NEW YORK – A team led by researchers at the German Cancer Research Center (DKFZ) has developed a targeted mass spectrometry workflow that could improve the sensitivity of immunopeptidomic experiments.

纽约——由德国癌症研究中心（DKFZ）研究人员领导的一个团队开发了一种有针对性的质谱工作流程，可以提高免疫肽组学实验的灵敏度。

Detailed in a paper published last month in Molecular & Cellular Proteomics, the approach, called optiPRM, could help researchers detect cancer neoantigens in patient tumor samples, aiding applications like the development of personalized cancer vaccines, said Angelika Riemer, head of the division of immunotherapy and immunoprevention at the DKFZ and senior author on the study..

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Immunopeptidomics is the study of peptides presented by human leukocyte antigen (HLA) molecules. These molecules play a key role in immunity, displaying peptide antigens at the cell surface that generate immune cell responses to various infections or diseases. Identification and manipulation of these antigens is key to research in areas like cancer immunotherapy, where scientists are working to trigger patients' immune systems to fight their cancers by presenting cancer- and patient-specific HLA antigens..

免疫肽组学是对人类白细胞抗原（HLA）分子呈递的肽的研究。这些分子在免疫中起关键作用，在细胞表面显示肽抗原，产生对各种感染或疾病的免疫细胞应答。鉴定和操纵这些抗原是癌症免疫治疗等领域研究的关键，科学家正在通过呈递癌症和患者特异性HLA抗原来触发患者的免疫系统以对抗癌症。。

One of the major challenges to developing effective cancer vaccines is determining which cancer-specific peptides are likely to bind to HLA molecules and thereby be presented at the cell surface. Currently, this is typically done using algorithms that use a combination of DNA, RNA, and immunopeptidomic data to predict peptides that are likely to be displayed..

开发有效的癌症疫苗的主要挑战之一是确定哪些癌症特异性肽可能与HLA分子结合，从而呈现在细胞表面。目前，这通常是使用结合DNA，RNA和免疫肽组数据的算法来预测可能显示的肽。。

In theory, immunopeptidomics could enable direct detection of cancer neoantigens presented by HLA molecules, allowing researchers and drug developers to identify neoantigens for inclusion in vaccines and confirm the presence of predicted neoantigens. In practice, however, the technical limitations of mass spec approaches, and a lack of sensitivity in particular, have limited such applications..

理论上，免疫肽组学可以直接检测由HLA分子呈递的癌症新抗原，使研究人员和药物开发人员能够鉴定新抗原以包含在疫苗中并确认预测的新抗原的存在。然而，在实践中，质谱方法的技术局限性，特别是缺乏灵敏度，限制了此类应用。。

In their work, the DKFZ researchers aimed to boost the sensitivity of immunopeptidomic workflows by developing targeted mass spec assays carefully optimized for the specific cancer neoantigens they hoped to detect.

在他们的工作中，DKFZ研究人员旨在通过开发针对他们希望检测的特定癌症新抗原精心优化的靶向质谱分析来提高免疫肽组学工作流程的敏感性。

They started with DNA and RNA sequencing of the samples of interest, identifying sample-specific mutations. With that data, they generated a list of altered protein sequences produced by these genetic mutations and used the MHCcombine prediction tool to identify a list of the neoantigen candidates most likely to be presented by the tumor cells.

他们从感兴趣的样品的DNA和RNA测序开始，确定样品特异性突变。有了这些数据，他们生成了由这些基因突变产生的改变的蛋白质序列的列表，并使用MHCcombine预测工具来识别最有可能由肿瘤细胞呈现的新抗原候选物的列表。

They then produced synthetic stable isotope labeled (SIL) peptides to each of the candidate neoantigens..

然后，他们为每种候选新抗原生产了合成的稳定同位素标记（SIL）肽。。

The team then analyzed the SIL peptides using mass spec, running them on a Thermo Fisher Scientific Orbitrap Exploris 480 instrument. In these experiments they identified the optimal parameters for collision energies to be used for the analysis of each peptide.

然后，该团队使用质谱分析了SIL肽，并在Thermo Fisher Scientific Orbitrap Exploris 480仪器上运行。在这些实验中，他们确定了用于分析每种肽的碰撞能量的最佳参数。

This optimization, Riemer said, allowed the researchers to significantly improve the sensitivity of their assays. Using a dilution series of synthetic peptides spiked into a HeLa cell digest, they found that the optimized assay could identify peptides at roughly 12-fold lower concentration than the unoptimized assay.

Riemer说，这种优化使研究人员能够显着提高检测的灵敏度。使用掺入HeLa细胞消化液中的一系列稀释的合成肽，他们发现优化的测定法可以比未优化的测定法低约12倍的浓度鉴定肽。

They also observed a boost in signal intensity for their target peptides using the optimized parameters. While the MCP study focused primarily on optimizing collision energy selection, Riemer noted that the same approach could be used to optimize a variety of mass spec parameters..

他们还观察到使用优化的参数可以提高目标肽的信号强度。虽然MCP的研究主要集中在优化碰撞能量选择上，但Riemer指出，同样的方法可用于优化各种质谱参数。。

Applying the approach to tumor biopsies from five patients, the researchers generated targeted assays against a total of 274 predicted neoantigens and were able to identify five mutation-derived neoantigens across three of the five patients. In a sample from a patient with osteosarcoma and lung metastasis they detected two neoantigens, both produced by a single-nucleotide variant (SNV) in the gene ARHGAP35.

将该方法应用于五名患者的肿瘤活检，研究人员针对总共274种预测的新抗原进行了靶向检测，并能够在五名患者中的三名患者中鉴定出五种突变衍生的新抗原。在骨肉瘤和肺转移患者的样本中，他们检测到两种新抗原，均由ARHGAP35基因中的单核苷酸变体（SNV）产生。

In a sample from a patient with a small intestine carcinoma, they detected one neoantigen produced by an SNV in the gene RNF111. In a liposarcoma patient sample, they identified two neoantigens, one produced by a fusion involving the genes TSPAN8 and CPM and another by a fusion involving PAPOLA and MGAT4C..

在一名小肠癌患者的样本中，他们检测到RNF111基因中SNV产生的一种新抗原。在脂肪肉瘤患者样本中，他们鉴定出两种新抗原，一种是由涉及TSPAN8和CPM基因的融合产生的，另一种是由涉及丘疹和MGAT4C的融合产生的。。

The researchers were not able to detect any neoantigens in two of the five patient samples. Riemer said that there was nothing obviously unique about those samples or the number or characteristics of neoantigens predicted for them.

研究人员无法在五个患者样本中的两个中检测到任何新抗原。。

In their work with patient samples, the researchers were able to detect neoantigens in tumor biopsies consisting of as little as 35 mg of tissue, which Riemer said is an amount compatible with clinical workflows. Ultimately, she said, she hopes the method could help improve selection of tumor neoantigens to include in therapies like cancer vaccines or T-cell receptor T-cell therapy (TCR-T)..

在他们对患者样本的研究中，研究人员能够在肿瘤活检组织中检测到新抗原，这些组织只有35毫克，Riemer说这与临床工作流程相符。她说，最终，她希望该方法可以帮助改进肿瘤新抗原的选择，以包括在癌症疫苗或T细胞受体T细胞疗法（TCR-T）等疗法中。。

“What we are heading at is to make it clinically useful so that clinicians can rationally choose targets,” she said. “Our whole approach is to truly validate the targets. Then you may be able to include fewer peptides in a vaccine because you know they are truly there.”

。“我们的整个方法是真正验证目标。然后你可能能够在疫苗中包含更少的肽，因为你知道它们确实存在。”

Sensitivity still remains a challenge, but Riemer said she expects newer generations of mass spectrometers to help in this area. The Thermo Fisher Orbitrap Exploris 480 used in the MCP study was introduced five years ago, and since then, a number of higher-powered systems have come to market. In particular, Bruker has targeted the immunopeptidomics market with its TimsTOF Ultra and Ultra 2 instruments.

灵敏度仍然是一个挑战，但里默说，她希望新一代的质谱仪能在这方面有所帮助。MCP研究中使用的Thermo Fisher Orbitrap Exploris 480是五年前推出的，从那时起，许多功率更高的系统已经上市。特别是，布鲁克凭借其TimsTOF Ultra和Ultra 2仪器瞄准了免疫肽组学市场。

Thermo Fisher's Orbitrap Astral has also shown promise for such work, and its new Stellar MS was designed specifically with targeted protein measurements in mind..

Thermo Fisher的Orbitrap Astral也显示出了这项工作的前景，其新的恒星MS是专门设计的，考虑到有针对性的蛋白质测量。。

Riemer said she and her colleagues have had the opportunity to test their method on some of these newer instruments.

里默说，她和她的同事有机会在一些较新的仪器上测试他们的方法。

'My view is absolutely hopeful that with the new generation of instruments the sensitivity will be even better,' she said.

她说：“我的观点是绝对有希望的，有了新一代仪器，灵敏度会更好。”。

Riemer said that with the MCP study as a proof of concept, she and her colleagues are now beginning to apply their approach more widely at DKFZ. Thus far, they have used it to retrospectively analyze around 30 patient samples and hope to begin using it prospectively.

里默说，以MCP研究作为概念验证，她和她的同事现在开始在DKFZ更广泛地应用他们的方法。到目前为止，他们已经使用它来回顾性分析大约30个患者样本，并希望开始前瞻性地使用它。

'Obviously, it needs to be part of the clinical pipeline,' she said, noting that sequencing and mutation calling is already done for all DKFZ patients. 'With the patients already sequenced and the mutations known and the HLAs known, you can start this process.'

“显然，它需要成为临床流程的一部分，”她说，并指出所有DKFZ患者都已经完成了测序和突变检测。”患者已经测序，突变已知，HLA已知，您可以开始这个过程。”