FDA的观点临床试验中基于MSC的产品特性

1、专注医疗咨询、专业医疗顾问 FDA的观点:临床试验中基于MSC的产品特性(E NINGMENG整理)IntroductionMSCs,commonly referred to as mesenchymal stem cells or mesenchymal stromal cells,are a diverse population of cells with a wide range of potential therapeuticapplications. Despite considerable interest and effort, there are currently noFDA

2、-approved Biologics License Applications (BLAs) for any MSC-based products.There is also no consistent nomenclature or definition of MSCs (Keating, 2012).In 2006, the International Society for Cellular Therapy (ISCT) proposed a setof minimal criteria to characterize MSCs (such as cell surface marker

3、expression). More recently, researchers in the MSC field have shown that thesecommonly described markers are not distinctive and therefore may not besufficient for defining the cellular composition and biological function orfunctions of an MSC-based product. Although not an FDA requirement, stakehol

4、derefforts toward generating consensus on MSC definitions would be a usefuldevelopment for the field, which would allow comparison across multiple studiesand could facilitate potential clinical use. In order to better understand thecomposition, phenotype, and range of bioactivity for MSCs, it may be

5、 useful tobe able to correlate MSC surface and/or secreted protein markers with theirin vitro and in vivo bioactivity. However, incomplete knowledge aboutMSC mechanisms-of-action (MOAs) and how these may relate to outcomes fordifferent clinical indications and routes of administration make suchcompa

6、risons especially difficult. It is important to note that the FDA reviewseach regulatory submission based on its own merits, and nomenclature is not aregulatory concern during early clinical development. Closer to licensure,however, nomenclature assumes greater significance for regulatory requiremen

7、ts,such as product labeling.简介MSCs，通常表述为间充质干细胞 或间质基质细胞，他们是一种不同的细胞种群 有着广泛治疗潜力的应用。尽管巨大的兴趣与努力， 目前还没有任何基于MSC的产品取得FDA批准应用生物制品许可证（BLAS）。 也没有对MSCs的命名和定义确定下来（基廷，2012年）。在2006年，TheInternatiuional Society forCellularTherapy（isct）提出了最低标准 表征间充质干细胞（如细胞表面标志物的表达）。最近，MSC领域的研究者表示这些普通的表述并不是独特的属性，所以无法足够的定义细胞的组成和生物功能和MS

8、C产品功能。虽然不是FDA的要求，利益相关者努力形成对MSC的定义的共识，有助于这个领域的发展。这将使不同的研究产生比较并促进潜在的临床应用。为了更好的理解MSCs的组成，表型和生物活性指标，将MSC表面形态和或者分泌的蛋白标记物与他们在体内货这体外的生物活性建立联系将是有帮助的。然而，对MSC机制-行动的不了解，以及对于不同临床适应症和给药途径会产生怎样的结果使这个比较相当困难。值得注意的是，美国FDA审查根据它自己的优点每个管制提交它是重要的，并命名法是不早期临床开发的调节问题。言归正传执照，然而，命名承担监管要求，如产品标签更大的意义.In order to track emerging

9、 trends in this rapidly expandingfield, we assessed initial filings of 66 Investigational New Drug (IND)submissions to the FDA for MSC-based products and MSC-related information fromworldwide clinical trial registries (as of December, 2012). We limited ourassessment to MSC-based products that were u

10、sed in similar ways to avoidpotential confusion that would arise from making less biologically relevantcomparisons. For example, we excluded whole bone marrow mononuclearpreparations even when the sponsor described their product using MSCterminology. We also excluded trials that only used MSCs durin

11、g manufacturingof a final cell therapy product (e.g., MSCs used for ex vivo culture only)in which MSCs were not administered to patients. Our analysis revealed a highdegree of variability in terms of MSC sources, manufacturing processes, andin vitro and in vivo product characterization. This lack of

12、 consensushighlights potential challenges to the clinical translation of MSC-based products.为了跟踪新出现的趋势在这个快速发展的领域，我们评估了提交给FDA（IND）的来自世界各地的临床试验注册的MSC为基础的产品和MSC相关的信息的66新药研究的初始申请。（如12月，2012）。为避免潜在的冲突，哪些不能产生多少生物相关性比较的使用类似方法的MSC产品，我们限制了评估。例如，我们排除了全骨髓单个核的提取，即使赞助商使用MSC术语描述他们的产品。我们也排除试验，只有制造的最终细胞治疗产品的过程中使用的M

13、SC（例如，MSC仅仅用于离体培养），其中的MSC未给予患者。我们的分析显示不同的MSC来源，不同制造工艺，以及是体外还是体内产品，变异性程度很高。这种缺乏共性，突出以MSC为基础的产品的临床转化有着潜在的挑战。Donor and Tissue Source Diversity in MSC-Based Product INDs捐助者和组织来源多样性的MSC-基于产品的INDThere was an approximately 3-fold increase in the number of MSC-based product IND submissions to the FDA betwee

14、n 2006 and 2012 in the set we assessed. In this period, there was also a substantial increase in registered MSC clinical trials initiated worldwide (246 trials; source: , “Mesenchymal Stem OR Mesenchymal Stromal,” queried in January, 2013). Despite this rapid expansion in

15、 clinical trials and calls for revised nomenclature, the original terminology (Mesenchymal Stem Cell) still appears to predominate. We found that the term “Mesenchymal Stem Cell” was used to describe 72% of MSC-based product registered clinical trials worldwide, and 76% of MSC-based products in orig

16、inal IND submissions prior to 2013. As the number of registered clinical trials worldwide and MSC-based product IND submissions increased, the diversity in donor and tissue source increased as well. Donor source diversity refers to whether the cells were isolated from an autologous (self) or allogen

17、eic (non-self) donor. It also refers to the variability observed between donors, which could be related to age and health of the donor among other factors. Almost all MSC-based product INDs prior to 2008 were sourced from allogeneic donor bone marrow (e.g., 100% in 2006). Since then, MSC donor and t

18、issue source diversity has significantly increased. For example, the percentage of allogeneic MSC-based products being evaluated under IND decreased to 42% in 2011 (the only year the percentage dropped below 50% prior to 2013) and increased to 73% allogeneic MSC-based product INDs in 2012.2006年和2012

19、年之间，我们评估的提交给FDA的MSC为基础的产品IND的数量大约增长了3倍。在此期间，也有全球发起注册的MSC临床试验的大幅增加（246试验;来源：，“间充质干或间充质基质，”一月份，2013年）。尽管在临床试验快速扩张和呼吁修订术语，原来术语（间充质干细胞）似乎仍然占主导地位。我们发现的是，术语“间充质干细胞”是用来描述的全球注册临床试验基于MSC的产品的72，同时76的MSC的产品是原IND提交，随着注册临床试验的次数全世界与基于MSC产品IND提交的增加，供体的多样性和组织来源也在增加。供体来源的多样性是指细胞是否是从自体（个体经

20、营）或异体（非自身）供体。它也指捐助者之间观察到的变化，这可能关联到年龄以及捐献者的健康等其他因素。从2008年以前，几乎所有的MSC为基础的产品来自的异体骨髓（例如，100，2006年）。此后，MSC捐助者和组织来源多样性显著增加。例如，2011同种异体基于MSC的产品的比例下降到42（2013前唯一年百分比下降至低于50），在2012年同种异体基于MSC的产品提高到73。One parameter that significantly affects how MSCs are described according to the literature is tissue source (P

21、hinney and Sensebé, 2013). Strikingly, the proportion of the 66 IND submissions that evaluated MSC-based products derived from bone marrow was 100% through 2007 but decreased to 55% by 2012 (Figure 1). Prior to 2013, the second most common source under IND was umbilical cord or placental tissue

22、; the third most common was adipose tissue. The number of adipose-derived MSC-based product INDs has increased significantly since 2011 (there was a 3-fold increase between 2011 and 2012 alone). This shift is consistent with clinical trials registered worldwide, where less than half of the MSC-based

23、 product trials registered in 2012 used bone marrow as a source.一个影响MSC如何描述的显著参数根据文献中描述取决于的是组织来源（菲尼和Sensebé，2013）。引人注目的是，2007年66 IND提交的评估骨髓来源的MSC为基础的产品的比例为100，到2012年下降到55。（图1）。此前2013年，IND下的第二个最常见的来源是脐带或胎盘组织; 第三个最常见的是脂肪组织。脂肪来源的MSC为基础的产品的IND的数量自2011年显著增加（单单2011和2012年之间就增加3倍）。这种转变是全球注册临床试验，其中只有不到一

24、半在2012年注册的MSC为基础的产品试验用骨髓作为源。Manufacturing Diversity in MSC-Based Product INDs在基于MSC产品的IND制造业多样性MSC researchers and manufacturers use a wide range of protocols to manufacture their MSC-based products. Of these manufacturing differences, we assessed four prominent parameters that have the potential to

25、 influence product characteristics: (1) fetal bovine serum (FBS), (2) atmospheric oxygen (21%), (3) cryopreservation of the final product, and (4) cell banking (working or master cell bank). The majority of regulatory submissions (over 80%) describe the use of FBS during manufacturing. The range of

26、FBS concentration in media ranges from approximately 2%20%, with 10% FBS the most common concentration. Different percentages of FBS result in different amounts of growth factors present in a culture, thus some MSC manufacturers emphasize the importance of qualifying FBS lots to facilitate product c

27、omparability between manufacturing runs. However, it may be challenging to determine which tests and product characteristics (i.e., critical quality attributes) are the most appropriate to select for comparison. The choice of assays and/or markers tested can be just as challenging for implementing m

28、anufacturing process changes with respect to demonstrating final product comparability (Carmen et al., 2012). The most common alternative for FBS described in regulatory submissions is human platelet lysate, where performing qualification and demonstrating comparability are similarly important. Many

29、 submissions describe the use of growth factors in addition to serum (25%). Importantly, many sponsors describe process development to include replacing the use of animal-derived serum during their manufacturing process.MSC研究人员和制造商使用多种协议来制造自己的MSC为基础的产品。这些制造差异，我们评估潜力有可能影响产品的特性的4个突出参数：（1）胎儿血清（FBS），（2）

30、大气中的氧（21）（3）冷冻保存（4）细胞银行（工作或主细胞库）。大多数监管意见书（超过80），说明生产过程中使用的FBS。FBS的浓度在媒体的范围内，从约2-20的范围内， 10FBS是最常见的浓度。不同的FBS比例产生大量不同的成长因素存在于培养物过程中，因而一些MSC厂家强调排位FBS的地段，以方便制造运行之间产品可比性的重要性。然而，它可以是具有挑战性的，以确定哪些测试和产品特性（即，关键质量属性）是最合适的选择进行比较。测试一样可以实现制造过程的变化就显示出最终产品的可比性挑战性试验和/或标记的选择（卡门等，2012）。在监管部门提交描述FBS最常见的选择是人类血小板裂解液，在那里表

31、演的资格，并展示可比性也同样重要。多份意见书描述了除血清（25）使用的生长因子。重要的是，很多赞助商描述工艺开发，包括在其制造过程中更换使用动物来源的血清。Most (90%) MSC-based product submissions describe the use of atmospheric oxygen during cell culture. Some groups have described the utility of more physiological oxygen conditions for MSC manufacture (e.g. low oxygen tensi

32、on, such as 5% O2).大部分（约90），基于MSC产品提交描述细胞培养过程中使用大气中的氧气。一些小组已经描述的多个生理氧条件MSC制造的效用（例如，低氧环境，如5）。The majority of MSC-based product regulatory submissions (over 80%) also describe the use of cryopreservation to store and transport their final product, which is usually thawed within a few hours of patient

33、infusion. Recently, MSC researchers have described challenges inherent in assessing the potential functionality of MSCs after thawing immediately before infusion, especially when bioactivity assays are often performed on MSCs prior to or without cryopreservation, or following culture rescue (Galipea

34、u, 2013). A common postthaw test described in an MSC-based product regulatory submission is viability, expected to exceed 70% for intravenously administered MSCs. However, it is unclear how relevant viability is for MSC functionality immediately postthaw especially when considering the potential for

35、 delayed cytotoxicity (e.g., 2472 hr). For example, there is no well-documented evidence that MSCs have the ability to produce and secrete factors produced de novo in response to microenvironmental cues immediately postthaw.大多数基于MSC的产物监管提交的（80以上）也描述了使用冷冻保存的储存和运输的最终产品，这通常是病人输液前几小时内解冻。最近研究人员描述了在评估充质干细

36、胞在解冻后立即注射的潜在功能的挑战，特别是在生物活性测定是在干细胞之前，或没有低温保存，或细胞增殖后（Galipeau，2013）。在MSC为基础的产品监管申请中，常见解冻后试验是可行，有望突破为静脉注射干细胞70。然而，目前还不清楚立即解冻后怎样关联MSC的存活率特别是考虑延迟的细胞毒性（例如，24-72小时）。例如，没有证据充分的证据表明，立即解冻后，干细胞具有产生和分泌产生从头响应于微环境线索因子的能力。A smaller proportion (35%) of regulatory submissions described the use of cell banking system

37、s. When cell banking is employed, a multitiered system (e.g., master cell bank and working cell bank) is described about one-third of the time. If a cell banking system is described, we have found that MSCs, in general, are grown to a higher range of passage numbers or population doublings to manufa

38、cture the final product. Even when no banking system is employed, there is a wide range of passages or population doublings described to manufacture the final product. For example, passage numbers approaching ten have been noted. Although population doubling data is more informative, it is often not

39、 described. Given the logistical requirements of a cell banking system, all banked MSCs are cryopreserved at one or more stages of manufacturing.监管意见书的比例较小（35）描述了使用电池的银行系统。当细胞库应用中，一个多层次的系统（例如，主细胞库和工作细胞库）中描述了大约三分之一的时间。如果一个细胞库系统进行说明，我们已发现，干细胞，在一般情况下，生长至更高的范围通路号码或群体倍增来制造最终产品。即使当没有银行系统时，存在一个宽范围描述来制造最终产品

40、通道或群体倍增。例如，接近10通道数字已悉。虽然群体倍增的数据是更多的信息时，常常不描述。给定的细胞银行系统的后勤需求，所有编组的MSC冷冻保存在制造一个或多个阶段。Cell Surface Marker Characterization Proposed in MSC-Based Product INDs基于MSC产品的中提出的细胞表面标志特征We found substantial variability in the panel of cell surface markers proposed for characterization of MSC-based products in F

41、DA regulatory submissions in terms of frequencies and ranges of expression (Table 1). The number of MSC markers used for characterization at different stages (i.e., in-process and/or lot release testing) is also variable (Table S1 available online). In general, even in cases where many markers are s

42、tudied “for information purposes only,” only a select few markers are proposed for lot release criteria. Although there is variability in the proposed marker criteria described in a given MSC-based product regulatory submission, seven of the nine initial ISCT-proposed markers (Dominici et al., 2006)

43、 are ranked at the top of the list (Table 1). The same top seven markers were specifically utilized for lot release criteria, such as for sponsor-proposed identity and purity (see FDAs guidance document for more information on identity and purity at /downloads/Bio . ation/ucm092705.

44、pdf). We found that most of the MSC-based product IND submissions propose some subset of seven of the initial ISCT-proposed marker criteria (CD105, CD73, CD90, CD45, CD34, CD14, and HLA class II), albeit with more loosely defined ranges of expression. For example, instead of CD105 expression levels

45、at 95% or greater as per ISCT-proposed criteria, MSC-based submissions propose CD105 expression levels as low as 80%. It is unclear whether the quantitative difference in expression levels proposed is relevant in terms of overall MSC product characterization. The same seven markers are arguably the

46、most commonly described in the literature as well (Mafi et al., 2011).表格1。基于MSC的产品表型标记表达建议的MSC-基于产品的IND使用RANK 常见的产品标记名称 用法 范围描述RANK 使用范围描述 平均。最小。±SD 平均。最大。±SD1 CD45 91 2 58 0±0 7±6.842 CD105 73 1 67 88±7.54 100±03 CD90 61 3 36 87±7.17 100±04 CD73 52 4 29 86

47、77;7.24 100±0五 CD34 48 7 21 0±0 9±6.566 CD14 47 6 24 0±0 7±7.007 HLA II类 44 五 27 0±0 9±7.158 CD44 三十 - - - -9 HLA I类 26 10 14 74±18.60 100±010 CD29 24 - - - -11 CD106 23 - - - -12 CD19 21 - - - -13 CD80 21 - - - -14 CD86 21 - - - -15 CD166 20 8 18 92±

48、;4.52 100±016 CD10 18 - - 17 CD146 15 9 15 67±4.83 100±018 CD40 15 - - - -19 的CD11b 14 - - - -20 CD200 12 - - - -前20名的产品标记，并显示在所有原来的MSC提交分析（N = 66）评为汇总数据的使用百分比表示。“用法”指的是频率特定标记定性提出在产物表征跨所有MSC提交分析（即，正或负）的任何阶段。此外，前10产物的标记，使用提出表达的定量范围，说明的显示，以及在所有原始MSC提交分析（N = 66）评为合计数据中使用百分比计。“具有所述范围”，是指一

49、个特定的标记物是如何经常提出具有表达的定量范围。“AV。最小。±SD“是指在表达的范围内的平均最小值，加或减标准差。“AV。最大。±SD“是指在表达的范围内的平均最大值，加或减标准差。标志物阳性是大胆而负标记为斜体。所有的百分比都四舍五入到最接近的整百分比值，和标准偏差是到小数点后两位。我们发现实质性变异的细胞表面标志物在频率和表达的范围（方面提出了在美国FDA监管申请基于MSC的产品特性的面板表1）。用于表征在不同阶段的MSC标记数（即，在进程和/或大量释放测试）也是可变（表S1提供在线）。在一般情况下，即使在许多标记研究“仅供参考”的情况下，提出了大量的发布标准只有一小

50、部分的标记。虽然有变异在一个给定的MSC基产品监管提交描述所提出的标记物的标准，七九初始ISCT提议的标志物（Dominici 等人，2006）都排在列表的顶部（表1）。同样排名前七位的标志是专为批签发标准，如赞助商提议的特性和纯度（见FDA的指导性文件的有关身份和纯度的更多信息利用 我们发现，大多数基于MSC的产物IND提交的提议的七初始ISCT提议的标记标准（CD105，CD73，CD90，CD45，CD34，CD14，和II类HLA）的一些子集，尽管更松散定义范围。的表达。例如，代替CD105表达水平在95或更高的按ISCT提议的标准的，基于MSC的提交建议CD105表达水平低约80，这

51、是不明确提出的表达水平定量差是否有关在整体的MSC产物表征而言，同样的7标记可以说在文献中最常描述以及（MAFI 等人，2011）。MSC-Based Product Bioactivity Characterization In Vitro and In Vivo基于MSC产品在体外和体内的活性分析We also found significant heterogeneity in descriptions of MSC bioactivity characterization in situations where a candidate marker for a given assay

52、has been defined. “Candidate marker” refers to a molecular marker that may be correlated with bioactivity. Examples include a secreted factor, or expression of proteins on the surface of either the MSCs or target cells (e.g., T cells) that may be related to a given biological activity. Such candidat

53、e markers are often proposed by the sponsor as the potential basis for a potency assay during clinical development (an industry perspective on cell therapy potency is reviewed in Bravery et al., 2013); also, the FDA has published a guidance document on cell therapy potency (see FDA website at http:/

54、/downloads/Bio . erapy/UCM243392.pdf). Our survey found that less than half of the initial MSC-based product IND submissions describe marker-based bioactivity assays. Of the regulatory submissions that did contain such descriptions, most were submitted by commercial sponsors using MSCs fr

55、om allogeneic donors. The described markers of bioactivity include factors secreted from MSCs, such as proangiogenic growth factors or anti-inflammatory/Th2 cytokines, as well as markers tested in MSC-leukocyte coculture proliferation assays. Another consideration that can be taken into account is e

56、merging evidence that MSC tissue source may impact bioactivity. In one example, adipose-derived MSCs were found to have a greater immunosuppressive capacity on T cells and monocytes via increased expression/secretion of anti-inflammatory factors in comparison to bone-marrow-derived MSCs (Melief et al., 2013).我们还发现显著异质性的情况下，MSC生物活性特征的描述，其中的候选标记为一个给定的实验已经确定。“候选标记”是指一种分子标记物可以与生物活性相关。例子包括分泌因子，或任一的MSC或靶细胞（例如，T细胞），其可以与一个给定生物活性的表面上的蛋白质的表达。这样的候选标记通常建议申办者为临床开发过程中效价测定的物质基础（对细胞疗法效力的行业观点进行审查勇敢等，2013。）; 同时，美国食品药品管理局发表了关于细胞疗法效力的指导性文件（见FDA网站 我们的调查发现，只有不到一半的初始基于MSC产品IND意见书描述了基于标