spheres, macrophages, and dendritic cells at different stages of differentiation. They accumulate in large amounts in cancer patients, and they have powerful immunosuppressive functions, inhibiting the cytotoxic activity of both natural killer cells (NK) and natural killer T cells (NKT), and are mediated by CD8 + T cells The adaptive immune response. Although the mechanism of NK cell inhibition is currently not well understood, MDSC-mediated T cell inhibition is responsible for multiple pathways, including the production of
MDSC係由促發炎細胞介素誘導,且在感染性及發炎性病理狀態中發現數目增加。其等聚積於荷瘤小鼠之血液、骨髓、及次級淋巴器官中,且其等在腫瘤微環境中的存在表明在促進腫瘤相關之免疫抑制中起致病作用。 MDSC is induced by pro-inflammatory cytokines, and the number is found to increase in infectious and inflammatory pathological conditions. They accumulate in the blood, bone marrow, and secondary lymphoid organs of tumor-bearing mice, and their presence in the tumor microenvironment indicates that they play a pathogenic role in promoting tumor-related immunosuppression.
MDSC已描述於患有結腸癌、黑色素瘤、肝細胞癌、頭部及頸部鱗狀細胞癌、非小細胞肺癌、腎細胞癌、胰腺癌、及乳癌之病患中(Mandruzzato等人,(2009)J Immunol 182:6562-6568;Liu等人,(2009)J Cancer Res Clin Oncol 136:35-45;Ko等人,(2009)Clin Cancer Res 15:2148-2157;Morse等人,(2009)Expert Opin Biol Ther 9:331-339;Diaz-Montero等人,(2009)Cancer Immunol Immunother 58:49-59;Corzo等人,(2009)J Immunol 182:5693-5701)。在癌症病患中,Diaz等人(Diaz-Montero等人,(2009)Cancer Immunol Immunother 58:49-59)提出MDSC之聚積與更加嚴重的疾病及不良的預後相關。 MDSC has been described in patients with colon cancer, melanoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, non-small cell lung cancer, renal cell carcinoma, pancreatic cancer, and breast cancer (Mandruzzato et al., ( 2009) J Immunol 182: 6562-6568; Liu et al. (2009) J Cancer Res Clin Oncol 136: 35-45; Ko et al. (2009) Clin Cancer Res 15: 2148-2157; Morse et al. (2009) ) Expert Opin Biol Ther 9:331-339; Diaz-Montero et al. (2009) Cancer Immunol Immunother 58:49-59; Corzo et al. (2009) J Immunol 182:5693-5701). Among cancer patients, Diaz et al. (Diaz-Montero et al. (2009) Cancer Immunol Immunother 58:49-59) proposed that the accumulation of MDSC is associated with more serious diseases and poor prognosis.
腫瘤浸潤Breg已在固態腫瘤中鑑定出,且Breg可藉由各種機制促進腫瘤生長及轉移,諸如抑制CD8+ T細胞及NK細胞之抗瘤活性,如例如Ding等人,(2015)Human Immunology 76:615-62中所述。 Tumor infiltrating Breg has been identified in solid tumors, and Breg can promote tumor growth and metastasis through various mechanisms, such as inhibiting the anti-tumor activity of CD8 + T cells and NK cells, such as, for example, Ding et al., (2015) Human Immunology 76 : Described in 615-62.
「抗體依賴性細胞毒性」、「抗體依賴性細胞媒介的細胞毒性」、或「ADCC」是一種誘導細胞死亡的機制,其取決於抗體包覆的目標細胞與具有裂解活性的效應細胞(諸如自然殺手細胞、單核球、巨噬細胞、及嗜中性球)之間經由效應細胞上表現的Fcγ受體(FcγR)的相互作用。例如,NK細胞表現FcγRIIIa,而單核球表現FcγRI、FcγRII、及FcvRIIIa。抗體包覆的目標細胞(諸如CD38表現細胞)的死亡會發生是由於效應細胞活性,其係透過分泌膜孔形成蛋白(membrane pore-forming protein)及蛋白酶。為了評估特異性結合CD38之抗體的ADCC活性,可將該抗體加入至CD38表現細胞與免疫效應細胞的組合,該等免疫效應細胞可被抗原抗體複合物活化而導致目標細胞的細胞裂解。細胞裂解一般是透過從裂解細胞中釋放的標記(例如放射性基質、螢光染料、或天然的細胞內蛋白質)來偵測。用於該等測定之例示性效應細胞包括周邊血液單核細胞(PBMC)及NK細胞。例示性目標細胞包括表現CD38之Treg或MDSC。在一例示性測定中,將目標細胞以20μCi的51Cr標記2小時並徹底清洗。可將該等目標細胞的細胞濃度調整至1×106細胞/ml,並加入各種濃度的抗 CD38抗體。以效應:目標細胞比率為40:1加入目標細胞以開始測定。於37℃培養3小時後,以離心停止測定,並在閃爍計數器中測量從裂解細胞中釋放出的51Cr。細胞毒性百分比可計算為加入3%過氯酸至目標細胞中可誘導的最大裂解百分比。 "Antibody-dependent cytotoxicity", "antibody-dependent cell-mediated cytotoxicity", or "ADCC" is a mechanism for inducing cell death, which depends on the target cells coated by the antibody and the effector cells with lytic activity (such as natural Killer cells, monocytes, macrophages, and neutrophils) interact via Fcγ receptors (FcγR) expressed on effector cells. For example, NK cells express FcyRIIIa, while monocytes express FcyRI, FcyRII, and FcvRIIIa. The death of antibody-coated target cells (such as CD38 expressing cells) occurs due to effector cell activity, which is through the secretion of membrane pore-forming proteins and proteases. In order to evaluate the ADCC activity of an antibody that specifically binds to CD38, the antibody can be added to a combination of CD38 expressing cells and immune effector cells, which can be activated by the antigen-antibody complex to cause cell lysis of target cells. Cell lysis is generally detected by the release of labels (such as radioactive substrates, fluorescent dyes, or natural intracellular proteins) from the lysed cells. Exemplary effector cells used in these assays include peripheral blood mononuclear cells (PBMC) and NK cells. Exemplary target cells include Tregs or MDSCs that express CD38. In an exemplary assay, target cells were labeled with 20 μCi of 51 Cr for 2 hours and washed thoroughly. The cell concentration of these target cells can be adjusted to 1×10 6 cells/ml, and various concentrations of anti-CD38 antibodies can be added. Start the measurement by adding target cells with an effect: target cell ratio of 40:1. After culturing at 37°C for 3 hours, the measurement was stopped by centrifugation, and the 51 Cr released from the lysed cells was measured in a scintillation counter. The percentage of cytotoxicity can be calculated as the maximum percentage of lysis that can be induced by adding 3% perchloric acid to the target cells.
「抗體依賴性細胞吞噬作用」(「ADCP」)係指一種透過吞噬細胞(諸如巨噬細胞或樹突細胞)內化(internalization)以消滅抗體包覆的目標細胞的機制。ADCP可藉由使用表現CD38之Treg或MDSC作為目標細胞(其等經工程改造以表現GFP或其他標記分子)來評估。效應:目標細胞比率可為例如4:1。可將效應細胞與目標細胞在抗CD38抗體存在或不存在的情況下一起培養4小時。培養後,可使用細胞剝離液(accutase)將細胞分離。巨噬細胞可用偶接螢光標記的抗CD11b及抗CD14抗體來鑑定,且吞噬作用百分比可基於在該等CD11+CD14+巨噬細胞中的GFP螢光%使用標準方法判定。 "Antibody-dependent cellular phagocytosis"("ADCP") refers to a mechanism through internalization of phagocytes (such as macrophages or dendritic cells) to destroy antibody-coated target cells. ADCP can be assessed by using CD38-expressing Treg or MDSC as target cells (which are engineered to express GFP or other marker molecules). Effect: The target cell ratio can be, for example, 4:1. The effector cells and target cells can be cultured for 4 hours in the presence or absence of anti-CD38 antibodies. After culturing, the cells can be separated using a cell stripping solution (accutase). Macrophages can be identified by coupling with fluorescently labeled anti-CD11b and anti-CD14 antibodies, and the percentage of phagocytosis can be determined based on the % GFP fluorescence in these CD11 + CD14 + macrophages using standard methods.
「補體依賴性細胞毒性(complement-dependent cytotoxicity)」或「CDC」係指一種誘導細胞死亡的機制,其中與目標結合之抗體的Fc效應域結合並活化補體成分C1q,其轉而再活化補體級聯反應而引起目標細胞死亡。補體的活化亦可導致補體成分沉積在該目標細胞表面上,藉由結合白血球上的補體受體(例如CR3)而促進ADCC。 "Complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death, in which the Fc effector domain of an antibody bound to the target binds and activates the complement component C1q, which in turn reactivates the complement level The linked reaction causes the death of the target cell. The activation of complement can also lead to the deposition of complement components on the surface of the target cells, and promote ADCC by binding to complement receptors (such as CR3) on white blood cells.
單株抗體誘導ADCC之能力可藉由工程改造其寡醣成分來增強。人類IgG1或IgG3係在Asn297處經N-醣基化並且大部分聚醣係呈熟知之雙觸角(biantennary)G0、G0F、G1、G1F、G2、或G2F形式。由未經工程改造之CHO細胞所生產之抗體通常具有約至少85%之聚醣海藻糖(glycan fucose)含量。自附接至Fc區之雙觸角複合型寡醣移除核心海藻糖經由改善FcγRIIIa結合且不改變抗原結合或CDC活性來增強抗體之ADCC。該等mAb可使用已報導會引起成功表現相對高量去海藻糖基化(defucosylated)抗體(帶有雙觸角複合型之Fc寡醣)的不同方法來達成,諸如控制培育滲透壓(Konno等人,(2012)Cytotechnology 64:249-65)、應用變異體CHO株Lec13作為宿主細胞系(Shields等人,(2002)J Biol Chem 277:26733-26740)、 應用變異體CHO株EB66作為宿主細胞系(Olivier等人,(2010)MAbs 2(4),Epub ahead of print;PMID:20562582)、應用大鼠融合瘤細胞系YB2/0作為宿主細胞系(Shinkawa等人,(2003)J Biol Chem 278:3466-3473)、引入專門針對α1,6-岩藻糖基轉移酶(1,6-fucosyltrasferase,FUT8)基因之短小干擾RNA(Mori等人,(2004)Biotechnol Bioeng 88:901-908)、或共表現β-1,4-N-乙醯葡糖胺基轉移酶III(β-1,4-N-acetylglucosaminyltransferase III)及高基氏α-甘露糖苷酶II(Golgi α-mannosidase II)或基夫鹼(kifunensine)(一種強效α-甘露糖苷酶I抑制劑)(Ferrara等人,(2006)J Biol Chem 281:5032-5036;Ferrara等人,(2006)Biotechnol Bioeng 93:851-861;Xhou等人,(2008)Biotechnol Bioeng 99:652-65)。由在本發明之方法中,以及在以下每一個編號實施例的一些實施例中所使用的抗CD38抗體所引發的ADCC亦可藉由在抗體Fc中的某些取代來增強。例示性取代例如為在胺基酸位置256、290、298、312、356、330、333、334、360、378、或430處之取代(殘基編號根據EU索引),如美國專利第6,737,056號中所述。 The ability of monoclonal antibodies to induce ADCC can be enhanced by engineering their oligosaccharide components. Human IgG1 or IgG3 is N-glycosylated at Asn297 and most of the glycans are in the well-known form of biantennary G0, G0F, G1, G1F, G2, or G2F. Antibodies produced by unengineered CHO cells usually have a glycan fucose content of at least 85%. Removal of core trehalose from biantennary complex oligosaccharides attached to the Fc region enhances the ADCC of antibodies by improving FcγRIIIa binding without changing antigen binding or CDC activity. These mAbs can be achieved using different methods that have been reported to cause the successful expression of relatively high amounts of defucosylated antibodies (Fc oligosaccharides with biantennary complex type), such as controlling the incubation osmotic pressure (Konno et al. , (2012) Cytotechnology 64: 249-65), using the variant CHO strain Lec13 as the host cell line (Shields et al., (2002) J Biol Chem 277: 26733-26740), using the variant CHO strain EB66 as the host cell line (Olivier et al., (2010) MAbs 2(4), Epub ahead of print; PMID: 20562582), using the rat fusion tumor cell line YB2/0 as the host cell line (Shinkawa et al., (2003) J Biol Chem 278 : 3466-3473), the introduction of a short interfering RNA specifically targeting α1,6-fucosyltrasferase (1,6-fucosyltrasferase, FUT8 ) gene (Mori et al. (2004) Biotechnol Bioeng 88:901-908), co-expression or β-1,4- N - acetyl glucosaminyl transferase III (β-1,4-N- acetylglucosaminyltransferase III) and the group's high α- mannosidase II (Golgi α-mannosidase II) or Keefe Kifunensine (a potent α-mannosidase I inhibitor) (Ferrara et al., (2006) J Biol Chem 281:5032-5036; Ferrara et al., (2006) Biotechnol Bioeng 93:851-861; Xhou Et al. (2008) Biotechnol Bioeng 99:652-65). The ADCC induced by the anti-CD38 antibody used in the method of the present invention and in some examples of each numbered example below can also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions are, for example, substitutions at positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 of the amino acid (residue numbering according to the EU index), such as U.S. Patent No. 6,737,056 As described in.
在一些實施例中,特異性結合CD38之抗體包含在抗體Fc中的一個取代。 In some embodiments, the antibody that specifically binds CD38 comprises a substitution in the antibody Fc.
在一些實施例中,特異性結合CD38之抗體包含在抗體Fc中胺基酸位置256、290、298、312、356、330、333、334、360、378、或430處的一個取代(殘基編號根據EU索引)。 In some embodiments, the antibody that specifically binds CD38 contains a substitution (residue Numbering is based on EU index).
在一些實施例中,特異性結合CD38之抗體具有雙觸角聚醣結構,其海藻糖含量係約介於0%至約15%之間,例如15%、14%、13%、12%、11% 10%、9%、8%、7%、6%、5%、4%、3%、2%、1%、或0%。 In some embodiments, the antibody that specifically binds to CD38 has a biantennary glycan structure, and its trehalose content is between about 0% and about 15%, such as 15%, 14%, 13%, 12%, 11 % 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.
在一些實施例中,特異性結合CD38之抗體具有雙觸角聚醣結構,其海藻糖含量係約50%、40%、45%、40%、35%、30%、25%、20%、15%、14%、13%、12%、11% 10%、9%、8%、7%、6%、5%、4%、3%、2%、1%、或0%。 In some embodiments, the antibody that specifically binds to CD38 has a biantennary glycan structure, and its trehalose content is about 50%, 40%, 45%, 40%, 35%, 30%, 25%, 20%, 15%. %, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.
Fc中的取代及減少的海藻糖含量可增強該特異性結合CD38之抗體的ADCC活性。 The substitution in Fc and the reduced trehalose content can enhance the ADCC activity of the antibody that specifically binds to CD38.
「海藻糖含量(Fucose content)」意指Asn297處糖鏈中海藻糖單醣的量。海藻糖的相對量係含海藻糖的結構相對於所有醣類結構的百分比。這些可藉由多種方法來表徵及定量,例如:1)使用經N-醣苷酶F處理過的樣本(例如複合、雜合、及寡與高甘露糖(oligo-and high-mannose)結構)的MALD1-TOF,如在國際專利公開號WO2008/077546中所述;2)酶促釋放Asn297聚醣,隨後衍生化並藉由HPLC(UPLC)以螢光偵測及/或HPLC-MS(UPLC-MS)來偵測/定量;3)天然或還原mAb的完整蛋白質分析,將Asn297聚醣以Endo S或其他會在第一與第二GlcNAc單醣之間切割而留下連接至第一GlcNAc的海藻糖的酵素處理或不經處理;4)以酶消化法(enzymatic digestion)(例如胰蛋白酶或內肽酶Lys-C)將mAb消化成構成分(constituent)肽,隨後以HPLC-MS(UPLC-MS)分離、偵測及定量;或5)用PNGase F在Asn 297處進行特異性酶促去醣基化(specific enzymatic deglycosylation)以將mAb寡醣自mAb蛋白分離。該等釋放出的寡醣可用螢光團標記,藉由各種互補的技術分離和鑑定,該等技術允許:藉由基質輔助雷射脫附遊離(MALDI)質譜術比較實驗質量與理論質量以精細表徵聚醣結構、藉由離子交換HPLC(GlycoSep C)判定唾液酸化(sialylation)程度、藉由正相HPLC(GlycoSep N)根據親水性標準(hydrophilicity criteria)分離及定量寡醣形式、及藉由高效毛細管電泳-雷射誘導螢光(HPCE-LIF)分離及定量寡醣。 "Fucose content" means the amount of trehalose monosaccharide in the sugar chain at Asn297. The relative amount of trehalose is the percentage of trehalose-containing structures relative to all carbohydrate structures. These can be characterized and quantified by a variety of methods, such as: 1) Using samples treated with N-glycosidase F (such as complex, hybrid, and oligo-and high-mannose structures) MALD1-TOF, as described in International Patent Publication No. WO2008/077546; 2) Enzymatic release of Asn297 glycan, followed by derivatization and fluorescence detection by HPLC (UPLC) and/or HPLC-MS (UPLC-MS) MS) to detect/quantify; 3) Intact protein analysis of natural or reduced mAb, using Endo S or other glycans that cleave Asn297 glycans between the first and second GlcNAc monosaccharides, leaving behind those connected to the first GlcNAc Trehalose enzyme treatment or no treatment; 4) Use enzymatic digestion (such as trypsin or endopeptidase Lys-C) to digest mAb into constituent peptides, and then use HPLC-MS (UPLC -MS) separation, detection and quantification; or 5) specific enzymatic deglycosylation at Asn 297 with PNGase F to separate mAb oligosaccharides from mAb proteins. The released oligosaccharides can be labeled with fluorophores, separated and identified by a variety of complementary technologies, which allow: the comparison of experimental and theoretical quality by matrix-assisted laser desorption free (MALDI) mass spectrometry (MALDI) mass spectrometry Characterize the structure of glycans, determine the degree of sialylation by ion exchange HPLC (GlycoSep C), separate and quantify oligosaccharide forms by normal phase HPLC (GlycoSep N) according to hydrophilicity criteria, and by high efficiency Capillary electrophoresis-laser induced fluorescence (HPCE-LIF) separation and quantification of oligosaccharides.
本文中所使用之「低海藻糖(Low fucose)」或「低海藻糖含量(low fucose content)」係指抗體的海藻糖含量為約0%至15%。 As used herein, "low fucose" or "low fucose content" means that the trehalose content of the antibody is about 0% to 15%.
本文中使用之「正常海藻糖(Normal fucose)」或「正常海藻糖含量(normal fucose content)」係指抗體的海藻糖含量約超過50%,通常約超過60%、70%、80%、或超過85%。 As used herein, "Normal fucose" or "normal fucose content" means that the trehalose content of the antibody exceeds about 50%, usually about 60%, 70%, 80%, or More than 85%.
在一些實施例中,特異性結合CD38之抗體可藉由細胞凋亡來誘導Treg、MDSC、及/或Breg之殺滅。評估細胞凋亡的方法係熟知的,且包括例如使用標準方法進行膜聯蛋白IV(annexin IV)染 色。在本發明之方法中所使用之抗CD38抗體可在約20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、或100%的細胞中誘導細胞凋亡。 In some embodiments, antibodies that specifically bind to CD38 can induce the killing of Treg, MDSC, and/or Breg through apoptosis. Methods of assessing apoptosis are well known and include, for example, annexin IV staining using standard methods. color. The anti-CD38 antibody used in the method of the present invention can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, or 100% of cells induce apoptosis.
在一些實施例中,Teff或免疫抑制細胞存在於骨髓中或周邊血液中。 In some embodiments, Teff or immunosuppressive cells are present in bone marrow or in peripheral blood.
在一些實施例中,Teff或免疫抑制細胞存在於骨髓中。 In some embodiments, Teff or immunosuppressive cells are present in the bone marrow.
在一些實施例中,Teff或免疫抑制細胞存在於周邊血液中。 In some embodiments, Teff or immunosuppressive cells are present in peripheral blood.
在一些實施例中,特異性結合CD38之抗體增加CD8+ T細胞對Treg之比率。 In some embodiments, antibodies that specifically bind to CD38 increase the ratio of CD8 + T cells to Treg.
在一些實施例中,特異性結合CD38之抗體增加CD8+中央記憶細胞對CD8+初始細胞之比率。CD8+中央記憶細胞可鑑定為CD45RO+/CD62L+高細胞。CD8+初始細胞可鑑定為CD45RO-/CD62L+細胞。 In some embodiments, antibodies that specifically bind to CD38 increase the ratio of CD8+ central memory cells to CD8 + naive cells. CD8 + central memory cells can be identified as CD45RO + /CD62L + tall cells. CD8 + naive cells can be identified as CD45RO-/CD62L + cells.
在一些實施例中,特異性結合CD38之抗體係非促效性抗體。 In some embodiments, the antibodies that specifically bind to CD38 are non-agonistic antibodies.
特異性結合CD38之非促效性抗體係指在結合至CD38之後,當相較於由同型對照抗體或僅有介質所誘導的增生時,不會誘導體外周邊血液單核細胞樣本之顯著增生的抗體。 A non-agonist anti-acting system that specifically binds to CD38 refers to a system that does not induce significant proliferation of peripheral blood mononuclear cell samples in vitro when compared to the proliferation induced by isotype control antibodies or only mediators after binding to CD38 Antibody.
在一些實施例中,特異性結合CD38之非促效性抗體以統計學不顯著的方式誘導周邊血液單核細胞(PBMC)之增生。PBMC增生可藉由自健康供體單離之PBMC並於200μl RPMI中在測試抗體存在或不存在之情況下以1×105個細胞/孔於平底96孔盤中培育該等細胞來評估。於37℃培養4天後,可添加30μl 3H-胸苷(16.7μCi/ml),且可繼續培育過夜。3H-胸苷併入可使用Packard Cobra加馬計數器(Packard Instruments,Meriden,DT,USA),根據製造商之說明評估。數據可計算為從若干供體獲得之PBMC的平均cpm(±SEM)。介於在測試抗體存在或不存在之情況下所培育之樣本間的統計學顯著性或不顯著性係使用標準方法來計算。 In some embodiments, non-agonistic antibodies that specifically bind to CD38 induce the proliferation of peripheral blood mononuclear cells (PBMC) in a statistically insignificant manner. PBMC proliferation can be assessed by culturing PBMC isolated from healthy donors in 200 μl RPMI with 1×10 5 cells/well in a flat-bottomed 96-well plate in the presence or absence of the test antibody. After culturing at 37°C for 4 days, 30 μl of 3 H-thymidine (16.7 μCi/ml) can be added, and the incubation can be continued overnight. 3 H-thymidine incorporation can be evaluated using Packard Cobra gamma counter (Packard Instruments, Meriden, DT, USA) according to the manufacturer's instructions. The data can be calculated as the average cpm (±SEM) of PBMC obtained from several donors. The statistical significance or insignificance between samples grown in the presence or absence of the test antibody is calculated using standard methods.
可用於本發明之方法中的例示性抗CD38抗體係DARZALEXTM(達拉單抗)。DARZALEXTM(達拉單抗)包含分別顯示於SEQ ID NO:4及5的重鏈可變區(VH)及輕鏈可變區(VL)胺基酸序列、分別為SEQ ID NO:6、7、及8的重鏈互補決定區1(HCDR1)、HCDR2、及HCDR3、及分別為SEQ ID NO:9、10、及11的輕鏈互補決定區1(LCDR1)、LCDR2、及LCDR3,且係IgG1/κ亞型並描述於美國專利第7,829,693號中。DARZALEXTM(達拉單抗)的重鏈胺基酸序列係顯示於SEQ ID NO:12,且輕鏈胺基酸序列係顯示於SEQ ID NO:13。 An exemplary anti-CD38 anti-system DARZALEX(TM) (dalazumab) that can be used in the method of the present invention. DARZALEX TM (dalatumumab) includes the heavy chain variable region (VH) and light chain variable region (VL) amino acid sequences shown in SEQ ID NOs: 4 and 5, respectively, which are respectively SEQ ID NO: 6, The heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 of 7, and 8, and the light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, and It is of the IgG1/κ subtype and is described in U.S. Patent No. 7,829,693. The heavy chain amino acid sequence of DARZALEX (TM) (dalazumab) is shown in SEQ ID NO: 12, and the light chain amino acid sequence is shown in SEQ ID NO: 13.
在一些實施例中,特異性結合CD38之抗體與包含SEQ ID NO:4之重鏈可變區(VH)及SEQ ID NO:5之輕鏈可變區(VL)的抗體競爭結合至CD38。 In some embodiments, an antibody that specifically binds CD38 competes with an antibody comprising the heavy chain variable region (VH) of SEQ ID NO: 4 and the light chain variable region (VL) of SEQ ID NO: 5 for binding to CD38.
在一些實施例中,特異性結合CD38之抗體至少結合至人類CD38(SEQ ID NO:1)之SKRNIQFSCKNIYR(SEQ ID NO:2)區及EKVQTLEAWVIHGG(SEQ ID NO:3)區。 In some embodiments, the antibody that specifically binds to CD38 binds to at least the SKRNIQFSCKNIYR (SEQ ID NO: 2) region and EKVQTLEAWVIHGG (SEQ ID NO: 3) region of human CD38 (SEQ ID NO: 1).
SEQ ID NO:1 SEQ ID NO: 1
SEQ ID NO:2 SKRNIQFSCKNIYR SEQ ID NO: 2 SKRNIQFSCKNIYR
SEQ ID NO:3 EKVQTLEAWVIHGG SEQ ID NO: 3 EKVQTLEAWVIHGG
SEQ ID NO:4 SEQ ID NO: 4
SEQ ID NO:5 SEQ ID NO: 5
SEQ ID NO:6 SFAMS SEQ ID NO: 6 SFAMS
SEQ ID NO:7 AISGSGGGTYYADSVKG SEQ ID NO: 7 AISGSGGGTYYADSVKG
SEQ ID NO:8 DKILWFGEPVFDY SEQ ID NO: 8 DKILWFGEPVFDY
SEQ ID NO:9 RASQSVSSYLA SEQ ID NO: 9 RASQSVSSYLA
SEQ ID NO:10 DASNRAT SEQ ID NO: 10 DASNRAT
SEQ ID NO:11 QQRSNWPPTF SEQ ID NO: 11 QQRSNWPPTF
SEQ ID NO:12 SEQ ID NO: 12
SEQ ID NO:13 SEQ ID NO: 13
可使用熟知的體外方法來評估抗體與參考抗體(諸如具有SEQ ID NO:4之VH及SEQ ID NO:5之VL的DARZALEXTM(達拉單抗))對CD38的競爭結合。在一例示性方法中,可將重組表現CD38的CHO細胞與未標記的參考抗體於4℃培養15分鐘,然後與過量的螢光標記測試抗體於4℃培養45分鐘。於PBS/BSA中清洗後,可藉由流動式細胞測量術(flow cytometry)使用標準方法來測量螢光。在另一例示性方法中,可將人類CD38的細胞外部分塗覆在ELISA盤的表面上。可將過量的未標記參考抗體加入約15分鐘,且隨後可加入經生物素化的測試抗體。在PBS/Tween中清洗後,可使用 共軛辣根過氧化酶(horseradish peroxidase,HRP)的鏈黴親和素(streptavidin)來偵測該測試生物素化抗體的結合並使用標準方法來偵測訊號。在該等競爭性測定(competition assay)中,顯而易見的是參考抗體可係標記的且該測試抗體可係未標記的。當參考抗體抑制測試抗體結合至CD38,或測試抗體抑制參考抗體結合至CD38達至少80%,例如81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%時,測試抗體與參考抗體競爭。該測試抗體的表位可進一步藉由例如胜肽圖譜技術(peptide mapping)或氫/氘保護測定使用已知方法來定義,或者藉由晶體結構判定來定義。 A well-known in vitro method can be used to evaluate the competition of the binding of an antibody to CD38 with a reference antibody (such as DARZALEX(TM ) with VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5). In an exemplary method, the recombinant CD38-expressing CHO cells can be incubated with an unlabeled reference antibody for 15 minutes at 4°C, and then incubated with an excess of fluorescently labeled test antibody at 4°C for 45 minutes. After washing in PBS/BSA, standard methods can be used to measure fluorescence by flow cytometry. In another exemplary method, the extracellular portion of human CD38 can be coated on the surface of an ELISA dish. An excess of unlabeled reference antibody can be added for about 15 minutes, and then the biotinylated test antibody can be added. After washing in PBS/Tween, use streptavidin (horseradish peroxidase, HRP) to detect the binding of the test biotinylated antibody and use standard methods to detect the signal . In these competition assays, it is obvious that the reference antibody can be labeled and the test antibody can be unlabeled. When the reference antibody inhibits the binding of the test antibody to CD38, or the test antibody inhibits the binding of the reference antibody to CD38 by at least 80%, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, the test antibody competes with the reference antibody. The epitope of the test antibody can be further defined by, for example, peptide mapping or hydrogen/deuterium protection assay using known methods, or by crystal structure determination.
結合至人類CD38(SEQ ID NO:1)之SKRNIQFSCKNIYR(SEQ ID NO:2)區及EKVQTLEAWVIHGG(SEQ ID NO:3)區的抗體可例如藉由下列產生:使用標準方法及本文中所述之方法利用具有顯示於SEQ ID NO:2及3中之胺基酸序列的胜肽免疫小鼠,且使用例如ELISA或突變誘發研究以定性用於結合至胜肽的所得抗體。 Antibodies that bind to the SKRNIQFSCKNIYR (SEQ ID NO: 2) and EKVQTLEAWVIHGG (SEQ ID NO: 3) regions of human CD38 (SEQ ID NO: 1) can be produced, for example, by the following: using standard methods and the methods described herein Mice were immunized with peptides having the amino acid sequences shown in SEQ ID NOs: 2 and 3, and the resulting antibodies bound to the peptides were qualitatively used, for example, ELISA or mutagenesis studies.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予抗CD38抗體,其結合至人類CD38(SEQ ID NO:1)之SKRNIQFSCKNIYR(SEQ ID NO:2)區及EKVQTLEAWVIHGG(SEQ ID NO:3)區。本發明之方法中所使用之抗體的表位包括具有顯示於SEQ ID NO:2或SEQ ID NO:3之序列的殘基中的一些或全部。在一些實施例中,抗體表位包含人類CD38(SEQ ID NO:1)之SKRNIQFSCKNIYR(SEQ ID NO:2)區中至少一個胺基酸及EKVQTLEAWVIHGG(SEQ ID NO:3)區中至少一個胺基酸。在一些實施例中,抗體表位包含人類CD38(SEQ ID NO:1)之SKRNIQFSCKNIYR(SEQ ID NO:2)區中至少兩個胺基酸及EKVQTLEAWVIHGG(SEQ ID NO:3)區中至少兩個胺基酸。在一些實施例中,抗體表位包含人類CD38(SEQ ID NO:1)之SKRNIQFSCKNIYR(SEQ ID NO:2)區中至少三個胺基酸及EKVQTLEAWVIHGG(SEQ ID NO:3)區中至少三個胺基酸。 The present invention also provides a method for treating a patient suffering from solid tumors, which comprises administering to the patient in need of an anti-CD38 antibody that binds to SKRNIQFSCKNIYR (SEQ ID NO: 1) of human CD38 (SEQ ID NO:1) NO: 2) region and EKVQTLEAWVIHGG (SEQ ID NO: 3) region. The epitope of the antibody used in the method of the present invention includes some or all of the residues having the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the antibody epitope comprises at least one amino acid in the SKRNIQFSCKNIYR (SEQ ID NO: 2) region of human CD38 (SEQ ID NO: 1) and at least one amino acid in the EKVQTLEAWVIHGG (SEQ ID NO: 3) region acid. In some embodiments, the antibody epitope comprises at least two amino acids in the SKRNIQFSCKNIYR (SEQ ID NO: 2) region of human CD38 (SEQ ID NO: 1) and at least two in the EKVQTLEAWVIHGG (SEQ ID NO: 3) region Amino acid. In some embodiments, the antibody epitope comprises at least three amino acids in the SKRNIQFSCKNIYR (SEQ ID NO: 2) region of human CD38 (SEQ ID NO: 1) and at least three in the EKVQTLEAWVIHGG (SEQ ID NO: 3) region Amino acid.
在一些實施例中,特異性結合CD38之抗體包含分別為SEQ ID NO:6、7、及8之HCDR1、HCDR2、及HCDR3胺基酸序列。 In some embodiments, the antibody that specifically binds to CD38 comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively.
在一些實施例中,特異性結合CD38之抗體包含分別為SEQ ID NO:9、10、及11之LCDR1、LCDR2、及LCDR3胺基酸序列。 In some embodiments, the antibody that specifically binds to CD38 includes the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NOs: 9, 10, and 11, respectively.
在一些實施例中,特異性結合CD38之抗體包含分別為SEQ ID NO:6、7、8、9、10、及11之HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、及LCDR3胺基酸序列。 In some embodiments, the antibody that specifically binds to CD38 comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NOs: 6, 7, 8, 9, 10, and 11, respectively.
在一些實施例中,特異性結合CD38之抗體包含VH與VL,該VH係與SEQ ID NO:4具有95%、96%、97%、98%、99%、或100%同一性,且該VL係與SEQ ID NO:5具有95%、96%、97%、98%、99%、或100%同一性。 In some embodiments, the antibody that specifically binds to CD38 comprises VH and VL, and the VH is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, and the The VL line is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5.
在一些實施例中,特異性結合CD38之抗體包含SEQ ID NO:4之VH及SEQ ID NO:5之VL。 In some embodiments, the antibody that specifically binds to CD38 includes the VH of SEQ ID NO:4 and the VL of SEQ ID NO:5.
在一些實施例中,特異性結合CD38之抗體包含SEQ ID NO:12之重鏈及SEQ ID NO:13之輕鏈。 In some embodiments, the antibody that specifically binds to CD38 comprises the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO: 13.
可用於本發明之任何實施例中的其他例示性抗CD38抗體係:mAb003,其包含分別為SEQ ID NO:14及15的VH及VL序列並描述於美國專利第7,829,693號中。mAb003的該VH及該VL可被表示為IgG1/κ。 Another exemplary anti-CD38 antibody system that can be used in any embodiment of the present invention: mAb003, which contains the VH and VL sequences of SEQ ID NOs: 14 and 15, respectively, and is described in US Patent No. 7,829,693. The VH and the VL of mAb003 can be expressed as IgG1/κ.
SEQ ID NO:14 SEQ ID NO: 14
SEQ ID NO:15 mAb024,其包含分別為SEQ ID NO:16及17的VH及VL序列並描述於美國專利第7,829,693號中。mAb024的該VH及該VL可被表示為IgG1/κ。 SEQ ID NO: 15 mAb024, which includes the VH and VL sequences of SEQ ID NOs: 16 and 17, respectively, and is described in US Patent No. 7,829,693. The VH and the VL of mAb024 can be expressed as IgG1/κ.
SEQ ID NO:16 SEQ ID NO: 16
SEQ ID NO:17 SEQ ID NO: 17
MOR-202(MOR-03087),其包含分別為SEQ ID NO:18及19的VH及VL序列並描述於美國專利第8,088,896號中。MOR-202的該VH及該VL可被表示為IgG1/κ。 MOR-202 (MOR-03087), which includes the VH and VL sequences of SEQ ID NOs: 18 and 19, respectively, and is described in US Patent No. 8,088,896. The VH and the VL of MOR-202 can be expressed as IgG1/κ.
SEQ ID NO:18 SEQ ID NO: 18
SEQ ID NO:19 伊沙妥昔單抗(Isatuximab);其包含分別為SEQ ID NO:20及21的VH及VL序列並描述於美國專利第8,153,765號中。伊沙妥昔單抗之VH及VL可被表示為IgG1/κ。 SEQ ID NO: 19 Isartuximab (Isatuximab); it contains the VH and VL sequences of SEQ ID NOs: 20 and 21, respectively, and is described in US Patent No. 8,153,765. The VH and VL of Isartuximab can be expressed as IgG1/κ.
SEQ ID NO 20: SEQ ID NO 20:
SEQ ID NO:21: SEQ ID NO: 21:
可用於本發明之方法中的其他例示性抗CD38抗體包括描述於國際專利公開號WO05/103083、國際專利公開號WO06/125640、國際專利公開號WO07/042309、國際專利公開號WO08/047242、或國際專利公開號WO14/178820中者。 Other exemplary anti-CD38 antibodies that can be used in the methods of the present invention include those described in International Patent Publication No. WO05/103083, International Patent Publication No. WO06/125640, International Patent Publication No. WO07/042309, International Patent Publication No. WO08/047242, or International Patent Publication No. WO14/178820.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體達一段足以治療該固態腫瘤的時間,該抗體包含SEQ ID NO:4之VH及SEQ ID NO:5之VL。 The present invention also provides a method for treating a patient suffering from a solid tumor, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need thereof for a period of time sufficient to treat the solid tumor, the The antibody includes the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體達一段足以治療該固態腫瘤的時間,該抗體包含SEQ ID NO:14之VH及SEQ ID NO:15之VL。 The present invention also provides a method for treating a patient suffering from a solid tumor, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need thereof for a period of time sufficient to treat the solid tumor, the The antibody includes the VH of SEQ ID NO:14 and the VL of SEQ ID NO:15.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體達一段足以治療該固態腫瘤的時間,該抗體包含SEQ ID NO:16之VH及SEQ ID NO:17之VL。 The present invention also provides a method for treating a patient suffering from a solid tumor, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need thereof for a period of time sufficient to treat the solid tumor, the The antibody includes the VH of SEQ ID NO:16 and the VL of SEQ ID NO:17.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體達一段足以治療該固態腫瘤的時間,該抗體包含SEQ ID NO:18之VH及SEQ ID NO:19之VL。 The present invention also provides a method for treating a patient suffering from a solid tumor, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need thereof for a period of time sufficient to treat the solid tumor, the The antibody includes the VH of SEQ ID NO:18 and the VL of SEQ ID NO:19.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體達一段足以治療該固態腫瘤的時間,該抗體包含SEQ ID NO:20之VH及SEQ ID NO:21之VL。 The present invention also provides a method for treating a patient suffering from a solid tumor, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need thereof for a period of time sufficient to treat the solid tumor, the The antibody includes the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
在一些實施例中,固態腫瘤係黑色素瘤。 In some embodiments, the solid tumor is melanoma.
在一些實施例中,固態腫瘤係肺癌。 In some embodiments, the solid tumor is lung cancer.
在一些實施例中,固態腫瘤係鱗狀非小細胞肺癌(NSCLC)。 In some embodiments, the solid tumor is squamous non-small cell lung cancer (NSCLC).
在一些實施例中,固態腫瘤係非鱗狀NSCLC。 In some embodiments, the solid tumor is non-squamous NSCLC.
在一些實施例中,固態腫瘤係肺腺癌。 In some embodiments, the solid tumor is lung adenocarcinoma.
在一些實施例中,固態腫瘤係腎細胞癌(RCC)(例如,腎臟透明細胞癌或腎臟乳突細胞癌)、或其轉移性病變。 In some embodiments, the solid tumor is renal cell carcinoma (RCC) (eg, renal clear cell carcinoma or renal papillary cell carcinoma), or metastatic lesions thereof.
在一些實施例中,固態腫瘤係間皮瘤。 In some embodiments, the solid tumor is mesothelioma.
在一些實施例中,固態腫瘤係鼻咽癌(NPC)。 In some embodiments, the solid tumor is nasopharyngeal carcinoma (NPC).
在一些實施例中,固態腫瘤係結腸直腸癌。 In some embodiments, the solid tumor is colorectal cancer.
在一些實施例中,固態腫瘤係前列腺癌或去勢抗性前列腺癌。 In some embodiments, the solid tumor is prostate cancer or castration resistant prostate cancer.
在一些實施例中,固態腫瘤係胃癌(stomach cancer)。 In some embodiments, the solid tumor is stomach cancer.
在一些實施例中,固態腫瘤係卵巢癌。 In some embodiments, the solid tumor is ovarian cancer.
在一些實施例中,固態腫瘤係胃癌(gastric cancer)。 In some embodiments, the solid tumor is gastric cancer.
在一些實施例中,固態腫瘤係肝癌。 In some embodiments, the solid tumor is liver cancer.
在一些實施例中,固態腫瘤係胰腺癌。 In some embodiments, the solid tumor is pancreatic cancer.
在一些實施例中,固態腫瘤係甲狀腺癌。 In some embodiments, the solid tumor is thyroid cancer.
在一些實施例中,固態腫瘤係頭部及頸部鱗狀細胞癌。 In some embodiments, the solid tumor is a squamous cell carcinoma of the head and neck.
在一些實施例中,固態腫瘤係食道或胃腸道癌。 In some embodiments, the solid tumor is a cancer of the esophagus or gastrointestinal tract.
在一些實施例中,固態腫瘤係乳癌。 In some embodiments, the solid tumor is breast cancer.
在一些實施例中,固態腫瘤係輸卵管癌。 In some embodiments, the solid tumor is fallopian tube cancer.
在一些實施例中,固態腫瘤係腦癌。 In some embodiments, the solid tumor is a brain cancer.
在一些實施例中,固態腫瘤係尿道癌。 In some embodiments, the solid tumor is urethral cancer.
在一些實施例中,固態腫瘤係泌尿生殖癌。 In some embodiments, the solid tumor is urogenital cancer.
在一些實施例中,固態腫瘤係子宮內膜異位。 In some embodiments, the solid tumor is endometriosis.
在一些實施例中,固態腫瘤係子宮頸癌。 In some embodiments, the solid tumor is cervical cancer.
在一些實施例中,固態腫瘤係癌症之轉移性病變。 In some embodiments, solid tumors are metastatic lesions of cancer.
在一些實施例中,固態腫瘤缺乏可偵測的CD38表現。 In some embodiments, solid tumors lack detectable CD38 manifestations.
當相較於對照,例如使用熟知方法以抗CD38抗體偵測的表現之於以同型對照抗體偵測的表現時,當固態腫瘤組織中或單離自固態腫瘤之細胞上的CD38表現係統計學不顯著的時候,固態腫瘤缺乏可偵測的CD38表現。 When compared to a control, for example, the expression detected with an anti-CD38 antibody using a well-known method is compared with the expression detected with an isotype control antibody, the expression of CD38 in solid tumor tissue or on cells isolated from the solid tumor is statistically When insignificant, solid tumors lack detectable CD38 manifestations.
本發明之方法中所使用之抗CD38抗體亦可重新選自例如噬菌體呈現庫,其中噬菌體係經工程改造以表現人類免疫球蛋白或其部分,諸如Fab、單鏈抗體(scFv)、或未配對或配對抗體可變區(Knappik等人,(2000)J Mol Biol 296:57-86;Krebs等人,(2001)J Immunol Meth 254:67-84;Vaughan等人,(1996)Nature Biotechnology 14:309-314;Sheets等人,(1998)PITAS(USA)95:6157-6162;Hoogenboom及Winter,(1991)J Mol Biol 227:381;Marks等人,(1991)J Mol Biol 222:581)。CD38結合可變域可自例如噬菌體呈現庫(表現抗體重鏈及輕鏈可變區)單離為具有噬菌體pIX外殼蛋白的融合蛋白,如描述於Shi等人,(2010)J Mol Biol 397:385-96及國際專利公開號WO09/085462中。抗體庫可用對於人類CD38細胞外域之結合進行篩選,所獲得之陽性殖株進一步表徵,自殖株溶解物(lysate)單離出Fab,且隨後選殖為全長抗體。此種用於單離人類抗體之噬菌體呈現法已於本領域中建立。參見例如:美國專 利第5,223,409號、美國專利第5,403,484號、美國專利第5,571,698號、美國專利第5,427,908號、美國專利第5,580,717號、美國專利第5,969,108號、美國專利第6,172,197號、美國專利第5,885,793號、美國專利第6,521,404號、美國專利第6,544,731號、美國專利第6,555,313號、美國專利第6,582,915號、及美國專利第6,593,081號。 The anti-CD38 antibody used in the method of the present invention can also be re-selected from, for example, a phage display library, where the phage system is engineered to express human immunoglobulin or parts thereof, such as Fab, single chain antibody (scFv), or unpaired Or paired antibody variable regions (Knappik et al., (2000) J Mol Biol 296:57-86; Krebs et al., (2001) J Immunol Meth 254:67-84; Vaughan et al., (1996) Nature Biotechnology 14: 309-314; Sheets et al., (1998) PITAS (USA) 95: 6157-6162; Hoogenboom and Winter, (1991) J Mol Biol 227:381; Marks et al., (1991) J Mol Biol 222:581). The CD38 binding variable domain can be isolated from, for example, a phage display library (expressing antibody heavy and light chain variable regions) as a fusion protein with a phage pIX coat protein, as described in Shi et al., (2010) J Mol Biol 397: 385-96 and International Patent Publication No. WO09/085462. The antibody library can be screened for binding to the extracellular domain of human CD38, and the obtained positive clones are further characterized. The lysate from the clones isolates the Fab, and is subsequently selected as a full-length antibody. This phage display method for isolating human antibodies has been established in the art. See for example: U.S. Patent No. 5,223,409, U.S. Patent No. 5,403,484, U.S. Patent No. 5,571,698, U.S. Patent No. 5,427,908, U.S. Patent No. 5,580,717, U.S. Patent No. 5,969,108, U.S. Patent No. 6,172,197, U.S. Patent No. 5,885,793 , U.S. Patent No. 6,521,404, U.S. Patent No. 6,544,731, U.S. Patent No. 6,555,313, U.S. Patent No. 6,582,915, and U.S. Patent No. 6,593,081.
在一些實施例中,抗CD38抗體係IgG1、IgG2、IgG3、或IgG4同型。 In some embodiments, the anti-CD38 antibody system IgG1, IgG2, IgG3, or IgG4 isotype.
該抗體的Fc部份可媒介抗體的效應功能(effector function),諸如抗體依賴性細胞媒介的細胞毒性(ADCC)、抗體依賴性細胞吞噬作用(ADCP)、或補體依賴性細胞毒性(CDC)。該功能可藉由Fc效應域(effector domain)與具有吞噬或裂解活性的免疫細胞上Fc受體的結合來媒介,或藉由Fc效應域與補體系統成分的結合來媒介。通常,由與Fc結合的細胞或補體成分所媒介的效應會導致目標細胞(例如CD38表現細胞)的抑制或損耗(depletion)。人類IgG同型IgG1、IgG2、IgG3、及IgG4在效應功能上顯示出差別能力。ADCC可由IgG1及IgG3媒介,ADCP可由IgG1、IgG2、IgG3、及IgG4媒介,且CDC可由IgG1及IgG3媒介。 The Fc part of the antibody can mediate the effector function of the antibody, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). This function can be mediated by the binding of Fc effector domains to Fc receptors on immune cells with phagocytic or lytic activity, or by the binding of Fc effector domains to components of the complement system. Generally, the effects mediated by cells or complement components that bind to Fc will result in the inhibition or depletion of target cells (for example, CD38 expressing cells). Human IgG isotypes IgG1, IgG2, IgG3, and IgG4 show differential ability in effector functions. ADCC can be mediated by IgG1 and IgG3, ADCP can be mediated by IgG1, IgG2, IgG3, and IgG4, and CDC can be mediated by IgG1 and IgG3.
與包含SEQ ID NO:4之VH及SEQ ID NO:5之VL的抗體實質上同一的抗體可在本發明之方法中使用。本文中所使用之用語「實質上同一(substantially identical)」意指所比較的兩個抗體VH或VL胺基酸序列係同一或具有「無實質差異(insubstantial differences)」。無實質差異係在抗體重鏈或輕鏈中1、2、3、4、5、6、7、8、9、10、11、12、13、14、或15個不會對抗體性能產生不利影響的胺基酸取代。同一性百分比可例如藉由使用Vector NTI v.9.0.0(Invitrogen,Carlsbad,CA)之AlignX模組的預設設定進行成對比對來判定。本發明的蛋白質序列可被用作查詢序列(query sequence)來執行針對公開或專利數據庫的檢索以(例如)鑑定相關序列。用來執行該等檢索之例示性程式係XBLAST或BLASTP程式(http_//www_ncbi_nlm/nih_gov),或使用預設設定的GenomeQuestTM (GenomeQuest,Westborough,MA)套件。可對特異性結合CD38的抗體進行的例示性取代係例如以具有類似電荷、疏水性、或立體化學特性的胺基酸進行的保守型取代(conservative substitution)。亦可進行保守型取代以改良抗體性質(例如穩定性或親和力),或改良抗體的效應功能。例如可對該抗CD38抗體的重鏈或輕鏈中進行1、2、3、4、5、6、7、8、9、10、11、12、13、14、或15個胺基酸取代。此外,該VH或VL中的任何天然殘基亦可經丙胺酸取代,如先前已針對丙胺酸掃描式突變誘發(alanine scanning mutagenesis)所描述者(MacLennan等人,Acta Physiol Scand Suppl 643:55-67,1998;Sasaki等人,Adv Biophys 35:1-24,1998)。所欲之胺基酸取代可在此等取代係所欲時由所屬領域中具有通常知識者判定。胺基酸取代可例如藉由PCR突變誘發(美國專利第4,683,195號)來進行。變異體庫可使用熟知方法來產生,例如使用隨機(NNK)或非隨機密碼子(例如DVK密碼子),其編碼11種胺基酸(Ala、Cys、Asp、Glu、Gly、Lys、Asn、Arg、Ser、Tyr、Trp),然後篩選變異體庫以找出具有所欲性質之變異體。所產生的變異體可使用本文所述之方法體外測試其等與CD38的結合、其等誘導ADCC、ADCP、或細胞凋亡、或調節CD38酶活性的能力。 An antibody that is substantially the same as the antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 can be used in the method of the present invention. The term "substantially identical" as used herein means that the VH or VL amino acid sequences of the two antibodies compared are identical or have "insubstantial differences". The insubstantial difference is that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 in the heavy or light chain of the antibody will not adversely affect the performance of the antibody Affected amino acid substitution. The identity percentage can be determined, for example, by using the default settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, CA) to make a comparison. The protein sequence of the present invention can be used as a query sequence to perform searches against public or patent databases to, for example, identify related sequences. Exemplary programs used to perform these searches are XBLAST or BLASTP programs (http_//www_ncbi_nlm/nih_gov), or GenomeQuest TM (GenomeQuest, Westborough, MA) package with default settings. Exemplary substitutions that can be made to an antibody that specifically binds to CD38 are, for example, conservative substitutions with amino acids with similar charge, hydrophobicity, or stereochemical properties. Conservative substitutions can also be made to improve antibody properties (such as stability or affinity), or to improve the effector function of the antibody. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions can be made in the heavy or light chain of the anti-CD38 antibody . In addition, any natural residues in the VH or VL can also be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., Acta Physiol Scand Suppl 643:55- 67, 1998; Sasaki et al., Adv Biophys 35: 1-24, 1998). The desired amino acid substitution can be determined by a person with ordinary knowledge in the field when these substitutions are desired. Amino acid substitution can be performed, for example, by PCR mutagenesis (U.S. Patent No. 4,683,195). The library of variants can be generated using well-known methods, such as using random (NNK) or non-random codons (such as DVK codons), which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp), and then screen the library of variants to find variants with desired properties. The generated variants can be tested in vitro for their binding to CD38, their ability to induce ADCC, ADCP, or apoptosis, or to regulate CD38 enzyme activity using the methods described herein.
在一些實施例中,特異性結合CD38之抗體可以一範圍之親和力(KD)結合人類CD38。在根據本發明之一實施例中,以及在以下每一個編號實施例的一些實施例中,特異性結合CD38之抗體以高親和力結合至CD38,例如KD等於或小於約10-7M,諸如但不限於,1至9.9(或其中任何範圍或值,諸如1、2、3、4、5、6、7、8、或9)x 10-8M、10-9M、10-10M、10-11M、10-12M、10-13M、10-14M、10-15M、或其中任何範圍或值,如藉由表面電漿共振或Kinexa方法所判定,如所屬技術領域中具有通常知識者所實踐。一例示性親和力係等於或小於1×10-8M。另一例示性親和力係等於或小於1×10-9M。 In some embodiments, antibodies that specifically bind to CD38 can bind to human CD38 with a range of affinities (K D ). In an embodiment according to the present invention, and in some embodiments of each of the following numbered embodiments, an antibody that specifically binds to CD38 binds to CD38 with high affinity, for example, K D is equal to or less than about 10 -7 M, such as But not limited to, 1 to 9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 -8 M, 10 -9 M, 10 -10 M , 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 M, or any range or value thereof, as determined by surface plasmon resonance or Kinexa method, as in the technical field Practiced by those with ordinary knowledge. An exemplary affinity is equal to or less than 1×10 -8 M. Another exemplary affinity is equal to or less than 1×10 −9 M.
在一些實施例中,特異性結合CD38之抗體係雙特異性抗體。現有的抗CD38抗體之VL及/或VH區或如本文所述重新鑑定 之VL及VH區可被工程改造至雙特異性全長抗體中。該等雙特異性抗體可藉由調節介於單特異性抗體重鏈間的CH3相互作用來製造以形成雙特異性抗體,其係使用諸如在以下文獻中所述之技術:美國專利第7,695,936號;國際專利公開號WO04/111233;美國專利公開號US2010/0015133;美國專利公開號US2007/0287170;國際專利公開號WO2008/119353;美國專利公開號US2009/0182127;美國專利公開號US2010/0286374;美國專利公開號US2011/0123532;國際專利公開號WO2011/131746;國際專利公開號WO2011/143545;或美國專利公開號US2012/0149876。可合併本發明之抗體之VL及/或VH區的另外雙特異性結構係例如雙可變域免疫球蛋白(國際專利公開號WO2009/134776)、或包括各種二聚化域以連接具有不同特異性的兩個抗體臂的結構,諸如白胺酸拉鍊(leucine zipper)或膠原蛋白二聚化域(國際專利公開號WO2012/022811、美國專利第5,932,448號;美國專利第6,833,441號)。 In some embodiments, the anti-system bispecific antibody that specifically binds to CD38. The VL and/or VH regions of existing anti-CD38 antibodies or re-identification as described herein The VL and VH regions can be engineered into bispecific full-length antibodies. These bispecific antibodies can be produced by modulating the CH3 interaction between the heavy chains of monospecific antibodies to form bispecific antibodies, using techniques such as those described in the following document: US Patent No. 7,695,936 ; International Patent Publication No. WO04/111233; U.S. Patent Publication No. US2010/0015133; U.S. Patent Publication No. US2007/0287170; International Patent Publication No. WO2008/119353; U.S. Patent Publication No. US2009/0182127; U.S. Patent Publication No. US2010/0286374; Patent Publication No. US2011/0123532; International Patent Publication No. WO2011/131746; International Patent Publication No. WO2011/143545; or US Patent Publication No. US2012/0149876. Other bispecific structures such as dual variable domain immunoglobulins (International Patent Publication No. WO2009/134776) that can incorporate the VL and/or VH regions of the antibody of the present invention, or include various dimerization domains to link with different specificities The structure of two antibody arms, such as leucine zipper or collagen dimerization domain (International Patent Publication No. WO2012/022811, U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441).
例如,雙特異性抗體可在無細胞環境中體外產生,此係藉由在兩個單特異性同二聚體抗體之CH3區中引入非對稱突變,且在還原條件中(以讓雙硫鍵異構化)以兩個親體單特異性同二聚體抗體形成該雙特異性異二聚體抗體,其係根據描述於國際專利公開號WO2011/131746中之方法。在該等方法中,第一單特異性雙價抗體(例如,抗CD38抗體)及第二單特異性雙價抗體係經工程改造以在CH3域具有某些促進異二聚體穩定性之取代;該等抗體係在足以讓絞鏈區中之半胱胺酸進行雙硫鍵異構化的還原條件下一起培養;從而藉由Fab臂交換來產生該雙特異性抗體。培養條件可最佳地被回復為非還原性(non-reducing)。可使用之例示性還原劑係2-巰基乙胺(2-MEA)、二硫蘇糖醇(dithiothreitol,DTT)、二硫赤蘚醇(dithioerythritol,DTE)、麩胱甘肽、參(2-羧乙基)膦(TCEP)、L-半胱胺酸及β-巰基乙醇,還原劑較佳係選自由下列所組成之群組:2-巰基乙胺、二硫蘇糖醇及參(2-羧乙基)膦。例如,可使用在至少20℃之溫度且有至少25mM之2-MEA存在下或於至少0.5mM之二硫蘇糖醇 存在且在5至8之pH下(例如在7.0之pH下或在7.4之pH下)培養至少90分鐘。 For example, bispecific antibodies can be produced in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies, and under reducing conditions (to allow the disulfide bond Isomerization) to form the bispecific heterodimeric antibody with two parent monospecific homodimeric antibodies according to the method described in International Patent Publication No. WO2011/131746. In these methods, the first monospecific bivalent antibody (eg, anti-CD38 antibody) and the second monospecific bivalent antibody system are engineered to have certain substitutions in the CH3 domain that promote heterodimer stability ; These antibodies are cultured together under reducing conditions sufficient to allow the cysteine in the hinge region to carry out disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange. The culture conditions can be optimally restored to non-reducing. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, ginseng (2- Carboxyethyl) phosphine (TCEP), L-cysteine and β-mercaptoethanol. The reducing agent is preferably selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and ginseng (2 -Carboxyethyl) phosphine. For example, it can be used in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol at a temperature of at least 20°C Exist and incubate at a pH of 5 to 8 (for example, at a pH of 7.0 or at a pH of 7.4) for at least 90 minutes.
可在該雙特異性抗體之第一重鏈中及在第二重鏈中使用的例示性CH3突變係K409R及/或F405L。 Exemplary CH3 mutant lines K409R and/or F405L that can be used in the first heavy chain and in the second heavy chain of the bispecific antibody.
本發明之方法可用來治療屬於任何分類之動物病患。此等動物之實例包括哺乳動物,諸如人類、鼠、犬、貓、及農畜(farm animal)。 The method of the present invention can be used to treat animal diseases belonging to any classification. Examples of such animals include mammals such as humans, mice, dogs, cats, and farm animals.
特異性結合CD38之抗體可以本發明之方法以合適的醫藥組成物之形式提供,該等醫藥組成物包含特異性結合CD38之抗體及醫藥上可接受之載劑。該載劑可係與特異性結合CD38之抗體一起投予之稀釋劑、佐劑、賦形劑、或媒劑。此等媒劑可為液體如水及油,包括來自石油、動物、蔬菜、或合成來源者,諸如花生油、大豆油、礦物油、芝麻油、及類似者。例如,可使用0.4%鹽水及0.3%甘胺酸。這些溶液係無菌且一般不含顆粒物質。其等可藉由習知、熟知的滅菌技術(例如過濾)來滅菌。該等組成物可含有如用以接近生理條件所需之醫藥上可接受的輔助物質,諸如pH調整及緩衝劑、穩定劑、增稠劑、潤滑劑、及著色劑等。在此類醫藥配方中特異性結合CD38之抗體的濃度可有廣泛變化,即從以重量計小於約0.5%,常達以重量計至少約1%至多達以重量計15或20%、25%、30%、35%、40%、45%、或50%,並且將主要基於所需劑量、流體體積、黏度等,根據所選擇之特定投予模式來選擇。合適的媒劑及調配物(包含其他的人類蛋白質,例如人類血清白蛋白),舉例而言,係被描述於例如Remington:The Science and Practice of Pharmacy,21st Edition,Troy,D.B.ed.,Lipincott Williams and Wilkins,Philadelphia,PA 2006,Part 5,Pharmaceutical Manufacturing pp 691-1092中,請特別參見pp.958-989。
Antibodies that specifically bind to CD38 can be provided by the method of the present invention in the form of suitable pharmaceutical compositions, which include antibodies that specifically bind to CD38 and a pharmaceutically acceptable carrier. The carrier can be a diluent, adjuvant, excipient, or vehicle administered together with an antibody that specifically binds CD38. These vehicles can be liquids such as water and oils, including those derived from petroleum, animal, vegetable, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional and well-known sterilization techniques (for example, filtration). These compositions may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and coloring agents, which are required to approximate physiological conditions. The concentration of antibodies that specifically bind to CD38 in such pharmaceutical formulations can vary widely, that is, from less than about 0.5% by weight, often up to at least about 1% by weight to as much as 15 or 20%, 25% by weight. , 30%, 35%, 40%, 45%, or 50%, and will be mainly based on the required dosage, fluid volume, viscosity, etc., according to the selected specific administration mode. Suitable vehicles and formulations (including other human proteins, such as human serum albumin), for example, are described in, for example, Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, DBed., Lipincott Williams and Wilkins, Philadelphia, PA 2006,
在本發明之方法中的特異性結合CD38的抗體之投予模式可係任何合適之路徑,諸如腸胃外投予,例如皮內、肌肉內、腹膜 內(intraperitoneal)、靜脈內或皮下、肺臟、經黏膜(口腔、鼻腔、陰道內、直腸)、或技藝人士所瞭解以及在所屬技術領域中所熟知之其他手段。特異性結合CD38的抗體可使用已知方法瘤內投予至淋巴結引流部位,用以局部遞送至腫瘤中。 The mode of administration of the antibody that specifically binds to CD38 in the method of the present invention may be any suitable route, such as parenteral administration, such as intradermal, intramuscular, and peritoneal administration. Intraperitoneal, intravenous or subcutaneous, lung, transmucosal (oral, nasal, intravaginal, rectal), or other methods known to those skilled in the art and well-known in the art. Antibodies that specifically bind to CD38 can be intratumorally administered to lymph node drainage sites using known methods for local delivery to the tumor.
特異性結合CD38的抗體可藉由任何合適的路徑投予至病患,例如非經腸道(parentally)投予(其藉由靜脈(i.v.)輸液或推注注射(bolus injection))、肌內或皮下或腹膜內。i.v.輸液可在例如15、30、60、90、120、180、240分鐘內給予,或者在1、2、3、4、5、6、7、8、9、10、11或12小時內給予。 Antibodies that specifically bind to CD38 can be administered to the patient by any suitable route, such as parentally administered (by intravenous (iv) infusion or bolus injection), intramuscular Or subcutaneously or intraperitoneally. iv infusion can be given within, for example, 15, 30, 60, 90, 120, 180, 240 minutes, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours .
給予病患之劑量係足以減輕或至少部分遏止所要治療之疾病(「治療有效量」)並且有時可係0.005mg至約100mg/kg,例如約0.05mg至約30mg/kg或約5mg至約25mg/kg、或約4mg/kg、約8mg/kg、約16mg/kg、或約24mg/kg,或者例如約1、2、3、4、5、6、7、8、9、或10mg/kg,但可甚至更高,例如約15、16、17、18、19、20、21、22、23、24、25、30、40、50、60、70、80、90、或100mg/kg。 The dose administered to the patient is sufficient to alleviate or at least partially curb the disease to be treated ("therapeutically effective amount") and can sometimes range from 0.005 mg to about 100 mg/kg, such as from about 0.05 mg to about 30 mg/kg or from about 5 mg to about 25mg/kg, or about 4mg/kg, about 8mg/kg, about 16mg/kg, or about 24mg/kg, or for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ kg, but can be even higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg/kg .
亦可給予固定單位劑量,例如50、100、200、500、或1000mg,或者劑量可基於病患之表面積,例如500、400、300、250、200、或100mg/m2。經常可投予介於1與8次間的劑量(例如1、2、3、4、5、6、7、或8次),但亦可給予9、10、11、12、13、14、15、16、17、18、19、20、或更多次劑量。 A fixed unit dose may also be given, such as 50, 100, 200, 500, or 1000 mg, or the dose may be based on the surface area of the patient, such as 500, 400, 300, 250, 200, or 100 mg/m 2 . Often doses between 1 and 8 times (for example, 1, 2, 3, 4, 5, 6, 7, or 8 times) can be administered, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses.
本發明之方法中之特異性結合CD38之抗體的投予可在一天、兩天、三天、四天、五天、六天、一週、兩週、三週、一個月、五週、六週、七週、兩個月、三個月、四個月、五個月、六個月、或更久之後重覆進行。重覆治療過程亦為可能者,如為慢性投予。重覆投予可在相同劑量或在不同劑量下。例如,本發明之方法中之特異性結合CD38之抗體可在8mg/kg或在16mg/kg下以每週間隔投予持續8週,接著在8mg/kg或在16mg/kg下每兩週投予持續另外16週,接著在8mg/kg或在16mg/kg下藉由靜脈輸液每四週投予。 The antibody that specifically binds to CD38 in the method of the present invention can be administered in one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, and six weeks. , Seven weeks, two months, three months, four months, five months, six months, or more later. It is also possible to repeat the treatment process, such as chronic administration. Repeated administration can be at the same dose or at different doses. For example, the antibody that specifically binds to CD38 in the method of the present invention can be administered at 8 mg/kg or at 16 mg/kg at weekly intervals for 8 weeks, and then at 8 mg/kg or at 16 mg/kg every two weeks. The administration continued for another 16 weeks, followed by intravenous infusion at 8 mg/kg or 16 mg/kg every four weeks.
特異性結合CD38之抗體可在本發明之方法中藉由維持療法投予,諸如(例如)一週一次,持續6個月或更長時間。 Antibodies that specifically bind to CD38 can be administered by maintenance therapy in the methods of the invention, such as, for example, once a week for 6 months or longer.
例如,本發明之方法中之特異性結合CD38的抗體可以約0.1至100mg/kg的量作為日劑量,諸如0.5、0.9、1.0、1.1、1.5、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、40、45、50、60、70、80、90、或100mg/kg,每天提供、於開始治療後的第1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、或40天中之至少一天提供、或者可替代地,於開始治療後的第1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、或20週中之至少一週提供、或其任何組合,並使用每24、12、8、6、4、或2小時之單次或分次劑量、或其任何組合。 For example, the antibody that specifically binds to CD38 in the method of the present invention can be used as a daily dose in an amount of about 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg/kg, provided daily, on the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, after starting treatment 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, Provided on at least one day of 39, or 40 days, or alternatively, on the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, after starting treatment Provide at least one week of 15, 16, 17, 18, 19, or 20 weeks, or any combination thereof, and use a single or divided dose every 24, 12, 8, 6, 4, or 2 hours, or Any combination.
本發明之方法中之特異性結合CD38的抗體亦可預防性投予以減少發展癌症之風險、延緩癌症進展事件之開始發生、及/或當癌症處於緩解時減少復發之風險。這可能在已知存在有腫瘤但因其他生物因素而很難定位腫瘤的病患中特別有用。 The antibody that specifically binds to CD38 in the method of the present invention can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of cancer progression events, and/or reduce the risk of recurrence when the cancer is in remission. This may be particularly useful in patients who are known to have tumors but are difficult to locate tumors due to other biological factors.
本發明之方法中之特異性結合CD38之抗體可凍乾貯存,並在使用前於合適的載劑中重構。此技術已顯示對於習知蛋白質製劑為有效者並且可使用熟知之凍乾及重構技術。 The antibody that specifically binds to CD38 in the method of the present invention can be lyophilized for storage, and reconstituted in a suitable carrier before use. This technique has been shown to be effective for conventional protein preparations and well-known freeze-drying and reconstitution techniques can be used.
本發明之方法中之特異性結合CD38之抗體可與第二治療劑組合投予。 The antibody that specifically binds to CD38 in the method of the present invention can be administered in combination with a second therapeutic agent.
在本發明之方法中,特異性結合CD38之抗體可與化學治療藥品或所屬技術領域中具有通常知識者已知的其他抗癌治療劑中之任何一或多者一起投予。化學治療劑係可用於治療癌症的化學化合物,且包括生長抑制劑或其他細胞毒性劑,並包括烷化劑、抗代謝藥、抗微管抑制劑、拓樸異構酶抑制劑(topoisomerase inhibitor)、受體酪胺酸激酶抑制劑、血管生成抑制劑、及類似者。化學治療劑之實例包括:烷化劑,諸如噻替派(thiotepa)及環磷醯胺(CYTOXAN®);
磺酸烷基酯,諸如白消安(busulfan)、英丙舒凡(improsulfan)、及哌泊舒凡(piposulfan);氮丙啶類(aziridine),諸如苯佐替派(benzodopa)、卡波醌(carboquone)、美妥替派(meturedopa)、及烏瑞替派(uredopa);乙烯亞胺(ethylenimine)及甲基三聚氰胺(methylamelamine),包括六甲蜜胺(altretamine)、三亞乙基密胺(triethylenemelamine)、三亞乙基磷醯胺(trietylenephosphoramide)、三亞乙基硫代磷醯胺(triethylenethiophosphaoramide)、及三羥甲基密胺(trimethylolomelamine);氮芥(nitrogen mustard),諸如氮芥苯丁酸(chlorambucil)、萘氮芥(chlornaphazine)、膽磷醯胺(cholophosphamide)、雌莫司汀(estramustine)、異環磷醯胺(ifosfamide)、雙氯乙基甲胺(mechlorethamine)、鹽酸雙氯乙基甲胺氧化物(mechlorethamine oxide hydrochloride)、黴法蘭(melphalan)、新氮芥(novembichin)、膽固醇苯乙酸氮芥(phenesterine)、松龍苯芥(prednimustine)、氯乙環磷醯胺(trofosfamide)、尿嘧啶氮芥(uracil mustard);硝基脲類(nitrosureas),諸如卡莫司汀(carmustine)、吡葡亞硝脲(chlorozotocin)、福莫司汀(fotemustine)、洛莫司汀(lomustine)、尼莫司汀(nimustine)、雷莫司汀(ranimustine);抗生素,諸如阿克拉黴素(aclacinomysin)、放線菌黴素(actinomycin)、氨茴黴素(authramycin)、偶氮絲胺酸(azaserine)、博萊黴素(bleomycin)、放線菌素(cactinomycin)、卡奇黴素(calicheamicin)、卡拉比黴素(carabicin)、洋紅黴素(carminomycin)、嗜癌黴素(carzinophilin)、色黴素(chromomycin)、更生黴素(dactinomycin)、道諾黴素(daunorubicin)、地托比星(detorubicin)、6-重氮基-5-側氧基-L-正白胺酸、多柔比星(doxorubicin)、表柔比星(epirubicin)、依索比星(esorubicin)、艾達魯比星(idarubicin)、麻西羅黴素(marcellomycin)、絲裂黴素(mitomycin)、黴酚酸(mycophenolic acid)、諾加黴素(nogalamycin)、橄欖黴素(olivomycin)、培洛黴素(peplomycin)、潑非黴素(potfiromycin)、嘌呤黴素(puromycin)、三鐵阿黴素(quelamycin)、羅多比星(rodorubicin)、鏈黑菌素(streptonigrin)、鏈脲黴素(streptozocin)、殺結核菌素(tubercidin)、烏苯美司
(ubenimex)、淨司他汀(zinostatin)、佐柔比星(zorubicin);抗代謝藥,諸如胺甲喋呤(methotrexate)及5-FU;葉酸類似物,諸如二甲葉酸(denopterin)、胺甲喋呤、蝶羅呤(pteropterin)、三甲曲沙(trimetrexate);嘌呤類似物,諸如氟達拉濱(fludarabine)、6-巰基嘌呤(6-mercaptopurine)、硫咪嘌呤(thiamiprine)、硫鳥嘌呤(thioguanine);嘧啶類似物,諸如環胞苷(ancitabine)、阿紮胞苷(azacitidine)、6-氮雜尿苷(6-azauridine)、卡莫氟(carmofur)、阿糖胞苷(cytarabine)、二脫氧尿苷(dideoxyuridine)、去氧氟尿苷(doxifluridine)、依諾他濱(enocitabine)、氟尿苷(floxuridine);雄性激素,諸如卡普睾酮(calusterone)、丙酸甲雄烷酮(dromostanolone propionate)、環硫雄醇(epitiostanol)、美雄烷(mepitiostane)、睾內酯(testolactone);抗腎上腺劑,諸如氨魯米特(aminoglutethimide)、米托坦(mitotane)、曲洛司坦(trilostane);葉酸補充劑,諸如亞葉酸(frolinic acid);醋葡醛內酯(aceglatone);醛磷醯胺糖苷(aldophosphamide glycoside);胺基酮戊酸(aminolevulinic acid);安吖啶(amsacrine);貝塔布辛(bestrabucil);比生群(bisantrene);依達曲沙(edatraxate);得弗伐胺(defofamine);地美可辛(demecolcine);亞胺醌(diaziquone);依洛尼塞(elfornithine);依利醋銨(elliptinium acetate);依託格魯(etoglucid);硝酸鎵;羥基脲(hydroxyurea);香菇多糖(lentinan);氯尼達明(lonidamine);米托胍腙(mitoguazone);米托蒽醌(mitoxantrone);莫匹丹莫(mopidanmol);二胺硝吖啶(nitracrine);噴司他汀(pentostatin);苯來美特(phenamet);吡柔比星(pirarubicin);鬼臼酸(podophyllinic acid);2-乙基醯肼(2-ethylhydrazide);丙卡巴肼(procarbazine);PSK®;雷佐生(razoxane);西索菲蘭(sizofiran);螺旋鍺(spirogermanium);細交鏈孢菌酮酸(tenuazonic acid);三亞胺醌(triaziquone);2,2',2"-三氯三乙胺;胺甲酸酯;長春地辛(vindesine);達卡巴嗪(dacarbazine);甘露醇氮芥(mannomustine);二溴甘露醇(mitobronitol);二溴衛矛醇(mitolactol);哌泊溴烷(pipobroman);格塞圖辛(gacytosine);阿拉伯糖苷(arabinoside)(「Ara-C」);環磷醯胺;噻替派(thiotepa);類紫杉
醇或紫杉烷家族之成員,諸如紫杉醇(TAXOL®)、剋癌易(docetaxel,TAXOTERE®)、及其類似物;氮芥苯丁酸(chlorambucil);吉西他濱(gemcitabine);6-硫鳥嘌呤;巰基嘌呤;胺甲喋呤;鉑類似物,諸如順氯氨鉑(cisplatin)及卡鉑(carboplatin);長春花鹼(vinblastine);鉑;依妥普賽(etoposide,VP-16);異環磷醯胺;絲裂黴素C;邁杜蔥酮(mitoxantrone);長春新鹼(vincristine);長春瑞濱(vinorelbine);溫諾平(navelbine);諾安托(novantrone);坦尼坡賽(teniposide);道紅鏈絲菌素(daunomycin);胺蝶呤(aminopterin);截瘤達(xeloda);伊班膦酸鹽(ibandronate);CPT-11;拓樸異構酶抑制劑RFS 2000;二氟甲基鳥胺酸(DMFO);視黃酸;埃斯培拉黴素(esperamicin);卡培他濱(capecitabine);受體酪胺酸激酶抑制劑及/或血管生成抑制劑,包括NEXAVAR®(索拉非尼(sorafenib))、SUTENT®(舒尼替尼(sunitinib))、VOTRIENTTM(帕唑帕尼(pazopanib))、PALLADIATM(托西尼布(toceranib))、ZACTIMATM(凡德他尼(vandetanib))、RECENTIN®(西地尼布(cediranib))、瑞格非尼(regorafenib)(BAY 73-4506)、阿西替尼(axitinib)(AG013736)、來他替尼(lestaurtinib)(CEP-701)、TARCEVA®(厄洛替尼(erlotinib))、IRESSATM(吉非替尼(gefitinib))、Gilotrif®(阿法替尼(afatinib))、TYKERB®(拉帕替尼(lapatinib))、來那替尼(neratinib)、及類似者、以及上述中任一者之醫藥上可接受之鹽、酸、或衍生物。此定義中亦包括用於調節或抑制激素對腫瘤作用之抗激素劑,諸如抗雌激素,包括例如他莫昔芬(tamoxifen)、雷洛昔芬(raloxifene)、芳香酶抑制性4(5)-咪唑、4-羥基他莫昔芬、曲沃昔芬(trioxifene)、雷洛昔芬(keoxifene)、LY 117018、奧那司酮(onapristone)、及FARESTON®(托瑞米芬(toremifene));及抗雄激素,諸如氟他胺(flutamide)、尼魯米特(nilutamide)、比卡魯胺(bicalutamide)、亮丙瑞林(leuprolide)、及戈舍瑞林(goserelin);及上述中任一者之醫藥上可接受之鹽、酸、或衍生物。如那些在Wiemann等人,1985,在Medical Oncology(Calabresi等人,eds.)中,Chapter 10,McMillan Publishing中所揭露的其他習知細胞毒性化合物亦可適用於本發明之方法。
In the method of the present invention, the antibody that specifically binds to CD38 can be administered together with any one or more of chemotherapeutic drugs or other anti-cancer therapeutic agents known to those with ordinary knowledge in the art. Chemotherapeutic agents are chemical compounds that can be used to treat cancer, and include growth inhibitors or other cytotoxic agents, and include alkylating agents, antimetabolites, anti-microtubule inhibitors, and topoisomerase inhibitors , Receptor tyrosine kinase inhibitors, angiogenesis inhibitors, and the like. Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN®); sulfonic acid alkyl esters, such as busulfan (busulfan), improsulfan (improsulfan), And piposulfan (piposulfan); aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; Ethyleneimine and methylamelamine, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylene thiophosphoramide ( triethylenethiophosphaoramide, and trimethylolomelamine; nitrogen mustard, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine (estramustine), ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin ), cholesterol phenesterine (phenesterine), prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard (uracil mustard); nitrosureas, such as carmus Carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as Aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, cactinomycin (Calicheamicin), carabicin (carabicin), carminomycin (carminomycin), oncinomycin (c arzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-n-leucine Acid, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin ), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, triiron Adriamycin (quelamycin), rhodoubicin (rodorubicin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), netstatin (zinostatin), zorubicin (zorubicin); antimetabolites, such as methotrexate and 5-FU; folate analogs, such as denopterin, methotrexate, pterorin ( pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs , Such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine ), doxifluridine, enocitabine, floxuridine; androgen, such as calusterone, dromostanolone propionate, sulfide Androstanol (epitiostanol), mepitiostane (mepitiostane), testolactone (testolactone); anti-adrenal agents, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); Supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; betabuxin (bestrabucil); bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; Elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone ( mitoxantrone; mopidanmol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid ; 2-ethylhydrazide (2-ethylhydrazide); procarbazine; PSK®; razoxane; sizofiran; spirogermanium; Alternaria tenuifonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2',2"-trichlorotriethylamine;carbamate; vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard ( mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); ring Phosphasamide; thiotepa; taxol-like or members of the taxane family, such as paclitaxel (TAXOL®), docetaxel (TAXOTERE®), and its analogs; chlorambucil ); gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; Etoposi de,VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; navelbine Novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; receptor tyrosine kinase inhibition Agents and/or angiogenesis inhibitors, including NEXAVAR® (sorafenib (sorafenib)), SUTENT® (sunitinib), VOTRIENT TM (pazopanib), PALLADIA TM (support Toceranib), ZACTIMA TM (vandetanib), RECENTIN® (cediranib), regorafenib (BAY 73-4506), axitinib (axitinib) (AG013736), Lestaurtinib (CEP-701), TARCEVA® (erlotinib), IRESSA TM (gefitinib), Gilotrif ® (Afatinib) Afatinib), TYKERB® (lapatinib), neratinib, and the like, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. This definition also includes antihormonal agents used to modulate or inhibit the effect of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitory 4(5) -Imidazole, 4-hydroxy tamoxifen, trioxifene (trioxifene), raloxifene (keoxifene), LY 117018, onapristone, and FARESTON® (toremifene) ; And antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and the above Any pharmaceutically acceptable salt, acid, or derivative. For example, other conventional cytotoxic compounds disclosed in Wiemann et al., 1985, in Medical Oncology (Calabresi et al., eds.),
可與本發明之方法中之特異性結合CD38之抗體組合使用的例示性藥劑包括酪胺酸激酶抑制劑及標靶抗癌療法,諸如IRESSATM(吉非替尼)及Tarceva®(厄洛替尼)、以及HER2、HER3、HER4、或VEGF之其他拮抗劑。例示性HER2拮抗劑包括CP-724-714、HERCEPTINTM(曲妥珠單抗(trastuzumab))、OMNITARGTM(帕妥珠單抗(pertuzumab))、TAK-165、TYKERB®(拉帕替尼)(EGFR及HER2抑制劑)、及GW-282974。例示性HER3拮抗劑包括抗Her3抗體(參見例如,美國專利公開號2004/0197332)。例示性HER4拮抗劑包括抗HER4 siRNA(參見例如,Maatta等人,Mol Biol Cell 17:67-79,2006)。例示性VEGF拮抗劑係AvastinTM(貝伐單抗(bevacizumab))。 Exemplary agents that can be used in combination with antibodies that specifically bind to CD38 in the method of the present invention include tyrosine kinase inhibitors and targeted anti-cancer therapies, such as IRESSA TM (gefitinib) and Tarceva ® (erlotinib) Ni), and other antagonists of HER2, HER3, HER4, or VEGF. Exemplary HER2 antagonists include CP-724-714, HERCEPTIN TM (trastuzumab (trastuzumab)), OMNITARG TM (pertuzumab (pertuzumab)), TAK-165 , TYKERB® ( lapatinib) (EGFR and HER2 inhibitors), and GW-282974. Exemplary HER3 antagonists include anti-Her3 antibodies (see, for example, U.S. Patent Publication No. 2004/0197332). Exemplary HER4 antagonists include anti-HER4 siRNA (see, eg, Maatta et al., Mol Biol Cell 17: 67-79, 2006). An exemplary VEGF antagonist is Avastin ™ (bevacizumab).
可與本發明之方法中之特異性結合CD38之抗體組合使用的例示性藥劑包括用於固態腫瘤之標準照護藥品、或免疫檢查點抑制劑。 Exemplary agents that can be used in combination with an antibody that specifically binds to CD38 in the method of the present invention include standard care drugs for solid tumors, or immune checkpoint inhibitors.
本發明之方法中之第二治療劑可係免疫檢查點抑制劑。 The second therapeutic agent in the method of the present invention may be an immune checkpoint inhibitor.
在一些實施例中,免疫檢查點抑制劑係抗PD-1抗體、抗PD-L1抗體、抗PD-L2抗體、抗LAG3抗體、抗TIM3抗體、或抗CTLA-4抗體。 In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, or an anti-CTLA-4 antibody.
在一些實施例中,免疫檢查點抑制劑係拮抗性抗PD-1抗體、拮抗性抗PD-L1抗體、拮抗性抗PD-L2抗體、拮抗性抗LAG3抗體、或拮抗性抗TIM3抗體。 In some embodiments, the immune checkpoint inhibitor is an antagonistic anti-PD-1 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, an antagonistic anti-LAG3 antibody, or an antagonistic anti-TIM3 antibody.
在一些實施例中,免疫檢查點抑制劑係抗PD-1抗體。 In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody.
在一些實施例中,免疫檢查點抑制劑係抗PD-L1抗體。 In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody.
在一些實施例中,免疫檢查點抑制劑係抗PD-L2抗體。 In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody.
在一些實施例中,免疫檢查點抑制劑係抗LAG3抗體。 In some embodiments, the immune checkpoint inhibitor is an anti-LAG3 antibody.
在一些實施例中,免疫檢查點抑制劑係抗TIM3抗體。 In some embodiments, the immune checkpoint inhibitor is an anti-TIM3 antibody.
在一些實施例中,免疫檢查點抑制劑係抗CTLA-4抗體。 In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody.
任何拮抗性抗PD-1抗體均可用於本發明之方法中。可使用之例示性抗PD-1抗體係OPVIDO®(尼沃魯單抗)及KEYTRUDA®(派立珠單抗)。OPVIDO®(尼沃魯單抗)係描述於例如美國專利第8,008,449號中(抗體5C4)且包含SEQ ID NO:24之VH及SEQ ID NO:25之VL。KEYTRUDA®(派立珠單抗)係描述於例如美國專利第8,354,509號中且包含SEQ ID NO:22之VH及SEQ ID NO:23之VL。尼沃魯單抗及派立珠單抗之胺基酸序列亦可透過CAS登錄取得。可使用之另外的PD-1抗體係描述於美國專利第7,332,582號、美國專利公開號2014/0044738、國際專利公開號WO2014/17966、及美國專利公開號2014/0356363中。 Any antagonistic anti-PD-1 antibody can be used in the method of the invention. Exemplary anti-PD-1 antibodies that can be used OPVIDO® (nivoluzumab) and KEYTRUDA® (pelizumab). OPVIDO® (nivolumab) is described in, for example, US Patent No. 8,008,449 (antibody 5C4) and includes the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25. KEYTRUDA® (Peclizumab) is described in, for example, US Patent No. 8,354,509 and includes the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23. The amino acid sequence of Nivolumab and Peclizumab can also be obtained through CAS registration. Additional PD-1 anti-systems that can be used are described in U.S. Patent No. 7,332,582, U.S. Patent Publication No. 2014/0044738, International Patent Publication No. WO2014/17966, and U.S. Patent Publication No. 2014/0356363.
「拮抗劑(Antagonist)」係指當結合至細胞蛋白質時抑制至少一種該蛋白質之天然配位體所誘導之反應或活性的分子。一分子,當至少一種反應或活性受到的抑制比該至少一種反應或活性在不存在拮抗劑之情況下(例如,陰性對照)受到的抑制多至少約30%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、或100%時,或當相較於在不存在拮抗劑之情況下的抑制,抑制係統計學顯著的時,係拮抗劑。拮抗劑可係抗體、可溶性配位體、小分子、DNA、或RNA諸如siRNA。例如藉由PD-1結合至其受體PD-L1或PD-L2所誘導之典型反應或活性可係抗原特異性CD4+或CD8+細胞增生的減少或T細胞之干擾素-γ(IFN-γ)產生的減少,其導致抑制針對例如腫瘤的免疫反應。藉由TIM-3結合至其受體(諸如半乳糖凝集素-9)所誘導之典型反應或活性可係抗原特異性CD4+或CD8+細胞增生的減少、T細胞之IFN-γ產生的減少、或CD4+或CD8+細胞上之CD137表面表現的減少,其導致抑制針對例如腫瘤的免疫反應。因此,特異性結合PD-1的拮抗性PD-1抗體、拮抗性PD-L2、特異性結合TIM-3的拮抗性抗體藉由對於抑制劑途徑加以抑制來誘導免疫反應。 "Antagonist" refers to a molecule that, when bound to a cellular protein, inhibits the reaction or activity induced by at least one of the protein's natural ligands. A molecule, when at least one reaction or activity is inhibited by at least about 30%, 40%, 45%, 50% more than the at least one reaction or activity in the absence of an antagonist (eg, negative control) , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or when compared to inhibition in the absence of an antagonist, inhibit When it is statistically significant, it is an antagonist. Antagonists can be antibodies, soluble ligands, small molecules, DNA, or RNA such as siRNA. For example, the typical response or activity induced by PD-1 binding to its receptor PD-L1 or PD-L2 can be the reduction of antigen-specific CD4 + or CD8 + cell proliferation or the interferon-γ (IFN-γ (IFN- γ) Reduction in production, which leads to suppression of immune responses against, for example, tumors. The typical response or activity induced by the binding of TIM-3 to its receptor (such as galectin-9) can be the reduction of antigen-specific CD4 + or CD8 + cell proliferation, and the reduction of IFN-γ production by T cells , Or a reduction in the surface expression of CD137 on CD4+ or CD8 + cells, which results in suppression of immune responses against, for example, tumors. Therefore, antagonistic PD-1 antibodies that specifically bind to PD-1, antagonistic PD-L2, and antagonistic antibodies that specifically bind to TIM-3 induce immune responses by inhibiting the inhibitor pathway.
SEQ ID NO:22 SEQ ID NO: 22
SEQ ID NO:23 SEQ ID NO: 23
SEQ ID NO:24 SEQ ID NO: 24
SEQ ID NO:25 SEQ ID NO: 25
增強免疫反應的抗PD-L1抗體可用於本發明之方法中(例如拮抗性抗PD-L1抗體)。可使用之例示性抗PD-L1抗體係德瓦魯單抗(durvalumab)、阿替珠單抗(atezolizumab)、及艾維路單抗(avelumab)、以及描述於例如美國專利公開號2009/0055944、美國專利第美國專利第8,552,154號、美國專利第8,217,149號、及美國專利第8,779,108號中者。 Anti-PD-L1 antibodies that enhance immune response can be used in the methods of the present invention (for example, antagonistic anti-PD-L1 antibodies). Exemplary anti-PD-L1 antibody systems that can be used, durvalumab, atezolizumab, and avelumab, and are described in, for example, U.S. Patent Publication No. 2009/0055944 , U.S. Patent No. 8,552,154, U.S. Patent No. 8,217,149, and U.S. Patent No. 8,779,108.
德瓦魯單抗包含SEQ ID NO:26之VH及SEQ ID NO:27之VL。 Devalumumab includes the VH of SEQ ID NO:26 and the VL of SEQ ID NO:27.
阿替珠單抗包含SEQ ID NO:28之VH及SEQ ID NO:29之VL。 Atezizumab includes the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29.
艾維路單抗包含SEQ ID NO:30之VH及SEQ ID NO:31之VL。 Aveluzumab includes the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31.
SEQ ID NO:26 SEQ ID NO: 26
SEQ ID NO:27 SEQ ID NO: 27
SEQ ID NO:28 SEQ ID NO: 28
SEQ ID NO:29 SEQ ID NO: 29
SEQ ID NO:30 SEQ ID NO: 30
SEQ ID NO:31 SEQ ID NO: 31
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-1抗體(其包含SEQ ID NO:24之VH及SEQ ID NO:25之VL)之組合達一段足以治療該固態腫瘤的時間。 The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-1 antibody (which includes the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25) is combined for a period of time sufficient to treat the solid tumor.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-1抗體(其包含SEQ ID NO:22之VH及SEQ ID NO:23之VL)之組合達一段足以治療該固態腫瘤的時間。 The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-1 antibody (which includes the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23) is combined for a period of time sufficient to treat the solid tumor.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-L1抗體(其包含SEQ ID NO:26之VH及SEQ ID NO:27之VL)之組合達一段足以治療該固態腫瘤的時間。 The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27) is combined for a period of time sufficient to treat the solid tumor.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗 體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-L1抗體(其包含SEQ ID NO:28之VH及SEQ ID NO:29之VL)之組合達一段足以治療該固態腫瘤的時間。 The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient who needs it. The combination of the body (which includes the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5) and the anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29) is sufficient for a period of time The time to treat the solid tumor.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-L1抗體(其包含SEQ ID NO:30之VH及SEQ ID NO:31之VL)之組合達一段足以治療該固態腫瘤的時間。 The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31) is combined for a period of time sufficient to treat the solid tumor.
本發明亦提供一種增強病患之免疫反應的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-1抗體(其包含SEQ ID NO:24之VH及SEQ ID NO:25之VL)之組合達一段足以增強免疫反應的時間。 The present invention also provides a method for enhancing the immune response of a patient, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH and SEQ ID of SEQ ID NO: 4 to the patient who needs it. NO: 5 VL) and anti-PD-1 antibody (which includes the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25) are combined for a period of time sufficient to enhance the immune response.
本發明亦提供一種增強病患之免疫反應的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-1抗體(其包含SEQ ID NO:22之VH及SEQ ID NO:23之VL)之組合達一段足以增強免疫反應的時間。 The present invention also provides a method for enhancing the immune response of a patient, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH and SEQ ID of SEQ ID NO: 4 to the patient who needs it. NO: 5 VL) and anti-PD-1 antibody (which includes the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23) are combined for a period of time sufficient to enhance the immune response.
本發明亦提供一種增強病患之免疫反應的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-L1抗體(其包含SEQ ID NO:26之VH及SEQ ID NO:27之VL)之組合達一段足以增強免疫反應的時間。 The present invention also provides a method for enhancing the immune response of a patient, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH and SEQ ID of SEQ ID NO: 4 to the patient who needs it. NO: 5 VL) and anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27) are combined for a period of time sufficient to enhance the immune response.
本發明亦提供一種增強病患之免疫反應的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-L1抗體(其包含SEQ ID NO:28之VH及SEQ ID NO:29之VL)之組合達一段足以增強免疫反應的時間。 The present invention also provides a method for enhancing the immune response of a patient, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH and SEQ ID of SEQ ID NO: 4 to the patient who needs it. NO: 5 VL) and anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29) are combined for a period of time sufficient to enhance the immune response.
本發明亦提供一種增強病患之免疫反應的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體 (其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-L1抗體(其包含SEQ ID NO:30之VH及SEQ ID NO:31之VL)之組合達一段足以增強免疫反應的時間。 The present invention also provides a method for enhancing the immune response of a patient, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need The combination of the anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5) and the anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31) is sufficient to enhance Time of immune response.
本發明亦提供一種治療患有結腸直腸癌之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體與拮抗性抗PID-1抗體之組合達一段足以治療該結腸直腸癌的時間。 The present invention also provides a method for treating a patient suffering from colorectal cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PID-1 antibody to the patient in need For a period of time sufficient to treat the colorectal cancer.
本發明亦提供一種治療患有結腸直腸癌之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體與拮抗性抗PD-L1抗體之組合達一段足以治療該結腸直腸癌的時間。 The present invention also provides a method for treating a patient suffering from colorectal cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-L1 antibody to the patient in need For a period of time sufficient to treat the colorectal cancer.
本發明亦提供一種治療患有結腸直腸癌之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體與拮抗性抗PD-L2抗體之組合達一段足以治療該結腸直腸癌的時間。 The present invention also provides a method for treating a patient suffering from colorectal cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-L2 antibody to the patient in need of it For a period of time sufficient to treat the colorectal cancer.
本發明亦提供一種治療患有肺癌之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體與拮抗性抗PD-1抗體之組合達一段足以治療該肺癌的時間。 The present invention also provides a method for treating a patient suffering from lung cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-1 antibody to the patient in need thereof for a period of time Enough time to treat this lung cancer.
本發明亦提供一種治療患有肺癌之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體與拮抗性抗PD-L1抗體之組合達一段足以治療該肺癌的時間。 The present invention also provides a method for treating a patient suffering from lung cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-L1 antibody to the patient who needs it for a period of time Enough time to treat this lung cancer.
本發明亦提供一種治療患有肺癌之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體與拮抗性抗PD-L2抗體之組合達一段足以治療該肺癌的時間。 The present invention also provides a method for treating a patient suffering from lung cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-L2 antibody to the patient who needs it for a period of time Enough time to treat this lung cancer.
本發明亦提供一種治療患有前列腺癌之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體與拮抗性抗PD-1抗體之組合達一段足以治療該前列腺癌的時間。 The present invention also provides a method for treating a patient suffering from prostate cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-1 antibody to the patient who needs it. A period of time sufficient to treat the prostate cancer.
本發明亦提供一種治療患有前列腺癌之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體與拮抗性抗PD-L1抗體之組合達一段足以治療該前列腺癌的時間。 The present invention also provides a method for treating a patient suffering from prostate cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-L1 antibody to the patient who needs it. A period of time sufficient to treat the prostate cancer.
本發明亦提供一種治療患有前列腺癌之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體與拮抗性抗PD-L2抗體之組合達一段足以治療該前列腺癌的時間。 The present invention also provides a method for treating a patient suffering from prostate cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-L2 antibody to the patient who needs it. A period of time sufficient to treat the prostate cancer.
增強免疫反應的抗LAG-3抗體可用於本發明之方法中。可使用之例示性抗LAG-3抗體係描述於例如國際專利公開號WO2010/019570中者。 Anti-LAG-3 antibodies that enhance immune response can be used in the methods of the present invention. Exemplary anti-LAG-3 resistance systems that can be used are described in, for example, International Patent Publication No. WO2010/019570.
增強免疫反應的抗CTLA-4抗體可用於本發明之方法中。可使用之例示性抗CTLA-4抗體係伊匹單抗。 Anti-CTLA-4 antibodies that enhance immune response can be used in the methods of the present invention. An exemplary anti-CTLA-4 antibody system that can be used Ipilimumab.
可用於本發明之方法中之抗PD-1、抗PD-L1、抗PD-L2、抗LAG3、抗TIM3、及抗CTLA-4抗體亦可使用本文中所述之方法重新產生。 Anti-PD-1, anti-PD-L1, anti-PD-L2, anti-LAG3, anti-TIM3, and anti-CTLA-4 antibodies that can be used in the method of the present invention can also be regenerated using the methods described herein.
在一些實施例中,可使用包含SEQ ID NO:32之VH及SEQ ID NO:33之VL的抗PD1抗體。 In some embodiments, an anti-PD1 antibody comprising the VH of SEQ ID NO:32 and the VL of SEQ ID NO:33 can be used.
在一些實施例中,可使用包含SEQ ID NO:34之VH及SEQ ID NO:35之VL的抗PD1抗體。 In some embodiments, an anti-PD1 antibody comprising the VH of SEQ ID NO:34 and the VL of SEQ ID NO:35 can be used.
在一些實施例中,可使用包含SEQ ID NO:36之VH及SEQ ID NO:37之VL的抗TIM-3抗體。 In some embodiments, an anti-TIM-3 antibody comprising the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 37 can be used.
在一些實施例中,可使用包含SEQ ID NO:38之VH及SEQ ID NO:39之VL的抗TIM-3抗體。 In some embodiments, an anti-TIM-3 antibody comprising the VH of SEQ ID NO: 38 and the VL of SEQ ID NO: 39 can be used.
SEQ ID NO:32 SEQ ID NO: 32
SEQ ID NO:33 SEQ ID NO: 33
SEQ ID NO:34 SEQ ID NO: 34
SEQ ID NO:35 SEQ ID NO: 35
SEQ ID NO:36 SEQ ID NO: 36
SEQ ID NO:37 SEQ ID NO: 37
SEQ ID NO:38 SEQ ID NO: 38
SEQ ID NO:39 SEQ ID NO: 39
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-1抗體(其包含SEQ ID NO:32之VH及SEQ ID NO:33之VL)之組合達一段足以治療該固態腫瘤的時間。 The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-1 antibody (which includes the VH of SEQ ID NO: 32 and the VL of SEQ ID NO: 33) is combined for a period of time sufficient to treat the solid tumor.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗PD-1抗體(其包含SEQ ID NO:34之VH及SEQ ID NO:35之VL)之組合達一段足以治療該固態腫瘤的時間。 The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-1 antibody (which includes the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 35) is combined for a period of time sufficient to treat the solid tumor.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗TIM-3抗體(其包含SEQ ID NO:36之VH及SEQ ID NO:37之VL)之組合達一段足以治療該固態腫瘤的時間。 The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-TIM-3 antibody (which includes the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 37) is combined for a period of time sufficient to treat the solid tumor.
本發明亦提供一種治療患有固態腫瘤之病患的方法,其包含向有彼之需要之該病患投予治療有效量之特異性結合CD38之抗體(其包含SEQ ID NO:4之VH及SEQ ID NO:5之VL)與抗TIM-3抗體(其包含SEQ ID NO:38之VH及SEQ ID NO:39之VL)之組合達一段足以治療該固態腫瘤的時間。 The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-TIM-3 antibody (which includes the VH of SEQ ID NO: 38 and the VL of SEQ ID NO: 39) for a period of time sufficient to treat the solid tumor.
在本發明之方法中,特異性結合CD38之抗體與第二治療劑之組合可在任何習知時段內投予。例如,特異性結合CD38之抗體及第二治療劑可在同一天,且甚至以同一靜脈輸液投予至病患。然而,特異性結合CD38之抗體及第二治療劑亦可在交替的日子、或交替的週或月、等等投予。在一些方法中,特異性結合CD38之抗體及第二治療劑可在足夠接近的時間投予,使其等在可偵測的水平下同時存在(例如,在血清中)於經治療的病患中。在一些方法中,以特異性結合CD38之抗體(其在一段時間內由數個劑量組成)治療之整個過程係在以第二治療劑(其由數個劑量組成)治療之過程之前或之 後。介於投予特異性結合CD38之抗體與第二治療劑之間可使用1、2、或若干天或週之恢復期。 In the method of the present invention, the combination of an antibody that specifically binds to CD38 and a second therapeutic agent can be administered within any conventional time period. For example, the antibody that specifically binds to CD38 and the second therapeutic agent can be administered to the patient on the same day and even by the same intravenous infusion. However, the antibody that specifically binds to CD38 and the second therapeutic agent can also be administered on alternate days, alternate weeks or months, and so on. In some methods, the antibody that specifically binds to CD38 and the second therapeutic agent can be administered at a sufficiently close time so that they are present at a detectable level (for example, in serum) in the treated patient. middle. In some methods, the entire course of treatment with an antibody that specifically binds to CD38 (which consists of several doses over a period of time) is before or after the course of treatment with the second therapeutic agent (which consists of several doses). Rear. A recovery period of 1, 2, or several days or weeks can be used between the administration of the antibody that specifically binds to CD38 and the second therapeutic agent.
特異性結合CD38之抗體或特異性結合CD38之抗體與第二治療劑之組合可與任何形式的放射療法(包括體外放射(external beam radiation),強度調控放射療法(IMRT)、聚焦放射、及任何形式的放射手術(包括γ刀(Gamma Knife)、射波刀(Cyberknife)、直線加速器(Linac)、及間質放射(例如植入的放射性種粒、GliaSite氣球)))、及/或與手術一起施用。 The combination of an antibody that specifically binds to CD38 or an antibody that specifically binds to CD38 and a second therapeutic agent can be combined with any form of radiation therapy (including external beam radiation, intensity-modulated radiation therapy (IMRT), focused radiation, and any Forms of radiosurgery (including Gamma Knife, Cyberknife, Linac, and Interstitial Radiation (e.g. implanted radioactive seeds, GliaSite balloon)), and/or surgery Apply together.
可使用的聚焦放射法包括立體定位放射手術、分次立體定位放射手術、及強度調控放射療法(IMRT)。顯而易見的是,立體定位放射手術涉及精確地遞送輻射至腫瘤組織(例如腦腫瘤),同時避開周圍的非腫瘤、正常組織。使用立體定位放射手術所施加的輻射劑量可能會變化,通常為1Gy至約30Gy,且可包含中間範圍,包括例如1至5、10、15、20、25、高至30Gy的劑量。因為非侵入性固定裝置的緣故,立體定位放射不需要在單次治療中被遞送。治療計劃可以日復一日確實地重複,藉此使多個分次輻射劑量得以被遞送。當用來在一段時間內治療腫瘤時,該放射手術被稱為「分次立體定位放射手術(fractionated stereotactic radiosurgery)」或FSR。相比之下,立體定位放射手術係指單次治療(one-session treatment)。分次立體定位放射手術可能導致高治療比率,即腫瘤細胞殺滅率高且對正常組織低影響。腫瘤及正常組織對高單次劑量輻射之於多次小劑量輻射的反應不同。單次大劑量輻射比起若干次小劑量輻射可能會殺滅更多的正常組織。因此,多次小劑量輻射可以殺滅更多的腫瘤細胞而不傷害正常組織。使用分次立體定位放射所施加的輻射劑量可能會在1Gy至約50Gy的範圍內變化,且可包含中間範圍,包括例如1至5、10、15、20、25、30、40、直到50Gy的低分次劑量。也可使用強度調控放射療法(IMRT)。IMRT係高精度三維順形放射療法(3DCRT)的進階模式,其使用電腦控制的直線加速器以遞送精確的輻射劑量至惡性腫瘤或腫瘤內的特定範圍。在3DCRT中,使用多葉式準直儀(MLC)將每個輻射束的輪廓成形為適合目標的輪廓(來自射束透視(beam's eye view, BEV)觀點),藉此產生若干光束。IMRT以多個小量來調節該輻射束的強度,使輻射劑量得以更精確地符合腫瘤的三維(3-D)形狀。因此,IMRT使更高的輻射劑量能集中到腫瘤內的區域,同時盡量減少對周圍正常關鍵結構的劑量。IMRT提高了使治療量符合凹腫瘤形狀的能力,例如,當腫瘤係纏繞在諸如脊髓或主要器官或血管的脆弱結構上。 The available focused radiation methods include stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity-modulated radiotherapy (IMRT). It is obvious that stereotactic radiosurgery involves precise delivery of radiation to tumor tissues (such as brain tumors) while avoiding surrounding non-tumor, normal tissues. The radiation dose applied using stereotactic radiosurgery may vary, usually from 1 Gy to about 30 Gy, and may include intermediate ranges, including, for example, doses ranging from 1 to 5, 10, 15, 20, 25, and up to 30 Gy. Because of the non-invasive fixation device, stereotactic radiation does not need to be delivered in a single treatment. The treatment plan can be reliably repeated day after day, thereby allowing multiple fractional radiation doses to be delivered. When used to treat tumors over a period of time, the radiosurgery is called "fractionated stereotactic radiosurgery" or FSR. In contrast, stereotactic radiosurgery refers to one-session treatment. Fractionated stereotactic radiosurgery may result in a high treatment rate, that is, a high tumor cell killing rate and low impact on normal tissues. Tumors and normal tissues respond differently to high single-dose radiation than multiple low-dose radiation. A single large dose of radiation may kill more normal tissues than several small doses of radiation. Therefore, multiple small doses of radiation can kill more tumor cells without harming normal tissues. The radiation dose applied using fractionated stereotactic radiation may vary from 1 Gy to about 50 Gy, and may include intermediate ranges, including, for example, 1 to 5, 10, 15, 20, 25, 30, 40, and up to 50 Gy. Low divided dose. Intensity-modulated radiation therapy (IMRT) can also be used. IMRT is an advanced mode of high-precision three-dimensional conformal radiotherapy (3DCRT), which uses a computer-controlled linear accelerator to deliver precise radiation doses to malignant tumors or specific areas within the tumor. In 3DCRT, a multi-leaf collimator (MLC) is used to shape the contour of each radiation beam into a contour suitable for the target (from beam perspective (beam's eye view, BEV) point of view), thereby generating several beams. IMRT adjusts the intensity of the radiation beam in multiple small amounts, so that the radiation dose can more accurately conform to the three-dimensional (3-D) shape of the tumor. Therefore, IMRT enables higher radiation doses to be concentrated to the area within the tumor, while minimizing the dose to the surrounding normal key structures. IMRT improves the ability to adapt the treatment volume to the shape of a concave tumor, for example, when the tumor is entangled in fragile structures such as the spinal cord or major organs or blood vessels.
特異性結合CD38之抗體可以醫藥組成物之形式皮下投予,該醫藥組成物包含特異性結合CD38之抗體及玻尿酸酶。 The antibody that specifically binds to CD38 can be administered subcutaneously in the form of a pharmaceutical composition comprising an antibody that specifically binds to CD38 and hyaluronidase.
特異性結合CD38之抗體在皮下投予之醫藥組成物中的濃度可係約20mg/ml。 The concentration of the antibody that specifically binds to CD38 in the pharmaceutical composition administered subcutaneously can be about 20 mg/ml.
皮下投予之醫藥組成物可包含介於約1,200mg至1,800mg之間的特異性結合CD38之抗體。 The pharmaceutical composition administered subcutaneously may contain between about 1,200 mg to 1,800 mg of an antibody that specifically binds to CD38.
皮下投予之醫藥組成物可包含約1,200mg的特異性結合CD38之抗體。 The pharmaceutical composition administered subcutaneously may contain about 1,200 mg of an antibody that specifically binds to CD38.
皮下投予之醫藥組成物可包含約1,600mg的特異性結合CD38之抗體。 The pharmaceutical composition for subcutaneous administration may contain about 1,600 mg of an antibody that specifically binds to CD38.
皮下投予之醫藥組成物可包含約1,800mg的特異性結合CD38之抗體。 The pharmaceutical composition administered subcutaneously may contain about 1,800 mg of an antibody that specifically binds to CD38.
皮下投予之醫藥組成物可包含介於約30,000U至45,000U之間的玻尿酸酶。 The pharmaceutical composition administered subcutaneously may contain between about 30,000 U and 45,000 U of hyaluronidase.
皮下投予之醫藥組成物可包含約1,200mg的特異性結合CD38之抗體及約30,000U的玻尿酸酶。 The pharmaceutical composition administered subcutaneously may include about 1,200 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase.
皮下投予之醫藥組成物可包含約1,800mg的特異性結合CD38之抗體及約45,000U的玻尿酸酶。 The subcutaneously administered pharmaceutical composition may include about 1,800 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.
皮下投予之醫藥組成物可包含約1,600mg的特異性結合CD38之抗體及約30,000U的玻尿酸酶。 The pharmaceutical composition administered subcutaneously may include about 1,600 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase.
皮下投予之醫藥組成物可包含約1,600mg的特異性結合CD38之抗體及約45,000U的玻尿酸酶。 The pharmaceutical composition for subcutaneous administration may include about 1,600 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.
皮下投予之醫藥組成物可包含玻尿酸酶rHuPH20,其具有SEQ ID NO:40之胺基酸序列。 The pharmaceutical composition for subcutaneous administration may include hyaluronidase rHuPH20, which has the amino acid sequence of SEQ ID NO:40.
rHuPH20係重組玻尿酸酶(HYLENEX®重組體),且描述於國際專利公開號WO2004/078140中。 rHuPH20 is a recombinant hyaluronidase (HYLENEX® recombinant) and is described in International Patent Publication No. WO2004/078140.
玻尿酸酶係降解玻尿酸(EC 3.2.1.35)的酶,並減低在細胞外基質中玻尿酸之黏度,從而增加組織滲透性。 Hyaluronidase is an enzyme that degrades hyaluronic acid (EC 3.2.1.35) and reduces the viscosity of hyaluronic acid in the extracellular matrix, thereby increasing tissue permeability.
SEQ ID NO:40 SEQ ID NO: 40
包含特異性結合CD38之抗體及玻尿酸酶的醫藥組成物之投予可在一天、兩天、三天、四天、五天、六天、一週、兩週、三週、四週、五週、六週、七週、兩個月、三個月、四個月、五個月、六個月、或更久之後重覆進行。重覆治療過程亦為可能者,如為慢性投予。重覆投予可在相同劑量或在不同劑量下。例如,包含特異性結合CD38之抗體及玻尿酸酶之醫藥組成物可每週投予一次持續八週,接著兩週投予一次持續16週,接著四週投予一次。待投予之醫藥組成物可包含約1,200mg的特異性結合CD38之抗體及約30,000U的玻尿酸酶,其中特異性結合CD38之抗體在醫藥組成物中之濃度係約20mg/ml。待投予之醫藥組成物可包含約1,800mg的特異性結合CD38 之抗體及約45,000U的玻尿酸酶。待投予之醫藥組成物可包含約1,600mg的特異性結合CD38之抗體及約30,000U的玻尿酸酶。待投予之醫藥組成物可包含約1,600mg的特異性結合CD38之抗體及約45,000U的玻尿酸酶。 The pharmaceutical composition containing the antibody that specifically binds to CD38 and hyaluronidase can be administered on one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, and six days. Repeat this after a week, seven weeks, two months, three months, four months, five months, six months, or longer. It is also possible to repeat the treatment process, such as chronic administration. Repeated administration can be at the same dose or at different doses. For example, a pharmaceutical composition comprising an antibody that specifically binds to CD38 and hyaluronidase can be administered once a week for eight weeks, then once every two weeks for 16 weeks, and then once every four weeks. The pharmaceutical composition to be administered may include about 1,200 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase, wherein the concentration of the antibody that specifically binds to CD38 in the pharmaceutical composition is about 20 mg/ml. The pharmaceutical composition to be administered may contain about 1,800 mg of specifically binding CD38 The antibody and about 45,000U of hyaluronidase. The pharmaceutical composition to be administered may include about 1,600 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase. The pharmaceutical composition to be administered may include about 1,600 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.
包含特異性結合CD38之抗體及玻尿酸酶之醫藥組成物可皮下投予至腹部區域。 A pharmaceutical composition containing an antibody that specifically binds to CD38 and hyaluronidase can be administered subcutaneously to the abdominal area.
包含特異性結合CD38之抗體及玻尿酸酶之醫藥組成物可以約80ml、90ml、100ml、110ml、或120ml之總體積投予。 The pharmaceutical composition containing an antibody that specifically binds to CD38 and hyaluronidase can be administered in a total volume of about 80ml, 90ml, 100ml, 110ml, or 120ml.
投予時,可將20mg/ml的特異性結合CD38之抗體(於25mM乙酸鈉、60mM氯化鈉、140mM D-甘露醇、0.04%聚山梨醇酯20,pH 5.5)與rHuPH20(1.0mg/mL(75-150kU/mL),於10mM L-組胺酸、130mM NaCl、10mM L-甲硫胺酸、0.02%聚山梨醇酯80,pH 6.5)混合,之後將該混合物投予至對象。
When administered, 20mg/ml of an antibody that specifically binds to CD38 (in 25mM sodium acetate, 60mM sodium chloride, 140mM D-mannitol, 0.04
雖然已用一般術語描述了本發明,本發明之實施例將進一步揭露於下列實例中,但其不應被解釋為限制權利要求的範圍。 Although the present invention has been described in general terms, the embodiments of the present invention will be further disclosed in the following examples, but they should not be construed as limiting the scope of the claims.
緊接在第一輸液之前的基期及在治療期間內指明的時間點,將周邊血液及骨髓抽出物收集於肝素化管(heparinized tube)中。大部分樣本係當其等到達中央實驗室時,在收集後24至48小時,使用即時流動式細胞測量術評估。周邊血液單核細胞(PBMC)係獲自全血,藉由密度梯度離心來單離,且冷凍儲存直至分析。針對T細胞活化、純系性、及CD38+ Treg抑制測定,使用冷凍PBMC樣本,同時對治療前及治療後之樣本進行分析。 The peripheral blood and bone marrow aspirates were collected in a heparinized tube at the base period immediately before the first infusion and at the specified time point during the treatment period. Most of the samples are evaluated using instant flow cytometry when they arrive at the central laboratory 24 to 48 hours after collection. Peripheral blood mononuclear cells (PBMC) were obtained from whole blood, isolated by density gradient centrifugation, and stored frozen until analysis. For the determination of T cell activation, homology, and CD38 + Treg inhibition, frozen PBMC samples are used, and samples before and after treatment are analyzed at the same time.
在BARC全球中心實驗室(BARC global central laboratory),對這些樣本進行流動式細胞測量分析,使用預先驗證的免疫分型測定來評估NK細胞、T細胞、B細胞、骨髓瘤細胞(CD138+)、及CD38表現。簡言之,將血液樣本及骨髓樣本利用下列多螢光染料抗體組染色:細胞譜系組:PerCPCy5.5α-CD19(純系 HIB19;Becton Dickinson[BD])、APCα-CD24(SN3;eBioscience)、PC7α-CD3(UCHT-1;Beckman Coulter)、V500α-CD16(3G8;BD)、及PEα-CD56(MY;BD);調節T細胞(Treg)組:APCα-CD25(2A3;BD)、PEα-CD127(HIL-7R-M21;BD)、APC-H7α-HLA-DR(G46-6;BD)、及PerCPα-CD4(L200;BD);初始(naive)/記憶T細胞組:APC-H7α-CD4(RPA-T4;BD)、PerCP-Cy5.5α-CD8(RPA-T4 BD)、PEα-CD62L(SK11;BD)、及APCα-CD45RA(HI100;BD)。CD38表現係使用Alexa 647標記之抗體mAb 003評估,其描述於美國專利第7,829,693號中,具有SEQ ID NO:14及SEQ ID NO:15之VH及VL序列。血液樣本係使用不同的裂解-清洗方法製備。利用各種抗體對骨髓抽出物樣本進行膜或細胞內染色。Becton Dickinson FACS裂解溶液用於裂解周邊血液樣本中之紅血球,且來自Invitrogen之Fix與Perm細胞滲透試劑用於骨髓抽出物樣本之細胞內染色。染色樣本係在FACS Canto II流動式細胞測量儀上獲得,且數據係使用FacsDiva軟體分析。在所測試之所有時間點判定免疫細胞群在血液樣本中的絕對計數及淋巴球在骨髓樣本中之百分比。 In the BARC global central laboratory (BARC global central laboratory), these samples are analyzed by flow cytometry, using pre-validated immunophenotyping assays to evaluate NK cells, T cells, B cells, myeloma cells (CD138 + ), And CD38 performance. In short, the blood samples and bone marrow samples were stained with the following multi-fluorescent dye antibody groups: Cell lineage group: PerCPCy5.5α-CD19 (purified HIB19; Becton Dickinson[BD]), APCα-CD24 (SN3; eBioscience), PC7α -CD3 (UCHT-1; Beckman Coulter), V500α-CD16 (3G8; BD), and PEα-CD56 (MY; BD); regulatory T cell (T reg ) group: APCα-CD25 (2A3; BD), PEα- CD127 (HIL-7R-M21; BD), APC-H7α-HLA-DR (G46-6; BD), and PerCPα-CD4 (L200; BD); naive/memory T cell group: APC-H7α- CD4 (RPA-T4; BD), PerCP-Cy5.5α-CD8 (RPA-T4 BD), PEα-CD62L (SK11; BD), and APCα-CD45RA (HI100; BD). CD38 expression was evaluated using Alexa 647-labeled antibody mAb 003, which is described in US Patent No. 7,829,693 and has the VH and VL sequences of SEQ ID NO: 14 and SEQ ID NO: 15. The blood samples are prepared using different lysis-washing methods. Various antibodies are used for membrane or intracellular staining of bone marrow aspirate samples. Becton Dickinson FACS lysis solution is used to lyse red blood cells in surrounding blood samples, and Fix and Perm cell permeation reagents from Invitrogen are used for intracellular staining of bone marrow aspirate samples. The stained samples were obtained on the FACS Canto II flow cytometer, and the data was analyzed using FacsDiva software. Determine the absolute count of the immune cell population in the blood sample and the percentage of lymphocytes in the bone marrow sample at all time points tested.
T細胞多樣性係藉由使用PBMC樣本之基因體DNA進行TCR重排之深度定序(deep sequencing)以評估CD8+ T細胞純系性來分析。TCR定序係使用Adaptive Biotechnologies市售的ImmunoseqTM測定進行,且分析係使用預審的多重聚合酶連鎖反應(PCR)測定(TR2015CRO-V-019)進行,其等係由正向引子及反向引子所構成,該等正向引子及反向引子直接靶向可變(V)基因(正向引子)及連接(J)基因(反向引子)之家族。各V及J基因引子係作為引導對(priming pair)以放大體細胞重組TCR,且各引子含有特定的通用DNA序列。在最初的PCR放大之後,將各放大物用正向引子及反向引子放大第二次,該等正向引子及反向引子含有通用序列及Illumina的DNA定序所需的轉接序列(adaptor sequence)。 T cell diversity is analyzed by using the genomic DNA of PBMC samples to perform deep sequencing of TCR rearrangement to assess CD8 + T cell homology. TCR sequencing was performed using the Immunoseq TM assay commercially available from Adaptive Biotechnologies, and the analysis was performed using a pre-qualified multiple polymerase chain reaction (PCR) assay (TR2015CRO-V-019), which was performed by a forward primer and a reverse primer As a result, these forward primers and reverse primers directly target the family of variable (V) genes (forward primers) and junction (J) genes (reverse primers). The primers of the V and J genes are used as a priming pair to amplify the recombined TCR of somatic cells, and each primer contains a specific universal DNA sequence. After the initial PCR amplification, each amplification was amplified a second time with forward primers and reverse primers. These forward primers and reverse primers contained universal sequences and adapter sequences required for Illumina DNA sequencing (adaptor sequence).
將病患PBMC接種於96孔盤上(2×105個細胞/孔),且用下列刺激5天:23種主要組織相容性基因複合體(MHC)I類限制性病毒胜肽之混合物(cocktail),其係來自人類巨細胞病毒(CMV)、艾司坦-巴爾病毒(EBV)、及流感病毒(2μg/ml;CEF胜肽池;PANATecs®);或相等數目的來自健康供體的25-Gy照射之同種異體PBMC。未經刺激之PBMC及經抗CD3/CD28塗覆珠粒刺激之PBMC分別充當陰性對照及陽性對照。在第5天,藉由三明治酶聯免疫吸附測定(ELISA;人類IFN γ ELISA Ready-SET-Go;eBioscience)測量無細胞上清液之干擾素γ(IFN-γ),且其充當T細胞活化之替代標誌。
The patient’s PBMC were inoculated on a 96-well plate (2×10 5 cells/well) and stimulated for 5 days with the following: 23 major histocompatibility gene complex (MHC) class I restricted viral peptide mixture (cocktail), which is derived from human cytomegalovirus (CMV), estan-Barr virus (EBV), and influenza virus (2μg/ml; CEF peptide pool; PANATecs ® ); or an equal number from healthy donors 25-Gy irradiated allogeneic PBMC. Unstimulated PBMC and PBMC stimulated with anti-CD3/CD28 coated beads served as negative control and positive control, respectively. On
將健康供體之PBMC用PerCP-Cy5.5α-CD3(SK7;BD)、KOα-CD45,(J33;Beckman Coulter)、V450α-CD4(SK3;BD)、PEα-CD25(M-A251,BD)、PE Cy7α-CD127(HIL-7R-M21;BD)、及APCα-CD38(HB-7;BD)標記並藉由FACS Aria(BD)分選。將經分選之效應細胞用羧基螢光素琥珀醯亞胺酯(CFSE;eBioscience)標記,並用抗CD3/CD28塗覆珠粒在CD38+Treg或CD38- Treg(Treg對效應細胞之比率係1:1)存在或不存在的情況下,於RPMI加10%胎牛血清中刺激。72小時後,進行流動式細胞測量術,且將CFSE之稀釋百分比用作T細胞增生之替代。 Use PerCP-Cy5.5α-CD3 (SK7; BD), KOα-CD45, (J33; Beckman Coulter), V450α-CD4 (SK3; BD), PEα-CD25 (M-A251, BD) for PBMC from healthy donors , PE Cy7α-CD127 (HIL-7R-M21; BD), and APCα-CD38 (HB-7; BD) labeled and sorted by FACS Aria (BD). The sorted effector cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; eBioscience) and coated with anti-CD3/CD28 beads on CD38 + Treg or CD38 - Treg (the ratio of Treg to effector cell is 1 : 1) In the presence or absence, stimulate in RPMI plus 10% fetal calf serum. After 72 hours, flow cytometry was performed, and the dilution percentage of CFSE was used as a substitute for T cell proliferation.
將三個正常健康供體之PBMC與骨髓瘤細胞系(RPMI8226,U266,H929)共培育六天,並評估顆粒球性MDSC(G-MDSC)之產生(CD11b+CD14-HLA-DR-CD15+CD33+),如Gorgun等人,Blood 121:2975-87,2013中所述。G-MDSC不存在於正常健康 PBMC中,然而在與所有三個骨髓瘤細胞系共培育之後,G-MDSC以總PBMC群之5至25%存在(數據未顯示)。針對G-MDSC之流動式細胞測量評估的圈選(gating)策略包括以CD11b+作為第一圈選,接著進行CD14-及HLA-DR-圈選,然後接著進行CD15+及CD33+圈選。G-MDSC經細胞分選且評估CD38表現水平及對DARZALEXTM(達拉單抗)媒介之ADCC的敏感度。為了評估DARZALEXTM(達拉單抗)對MDSC之ADCC/CDC的效應,將含有補體或同型對照之血清加至ADCC測定中。 PBMC from three normal healthy donors were incubated with myeloma cell lines (RPMI8226, U266, H929) for six days, and the production of granular spherical MDSC (G-MDSC) (CD11b + CD14 - HLA - DR - CD15 +) CD33 + ), as described in Gorgun et al., Blood 121:2975-87, 2013. G-MDSC is not present in normal healthy PBMC, but after co-cultivation with all three myeloma cell lines, G-MDSC is present in 5 to 25% of the total PBMC population (data not shown). The gating strategy for flow cytometric evaluation of G-MDSC includes CD11b + as the first circle selection, followed by CD14 - and HLA - DR - circle selection, and then CD15 + and CD33 + circle selection. G-MDSC was cell sorted and evaluated CD38 expression level and sensitivity to ADCC mediated by DARZALEX TM (dalazumab). In order to evaluate the effect of DARZALEX TM (dalazumab) on ADCC/CDC of MDSC, serum containing complement or isotype control was added to the ADCC assay.
自病患獲得肝素化周邊血液樣本,之後各自輸液DARZALEXTM(達拉單抗)。將周邊血液單核細胞(PBMC)藉由Ficoll-Hypaque密度梯度離心來單離,並儲存在液態氮中之冷凍保存(cryopreservation)培養基(添加10%人類血清及10%二甲亞碸之RPMI)。針對FACS分析,將PBMC解凍,且將2×106個細胞/組再懸浮於具有0.05%疊氮化物及0.1% HAS之磷酸鹽緩衝鹽水(PBS)中。 Heparinized peripheral blood samples were obtained from the patients, and then each was infused with DARZALEX TM (dalazumab). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation, and stored in cryopreservation medium in liquid nitrogen (RPMI supplemented with 10% human serum and 10% dimethylsulfoxide) . For FACS analysis, PBMC were thawed, and 2×10 6 cells/group were resuspended in phosphate buffered saline (PBS) with 0.05% azide and 0.1% HAS.
數據分析data analysis
所有數據分析及關聯圖表之產生僅使用R軟體進行(R:A Language and Environment for Statistical Computing,R Development Core Team,R Foundation for Statistical Computing,Vienna,Austria,2011,ISBN 3-900051-07-0 http_//_www_R-project_org/)。所有具有可評估反應的經治療之對象均包括在數據分析中。通篇一致地,反應者定義為按照IRC具有sCR、VGPR、及PR之最佳反應的對象,且無反應者定義為按照IRC具有MR、SD、及PD之最佳反應的對象。 All data analysis and the generation of associated graphs are only performed using R software (R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2011, ISBN 3-900051-07-0 http_ //_www_R-project_org/). All treated subjects with evaluable responses were included in the data analysis. Consistently throughout the text, responders are defined as subjects with the best responses of sCR, VGPR, and PR according to IRC, and non-responders are defined as subjects with the best responses of MR, SD, and PD according to IRC.
不同的統計學比較包括:(i)反應者與無反應者之間的基期水平,(ii)反應者及無反應者的基期之於治療中,(iii)反應者與無反應者之間的變化百分比,(iv)基期之於治療中的比率變化。各比較首先包括利用夏皮羅-威爾克(Shapiro-Wilk)檢定之常態性檢定(Royston (1995)Remark AS R94:A remark on Algorithm AS 181:The W test for normality.Applied Statistics,44,547-551)。發現到,幾乎所有數據都不具有常態分佈。差異水平檢定包括進行非參數威爾卡森等級和檢定(Wilcox rank sum test)(Hollander及Wolfe(1973),Nonparametric Statistical Methods.New York:John Wiley & Sons.Pages 27-33(單樣本),68-75(雙樣本))及在博克斯-卡克斯(Box Cox)轉換之後的t檢定(Weisberg,S.(2014)Applied Linear Regression,Fourth Edition,Wiley Wiley,Chapter 7)。針對博克斯-卡克斯轉換,將小數(1e-07)加至等於0的值。在所有情況下,兩個檢定一致。除非另外指示,否則威爾卡森等級和檢定p-值係顯示於本說明書通篇的表中。當測試反應者及無反應者之基期之於治療中的差異時,每個對象進行雙組配對測試,在所有其他情況下,進行雙組非配對測試。 Different statistical comparisons include: (i) the baseline level between responders and non-responders, (ii) the baseline level of responders and non-responders during treatment, and (iii) the baseline level between responders and non-responders Percentage change, (iv) The ratio of base period to treatment change. The comparisons first include the normality test using the Shapiro-Wilk test (Royston (1995) Remark AS R94: A remark on Algorithm AS 181: The W test for normality. Applied Statistics, 44, 547-551). It is found that almost all data does not have a normal distribution. The difference level test includes the non-parametric Wilcox rank sum test (Hollander and Wolfe (1973), Nonparametric Statistical Methods. New York: John Wiley & Sons. Pages 27-33 (single sample), 68 -75 (double sample)) and t test after Box Cox conversion (Weisberg, S. (2014) Applied Linear Regression, Fourth Edition, Wiley Wiley, Chapter 7). For Box-Carks conversion, the decimal (1e-07) is added to a value equal to 0. In all cases, the two checks agree. Unless otherwise instructed, Wil Carson's grades and verified p-values are shown in tables throughout this manual. When testing the difference in treatment between responders and non-responders, each subject undergoes a two-group paired test, and in all other cases, a two-group unpaired test.
因為針對不同的給藥排程,分析各種淋巴球群之樣本不是在同一時間點取得的,所以進行族群模型化。模型擬合係對訪視之等級進行。對總體及活化NK細胞的族群模型化涉及擬合折棒模型(broken stick model)(Lutz等人,「Statistical model to estimate a threshold dose and its confidence limits for the analysis of sublinear dose-response relationships,exemplified for mutagenicity data.」Mutation Research/Genetic Toxicology and Environmental Mutagenesis 678.2(2009):118-122.)。將具有隨機截距及斜率之線性混合效應模型對B細胞、T細胞亞群、及白血球、單核球、嗜中性球、及淋巴球病患族群之數據進行擬合(Bates等人,(2014)。「lme4:Linear mixed-effects models using Eigen and S4.」ArXiv e-print;提交至Journal of Statistical Software,http:_//_arxiv_org/abs/_1406.5823)。此線性混合模型化係對治療開始後的相對日子進行(ADY)。此線性混合模型擬合係對經對數轉換的反應變數進行。在反應變數值等於0之情況下,將0.1加至所有反應變數值以允許在對數尺度下的模型化。 Because samples of various lymphocyte populations were not obtained at the same time point for different dosing schedules, the population modeling was carried out. Model fitting is performed on the level of visits. Population modeling of the population and activated NK cells involves fitting a broken stick model (Lutz et al., "Statistical model to estimate a threshold dose and its confidence limits for the analysis of sublinear dose-response relationships, implemented for mutagenicity data." Mutation Research/Genetic Toxicology and Environmental Mutagenesis 678.2(2009):118-122.). A linear mixed-effect model with random intercept and slope was fitted to the data of B cells, T cell subpopulations, and leukocytes, monocytes, neutrophils, and lymphocytic disease populations (Bates et al., ( 2014). "lme4: Linear mixed-effects models using Eigen and S4." ArXiv e-print; submitted to Journal of Statistical Software , http:_//_arxiv_org/abs/_1406.5823). This linear mixed modeling is performed on the relative days after the start of treatment (ADY). This linear mixed model fitting system is performed on the logarithmic transformed response variable. In the case where the response variable value is equal to 0, 0.1 is added to all response variable values to allow modeling on a logarithmic scale.
研究54767414MMY2002(SIRIUS)之目標族群係患有晚期多發性骨髓瘤之病患,其等之前接受過至少3種療法,包括蛋白酶體抑制劑(PI)及免疫調節藥品(IMiD),或其等對於PI及IMiD而言係雙重難治。主要終點/最終分析之反應評估係基於獨立評審委員會(independent review committee,IRC)及電腦化演算法之評估,其使用2011 IMWG準則(臨床研究報告:一項開放標籤、多中心、第2期試驗,其探究在患有多發性骨髓瘤之對象中DARZALEXTM(達拉單抗)的功效及安全性,該等對象之前已接受至少3種療法(包括蛋白酶體抑制劑及IMiD)或該等對象對於蛋白酶體抑制劑及IMiD而言係雙重難治(EDMS-ERI-92399922;de Weers等人,(2011)J Immunol 186(3):1840-1848))。
Study 54767414MMY2002 (SIRIUS) target group of patients with advanced multiple myeloma, who have received at least 3 therapies before, including proteasome inhibitors (PI) and immunomodulatory drugs (IMiD), or the like PI and IMiD are double intractable. The primary endpoint/final analysis of the response evaluation is based on the independent review committee (IRC) and computerized algorithm evaluation, which uses the 2011 IMWG criteria (clinical study report: an open-label, multi-center,
這些評估包括:總體反應率(ORR)、反應持續時間、反應時間及最佳反應、臨床受益率、進展時間(TTP)、無進展存活期(PFS)、及總體存活期(OS)。 These assessments include: overall response rate (ORR), duration of response, response time and best response, clinical benefit rate, time to progression (TTP), progression-free survival (PFS), and overall survival (OS).
此研究中,總計124個對象經DARZALEXTM(達拉單抗)治療(de Weers等人,(2011)J Immunol 186(3):1840-1848)。18個對象係以8mg/kg治療,且106個對象係以16mg/kg治療。給藥排程係如下:A組:DARZALEXTM(達拉單抗)16mg/kg:週期1及週期2:第1、8、15、及22天(每週),週期3至週期6:第1及15天(每隔一週),週期7+:第1天(每4週)。各週期係4週。
In this study, a total of 124 subjects were treated with DARZALEX ™ (dalazumab) (de Weers et al., (2011) J Immunol 186(3): 1840-1848). 18 subjects were treated at 8 mg/kg, and 106 subjects were treated at 16 mg/kg. The dosing schedule is as follows: Group A: DARZALEX TM (dalazumab) 16 mg/kg:
B組:DARZALEXTM(達拉單抗)8mg/kg:週期1+:第1天(每4週)。
Group B: DARZALEX TM (Dalamumab) 8 mg/kg:
研究之主要目標係判定DARZALEXTM(達拉單抗)之2個治療方案的功效,如藉由ORR(CR+PR)所測量,在患有多發性骨髓瘤的對象中,該等對象之前已接受至少3種療法,包括PI及IMiD,或該等對象之疾病對於PI及IMiD而言係雙重難治(臨床研究報告:一項開放標籤、多中心、第2期試驗,其探究在患有多發性骨髓瘤之對象中DARZALEXTM(達拉單抗)的功效及安全性,該等對
象之前已接受至少3種療法(包括蛋白酶體抑制劑及IMiD)或該等對象對於蛋白酶體抑制劑及IMiD而言係雙重難治。EDMS-ERI-92399922)。
The main goal of the study is to determine the efficacy of the two treatment regimens of DARZALEX TM (dalazumab). As measured by ORR (CR+PR), in subjects with multiple myeloma, these subjects have previously Receiving at least 3 therapies, including PI and IMiD, or the diseases of these subjects are double refractory to PI and IMiD (clinical research report: an open-label, multi-center,
此研究之次級目標包括評估DARZALEXTM(達拉單抗)之安全性及耐受性、展示功效之額外量度(例如,臨床受益、TTP、PFS、及OS)連同評估藥物動力學、免疫原性、藥效動力學、以及探究預示對DARZALEXTM(達拉單抗)之反應的生物標誌。額外研究相關的資訊可得自臨床研究規程(臨床研究報告:一項開放標籤、多中心、第2期試驗,其探究在患有多發性骨髓瘤之對象中DARZALEXTM(達拉單抗)的功效及安全性,該等對象之前已接受至少3種療法(包括蛋白酶體抑制劑及IMiD)或該等對象對於蛋白酶體抑制劑及IMiD而言係雙重難治。EDMS-ERI-92399922)。
The secondary goals of this study include assessing the safety and tolerability of DARZALEX TM (dalazumab), demonstrating additional measures of efficacy (for example, clinical benefit, TTP, PFS, and OS), as well as assessing pharmacokinetics, immunogen Properties, pharmacodynamics, and exploration of biomarkers predicting response to DARZALEX TM (dalazumab). Additional research-related information can be obtained from the clinical research protocol (clinical research report: an open-label, multi-center,
在第1部分之第1階段中,8mg/kg組中1個對象(6%)有反應,且在16mg/kg組中5個對象(31%)有反應。因此,在第1部分之第2階段及第2部分中,僅16mg/kg組擴增。
In the first stage of
在16mg/kg組中,基於IRC評估,31個對象達成PR或更好的反應;ORR係29%(95% CI:21%,39%)。三個對象(3%)達成sCR,且13個對象(12%)達成VGPR或更好。 In the 16mg/kg group, 31 subjects achieved PR or better response based on IRC assessment; ORR was 29% (95% CI: 21%, 39%). Three subjects (3%) achieved sCR, and 13 subjects (12%) achieved VGPR or better.
CD38表現在多種免疫細胞及造血細胞上。藉由流動式細胞測量術進行廣泛的免疫剖析,以檢驗DARZALEXTM(達拉單抗)對免疫細胞子集的效應及這些細胞的基期水平與臨床反應的關聯。在基期及DARZALEXTM(達拉單抗)治療之後藉由流動式細胞測量術評估周邊血液及骨髓抽出物中之各種細胞群,包括T細胞(CD3+、CD4+、CD8+、及調節T細胞(Treg))、B細胞(CD19+)、NK細胞、單核球(CD14+)、白血球、及嗜中性球,以監測反應者及無反應者中這些細胞群之變化。 CD38 is expressed on a variety of immune cells and hematopoietic cells. Extensive immunoassays were performed by flow cytometry to test the effect of DARZALEX TM (dalazumab) on a subset of immune cells and the correlation between the baseline level of these cells and clinical response. After the base phase and DARZALEX TM (dalazumab) treatment, flow cytometry is used to evaluate various cell populations in peripheral blood and bone marrow aspirates, including T cells (CD3 + , CD4 + , CD8 + , and regulatory T cells) (Treg)), B cells (CD19 + ), NK cells, monocytes (CD14 + ), white blood cells, and neutrophils to monitor changes in these cell populations in responders and non-responders.
研究反應者及無反應者中周邊血液中之白血球、淋巴球、單核球、及嗜中性球計數。在以8mg/kg及16mg/kg劑量之病患中均發現總淋巴球隨著DARZALEXTM(達拉單抗)治療而增加(圖1)。線性混合效應模型化揭示每100天在對數尺度下增加0.8×106個細胞/μL(CI=0.06,0.11)。發現單核球及白血球之略微增加,其係於各100天在對數尺度下分別顯著增加0.03×106個細胞/μL(CI=0.01,0.04)及0.03×106個細胞/μL(CI=0.01,0.05)。儘管注意到在一些病患中嗜中性球減少,但是嗜中性球計數中位數與基期一致,且變化不顯著。 Study the counts of leukocytes, lymphocytes, monocytes, and neutrophils in peripheral blood in responders and non-responders. In patients at doses of 8 mg/kg and 16 mg/kg, total lymphocytes were found to increase with DARZALEX TM (dalazumab) treatment (Figure 1) . Linear mixed effect modeling revealed an increase of 0.8×10 6 cells/μL on a logarithmic scale every 100 days (CI=0.06, 0.11). It was found that monocytes and white blood cells increased slightly, which were significantly increased by 0.03×10 6 cells/μL (CI=0.01, 0.04) and 0.03×10 6 cells/μL (CI= 0.01, 0.05). Although it was noted that neutrophils decreased in some patients, the median neutrophil count was consistent with the base period, and the change was not significant.
比較反應組之間這些細胞群之各者的基期水平。使用威爾卡森符號等級檢定(Wilcoxon signed-rank test)並未發現跨反應組的這些細胞類型之任一者的基期水平不同的證據(表1)。 Compare the baseline level of each of these cell populations between the response groups. Using the Wilcoxon signed-rank test did not find evidence that the baseline levels of any of these cell types across the response group were different ( Table 1 ).
總NK細胞(CD16+CD56+)及活化NK細胞(CD16+CD56dim)在DARZALEXTM(達拉單抗)治療之情況下隨著時間推移而減少(數據未顯示)。 Total NK cells (CD16 + CD56 + ) and activated NK cells (CD16 + CD56 dim ) decreased over time under DARZALEX™ (data not shown).
在反應者及無反應者中測量周邊血液或骨髓抽出物中之B細胞(CD45+CD3-CD19+)在DARZALEXTM(達拉單抗)治療期間隨時間推移的絕對計數。B細胞在全血中略微增加,且在骨髓抽出物中維持不變。周邊血液中之B細胞的線性混合模型化揭示,在DARZALEXTM(達拉單抗)治療之過程內於各100天在對數尺度下最小增加0.1×106個細胞/μL[CI=0.04,0.16]。在達拉單抗治療期間骨髓抽出物中之B細胞(CD45+CD3-CD19+/淋巴球)之百分比在反應者或無反應者中皆沒有變化(分別係p=0.1及0.4)。另外,在反應者與無反應者之間,沒有發現在基期之B細胞計數不同的證據(p=0.5)。 The absolute counts of B cells (CD45 + CD3 - CD19 + ) in peripheral blood or bone marrow aspirates during DARZALEX TM (dalazumab) treatment were measured in responders and non-responders over time. B cells increased slightly in whole blood and remained unchanged in bone marrow aspirates. The linear mixed modelling of B cells in the peripheral blood revealed that the minimum increase of 0.1×10 6 cells/μL on the logarithmic scale during each 100 days of DARZALEX TM (dalazumab) treatment process [CI=0.04,0.16 ]. During dalamumab treatment, the percentage of B cells (CD45 + CD3 - CD19 + /lymphocytes) in bone marrow aspirates did not change among responders or non-responders (p=0.1 and 0.4, respectively). In addition, between responders and non-responders, there was no evidence of differences in B cell counts in the base phase (p=0.5).
在DARZALEXTM(達拉單抗)治療之後,周邊血液中之CD3+、CD4+、及CD8+ T細胞增加(淋巴球之絕對計數/μl及百分比兩者)。圖2顯示隨時間推移每個病患的周邊血液中之CD3+ T細胞(CD45+CD3+)之絕對計數自基期的變化百分比。圖中之黑色線顯示所有病患之絕對計數中位數×106個細胞/μL。圖中僅包括多於2次觀察的訪視。圖3顯示隨時間推移每個病患的周邊血液中之CD4+ T細胞(CD45+CD3+CD4+)之絕對計數自基期的變化%。圖中之黑色線顯示所有病患之中位數。圖中僅包括多於2次觀察的訪視。圖4顯示隨時間推移每個病患的周邊血液中之CD8+ T細胞(CD45+CD3+CD8+)之絕對計數自基期的變化%。圖中之黑色線顯示所有病患之中位數。圖中僅包括多於2次觀察的訪視。對周邊血液中之絕對計數的線性混合模型化揭示,平均來說,總T細胞(CD45+CD3+)在DARZALEXTM(達拉單抗)治療之後係於各100天在對數尺度下增加0.13×106個細胞/μl (CI=0.1,0.15)。發現CD8+ T細胞於各100天在對數尺度下顯著增加0.16×106個細胞/μl(CI=0.13,0.19)。發現CD4+細胞於各100天在對數尺度下中等增加0.11×106個細胞/μl(CI=0.09,0.13)。 After DARZALEX TM (dalazumab) treatment, CD3 + , CD4 + , and CD8 + T cells in the peripheral blood increase (both absolute count of lymphocytes/μl and percentage). Figure 2 shows the percentage change of the absolute count of CD3 + T cells (CD45 + CD3 + ) in the peripheral blood of each patient from the baseline over time. The black line in the figure shows the median absolute count of all patients×10 6 cells/μL. The figure only includes visits with more than 2 observations. Figure 3 shows the% change in the absolute count of CD4 + T cells (CD45 + CD3 + CD4 + ) in the peripheral blood of each patient over time from the baseline. The black line in the figure shows the median of all patients. The figure only includes visits with more than 2 observations. Figure 4 shows the% change in the absolute count of CD8 + T cells (CD45 + CD3 + CD8 + ) in the peripheral blood of each patient over time from the baseline. The black line in the figure shows the median of all patients. The figure only includes visits with more than 2 observations. Linear mixed modeling of absolute counts in peripheral blood reveals that, on average, total T cells (CD45 + CD3 + ) after DARZALEX TM (dalazumab) treatment increased by 0.13× on a logarithmic scale every 100 days. 10 6 cells/μl (CI=0.1,0.15). It was found that CD8 + T cells increased significantly by 0.16×10 6 cells/μl (CI=0.13, 0.19) on a logarithmic scale on each 100 days. It was found that CD4 + cells increased by 0.11×10 6 cells/μl on a logarithmic scale every 100 days (CI=0.09, 0.13).
對於T細胞亞群之各者,反應者顯示絕對計數自基期的最大變化百分比高於無反應者(CD3+ p=3.2993e-05;CD4+ p=3.486e-05;CD8+ p=2.7172e-05;調節T細胞p=0.002)。表2顯示威爾卡森符號等級檢定結果,用以比較反應者與無反應者之間周邊血液中之各T細胞亞群之絕對計數自基期的變化百分比。 For each of the T cell subpopulations, the responders showed that the maximum percentage change in absolute counts from the base period was higher than that of the non-responders (CD3 + p=3.2993e-05; CD4 + p=3.486e-05; CD8 + p=2.7172e -05; regulatory T cells p=0.002). Table 2 shows the results of Wilkason's sign rank test to compare the percentage change from the baseline of the absolute count of each T cell subpopulation in the peripheral blood between responders and non-responders.
類似地在骨髓中,發現反應者及無反應者之總T細胞(呈淋巴球之百分比的CD45+CD3+)及CD8+ T細胞(呈淋巴球之百分比的CD45+CD3+CD8+)在DARZALEXTM(達拉單抗)治療期間皆顯著地增加(CD3+反應者p=3.8147e-06,無反應者p=9.8225e-05;CD8+反應者p=3.8147e-06,無反應者p=0.0003)。骨髓中之CD4+ T細胞中位數在任一臨床反應組中皆沒有變化。表3顯示呈骨髓中淋巴球%之各種T細胞的威爾卡森符號等級檢定結果。圖5顯示在DARZALEXTM(達拉單抗)治療期間CD45+CD3+細胞隨時間推移的百分比(%)(反應者及無反應者均包括在圖表中)。圖6顯示在 DARZALEXTM(達拉單抗)治療期間CD45+CD3+CD8+細胞隨時間推移的%(反應者及無反應者均包括在圖表中)。 Similarly, in the bone marrow, the total T cells (CD45 + CD3 + as the percentage of lymphocytes) and CD8 + T cells (CD45 + CD3 + CD8 + as the percentage of lymphocytes) of responders and non-responders are found in DARZALEX TM (dalazumab) increased significantly during treatment (CD3 + responders p=3.8147e-06, non-responders p=9.8225e-05; CD8 + responders p=3.8147e-06, non-responders p =0.0003). The median number of CD4 + T cells in the bone marrow did not change in any clinical response group. Table 3 shows the results of Wilcarson's sign rank test of various T cells showing% of lymphocytes in bone marrow. Figure 5 shows the percentage (%) of CD45 + CD3 + cells over time during DARZALEX TM (Dalamumab) treatment (both responders and non-responders are included in the chart). Figure 6 shows the% of CD45 + CD3 + CD8 + cells over time during DARZALEX TM (Dalamumab) treatment (both responders and non-responders are included in the chart).
儘管反應者及無反應者均展示周邊血液及骨髓中之T細胞增加,但是反應者具有最大的自基期的變化百分比。為了區別在DARZALEXTM(達拉單抗)治療之前,是反應者還是無反應者具有不同水平的CD3+、CD4+、及CD8+ T細胞,比較周邊血液中各亞組之基期測量值。 Although both responders and non-responders showed increased T cells in the peripheral blood and bone marrow, the responders had the largest percentage change from the baseline. In order to distinguish whether the responders or non-responders had different levels of CD3 + , CD4 + , and CD8 + T cells before DARZALEX TM (Dalamumab) treatment, the baseline measurements of each subgroup in the peripheral blood were compared.
根據威爾卡森符號等級檢定,在周邊血液中之基期的絕對T細胞計數(表4)或骨髓中之自總淋巴球的T細胞百分比中(表5),反應者與無反應者之間不具有統計學顯著的差異。 According to Wilcarson's symbolic grade test, the absolute T cell count in the peripheral blood at the base period ( Table 4 ) or the percentage of T cells from the total lymphocytes in the bone marrow ( Table 5 ), between responders and non-responders There is no statistically significant difference.
Treg細胞係鑑定為樣本中之CD3+CD4+CD25+CD127dim細胞群。評估經DARZALEXTM(達拉單抗)治療的病患中周邊血液及骨髓中隨時間推移的CD8+ T細胞對Treg之比率。周邊血液及骨髓 中之比率均增加。圖7A顯示每個時間點所有病患之周邊血液中CD8+/Treg及CD8+/CD4+細胞比率之中位數值。圖7B顯示每個時間點所有病患之骨髓中CD8+/Treg及CD8+/CD4+ T細胞比率之中位數值。根據威爾卡森符號等級檢定,周邊血液中隨治療時間推移(表6)及骨髓(表7)中之CD8+Treg及CD8+/CD4+的絕對計數比率之變化係顯著的。 The Treg cell line was identified as the CD3 + CD4 + CD25 + CD127 dim cell population in the sample. To evaluate the ratio of CD8 + T cells to Tregs in peripheral blood and bone marrow over time in patients treated with DARZALEX TM (dalazumab). The ratio in peripheral blood and bone marrow increased. Figure 7A shows the median values of the CD8 + /Treg and CD8 +/ CD4 + cell ratios in the peripheral blood of all patients at each time point. Figure 7B shows the median CD8 + /Treg and CD8 + /CD4 + T cell ratios in the bone marrow of all patients at each time point. According to Wilcarson's symbolic grade test, the changes in the absolute count ratios of CD8 + Treg and CD8 + /CD4 + in peripheral blood with the passage of treatment time ( Table 6 ) and bone marrow ( Table 7 ) are significant.
在SIRIUS及GEN501研究(實例6)之組合數據分析中,周邊血液中之CD8+/CD4+及CD8+/Treg細胞之比率中位數在第8週(CD8+/CD4+之p=5.1×10-5,且CD8+/Treg之p=1.8×10-7)及在第16週(CD8+/CD4+之p=0.00017,且CD8+/Treg之p=4.1×10-7)係增加的。類似地,在骨髓中,CD8+/CD4+及CD8+/Treg細胞之比率中位數在治療中(第12+1週週期)相較於基期係增加的(CD8+/CD4+之p=0.00016,且CD8+/Treg之p=2.8×10-7)。反應者與無反應者之間未觀察到顯著差異。 In the combined data analysis of the SIRIUS and GEN501 study (Example 6) , the median ratio of CD8 + /CD4 + and CD8 + /Treg cells in peripheral blood was at week 8 (CD8 + /CD4 + p=5.1× 10 -5 and CD8 + /Treg p=1.8×10 -7 ) and increase in week 16 (CD8 + /CD4 + p=0.00017 and CD8 + /Treg p=4.1×10 -7 ) of. Similarly, in the bone marrow, the median ratio of CD8 + /CD4 + and CD8 + /Treg cells increased during treatment (the 12th + 1 week cycle) compared to the baseline (CD8 + /CD4 + p= 0.00016, and CD8 + /Treg's p=2.8×10 -7 ). No significant difference was observed between responders and non-responders.
研究GEN501(NCT00572488)評估雙重難治MM病患中作為單一療法的DARZALEXTM(達拉單抗)。樣本單離、處理、及統計分析係如實例1及實例2中所述。該研究已描述於Lokhorst等人,N Eng J Med 373:1207-19,2005中。 Study GEN501 (NCT00572488) evaluates DARZALEX TM (dalazumab) as a monotherapy in patients with dual refractory MM. Sample isolation, processing, and statistical analysis are as described in Example 1 and Example 2. This study has been described in Lokhorst et al., N Eng J Med 373:1207-19, 2005.
簡言之,研究GEN501係DARZALEXTM(達拉單抗)在患有MM的對象中之首次應用於人類(first-in-human)的臨床研究。其係第1/2期劑量逐步升高的安全性研究,該研究分成2個部分。第1部分係開放標籤劑量逐步升高的研究;第2部分係開放標籤以多個分群的單臂研究,其係基於第1部分中所確立之劑量水平。
In short, the study of GEN501 is the first-in-human clinical study of DARZALEXTM (darazumab) in subjects suffering from MM. It is the
在第1部分中,評估DARZALEXTM(達拉單抗)之10個劑量水平:0.005、0.05、0.10、0.50、1、2、4、8、16、及24mg/kg。兩個最低劑量分群各配給1(+3)個對象,且標準3(+3)個對象配給應用至剩餘8個劑量分群。第2部分係開放標籤單研究,其包括兩個劑量水平,8mg/kg及16mg/kg。第1部分包括32個對象,且第2部分包括72個對象。
In
鑒於在MY2002研究中在周邊血液及骨髓中注意到的CD8+ T細胞之擴增,使用ImmunoseqTM測定進行T細胞受體(TCR)之高通量次世代定序,以判定擴增CD8+ T細胞是否在本質上係純系的,其係適應性免疫反應之指示。對登記GEN501研究之對象之總計17個病患樣本進行評估(反應者n=6,即,PR;無反應者n=11,即,MR、SD、PD)。 In view of the expansion of CD8 + T cells noted in the peripheral blood and bone marrow in the MY2002 study, the Immunoseq TM assay was used to perform high-throughput next-generation sequencing of T cell receptors (TCR) to determine the expansion of CD8 + T Whether a cell is pure in nature is an indicator of an adaptive immune response. Evaluate a total of 17 patient samples from subjects registered in the GEN501 study (respondents n=6, that is, PR; non-responders n=11, that is, MR, SD, PD).
TCR定序揭示,DARZALEXTM(達拉單抗)治療顯著地增加病患之純系性。圖8A顯示DARZALEXTM(達拉單抗)治療之前之於之後的T細胞純系性之間的相關性(p=0.0056)。圖8B顯示個 體病患中之純系性之倍數變化。反應者係以星標出。此數據表明,在DARZALEXTM(達拉單抗)治療之情況下所注意到的T細胞擴增可在本質上係純系的。 TCR sequencing revealed that DARZALEX (TM ) treatment significantly increased the homology of the patient. Figure 8A shows the correlation between T cell homotypic before and after DARZALEX™ (dalumab) treatment (p=0.0056). Figure 8B shows the fold change of homology in individual patients. Respondents are marked with stars. This data indicates that the T cell expansion noted in the context of DARZALEX(TM) treatment can be pure-line in nature.
當相較於無反應者時,反應者在TCR貯庫中具有較大的總擴增(如藉由豐度變化所測量;CIA)。圖8C顯示個別病患之CIA%。A組:反應者,B組:無反應者。於反應者與無反應者之間觀察到統計學顯著的差異(p=0.037)。圖8D顯示反應者及無反應者中各擴增T細胞殖株之絕對豐度變化(CIA)之和。圖8E顯示各個別病患之最大CIA%。A組:反應者,B組:無反應者。於反應者與無反應者之間觀察到統計學顯著的差異(p=0.048)。圖8F顯示反應者(A組)及無反應者(B組)中之單個細胞殖株的最大CIA。 When compared to non-responders, responders have greater total amplification in the TCR reservoir (as measured by changes in abundance; CIA). Figure 8C shows the CIA% of individual patients. Group A: responders, group B: non-responders. A statistically significant difference was observed between responders and non-responders (p=0.037). Figure 8D shows the sum of the absolute abundance change (CIA) of each expanded T cell clone in responders and non-responders. Figure 8E shows the maximum CIA% for each individual patient. Group A: responders, group B: non-responders. A statistically significant difference was observed between responders and non-responders (p=0.048). Figure 8F shows the maximum CIA of single cell clones in responders (group A) and non-responders (group B).
CIA係藉由使用費雪精確檢定(Fisher’s exact test)(DeWit等人J.Virol.2015)鑑定介於兩個樣本之間的純系豐度之顯著差異且對各擴增殖株之絕對豐度變化求和來獲得。 The CIA line uses Fisher's exact test (DeWit et al. J.Virol. 2015) to identify significant differences in the abundance of pure lines between the two samples and changes in the absolute abundance of each expanded strain Sum to get.
評估登記GEN501之反應者及無反應者中的各種T及B細胞群。 Assess various T and B cell populations in responders and non-responders registered with GEN501.
類似於SIRIUS(MMY2002)研究,在DARZALEXTM(達拉單抗)治療期間,周邊血液及骨髓中之淋巴球均增加。此增加係歸因於CD4+及CD8+細胞兩者的數目增加。 Similar to the SIRIUS (MMY2002) study, during the DARZALEX TM (dalazumab) treatment, lymphocytes in the peripheral blood and bone marrow increased. This increase is due to the increase in the number of both CD4+ and CD8 + cells.
在登記GEN 501研究之17個病患之子集中研究經DARZALEXTM(達拉單抗)治療的病患中隨時間推移之CD8+ T細胞表型。使用標準規程,來自病患之CD8+細胞被鑑定為初始(CD45RO-/CD62L+)(TN)細胞或中央記憶(TCM)(CD45RO+/CD62L+高)細胞。 A subset of 17 patients who registered for the GEN 501 study focused on the CD8 + T cell phenotype over time in patients treated with DARZALEX TM (dalazumab). Using standard procedures, CD8 + cells from the patient were identified as naive (CD45RO-/CD62L + ) (T N ) cells or central memory (T CM ) (CD45RO + /CD62L + high ) cells.
圖9A顯示CD8+初始細胞之%(CD8+細胞之%),且圖9B顯示CD8+中央記憶細胞之%。DARZALEXTM(達拉單抗)治療顯著地降低初始CD8+ T細胞之量(第8週之p=1.82×10-4),且增加CD8+記憶T細胞之量(第8週之p=4.88×10-2)。這表明,初始細胞毒性T細胞轉變成記憶T細胞,其可針對特定抗原被活化。白色方形指示至少達成最小反應(MR)之病患,且黑色方形指示疾病穩定或疾病進展之病患。對治療有反應的病患中,CD8+初始T細胞之顯著地較大的降低係明顯的(數據未顯示)。圖9C顯示,DARZALEXTM(達拉單抗)治療增加HLA I類-限制性T細胞之百分比,該等細胞部分驅動病毒特異性及同種反應性T細胞反應。圖9D顯示,擴增效應記憶T細胞表現低水平的CD38。重要的是要注意,這些T細胞呈現正常且甚至增加的針對病毒胜肽及同種抗原的功能活性(參見實例8)。根據這些功能結果,我們推斷,在DARZALEXTM(達拉單抗)治療期間存在針對病毒及同種抗原的經歷抗原之T細胞的擴增、或活性改善。這些數據表明,不同於調節細胞子集,效應T細胞不需要CD38表現以適當地起作用並擴增。 Figure 9A shows the% of CD8 + naive cells (% of CD8 + cells), and Figure 9B shows the% of CD8 + central memory cells. DARZALEX TM treatment significantly reduced the amount of initial CD8 + T cells (p = 1.82×10 -4 at the 8th week) and increased the amount of CD8 + memory T cells ( p = 4.88 at the 8th week) ×10 -2 ). This indicates that naive cytotoxic T cells are transformed into memory T cells, which can be activated against specific antigens. The white square indicates that at least the minimum response has been achieved ( MR) patients, and the black squares indicate patients with stable disease or disease progression. In patients who responded to treatment, a significantly greater reduction in CD8+ naive T cells was evident (data not shown). Figure 9C shows that DARZALEX (TM ) treatment increases the percentage of HLA class I-restricted T cells, which partially drive virus-specific and alloreactive T cell responses. Figure 9D shows that expanded effector memory T cells exhibit low levels of CD38. It is important to note that these T cells exhibit normal and even increased functional activity against viral peptides and homologous antigens (see Example 8 ). Based on these functional results, we infer that there is an expansion of antigen-experienced T cells against the virus and alloantigens, or an improvement in activity during DARZALEX TM (dalazumab) treatment. These data indicate that, unlike a subset of regulatory cells, effector T cells do not require CD38 expression in order to function and expand properly.
細胞毒性T細胞之穩健擴增及活性增加之觀察,連同指示若干免疫抑制性細胞子集表現CD38的最近文獻一起,促進檢驗DARZALEXTM(達拉單抗)對調節細胞群調節T細胞(Treg)、骨髓衍生抑制細胞(MDSC)、及調節B細胞(Breg)的效應。 The observation of the robust expansion and increased activity of cytotoxic T cells, together with recent literature indicating that a subset of immunosuppressive cells express CD38, facilitates the examination of DARZALEX TM (dalazumab) on regulatory cell populations regulatory T cells (Treg) , Bone marrow-derived suppressor cells (MDSC), and regulate the effects of B cells (Breg).
使用標準規程單離調節T細胞(Treg)(CD3+CD4+CD25+CD127dim)。使用流動式細胞測量術分析Treg之頻率。 Isolate regulatory T cells (Treg) using standard procedures (CD3 + CD4 + CD25 + CD127 dim ). Use flow cytometry to analyze the frequency of Treg.
在Treg活化之前,周邊Treg之亞群(10%+10%)表現高水平的CD38。圖10A,上圖顯示在基期時Treg在CD3+CD4+細胞群(P4細胞群)中之頻率。圖10A,下圖顯示表現高CD38之Treg之子集(P5細胞群)。這些CD38+ Treg對DARZALEXTM(達拉單抗)治療高度敏感,且在DARZALEXTM(達拉單抗)之第一劑量之
後展現出顯著的且幾乎即時的衰退(病患n=17;第1週之於基期P=8.88×10-16)。DARZALEXTM(達拉單抗)治療之後Treg之頻率顯示於圖10B,上圖(P4細胞群)中。圖10B,下圖顯示,在第一(1st)DARZALEXTM(達拉單抗)輸液之後,CD38高Treg(P5細胞)係最顯著損耗的Treg群。這些CD38+ Treg在整個DARZALEXTM(達拉單抗)治療中係維持損耗的(在第1、4、及8週之於基期分別係p=8.88×10-16、1.11×10-15、及1.50×10-11)。圖10C顯示在基期、第1週、第4週、第8週、復發、及治療結束(EOT)6個月後的總CD3+細胞之CD38高Treg之%。CD38高Treg在該時間點恢復至基期。對治療有反應與沒有反應之病患之間的CD38+ Treg之變化係類似的,然而,顯示對DARZALEXTM(達拉單抗)治療有反應的病患在第8週的CD8+ T細胞:Treg比率顯著地較高(P=0.00955;圖10D)。
Before Treg activation, a subset of peripheral Tregs (10%+10%) showed high levels of CD38. Figure 10A, the upper panel shows the frequency of Treg in the CD3 + CD4 + cell population (P4 cell population) at the base phase. Figure 10A , the bottom panel shows a subset of Tregs (P5 cell population) that exhibit high CD38. These CD38 + Tregs are highly sensitive to DARZALEX TM (dalazumab) treatment, and showed significant and almost immediate decline after the first dose of DARZALEX TM (dalazumab) (patient n=17; 1st Weekly to the base period P = 8.88×10 -16 ). The frequency of Treg after DARZALEX (TM ) treatment is shown in Figure 10B , in the upper panel (P4 cell population). Figure 10B, the bottom panel shows the Treg population with the most significant loss of the CD38 high Treg (P5 cell) line after the first (1 st ) DARZALEX ™ (dalumab) infusion. These CD38 + Tregs were maintained attrition during the entire DARZALEX TM (dalazumab) treatment (p =8.88×10 -16 , 1.11×10 -15 , and p = 8.88×10 -16, 1.11×10 -15, and at the base period at the first, 4, and 8 weeks, respectively 1.50×10 -11 ). Figure 10C shows the% of CD38 high Treg of total CD3+ cells 6 months after base period,
為了評估CD38+ Treg之損耗與DARZALEXTM(達拉單抗)治療之可能的生物相關性,對自體CD3+ T細胞上的CD38+ Treg之於CD38- Treg的抑制能力進行評估。在利用多個健康供體的樣本進行的一系列實驗中,CD38+ Treg對T細胞增生的抑制(觀察到9.9%細胞增生)比CD38- Treg(觀察到53.2%細胞增生)或陰性對照(觀察到74.9%細胞增生)更穩健(圖10E)。 In order to evaluate the possible biological correlation between the loss of CD38 + Treg and DARZALEX TM (darumab) treatment, the ability of CD38 + Treg on autologous CD3 + T cells to inhibit CD38- Treg was evaluated. In a series of experiments using samples from multiple healthy donors, CD38 + Treg inhibited T cell proliferation (9.9% cell proliferation was observed) than CD38 - Treg (53.2% cell proliferation was observed) or a negative control (observed To 74.9% cell proliferation) is more robust ( Figure 10E ).
因為MDSC在冷凍PBMC樣本中不易偵測,所以CD38+顆粒球性MDSC(CD11b+CD14-HLA-DR-CD15+CD33+)係自PBMC體外產生,該等PBMC係從在基期的病患及已接受一次DARZALEXTM(達拉單抗)輸液的病患而單離。圖11顯示經鑑定之MDSC之流動式細胞測量術分佈圖(圖11,上方分佈圖,加框的細胞群)。大約一半的MDSC表現CD38(圖11,中間圖表;圈起來的P7細胞群)。在經DARZALEXTM(達拉單抗)治療的病患中,CD38高MDSC係幾乎耗盡的(圖11,下方圖表;圈起來的P7細胞群)。 Because MDSC is not easy to detect in frozen PBMC samples, CD38 + granular spherical MDSC (CD11b + CD14 - HLA-DR - CD15 + CD33 + ) is generated from PBMC in vitro. Patients who received a DARZALEX TM (dalazumab) infusion were isolated. Figure 11 shows the flow cytometry distribution map of the identified MDSCs ( Figure 11 , upper distribution map, boxed cell population). Approximately half of MDSCs express CD38 ( Figure 11 , middle graph; circled P7 cell population). In patients treated with DARZALEX TM (dalazumab), the CD38 high MDSC line was almost depleted ( Figure 11 , bottom graph; circled P7 cell population).
在無反應者及對治療有至少最小休止(Minimal Repose)的病患中,CD38高譜系非特異性MDSC在DARZALEXTM(達拉單抗)治療之情況下隨著時間推移而損耗。圖12顯示,在治療1週、4 週、或8週時,在病患中CD38高MDSC之百分比減少至接近0%。CD38高譜系非特異性MDSC在治療結束後返回至基期。 Among non-responders and patients with at least minimal repose to treatment, CD38 high- spectrum non-specific MDSCs are depleted over time under DARZALEX TM (dalazumab) treatment. Figure 12 shows that at 1 week, 4 weeks, or 8 weeks of treatment, the percentage of CD38 high MDSC in the patients decreased to nearly 0%. CD38 high- lineage non-specific MDSC returned to the base stage after the treatment.
具有在譜系非特異性MDSC內的最大CD38+群的病患展示對DARZALEXTM(達拉單抗)治療的最佳且最持久的反應。圖13顯示,具有最高百分比的CD38高MDSC(如圖11中所示)且分類為具有PR或MR之病患的病患2、4、15、16、或17具有至少8個月的無進展存活期(Progression-Free Survival,PFS)。
Patients with the largest CD38+ population within the lineage non-specific MDSC exhibited the best and most durable response to DARZALEX (TM) treatment. Figure 13 shows that
CD38高譜系非特異性MDSC亦對體外DARZALEXTM(達拉單抗)誘導的ADCC敏感。使用來自兩個供體的CD38高MDSC及道迪細胞作為對照目標細胞進行ADCC測定,其中效應細胞:目標細胞比率係50:1。圖14顯示來自一個供體的實驗之結果。DARZALEXTM(達拉單抗)誘導MDSC細胞之裂解。 CD38 high- lineage non-specific MDSCs are also sensitive to ADCC induced by DARZALEX TM in vitro. CD38 high MDSC and Daudi cells from two donors were used as control target cells for ADCC determination, in which the ratio of effector cells: target cells was 50:1. Figure 14 shows the results of an experiment from a donor. DARZALEX (TM) (dalazumab) induces the lysis of MDSC cells.
在DARZALEXTM(達拉單抗)治療的病患(n=16)中測量CD38+ Breg,且類似於CD38+ Treg,CD38+ Breg在DARZALEXTM(達拉單抗)之第一劑量之後係損耗的(第1週與基期比較,p=0.0018;成對威爾卡森等級檢定)且當病患在治療中時持續係低的(圖15A)。當刺激時,FACS分選之Breg產生IL-10(圖15B)。
CD38 + Breg was measured in patients (n=16) treated with DARZALEX TM (dalazumab), and similar to CD38 + Treg, CD38 + Breg was depleted after the first dose of DARZALEX TM (dalazumab) (Compared with base period in
總而言之,這些觀察表明,免疫抑制性CD38+ MDSC、Breg、及Treg之損耗係DARZALEXTM(達拉單抗)誘導的T細胞群及純系性之變化的顯著促成機制。 In summary, these observations indicate that the depletion of immunosuppressive CD38 + MDSCs, Bregs, and Tregs is a significant contributory mechanism for the changes in T cell populations and clones induced by DARZALEX(TM).
使用流動式細胞測量術研究患有NSCLC或前列腺癌之病患之周邊血液中MDSC(Lin-CD14-HLADR低/-)及其等CD38表現之百分比。 Use flow cytometry to study the percentage of MDSC (Lin- CD14 - HLADR low/- ) and other CD38 manifestations in the peripheral blood of patients with NSCLC or prostate cancer.
在來自NSCLC及前列腺癌病患之分析樣本中,MDSC之百分比分別係PBMC之介於約10%至37%之間及介於約10%至27%之間。CD38表現在NSCLC病患之PBMC之80至100%的Lin- CD14+HLADR-/低MDSC中、且在前列腺癌病患的PBMC之70至100%的MDSC中經鑑定。 In the analyzed samples from NSCLC and prostate cancer patients, the percentage of MDSC is between about 10% and 37% and between about 10% and 27% of PBMC, respectively. CD38 is expressed in 80 to 100% of Lin - CD14 + HLADR -/low MDSC of PBMC of NSCLC patients, and is identified in 70 to 100% of MDSC of PBMC of prostate cancer patients.
為了進一步評估DARZALEXTM(達拉單抗)對T細胞活化及功能性的效應,在DARZALEXTM(達拉單抗)治療的具有一定範圍臨床結果之病患(n=7)中,測量反應於病毒及同種抗原中之周邊T細胞的IFN-γ產生。具有PR或更佳反應的病患在DARZALEXTM(達拉單抗)治療之後,相較於基期展示出反應於病毒及同種抗原的IFN-γ分泌之顯著增加,至少在治療期間的一個時間點係如此,其表明T細胞功能不受低CD38表現而受損(參見實例6,圖9C)。類似於TCR純系性數據,此增加在對DARZALEXTM(達拉單抗)有反應的病患中,比在那些沒有反應的病患中更加明顯。圖16A顯示具有VGPR的一個代表性病患之抗病毒反應。圖16B顯示具有CR的一個代表性病患之抗病毒反應。圖16C顯示具有PD的一個代表性病患之抗病毒反應。圖16D顯示具有MR的一個代表性病患之抗病毒反應。在圖式中,誤差條代表雙份培育物之平均值之標準誤差。星號標示介於所指比較之間統計學顯著的變化。顯示按照獨立評審委員會標準的最佳反應。與這些結果一致,具有VGPR(圖16E)或CR(圖16F)之病患中之病毒反應性T細胞展示出在DARZALEXTM(達拉單抗)治療期間增生能力的增加。 In order to further evaluate the effect of DARZALEX TM (dalazumab) on T cell activation and function, in patients (n=7) treated with DARZALEX TM (dalazumab) with a certain range of clinical outcomes, the measurement response was IFN-γ production by surrounding T cells in viruses and allogeneic antigens. Patients with PR or better response after DARZALEX TM (dalazumab) treatment showed a significant increase in IFN-γ secretion in response to the virus and alloantigens compared to the base phase, at least at one time point during the treatment period This is so, which indicates that T cell function is not impaired by low CD38 manifestations (see Example 6, Figure 9C). Similar to the TCR pure lineage data, this increase was more pronounced in patients who responded to DARZALEX TM (dalazumab) than in patients who did not respond. Figure 16A shows the antiviral response of a representative patient with VGPR. Figure 16B shows the antiviral response of a representative patient with CR. Figure 16C shows the antiviral response of a representative patient with PD. Figure 16D shows the antiviral response of a representative patient with MR. In the diagram, the error bars represent the standard error of the average of the duplicate cultures. Asterisks indicate statistically significant changes between the indicated comparisons. Shows the best response in accordance with the standards of the independent review committee. Consistent with these results, virus-reactive T cells in patients with VGPR (Figure 16E) or CR (Figure 16F) showed an increase in proliferative capacity during DARZALEX(TM) treatment.
GEN501及SIRIUS研究之數據指示,在DARZALEXTM(達拉單抗)療法下,有一些表現CD38的免疫細胞係損耗的(NK細胞、調節T細胞(Treg)、調節B細胞(Breg)、及骨髓衍生抑制細胞(MDSC)),然而其他表現CD38的免疫細胞之數目卻是增加的(細胞毒性T細胞及輔助T細胞)。 The data from the GEN501 and SIRIUS studies indicate that under DARZALEX TM (dalazumab) therapy, there are some immune cell lines that exhibit CD38 depletion (NK cells, regulatory T cells (Treg), regulatory B cells (Breg), and bone marrow). Derived suppressor cells (MDSC)), however, the number of other immune cells that express CD38 is increased (cytotoxic T cells and helper T cells).
為了解決敏感度之機制,評估健康供體中及GEN501或SIRIUS研究中所登記之多發性骨髓瘤病患中之各種免疫細胞亞群中之CD38的表現水平。圖17A顯示健康供體之免疫細胞中CD38表現之分佈圖,且圖17B顯示多發性骨髓瘤病患之免疫細胞中CD38表現之分佈圖。在健康供體中,CD38表現在NK細胞上係最高,接著係單核球、B細胞、及T細胞。在多發性骨髓瘤病患中,CD38表現在漿細胞上最高,接著係B細胞之一子集、NK細胞、單核球、B細胞、及T細胞。圖17C顯示復發性及難治性骨髓瘤病患之NK細胞、Treg、Breg、B細胞、及T細胞之CD38的平均螢光強度(MFI)的比較,其展示在漿細胞之後,NK細胞表現最高水平的CD38,接著係調節T細胞(Treg)及調節B細胞(Breg)。 In order to solve the mechanism of sensitivity, the expression level of CD38 in various immune cell subpopulations in healthy donors and in patients with multiple myeloma registered in the GEN501 or SIRIUS study was evaluated. Figure 17A shows the distribution of CD38 expression in immune cells of healthy donors, and Figure 17B shows the distribution of CD38 expression in immune cells of patients with multiple myeloma. Among healthy donors, CD38 was highest on NK cells, followed by monocytes, B cells, and T cells. In patients with multiple myeloma, CD38 is highest on plasma cells, followed by a subset of B cells, NK cells, monocytes, B cells, and T cells. Figure 17C shows the comparison of the CD38 mean fluorescence intensity (MFI) of NK cells, Treg, Breg, B cells, and T cells in patients with relapsed and refractory myeloma. It shows that after plasma cells, NK cells have the highest performance The level of CD38 is followed by regulatory T cells (Treg) and regulatory B cells (Breg).
除了CD38表現,其他細胞表面蛋白諸如補體抑制蛋白(CIP;CD46,CD55,CD59)亦可能導致對DARZALEXTM(達拉單抗)的敏感性或抗性。體外評估免疫細胞亞群中之CIP發現到,NK細胞表現非常低水平的CD59及CD55,而其他T細胞群及B細胞群則表現出遠高得多的水平。這也可能導致免疫細胞亞型之DARZALEXTM(達拉單抗)敏感性之變異性(數據未顯示)。 In addition to CD38 expression, other cell surface proteins such as complement inhibitory proteins (CIP; CD46, CD55, CD59) may also lead to sensitivity or resistance to DARZALEX TM (dalazumab). In vitro evaluation of CIP in immune cell subpopulations revealed that NK cells showed very low levels of CD59 and CD55, while other T cell populations and B cell populations showed much higher levels. This may also lead to variability in the sensitivity of immune cell subtypes to DARZALEX TM (darumab) (data not shown).
此研究透過減少CD38+免疫抑制性細胞群及伴隨之誘導輔助及細胞毒性T細胞擴增、反應於病毒胜肽的IFN-γ之產生、及TCR純系性之增加而描述先前未知的DARZALEXTM(達拉單抗)之免疫調節效應,其指示出適應性免疫反應改善。 This study by reducing CD38 + immunosuppressive cell population and accompanying the induced secondary and cytotoxic T cell amplification reaction to produce IFN-γ of virus peptides of and increases TCR homogenous nature of the described previously known DARZALEX TM ( The immunomodulatory effect of dalamumab), which indicates an improvement in the adaptive immune response.
此研究展示出,MDSC及Breg表現CD38且易受DARZALEXTM(達拉單抗)治療影響。這些細胞已知存在於腫瘤微環境中,且導致腫瘤生長、免疫逃避(immune evasion)、血管生成、轉移、及抑制性細胞介素之產生。除了這些CD38+抑制性細胞子集,亦鑑定出新穎的調節T細胞亞群(CD4+CD25+CD127dim),其亦表現高水平的CD38且展示優異的自體T細胞抑制性能力。這些細胞亦對DARZALEXTM(達拉單抗)敏感,且在接受治療的病患中,這些細胞 顯著地減少。由DARZALEXTM(達拉單抗)媒介之該些CD38+免疫調節細胞的消除,可減少骨髓瘤微環境內的局部免疫抑制,且允許陽性免疫效應細胞擴增並導致抗瘤反應。 This study demonstrates that MDSC and Breg express CD38 and are susceptible to DARZALEX TM (dalazumab) treatment. These cells are known to exist in the tumor microenvironment and cause tumor growth, immune evasion, angiogenesis, metastasis, and the production of inhibitory cytokines. In addition to these CD38 + inhibitory cell subsets, a novel regulatory T cell subset (CD4 + CD25 + CD127 dim ) has also been identified, which also exhibits high levels of CD38 and exhibits excellent autologous T cell inhibitory ability. These cells are also sensitive to DARZALEX(TM) (darumab), and in patients receiving treatment, these cells are significantly reduced. The elimination of these CD38 + immunoregulatory cells mediated by DARZALEX TM (dalazumab) can reduce local immunosuppression in the myeloma microenvironment, and allow positive immune effector cells to expand and lead to anti-tumor responses.
實際上,周邊血液中及骨髓內(即,腫瘤)皆觀察到包括了CD4+及CD8+的廣泛T細胞群之顯著增加。特定CD8+亞群經DARZALEXTM(達拉單抗)療法改變,包括顯著地降低初始T細胞及伴隨的顯著增加之效應記憶CD8+ T細胞,其指示效應T細胞向經歷抗原之表型偏移,該表型保留了免疫記憶且可對腫瘤抗原具有反應性。CD8+:CD4+及CD8+:Treg之比率亦在治療之下顯著增加,其展示出陽性免疫調節物之於陰性免疫調節物的偏移。 In fact, a significant increase in a broad T cell population including CD4 + and CD8 + was observed in the peripheral blood and in the bone marrow (i.e., tumor). Specific CD8 + subpopulations have been altered by DARZALEX TM (dalazumab) therapy, including a significant reduction in naive T cells and a concomitant significant increase in effector memory CD8 + T cells, which indicate a shift of effector T cells to a phenotype experiencing antigen This phenotype retains immune memory and can be reactive to tumor antigens. The ratio of CD8 + :CD4 + and CD8 + : Treg also increased significantly under treatment, which showed a shift of positive immunomodulator from negative immunomodulator.
為了評估擴增CD4+及CD8+ T細胞在本質上係純系的,檢驗病患子集中之T細胞貯庫。即使在具有SD之最佳反應的病患或有進展的病患中,T細胞純系性也在DARZALEXTM(達拉單抗)治療之情況下顯著增加。因此,T細胞純系性增加不能簡單地歸因於腫瘤負荷的減少。然而,T細胞純系性的偏差在具有良好臨床反應的病患中較大,且與CD8+ T細胞增加相關,其表明在DARZALEXTM(達拉單抗)治療之下所觀察到的T細胞擴增係抗原驅動的。這在該病患族群中係顯著的,該病患群體經大量預治療(中位數係5個先前療法數)且不預期能夠建立強烈的抗瘤免疫反應。除了TCR純系性增加,對DARZALEXTM(達拉單抗)具有反應性的病患展示對先前存在的病毒及同種抗原的T細胞反應增加,其表明免疫系統從免疫抑制性狀態回復的救援。 In order to assess whether the expanded CD4 + and CD8 + T cells are pure in nature, examine the T cell reservoir in a subset of patients. Even in patients with the best response to SD or patients with progression, T cell homology is significantly increased under DARZALEX TM (dalazumab) treatment. Therefore, the increase in homologous T cells cannot be simply attributed to the decrease in tumor burden. However, the deviation of T cell homology is greater in patients with good clinical response and is associated with an increase in CD8 + T cells, which indicates the expansion of T cells observed under DARZALEX TM (dalazumab) treatment. Proliferation of antigen-driven. This is significant in this patient population, which has undergone a large number of pre-treatments (median is 5 previous therapies) and is not expected to be able to establish a strong anti-tumor immune response. In addition to increased TCR homology , patients who are responsive to DARZALEX TM (dalazumab) exhibit increased T cell responses to pre-existing viruses and alloantigens, which indicates the rescue of the immune system from an immunosuppressive state.
利用DARZALEXTM(達拉單抗)治療造成免疫抑制性MDSC以及調節T細胞及B細胞的減少。這些減少伴隨有CD4+ T輔助細胞及CD8+細胞毒性T細胞的擴增。如藉由IFN-γ產生所測量之T細胞純系性及功能性抗病毒反應亦在DARZALEXTM(達拉單抗)治療之下增加。這些觀察指示,儘管CD38表現低但T細胞仍適當地繼續起作用,且表明T細胞反應增加可能係因為調節細胞之損耗。此外這些T細胞擴增、活性、及純系性變化,在對DARZALEXTM(達拉單抗)有反應的病患中相較於在那些沒有反應的病患中,可能係更顯 著的。從DARZALEXTM(達拉單抗)療法復發與許多這些變化之逆轉相關聯。這表明透過免疫調節的DARZALEXTM(達拉單抗)之額外、先前未表徵機制之作用,其可導致臨床反應及DARZALEXTM(達拉單抗)之功效。 The use of DARZALEX TM (Dalazumab) treatment resulted in a reduction of immunosuppressive MDSC and regulatory T cells and B cells. These reductions are accompanied by the expansion of CD4 + T helper cells and CD8 + cytotoxic T cells. The T cell homologous and functional antiviral response as measured by IFN-γ production was also increased under DARZALEX TM (dalazumab) treatment. These observations indicate that T cells continue to function properly despite low CD38 performance, and indicate that the increased T cell response may be due to the depletion of regulatory cells. In addition, these T cell expansion, activity, and homologous changes may be more significant in patients who responded to DARZALEX TM (dalazumab) than in patients who did not respond. Recurrence from DARZALEX TM (dalazumab) therapy is associated with the reversal of many of these changes. This indicates the effect of the additional, previously uncharacterized mechanism of DARZALEX TM (dalazumab) through immunomodulation, which can lead to clinical response and the efficacy of DARZALEX TM (dalazumab).
最近,促進抗瘤免疫反應(而非直接靶向癌症)的抗體已在一系列環境中展示功效。抑制CTLA-4及PD-1的抗體促進T細胞擴增且增強T細胞活化,其導致在患有晚期固態腫瘤及血液學惡性腫瘤(諸如霍奇金氏淋巴瘤)之病患中延長的存活期且延緩疾病復發。藉由增強抗癌免疫,這些免疫調節抗體可能不僅誘導臨床反應,且亦預防疾病復發。 Recently, antibodies that promote anti-tumor immune responses (rather than directly target cancer) have demonstrated efficacy in a range of environments. Antibodies that inhibit CTLA-4 and PD-1 promote T cell expansion and enhance T cell activation, which leads to prolonged survival in patients with advanced solid tumors and hematological malignancies (such as Hodgkin's lymphoma) Period and delay the recurrence of the disease. By enhancing anti-cancer immunity, these immunomodulatory antibodies may not only induce clinical response, but also prevent disease recurrence.
將周邊血液樣本收集於標準血清分離管(2.5mL至5mL)中,且將血清試樣等分冷凍運送SomaLogic,Inc(Boulder,CO)以用於多分析物血清蛋白剖析。 The peripheral blood samples were collected in standard serum separation tubes (2.5 mL to 5 mL), and the serum samples were aliquoted frozen and transported to SomaLogic, Inc (Boulder, CO) for multi-analyte serum protein analysis.
血清蛋白剖析係在SomaLogic處使用預先驗證的SOMAscan測定進行,該SOMAscan測定藉由使用以SOMAmer親和力為基礎之分子測量1129種蛋白質分析物。SOMAmer試劑係以單鏈DNA為基礎之蛋白質親和力試劑。該測定使用少量的輸入樣本(150μL血漿)且將蛋白質訊號轉變成SOMAmer訊號,該SOMAmer訊號係藉由客製化DNA微陣列定量。 Serum protein profiling is performed at SomaLogic using the pre-validated SOMAscan assay, which measures 1129 protein analytes using SOMAmer affinity-based molecules. SOMAmer reagent is a protein affinity reagent based on single-stranded DNA. The assay uses a small amount of input sample (150 μL of plasma) and converts the protein signal into a SOMAmer signal, which is quantified by a customized DNA microarray.
各SOMAmer含有4個功能部分: Each SOMAmer contains 4 functional parts:
1. 一個獨特蛋白質識別序列 1. A unique protein recognition sequence
2. 用於捕捉的生物素 2. Biotin for capture
3. 光可裂解連接子 3. Photocleavable linker
4. 用於偵測之螢光分子 4. Fluorescent molecules for detection
該獨特蛋白質識別序列使用DNA且併入模擬胺基酸側鏈的化學修飾核苷酸,其擴增標準適體之多樣性且增強蛋白質-核酸相互作用的特異性及親和力(Gold等人,PLoS One 5:e15004,2010)。適體係藉由SELEX來選擇。SOMAmer試劑係使用呈現原始構形的蛋白質來選擇。因為此類SOMAmer試劑需要完整的三級蛋白質結構來結合。未折疊或變性的推測上非活性蛋白質不為SOMAmer試劑所偵測。 The unique protein recognition sequence uses DNA and incorporates chemically modified nucleotides that mimic amino acid side chains, which amplify the diversity of standard aptamers and enhance the specificity and affinity of protein-nucleic acid interactions (Gold et al., PLoS One 5: e15004, 2010). The suitable system is selected by SELEX. The SOMAmer reagent is selected using the protein in the original configuration. Because this type of SOMAmer reagent requires a complete tertiary protein structure to bind. Unfolded or denatured presumably inactive proteins are not detected by SOMAmer reagent.
針對樣本類型及稀釋,將SOMAmer試劑之主要混合物分組。在樣本培養之前,將試劑預結合至鏈黴親和素珠粒。蛋白質在平衡期間將樣本中之結合至同源SOMAmer、清洗、用NHS-生物素培養、清洗、然後將珠粒暴露於UV光以裂解該光可裂解連接子。洗出液含有SOMAmer試劑,該等試劑結合至其等的生物素所標記之蛋白質。鏈黴親和素捕捉且後續清洗移除未結合的SOMAmer試劑。在最終洗提中,SOMAmer分子透過變性條件從其等之同源蛋白質釋放。將最終洗出液混成至客製化Agilent DNA微陣列,而SOMAmer分子之螢光團係藉由相對螢光單位(RFU)來定量。RFU與樣本中之蛋白質之量成比例。 According to the sample type and dilution, the main mixture of SOMAmer reagent is grouped. Before the sample is incubated, the reagent is pre-bound to the streptavidin beads. The protein in the sample binds to the homologous SOMAmer during equilibration, washes, incubates with NHS-biotin, washes, and then exposes the beads to UV light to cleave the photocleavable linker. The eluate contains SOMAmer reagents, which bind to their biotin-labeled proteins. Streptavidin captures and subsequent washing removes unbound SOMAmer reagent. In the final elution, SOMAmer molecules are released from their homologous proteins through denaturing conditions. The final eluate is mixed into a customized Agilent DNA microarray, and the fluorophore of the SOMAmer molecule is quantified by relative fluorescence units (RFU). RFU is proportional to the amount of protein in the sample.
在兩個主要批次中測試MMY2002研究之樣本。第一批次的180個樣本含有來自90個對象的成對第1週期第1天(C1D1,基期)及C3D1(第3週期第1天)之血清樣本。將180個樣本一起在3個分開的SomaScan盤上分析。第二批次樣本包括50個C1D1樣本,其包括來自第1批次的35個重複樣本。
The samples from the MMY2002 study were tested in two main batches. The 180 samples in the first batch contained a pair of serum samples from 90 subjects on Day 1 (C1D1, Base Period) and C3D1 (
具有可評估反應的經治療之對象均包括在數據分析中。在報導全文中,反應者係定義為具有sCR、VGPR、及PR之總體最佳反應(按照IRC,針對MMY2002)的對象,疾病穩定(SD)對象係定義為具有最小反應(MR)或SD的對象,且無反應者係定義為具有疾病進展(PD)之總體最佳反應(按照IRC,針對MMY2002)的對象。 Treated subjects with evaluable responses were included in the data analysis. In the full text of the report, responders are defined as subjects with the overall best response of sCR, VGPR, and PR (according to IRC, for MMY2002), and subjects with stable disease (SD) are defined as subjects with minimal response (MR) or SD Subjects and non-responders are defined as subjects with the overall best response of disease progression (PD) (according to IRC, for MMY2002).
將第1批及第2批的MMY2002樣本於兩個不同版本的SOMAscan平臺上進行測試。兩個版本間的差異微小,且包括三個SOMAmer序列,該等序列於版本之間有所改變(CTSE:3594-6_1->3594-6_5,FCN1:3613-62_1->3613-62_5,BMPER:3654-27_1->3654-27_4)。該等均自分析移除。 The first and second batch of MMY2002 samples were tested on two different versions of the SOMAscan platform. The differences between the two versions are small and include three SOMAmer sequences, which have changed between versions (CTSE: 3594-6_1->3594-6_5, FCN1: 3613-62_1->3613-62_5, BMPER: 3654-27_1->3654-27_4). These are removed from the analysis.
根據SomaLogic之標準盤間校準工作流程,藉由透過計算主混合物(Master-mix)特定整體參考值對7個盤內對照校準物測量值之比率,來定義各SOMAmer之盤寬(plate-wide)校準比例因數,因此校準三個第1批次盤之測量值。將各SOMAmer試劑之盤特定(plate-specific)比例因數同等地應用於盤上的各樣本。 According to SomaLogic's standard inter-plate calibration workflow, the plate-wide of each SOMAmer is defined by calculating the ratio of the specific overall reference value of the master mix (Master-mix) to the measured values of the 7 intra-plate control calibrators. The scale factor is calibrated, so the measured values of the three first batch disks are calibrated. The plate-specific scale factor of each SOMAmer reagent is equally applied to each sample on the plate.
給定第1批次及第2批次之不同的SOMAscan平臺版本,藉由考慮跨批次的35個樣本之重複測量值的比率,利用SomaLogic之標準盤間校準工作流程之修改實施方案,進行系統性批次間變異性校正。針對各SOMAmer,計算35個重複樣本之每一者的第1批次校準後測量值除以第2批次校準前測量值之比率(ri,j)。將這35個比率之中位數用於定義第2批次樣本之修正SOMAmer特定校準比例因數。然後將這些校準比例因數同樣地實施於標準SOMAscan程序。 Given the different SOMAscan platform versions of the first batch and the second batch, by considering the ratio of the repeated measurement values of 35 samples across batches, using the modified implementation scheme of SomaLogic's inter-standard calibration workflow Systematic correction for batch-to-batch variability. For each SOMAmer, calculate the ratio (ri,j ) of the measured value after the first batch of calibration divided by the measured value of the second batch of each of the 35 replicate samples. Use the median of these 35 ratios to define the modified SOMAmer specific calibration ratio factor for the second batch of samples . Then these calibration scale factors are also implemented in the standard SOMAscan program.
一旦計算出修正校準比例因數,就繪製分析之各批次的所有比例因數之分佈,以評估離群值之存在狀況。由於再現性不良,因此將9個具有極大或極小校準值(>0.25且<3)的SOMAmer從分析移除。 Once the corrected calibration scale factor is calculated, the distribution of all scale factors for each batch of analysis is drawn to assess the existence of outliers. Due to poor reproducibility, 9 SOMAmers with very large or very small calibration values (>0.25 and <3) were removed from the analysis.
在MMY2002之批次校準及SOMAmer過濾完成之後,將log2轉換應用於MMY2002之所有蛋白質濃度值,以使數據更合乎常態分佈且改善參數統計檢定之表現。 After batch calibration of MMY2002 and SOMAmer filtering are completed, log2 conversion is applied to all protein concentration values of MMY2002 to make the data more in line with the normal distribution and improve the performance of parameter statistical verification.
藉由對定中心且成比例的數據集的主成份分析,進行數據集變異數之藉由元變數(meta-variables)所解釋的部分之估計(如人口統計、反應級別、及樣本時間點)及可能混雜因子之鑑定。將簡單的線性模型擬合以鑑定與感興趣的各變數顯著相關聯的最高等級PC。這些關聯之顯著性係使用渥得檢定(Wald test)來判定,且由該模型解釋的PC變異性之部分係藉由擬合之R2估計。針對MMY2002數據,部位ID被發現到與PC1相關,且解釋了數據集變異性之最大部分(7.37%,p-值=3.71×10-9)。為了減少樣本獲得部位相關效應在數據內的影響,利用ComBat28校正部位ID效應。 By principal component analysis of a centered and proportional data set, estimate the part of the data set variance explained by meta-variables (such as demographics, response levels, and sample time points) And the identification of possible confounding factors. A simple linear model is fitted to identify the highest grade PC that is significantly associated with each variable of interest. The significance of these associations was determined using the Wald test, and the part of the PC variability explained by the model was estimated by the fitted R2. For the MMY2002 data, the part ID was found to be related to PC1 and explained the largest part of the variability of the data set ( 7.37%, p-value = 3.71×10-9). In order to reduce the influence of the position-related effects of sample acquisition in the data, ComBat28 is used to correct the position ID effect.
藉由計算各蛋白質之平均值而合併在MMY2002第1批次與第2批次之間重複的35個樣本之數據。 The data of 35 samples repeated between the first batch and the second batch of MMY2002 were combined by calculating the average value of each protein.
對於在DARZALEXTM(達拉單抗)反應者之於無反應者中於基期以及治療中之蛋白質濃度分佈的統計學比較係使用兩個互補的方法進行:(i)威爾卡森等級和檢定(Hollander及Wolfe,Ninparametirc Statistical Methods.New York:John Wiley & Sons.1973.27-33(一個樣本),68-75(兩個樣本),對各個別SOMAmer進行,及(ii)Limma分析(Ritchie,M.E.等人,Nucleic Acids Res.2015;20:43(7):e47),對所有SOMAmer同時進行。所有p-值係使用針對多假說校正之班傑明-哈克伯格(Benjamini-Hochberg;BH)方法而進行調整(Benjamini及Hochberg,(1995)J.R.Statist.Soc.B.57: 289-300;R:A Language and Environment for Statistical Computing,R Development Core Team,R Foundation for Statistical Computing,Vienna,Austria.2011;ISBN 3-900051-07-0)。當調整的p-值<0.05時,拒絕無差異表現之歸零假說。 The statistical comparison of the protein concentration distribution between DARZALEXTM responders and non-responders in the base phase and during treatment was carried out using two complementary methods: (i) Wilkason rank and test ( Hollander and Wolfe, Ninparametirc Statistical Methods. New York: John Wiley & Sons. 1973.27-33 (one sample), 68-75 (two samples), performed on each SOMAmer, and (ii) Limma analysis (Ritchie, ME, etc.) Human, Nucleic Acids Res. 2015; 20: 43(7): e47), performed simultaneously for all SOMAmers. All p-value systems use Benjamini-Hochberg (Benjamini-Hochberg; BH) for multi-hypothesis correction Method to adjust (Benjamini and Hochberg, (1995) JRStatist.Soc. B.57: 289-300; R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria. 2011 ; ISBN 3-900051-07-0). When the adjusted p-value is <0.05, the zero hypothesis of no difference in performance is rejected.
使用三個替代統計學方法來比較基期蛋白質水平之於治療中蛋白質水平:(i)雙向重複測量ANOVA6、(ii)威爾卡森符號等級檢定、及(iii)傅里德曼檢定(Friedman test)(Johnson等人,(2007)Biostatistics 8(1):118-127)。所有p-值係經調整以控制FDR,該調整係使用針對多假說校正之BH方法(Benjamini及Hochberg,J.R.Statist.Soc.B.57:289-300,1995)。除了治療顯著性,雙向重複測量ANOVA(Chambers等人,Analysis of variance;designed experiments:Chapter 5.Statistical Models in S,Editors J.M Chambers and T.J Hastie.Wadsworth & Brookes/Cole.1992)亦應用於判定各SOMAmer是否發生顯著的時間點:反應級別相互作用。將修改的威爾卡森等級和檢定作為事後測試應用,以明確判定反應者及無反應者是否顯示不同的治療效應,其藉由計算每個對象之治療中蛋白質濃度值與基期蛋白質濃度值間的差異且進行威爾卡森等級和檢定。使用BH方法調整顯著性值,且當調整的p-值<0.05時,拒絕歸零假說。 Three alternative statistical methods were used to compare the baseline protein level to the protein level in treatment: (i) two-way repeated measures ANOVA6, (ii) Wilkason sign rank test, and (iii) Friedman test (Friedman test) ( Johnson et al. (2007) Biostatistics 8(1):118-127). All p-values were adjusted to control FDR, and the adjustment was performed using the BH method for multi-hypothesis correction (Benjamini and Hochberg, JR Statist. Soc. B. 57: 289-300, 1995). In addition to treatment significance, two-way repeated measures ANOVA (Chambers et al., Analysis of variance; designed experiments: Chapter 5.Statistical Models in S, Editors JM Chambers and TJ Hastie.Wadsworth & Brookes/Cole.1992) is also used to determine each SOMAmer Does a significant point in time occur: reaction level interaction. The modified Wilkason level and test are used as post-mortem tests to clearly determine whether responders and non-responders show different therapeutic effects by calculating the difference between the protein concentration value during treatment and the base-phase protein concentration value for each subject The difference and perform Wil Carson's level and verification. Use the BH method to adjust the significance value, and when the adjusted p-value is <0.05, the zero hypothesis is rejected.
將基期蛋白質水平MMY2002數據用於建立反應預測分級系統。巢套迴路分層的10折交叉驗證方法(10-fold cross-validation approach)重複30次,其使用4個不同的機器學習者:支持向量機(Support Vector Machine,SVM)、隨機森林(Random Forest,RF)、單純貝氏(Naïve Baye;NB)、及j48決策樹。針對各學習者,訓練程序以產生數據集之平衡的10個折開始(外迴路)。這些折之一者提供為測試分群,而剩餘的9個傳至內迴路作為訓練分群。在內迴路內,訓練分群被再次被分成平衡的10個折,其產生訓練內集及測試內集。對這 些訓練內集之各者進行學習者訓練,且針對外迴路內之各分群重複此程序30次。各內迴路學習者在預測測試內集時之準確度係用於選擇特徵及最佳化模型參數。一旦各訓練分組之30次(30x)內循環完成,就對各對應的測試分群進行外迴路之表現(使用最佳化的參數及特徵)之評估。然後將整個外循環程序重複30次,針對數據集內之每一樣本產生30次反應預測。獲自此循環方法的AUC、靈敏度、及特異度統計學係最終模型(以完整原始數據集所訓練)對新測試案例之表現的近似值。 The baseline protein level MMY2002 data was used to establish a response prediction grading system. The 10-fold cross-validation approach of nesting loop layering is repeated 30 times, using 4 different machine learners: Support Vector Machine (SVM), Random Forest (Random Forest) ,RF), Naïve Baye (NB), and j48 decision tree. For each learner, the training program starts with 10 folds that generate the balance of the data set (outer loop). One of these folds is provided as a test cluster, and the remaining 9 are passed to the inner loop as a training cluster. In the inner loop, the training group is again divided into 10 balanced folds, which generate the training inner set and the test inner set. Right this Each of these training inner sets performs learner training, and this procedure is repeated 30 times for each group in the outer loop. The accuracy of each inner loop learner in predicting the inner set of the test is used to select features and optimize model parameters. Once the 30 times (30x) inner loops of each training group are completed, the performance of the outer loop (using optimized parameters and features) is evaluated for each corresponding test group. Then the entire outer loop procedure was repeated 30 times, and 30 reaction predictions were generated for each sample in the data set. The AUC, sensitivity, and specificity statistics obtained from this round-robin method are the approximate values of the performance of the final model (trained with the complete original data set) on the new test case.
進行各種比較,包括蛋白質表現中治療誘導的反應依賴性變化。在反應者中顯示隨時間推移表現減小的蛋白質中之一者係PD-L1;然而在無反應者中,PD-L1蛋白質表現隨時間推移而增加。PD-L1在T細胞上的接合引起T細胞功能減少及Treg發展增加。圖18顯示在第1週期及第3週期,反應者、無反應者、及疾病穩定的病患中PD-L1之蛋白質表現概況。
Various comparisons were made, including treatment-induced response-dependent changes in protein expression. Among the responders, one of the proteins that showed decreased performance over time was PD-L1; however, among non-responders, the protein performance of PD-L1 increased over time. The engagement of PD-L1 on T cells causes a decrease in T cell function and an increase in Treg development. Figure 18 shows the profile of PD-L1 protein expression in responders, non-responders, and patients with stable disease in
PD-L1與其受體PD-1的接合抑制了抗瘤反應,且驅使T細胞無反應性及耗盡。儘管不希望受任何特定理論束縛,但是在CD38治療之後,PD-L1之下調亦可能導致固態腫瘤之抗瘤免疫反應增強之改善。 The engagement of PD-L1 with its receptor PD-1 inhibits the anti-tumor response and drives T cell anergy and depletion. Although not wishing to be bound by any specific theory, after CD38 treatment, PD-L1 down-regulation may also lead to an improvement in the anti-tumor immune response of solid tumors.
<110> 美商健生生物科技公司(Janssen Biotech,Inc.) <110> Janssen Biotech, Inc.
<120> 使用特異性結合CD38之抗體免疫調節及治療固態腫瘤 <120> Immunomodulation and treatment of solid tumors with antibodies that specifically bind to CD38
<140> 105134914 <140> 105134914
<141> 2016-10-28 <141> 2016-10-28
<150> US 15/191808
<150>
<151> 2016-06-24 <151> 2016-06-24
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<150> PCT/
<151> 2016-06-24 <151> 2016-06-24
<150> US 62/331489 <150> US 62/331489
<151> 2016-05-04 <151> 2016-05-04
<150> US 62/263307 <150> US 62/263307
<151> 2015-12-04 <151> 2015-12-04
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<150> US 62/263199 <150> US 62/263199
<151> 2015-12-04 <151> 2015-12-04
<160> 40 <160> 40
<170> PatentIn version 3.5 <170> PatentIn version 3.5
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<211> 300 <211> 300
<212> PRT <212> PRT
<213> 智人 <213> Homo sapiens
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<210> 2 <210> 2
<211> 14 <211> 14
<212> PRT <212> PRT
<213> 智人 <213> Homo sapiens
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<212> PRT <212> PRT
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<220> <220>
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<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
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<223> 抗CD38抗體之VL <223> VL of anti-CD38 antibody
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<211> 5 <211> 5
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
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<210> 7 <210> 7
<211> 17 <211> 17
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
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<400> 7 <400> 7
<210> 8 <210> 8
<211> 13 <211> 13
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
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<400> 8 <400> 8
<210> 9 <210> 9
<211> 11 <211> 11
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗CD38抗體之LCDR1 <223> LCDR1 of anti-CD38 antibody
<400> 9 <400> 9
<210> 10 <210> 10
<211> 7 <211> 7
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗CD38抗體之LCDR2 <223> LCDR2 of anti-CD38 antibody
<400> 10 <400> 10
<210> 11 <210> 11
<211> 10 <211> 10
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
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<400> 11 <400> 11
<210> 12 <210> 12
<211> 452 <211> 452
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
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<210> 13 <210> 13
<211> 214 <211> 214
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗CD38抗體之輕鏈 <223> Light chain of anti-CD38 antibody
<400> 13 <400> 13
<210> 14 <210> 14
<211> 122 <211> 122
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
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<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
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<210> 16 <210> 16
<211> 122 <211> 122
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
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<210> 17 <210> 17
<211> 107 <211> 107
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
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<211> 120 <211> 120
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗CD38抗體之VH MOR202 <223> VH MOR202 of anti-CD38 antibody
<400> 18 <400> 18
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<211> 109 <211> 109
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗CD38抗體之VL MOR202 <223> VL MOR202 of anti-CD38 antibody
<400> 19 <400> 19
<210> 20 <210> 20
<211> 120 <211> 120
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗CD38 mAb之VH伊沙妥昔單抗 <223> VH Isartuximab with anti-CD38 mAb
<400> 20 <400> 20
<210> 21 <210> 21
<211> 107 <211> 107
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗CD38 mAb之VL伊沙妥昔單抗 <223> VL Isartuximab against CD38 mAb
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<210> 22 <210> 22
<211> 120 <211> 120
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-1 mAb之VH Keytruda <223> VH Keytruda of anti-PD-1 mAb
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<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-1 mAb之VL Keytruda <223> VL Keytruda of anti-PD-1 mAb
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<210> 24 <210> 24
<211> 113 <211> 113
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-1 mAb之VH Opdivo <223> VH Opdivo of anti-PD-1 mAb
<400> 24 <400> 24
<210> 25 <210> 25
<211> 107 <211> 107
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-1 mAb之VL Opdivo <223> VL Opdivo of anti-PD-1 mAb
<400> 25 <400> 25
<210> 26 <210> 26
<211> 121 <211> 121
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-L1 mAb之VH德瓦魯單抗 <223> VH Devalumab against PD-L1 mAb
<400> 26 <400> 26
<210> 27 <210> 27
<211> 108 <211> 108
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-L1 mAb之VL德瓦魯單抗 <223> VL Devalumab for anti-PD-L1 mAb
<400> 27 <400> 27
<210> 28 <210> 28
<211> 118 <211> 118
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-L1 mAb之VH阿替珠單抗 <223> VH atezizumab against PD-L1 mAb
<400> 28 <400> 28
<210> 29 <210> 29
<211> 107 <211> 107
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-L1 mAb之VL阿替珠單抗 <223> VL atezizumab for anti-PD-L1 mAb
<400> 29 <400> 29
<210> 30 <210> 30
<211> 120 <211> 120
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-L1 mAb之VH艾維路單抗 <223> Anti-PD-L1 mAb of VH Avilizumab
<400> 30 <400> 30
<210> 31 <210> 31
<211> 110 <211> 110
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-L1 mAb之VL艾維路單抗 <223> Anti-PD-L1 mAb VL Avillumab
<400> 31 <400> 31
<210> 32 <210> 32
<211> 123 <211> 123
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-1 mAb之VH <223> VH of anti-PD-1 mAb
<400> 32 <400> 32
<210> 33 <210> 33
<211> 107 <211> 107
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-1 mAb之VL <223> VL of anti-PD-1 mAb
<400> 33 <400> 33
<210> 34 <210> 34
<211> 117 <211> 117
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-1 mAb之VH <223> VH of anti-PD-1 mAb
<400> 34 <400> 34
<210> 35 <210> 35
<211> 107 <211> 107
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗PD-1 mAb之VH <223> VH of anti-PD-1 mAb
<400> 35 <400> 35
<210> 36 <210> 36
<211> 117 <211> 117
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
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<400> 36 <400> 36
<210> 37 <210> 37
<211> 107 <211> 107
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗TIM-3 mAb之VL <223> VL of anti-TIM-3 mAb
<400> 37 <400> 37
<210> 38 <210> 38
<211> 124 <211> 124
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗TIM-3 mAb之VH <223> VH of anti-TIM-3 mAb
<400> 38 <400> 38
<210> 39 <210> 39
<211> 106 <211> 106
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 抗TIM-3 mAb之VL <223> VL of anti-TIM-3 mAb
<400> 39 <400> 39
<210> 40 <210> 40
<211> 509 <211> 509
<212> PRT <212> PRT
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 重組玻尿酸酶 <223> Recombinant Hyaluronidase
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