Izgutdina Adila, Rashid Tasfia, Temple William C, Patiño-Escobar Bonell, Walunj Sujata, Geng Huimin, Takamatsu Hiroyuki, Gil-Alós Daniel, Kang Amrik S, Ramos Emilio, Chen Szu-Ying, Johnson Haley, Nix Matthew A, Naik Akul, Yuan Constance M, Wang Hao-Wei, Aminov Sarah, Sahu Srabani, Larson Rebecca C, Carpenter Christopher, Salangsang Fernando, Phojanakong Paul, Serrano Juan Antonio Camara, Tariq Isa, Zakraoui Ons, Steri Veronica, Valeri Antonio, Martinez-Lopez Joaquin, Maus Marcela V, Parekh Samir, Verma Amit, Shah Nirali N, Wiita Arun P
Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA.
Department of Pediatrics, Division of Oncology, UCSF Benioff Children's Hospitals, University of California, San Francisco, San Francisco, CA.
bioRxiv. 2025 May 15:2025.05.09.653155. doi: 10.1101/2025.05.09.653155.
Chimeric antigen receptor (CAR) T-cell therapies are highly efficacious for several different hematologic cancers. However, for most CAR T targets it is observed that low surface antigen density on tumors can significantly reduce therapeutic efficacy. Here, we explore this dynamic in the context of CD72, a surface antigen we recently found as a promising target for refractory B-cell cancers, but for which CD72 low antigen density can lead to therapeutic resistance in preclinical models.
Primary samples were accessed via institutional review board-approved protocols. Affinity-matured and humanized nanobody clones were previously described in Temple et al. CAR T-cells were generated via lentiviral transduction. cytotoxicity assays were performed using luciferase-labeled cell lines. studies were performed using cell line- or patient-derived xenografts implanted in NOD gamma (NSG) mice.
We first confirmed ubiquitous CD72 expression across a range of primary B-cell non-Hodgkin lymphomas. We further found that after resistance to CD19-directed therapies, across both B-cell acute lymphoblastic leukemia (B-ALL) models and primary tumor samples, surface CD72 expression was largely preserved while CD22 expression was significantly diminished. Affinity maturation of a nanobody targeting CD72, when incorporated into chimeric antigen receptor (CAR) T-cells, led to more effective elimination of isogenic models of CD72 low-expressing tumors. These results suggested that nanobody-based CAR T-cells (nanoCARs) may exhibit a similar relationship between binder affinity, antigen expression, and efficacy as previously demonstrated only for scFv-based CAR T-cells. Surprisingly, however, this significantly improved efficacy only translated to modest survival benefit. As a parallel strategy to enhance CAR T function, we found that the small molecule bryostatin could also significantly increase CD72 surface antigen density on B-cell malignancy models. Structural modeling and biochemical analysis identified critical residues improving CD72 antigen recognition of our lead affinity-matured nanobody.
Together, these findings support affinity-matured CD72 nanoCARs as a potential immunotherapy product for CD19-refractory B-cell cancers. Our results also suggest that for B-ALL in particular, CD72 may be a preferable second-line immunotherapy target over CD22.
嵌合抗原受体（CAR）T细胞疗法对几种不同的血液系统癌症具有高度疗效。然而，对于大多数CAR T靶点，观察到肿瘤表面抗原密度低会显著降低治疗效果。在此，我们在CD72的背景下探讨这种动态变化，CD72是一种表面抗原，我们最近发现它是难治性B细胞癌症的一个有前景的靶点，但在临床前模型中，CD72低抗原密度可导致治疗抗性。
通过机构审查委员会批准的方案获取原发性样本。亲和力成熟和人源化的纳米抗体克隆先前已在坦普尔等人的研究中描述。通过慢病毒转导产生CAR T细胞。使用荧光素酶标记的细胞系进行细胞毒性测定。使用植入NODγ（NSG）小鼠体内的细胞系或患者来源的异种移植物进行研究。
我们首先证实了CD72在一系列原发性B细胞非霍奇金淋巴瘤中普遍表达。我们进一步发现，在对CD19导向疗法产生抗性后，在B细胞急性淋巴细胞白血病（B-ALL）模型和原发性肿瘤样本中，表面CD72表达基本保留，而CD22表达显著降低。将靶向CD72的纳米抗体进行亲和力成熟，当将其整合到嵌合抗原受体（CAR）T细胞中时，可更有效地消除CD72低表达肿瘤的同基因模型。这些结果表明，基于纳米抗体的CAR T细胞（nanoCARs）可能在结合亲和力、抗原表达和疗效之间呈现出与先前仅在基于单链抗体片段（scFv）的CAR T细胞中所证明的类似关系。然而，令人惊讶的是，这种显著提高的疗效仅转化为适度的生存获益。作为增强CAR T功能的并行策略，我们发现小分子苔藓抑素也可显著增加B细胞恶性肿瘤模型上的CD72表面抗原密度。结构建模和生化分析确定了关键残基，可改善我们领先的亲和力成熟纳米抗体对CD72抗原的识别。
总之，这些发现支持亲和力成熟的CD72 nanoCARs作为CD19难治性B细胞癌症的潜在免疫治疗产品。我们的结果还表明，特别是对于B-ALL，CD72可能是比CD22更优选的二线免疫治疗靶点。
