嵌合抗原受体T细胞（CAR-T）疗法在血液系统肿瘤中取得了瞩目的成就，但在实体瘤中的报道较少。2022年美国癌症研究协会（AACR）年会上，来自约翰斯·霍普金斯大学的Vincent K. Lam博士为大家汇报了BioNTech公司的BNT211在晚期实体瘤患者中的安全性和初步疗效。

As many of you are very well aware CAR-T progress, and solid tumors has been hampered by several key challenges, including difficulty in identifying a safe and effective target in tumors that are often heterogeneous, as well as maintaining inadequate T-cell response in the setting of a hostile tumor micro-environment and significant tumor burden. 

许多人非常清楚，CAR-T在实体瘤中的进展面临以下几个关键性挑战：难以在异质性肿瘤中确定安全且有效的靶点；在肿瘤微环境以及显著的肿瘤负荷下，T细胞应答持续不充分。

BNT211 brings to bear two key major innovations that address some of these challenges, namely, the identification of CLDN6 as a novel CAR-T target, as well as the use of an RNA vaccine to enhance CAR-T persistence.

面对这些挑战，BNT211带来两项重大创新：1、将CLDN6作为CAR-T新靶点；2、使用RNA疫苗来增强CAR-T应答的持续性。

CLDN6 is one of 27 members of the CLDN family of tight junction transmembrane proteins. These proteins are essential for maintaining epithelial homeostasis. But it's also increasingly recognized that they play an important role in mediating cancer metastasis. 

紧密连接蛋白6（claudin 6，CLDN6）是紧密连接跨膜蛋白CLDN家族的27个成员之一。这些蛋白对维持上皮稳态至关重要。但是人们也越来越认识到，它们在调节癌症转移的过程中发挥了重要作用。

CLDN6 is thought to be an oncofetal protein as it's not significantly expressed in normal adult tissue, but is aberrantly expressed in various cancers, including testicular, ovarian, uterine and lung cancer. There have been previous efforts to target CLDN6 in the clinic. In particular, a monoclonal antibody that was studied in ovarian and testicular cancer. 

目前认为，CLDN6是一种癌胚蛋白，它在成人正常组织中没有显著的表达，但在各类癌症中异常表达，包括睾丸癌、卵巢癌、子宫癌和肺癌。既往已在临床中对CLDN6靶点进行了探索，尤其是在卵巢癌和睾丸癌中进行了单克隆抗体的研究。

Unfortunately, there was no meaningful clinical signal detected in that early phase study, though they did report grade three and higher elevated liver and pancreatic enzymes. Multiple groups have ongoing efforts to further develop CLDN6 therapeutics, including multiple bispecifics, as well as antibody drug conjugates. 

遗憾的是，早期研究中没有显示出有意义的临床数据，同时还报告了≥3级肝酶和胰腺酶的升高。多个研究团队在努力进一步开发CLDN6疗法，包括多种双特异性抗体、抗体偶联药物（ADC）等。

As for the second key innovation of the BNT-211 over the past decade or so, several groups have shown in a pre-clinical setting that the concept of using vaccination to enhance in-vivo CAR-T expansion is feasible. 

过去的十年中，BNT211出现了第二个关键性创新，数个研究小组在临床前试验中观察到，使用疫苗增强体内CAR-T扩增的理论是可行的。

A more recent evolution of this entails, for example, the addition of a CAR peptide or a small molecule attached to an amphiphilic polymer that preferentially traffic to lymph nodes, and that CAR-T ligand is then transferred to the cell surface of antigen presenting cells, therefore, activating the cognate CAR-T. This approach has the benefit of not being HLA restricted or dependent on endogenous TCR. 

具体来说，将CAR肽或小分子连接至可优先运输到淋巴结的两亲性聚合物，即设计出两亲性CAR-T配体（amph-ligands）。注射后，它被运送至抗原递呈细胞（APC）的表面，从而激活同源CAR-T细胞。这种方法的优势是不受人类白细胞抗原（HLA）限制或不依赖于内源性T细胞受体（TCR）。

疫苗作用机制示意图. 图片来自：Science. 2019; 365(6449): 162-168.

BNT211 extends this concept using a liposomal RNA vaccine. And they have shown in their preclinical data that this vaccine construct does activate CLDN6 CAR-T in a dose dependent manner. That sub-therapeutic doses of CAR-T can be boosted with vaccination, therefore facilitating this notion of a tunable CAR-T.

BNT211使用脂质体RNA疫苗扩展了这一理论。临床前研究数据显示，该疫苗能以剂量依赖性方式激活CLDN6 CAR-T。CAR-T亚治疗剂量可通过疫苗接种得到增强，实现了这种可调式CAR-T的概念。

Now let's turn to the clinical study results as reported for the four patient groups highlighted in red at the bottom. All eligible patients were manufactured successfully, notably about 30% of patients met criteria for CLDN6 expression by IHC, most were homogeneous and at a fairly high level of expression. The study had relatively younger patients, likely due to the testicular cancer enrichment.

让我们看一下底部红色方框内四组患者的临床研究结果。所有符合条件的患者中，约30%符合免疫组化（IHC）CLDN6的表达标准，大部分是同质性的，且处于较高的表达水平。受试者相对年龄较轻，可能是睾丸癌所占比例较高的原因。

Just something to note as we start potentially thinking about the generalized ability of the safety and other results of the study, there was no unexpected safety signal, and the toxicity reported are generally in line with what we are used to in CAR-T therapy. Half of the patients experienced CRS, none worse than grade 2. And CRS was indeed more frequent at the higher dose levels. No treatment related neural toxicity was observed. 

研究结果方面，没有观察到额外的安全信号，报告的不良反应与CAR-T治疗中观察到的基本一致。半数患者经历了细胞因子释放综合征（CRS），未出现＞2级者。CRS在较高剂量组中的发生率更高。未观察到治疗相关性神经毒性。

Interestingly, there was a significant incidence of acute lipase elevation in the study, even in patients without CRS. several of these patients had quite severe elevations. Fortunately, all it was reported that all patients were asymptomatic, and without evidence of pancreatitis. This acute lipase elevation is worth touching upon as it may represent potential on-target off-tumor effect in the pancreas. Recall that the early CLDN6 monoclonal antibodies study did report elevated pancreatic enzymes as well. 

研究数据显示，急性脂肪酶升高的发生率显著增加，甚至在未发生CRS的患者中，部分患者为重度升高。幸运的是，这些患者都没有症状，无胰腺炎的表现。这种急性脂肪酶升高的现象值得探讨，它代表了胰腺中可能存在的靶向相关的、非肿瘤作用。回顾早期CLDN6单克隆抗体试验，也有胰酶升高的报告。

This is a consensus data from the human protein atlas, showing that indeed there can be very low levels of CLDN6 transcripts in various adult normal tissue, including the pancreas. Also, a separate group has reported rare CLDN6 immunostaining in various adult tissue. How do we reconcile this with the CLDN6 screening data that we had seen earlier? Could it be that CLDN6 protein expression is transient in some way? Or are there meaningful differences in the IHC antibodies that have been used? We look forward to learning more about this. 

这是来自人类蛋白质图谱的数据，显示了包括胰腺在内的各种成人正常组织中，的确可能存在非常低水平的CLDN6转录产物。另有一个研究团队报道，在各种成人组织中可见CLDN6免疫染色，尽管非常罕见。我们如何把现有数据和既往CLDN6的相关数据来综合分析？在某种程度上，CLDN6蛋白表达是短暂的？或使用的IHC抗体之间是否存在有意义的差异？我们期待能了解更多。

In terms of CAR-T kinetics, we see robust engraftment and persistence of cells through the first 5 months at both dose levels. If we focus in on dose level one（part 2）, we do see what appears to be the vaccine, administration of the vaccine at the time of CAR-T infusion being able to boost CAR-T level. If we follow this patient through it does appear as well, that the vaccine also is able to boost the CAR-T level, but it does still appear that there's still a gradual decline of the CAR-T transgene over time. We'll be interested to see whether or not this stabilizes with further vaccination. And notably, this boosting effect is not consistently seen in other patients in the study that received vaccine.

在CAR-T动力学方面，两个治疗剂量组的前5个月内，细胞移植和持续性都很强。我们再来看这一剂量组，在CAR-T输注时给予疫苗能提高CAR-T水平，继续观察这位患者的情况，疫苗虽然能提高CAR-T水平，但随着时间的推移，仍会逐渐出现下降。进一步接种疫苗是否可以稳定CAR-T水平，我们非常有兴趣去进一步探索。但是，此研究中接受疫苗的其他患者并没有观察到持续的疫苗促进作用。

Summarizing the preliminary efficacy, we see an overall response rate of 43 % with one Complete response and promising activity in testicular and ovarian cancer. This activity was seen at both those levels and in patients with and without vaccine. These early responses, unfortunately, don't appear to be as durable as we would have hoped, because only one patient in the analysis set had a response past 18 weeks, and that was the Complete responder. We do know, though, that there's another patient with ongoing PR at about 6 weeks, and we await further follow up. We also are eager to learn about potential clinical features or biomarkers that may be associated with response. For example, are there specific memory T cell subsets, and the infusion of product that may be most beneficial for maximizing the vaccine boosting effect?

总结一下初步疗效，ORR为43%，1名患者为CR。在睾丸癌和卵巢癌患者中，均显示出较为理想的活性。无论患者是否接种疫苗，均可看到这种活性。遗憾的是，早期应答水平的维持并没有如我们所愿。可分析数据中，只有1名患者的应答时间超过18周，就是那名CR患者。另有1名患者在大约6周时仍保持PR，我们将进一步观察。我们也希望能了解可能与治疗应答相关的潜在的临床特征或生物标志物，例如特定的记忆T细胞亚群；以及最有利于疫苗增强效果最大化的产品。

Just a quick note about the significance of these results in testicular and ovarian cancer. These tumor types generally have low TMB and minimal response to approve the immune checkpoint inhibitors. To my knowledge, there's no prior significant adopted cellular therapy, clinical study that has targeted testicular cancer and in ovarian cancer, there's been a long-standing exploration of TILs in the space, though, with modest clinical efficacy in the advanced stage setting. Multiple CAR-T targets and TCR targets have also been explored. Most recently promising results have been reported for MAGE-A4-CD8α co-receptor TCR.

快速总结一下这些结果在睾丸癌和卵巢癌中的重要性。这两种肿瘤类型，通常肿瘤突变负荷（TMB）低且对免疫检查点抑制剂的应答不佳。据我所知，目前尚没有重要的、可采用的细胞疗法。针对睾丸癌和卵巢癌的临床研究，对肿瘤浸润淋巴细胞（TIL）进行过长期的探索，但在晚期肿瘤中仅有适度的临床疗效。针对多个CAR-T靶点和TCR靶点也进行过探索。近期，MAGE-A4-CD8α共受体TCR显示出有希望的数据报告。

In summary, these updated results from BNT-211 are an important and exciting advance for our rapidly growing field. For the first time, CLDN6 is clinically validated as a novel CAR-T target with promising clinical activity in testicular and ovarian cancer. The elevated pancreatic enzymes of special interest and require further evaluation. Car-VAC appears safe in combination with CLDN6 CAR-T, but CAR-T persistence does not appear to be consistently enhanced yet. 

总结一下，BNT211的更新结果对这个快速发展的领域来说，是一个重要且令人兴奋的进步。作为一个新型的CAR-T靶点，CLDN6首次被临床验证在睾丸癌和卵巢癌中具有良好的临床活性。胰酶水平的升高需要进一步评估。Car-VAC（疫苗）与CLDN6 CAR-T联合似乎是安全的，但CAR-T水平似乎并未得到持续增强。

Open questions include what is the optimal dose, timing and duration of Car-VAC? Could the vaccine boosting effect, for example, be even more apparent in a lower antigen density tumor? What is the contribution, if any, of endogenous CLDN6-specific T cells to the clinical responses that we observed? And we do know that anti-PD1 can enhance persistence in both the CAR-T and vaccine setting. So how might we best utilize immune checkpoint blockade in this combined setting? Finally, we eagerly wait for further clinical studies in particularly on treatment biopsies that may be able to demonstrate CAR-T interactions at the tumor micro environment and potential mechanisms of resistance. 

开放性问题包括：Car-VAC的最佳剂量，使用时间和持续时间是多少？疫苗增强作用是否会在抗原密度较低的肿瘤中更加明显？内源性CLDN6特异性T细胞在我们观察到的临床应答中发挥了怎样的作用？抗PD-1能增强CAR-T以及疫苗的持续性，如何在联合方案中最好的利用免疫检查点阻断？最后，我们热切地等待进一步的临床研究，特别是治疗后活检，或许能证明CAR-T在肿瘤微环境中的相互作用以及潜在的耐药机制。