Negative Tumor Cells Research Articles

Synthesis and Characterization of a Novel PET Tracer for Noninvasive Evaluation of FGL1 Status in Tumors.

Fibrinogen-like protein 1 (FGL1) is a potential novel immune checkpoint target for malignant tumor diagnosis and therapy. Accurate detection of FGL1 levels in tumors via noninvasive PET imaging might be beneficial for managing the disease. To achieve this, multiple FGL1-targeting peptides (FGLP) were designed, and a promising candidate, 68Ga-NOTA-FGLP2, was identified through a high-throughput screening approach using microPET imaging of 68Ga-labeled peptides. Subsequent in vitro cell experiments showed that uptake values of 68Ga-NOTA-FGLP2 in FGL1 positive Huh7 tumor cells were significantly higher than those in FGL1 negative U87 MG tumor cells. Further microPET imaging showed that the Huh7 xenografts were clearly visualized with a favorable contrast. ROI analysis showed that the uptake values of the tracer in Huh7 xenografts were 2.63 ± 0.07% ID/g at 30 min p.i.. After treatment with an excess of unlabeled FGLP2, the tumor uptake significantly decreased to 0.54 ± 0.05% ID/g at 30 min p.i.. Moreover, the uptake in U87 MG xenografts was 0.44 ± 0.06% ID/g at the same time point. The tracer was excreted mainly through the renal system. 18F-FDG PET imaging was also performed in mice bearing Huh7 and U87 MG xenografts, respectively. However, there was no significant difference in the uptake between the tumors with different FGL1 expressions. Preclinical data indicated that 68Ga-NOTA-FGLP2 might be a suitable radiotracer for in vivo noninvasive visualization of tumors with abundant expression of FGL1. Further investigation of 68Ga-NOTA-FGLP2 for tumor diagnosis and therapy is undergoing.

Digital Image Comparison of Cellular Yield in Bronchial Brushing: Pre- and Post-Biopsy Lavage Cytology

Introduction: Bronchoscopy is a useful diagnostic tool capable of performing core biopsy, forceps biopsy, bronchoalveolar lavage, and bronchial brushing. This study compares the cellularity of bronchial cytology including pre- and post-biopsy lavage by digital image analysis, aiming to increase diagnostic and tumor yield by optimizing the sequence and combination of bronchial biopsy and cytology. Methods: Alveolar macrophage, bronchial epithelium, and tumor cell cellularity from liquid-based cytology preparations of bronchial brushing and pre-biopsy and post-biopsy bronchoalveolar lavage were annotated on digitized whole-slide images and compared. Secondary analysis on the relationship of tumor cell and non-lesional cell yield was performed. Results: Overall, 118 cytology specimens from 43 patients were retrieved in total. Bronchial epithelium count was higher in pre-biopsy than post-biopsy lavage (p < 0.01) but not for alveolar macrophages nor tumor cell (p > 0.05). Tumor cell count was higher for bronchial brushing cytology samples than lavage (p = 0.018). The alveolar macrophage count was higher in post-biopsy lavage than bronchial brushing (p = 0.033); otherwise, brushing showed consistently higher bronchial epithelium and tumor cell counts. There were 33 false negative (tumor cell absent) specimens, and the combination of bronchial brushing and pre-biopsy lavage yielded the lowest false negative cases. Correlation between bronchial epithelium and alveolar macrophage counts with tumor cell count was weak (correlation coefficient = −0.168–0.203) except for post-biopsy lavage (correlation coefficient = 0.412–0.479, p < 0.05). Conclusion: Bronchial brushing yields a greater amount of tumor cell than lavage, and timing lavage before or after core biopsy does not affect tumor cell yield. Combining bronchial brushing and pre-biopsy lavage results in the lowest false negative rate.

Comparing deep learning and pathologist quantification of cell-level PD-L1 expression in non-small cell lung cancer whole-slide images

Programmed death-ligand 1 (PD-L1) expression is currently used in the clinic to assess eligibility for immune-checkpoint inhibitors via the tumor proportion score (TPS), but its efficacy is limited by high interobserver variability. Multiple papers have presented systems for the automatic quantification of TPS, but none report on the task of determining cell-level PD-L1 expression and often reserve their evaluation to a single PD-L1 monoclonal antibody or clinical center. In this paper, we report on a deep learning algorithm for detecting PD-L1 negative and positive tumor cells at a cellular level and evaluate it on a cell-level reference standard established by six readers on a multi-centric, multi PD-L1 assay dataset. This reference standard also provides for the first time a benchmark for computer vision algorithms. In addition, in line with other papers, we also evaluate our algorithm at slide-level by measuring the agreement between the algorithm and six pathologists on TPS quantification. We find a moderately low interobserver agreement at cell-level level (mean reader-reader F1 score = 0.68) which our algorithm sits slightly under (mean reader-AI F1 score = 0.55), especially for cases from the clinical center not included in the training set. Despite this, we find good AI-pathologist agreement on quantifying TPS compared to the interobserver agreement (mean reader-reader Cohen’s kappa = 0.54, 95% CI 0.26–0.81, mean reader-AI kappa = 0.49, 95% CI 0.27—0.72). In conclusion, our deep learning algorithm demonstrates promise in detecting PD-L1 expression at a cellular level and exhibits favorable agreement with pathologists in quantifying the tumor proportion score (TPS). We publicly release our models for use via the Grand-Challenge platform.

Abstract 6025: Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display

Abstract Radioligand therapeutics (RLT) aim to selectively deliver radioisotopes to cancer tissues to eradicate tumor cells while limiting the damage to surrounding tissues. They are composed of a tumor-targeting ligand and a radioactive payload. The therapeutic potential of RLT strongly depends on their biostability and residence time in tumor and healthy tissue after injection. Macrocyclic peptides are effective tumor-targeting ligands because their size and molecular characteristics confer optimal pharmacologic properties that can be fine-tuned during the development process. mRNA display is a powerful platform for macrocyclic peptide drug discovery that identifies high affinity peptide ligands through in vitro selection. Here, we describe the selection and initial characterization of macrocyclic peptides discovered by mRNA display that bind to the extracellular domain of a tumor target. Enrichment of the library for peptides that bound to the recombinant target protein was observed after four rounds of selection with no further enrichment in round five. The library of round 5 was sequenced by Illumina sequencing and the top 1,000 sequences (highest counts) were used to perform cluster analysis. We tested the top sequences of 43 different clusters in in vitro translation binding assays to validate the selection output and identify sequences for hit confirmation. Most of the sequences tested showed clear binding to the target protein and only a few peptides showed binding to a closely related protein from the same protein family. No non-specific binding of the discovered peptides to an irrelevant protein was observed showing the high specificity of the generated library. From this confirmatory selection data, we chose a subset of peptides for chemical synthesis. Binding affinity measurements were determined by surface plasmon resonance (SPR) and cellular binding of selected candidates to target-positive and -negative tumor cells was analyzed by flow cytometry. From this hit confirmation data, in vitro internalization and in vitro anti-tumor efficacy of radiolabeled peptide candidates for RLT are assessed. Our data highlights the potential of mRNA display platforms for fast and efficient discovery of highly specific macrocyclic peptides with optimal binding properties suitable for RLT. Citation Format: Anika Jaekel, Kathryn Sherry, Prachi Desai, David Fox, Tang Guo-Qing, Manuel Sturzbecher-Hoehne, Dennis Mewis, Kaori Noridomi, Liam Rayman, Ping Ye. Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6025.

Abstract 5805: A novel platform of diversified payloads to drive ADC innovation

Abstract First generation of antibody drug conjugates (ADCs) for anticancer therapy has been described more than two decades ago with gemtuzumab-ozogamicin receiving the first approval in 2000. Over the last 4 years, 8 new ADCs got FDA approval essentially due to the innovation on the linker improving the disposition in patients and the manufacturability together with the technical advantages in the engineering of biologics and the introduction of new cytotoxic payloads such as deruxtecan. In the future, next generation ADCs will be developed exploring payloads beyond the currently used cytotoxins to improve safety, efficacy and durability of responses and therefore the short- and long-term therapeutic benefit to patients. Nerviano Medical Sciences (NMS) generated a portfolio of novel and diversified payload linkers leveraging on its proprietary chemical collection that includes cytotoxic and targeted payloads. A rigorous screening funnel has been applied considering diversification in terms of MoAs, pharmacokinetic properties, mechanism of resistance and combinability with other therapies to identify the optimal match with specific tumor types and targets, generating a new class of ADCs with improved selectivity and efficacy for hard-to-treat cancers even in chemoresistant settings. NMS ADC platform incorporates 1) our patent protected payload linker NMS-P945, a duocarmycin like DNA minor groove binder and alkylating agent inducing DNA damage, acting in chemoresistant cells with bystander effect and immunogenic cell death properties and generating ADCs highly efficacious in mouse xenograft models and safe when administered in NHP, 2) novel anthracyclines (PNU-682 derivatives) with an improved stability and safety profile and 3) targeted molecules with different mechanism of action including payloads active in synthetic lethality contexts. Payload linkers with different functionalization generated from each payload have been evaluated for chemical stability, sensitivity to lysosomal protease cleavage, and conjugability to monoclonal antibodies. The ADCs, generated with proof-of-concept antibodies (e.g. Trastuzumab), were characterized in terms of Drug to Antibody Ratio (DAR), aggregation level, plasma stability, antigen binding and differential antiproliferative activity in target positive vs target negative tumor cells. The best products resulting from the screening funnel have been subjected to a multidimensional analysis taking into consideration payload properties, tumor features and antigen expression in selected populations with the aim of identifying tumor types particularly sensitive to the payload. Our multidimensional approach coupling new payload linker with innovative MoA to careful selection of patients will allow to reach unprecedented efficacy and safety profile. Citation Format: Paolo Orsini, Matteo Salsa, Simona Rizzi, Ulisse Cucchi, Daniela Faiardi, Alessia Burocchi, Antonella Ciavolella, Rosita Lupi, Fabio Gasparri, Barbara Valsasina. A novel platform of diversified payloads to drive ADC innovation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5805.

Abstract 1329: Development of the first optimised “off the shelf” MAGE-A4 targeting TCR-NK cells for advancement into the clinic for the treatment of solid tumors

Abstract T cell receptor (TCR)-T based therapies, such as MAGE-A4 TCR-T cells, have shown compelling data demonstrating effective infiltration into and targeting of solid tumors with clinical responses across various solid cancers. However, heterogeneity and down-regulation of target antigen and HLA expression limit the durability and curative potential of these types of treatments. On the other hand, owing to their clinical potency, favorable safety profile and applicability as “off the shelf” therapy, natural killer (NK) cells have emerged as promising modalities in recent years. Unguided NK cells have shown limited potential against solid cancers, due to lacking infiltration into tumors. With our proprietary TCR-NK platform, we combine the solid tumor targeting capabilities of TCRs with the pan-cancer sensing and cytotoxic potential of highly potent killer cells, redirecting and arming NK cells with a fully functional TCR-CD3 complex. Here we present our lead product, an optimized TCR-NK cell product, ZI-MA4-1a, expressing an affinity enhanced TCR (KVL-a) directed to the HLA class I-restricted clinically validated cancer-testis antigen MAGE-A4, that is broadly expressed across solid tumors. The KVL-a TCR was successfully engineered from a naturally occurring wild-type TCR, increasing the affinity to MAGE-A4-peptide-HLA complex and resulting in enhanced recognition of tumor cells expressing the target antigen. ZI-MA4-1a is produced by transducing healthy peripheral blood derived NK cells with the full CD3 complex, the KVL-a TCR and a CD8 coreceptor. The process has been optimized to enrich TCR-NK cells, increasing process robustness, diminishing the impact of donor variability, and producing a higher performing TCR-NK cell product. We show the dual functionality of ZI-MA4-1a: TCR driven cytotoxicity as well as innate NK sensing of antigen knockout or HLA negative cancers. We demonstrate killing of heterogenous tumor cell models in vitro, as well as the multifunctionality of ZI-MA4-1a with killing, degranulation and multicytokine secretion. Interestingly, we observe enhanced proliferation of ZI-MA4-1a cells after activation by MAGE-A4 expressing tumor cells, indicating that TCR-NK cells acquire further proliferative capacity when the TCR is triggered. Finally, we demonstrate higher activity of ZI-MA4-1a compared to KVL-a and benchmark TCR transduced T cells against antigen positive targets, and activity against antigen negative tumor cells escaping TCR-T cell recognition. Citation Format: Sarah Vollmers, Artur Cieslar-Pobuda, Justyna Kmiecik, Giulia Malachin, Michelle L. Sætersmoen, Frida Høsøien Haugen, Margherita Boieri, Maja Sandve, Dennis Clement, Ines Cardoso, Pimthanya Wanichawan, Martha E. Haugstøyl, Anja Oldenburg, Liliane Christ, Pranav Oberoi, Luz Mora-Velandia, Anders Holm, Namir J. Hassan, Emilie Gauthy, Julia Ino, Sylvie Pollmann, Luise U. Weigand. Development of the first optimised “off the shelf” MAGE-A4 targeting TCR-NK cells for advancement into the clinic for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1329.

Abstract 4006: IL12-secreting CAR-T cells eliminate the requirement for lymphodepletion and promote epitope spreading via local antigen presenting cells

Abstract Introduction: EGFRvIII is a commonly studied tumor-specific antigen in glioblastoma (GBM) and poses an enticing target for immunotherapy. Its varied expression, however, complicates its effectiveness as a Chimeric Antigen Receptor (CAR) T cell target. Given the increasing prominence of Interleukin-12 (IL12) in treating solid tumors, its synergy with EGFRvIII specific CARs in treating preclinical models of GBM is explored. Methods: To simulate tumor heterogeneity, mice were implanted with equal ratios of CT2A and CT2A EGFRvIII+ cells. We developed EGFRvIII-specific third-generation CAR T cells, both with and without IL12 using a MSGV retrovirus backbone and transduced them into murine T cells. Their antitumor effectiveness was gauged by evaluating survival rates, and their capability to enhance and modify the tumor environment was determined using single-cell sequencing. Results: Standalone EGFRvIII CARs were unsuccessful in curing mice with heterogeneous tumors. However, 60% of mice treated with EGFRvIII CARs combined with IL12 were long term survivors. IL12 secreting CARs exhibited prolonged persistence in vivo, and there was no need for prior lymphodepletion before ALT introduction. Our studies reveal that EGFRvIII CARs secreting IL12 can eliminate EGFRvIII negative tumor cells by drawing on the inherent CD8 T cell compartment. Moreover, IL12 prominently impacted microglia among all Antigen Presenting Cells in the tumor environment, amplifying antigen presentation processes and broadening epitope exposure. Conclusion: By leveraging IL12, EGFRvIII CARs can counteract antigen heterogeneity in tumors by recruiting endogenous CD8 T cells and polarizing microglia. These IL12-secreting CARs outlast their traditional counterparts without requiring lymphodepletion. Harnessing CAR-T cells that secrete IL12 represents a novel strategy for overcoming tumor heterogeneity and treating GBM in a clinical context. Citation Format: Aditya Mohan, Steven Shen, Kelly Hotchkiss, Sarah Quackenbush, John Sampson, Peter Fecci, Anoop Patel. IL12-secreting CAR-T cells eliminate the requirement for lymphodepletion and promote epitope spreading via local antigen presenting cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4006.

Abstract 7418: LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data

Abstract Gene fusion resulted from genomic translocation plays important oncogenic roles including but not limited to gene activation and changing protein coding sequences. Historically, fusion gene analysis has been limited to bulk levels resulting in mixed oncogene activation models. Single cell Nanopore RNA sequencing (scNanoRNAseq) has the unique strength in detecting full-length chimeric transcripts in thousands of single cells in parallel. However, robust computational tools for detecting fusion genes in single cells from scNanoRNAseq data are missing. Here, we developed LongFuse, a fast and accurate tool, to detect fusion genes and their splicing isoforms in single cells from scNanoRNAseq data. LongFuse implements an XOR logic gate algorithm to speed up fusion candidate searching and stringent criteria to eliminate false discoveries due to trans-splicing events and randomly ligated chimeras. We applied LongFUSE onto scNanoRNAseq data of both human prostate cancer cell line, VCaP, and tumor tissues with known TMPRSS2-ERG fusion. Our results showed that both cancer cell line and prostate tumors harbored a mixture of fusion positive and fusion negative tumor cells. Gene expression analysis revealed transcriptional differences between fusion positive and negative cells including genes involved in cancer cell proliferation and invasion programs such as ELL2, ZBTB16, IGF1R, ANK3, MYPOP and PLPP1 genes. LongFUSE also calculates the splicing isoforms of fusion gene transcripts, allowing prediction of protein sequences of fusion gene transcripts. Taken together, our data demonstrated that LongFUSE is a robust computational tool for detecting gene fusions and their splicing isoforms at single cell level from scNanoRNAseq data. Citation Format: Cheng-Kai Shiau, Yueying He, Hsiao-Yun Lin, Lina Lu, Xiao-Dong Lu, Jindan Yu, Ruli Gao. LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7418.

Abstract 1339: Pre-clinical development of IAMA-004, a tri-specific antibody targeting innate immune receptors and tumor targets

Abstract Immune check-point inhibitor (ICI) such as anti-PD1 or anti-PDL1 antibody, which enhances the adaptive immunity via re-energizing exhausted T cells, has become the foundational therapy for many cancers. However, due to variety of other immunosuppressive tumor microenvironments, it is efficacious only in 15-30% of the patients. It is well known both the innate immunity and the adaptive immunity are required for full control of cancer growth. Thus, therapeutics harnessing the innate immunity will be complementary to ICI and improve the cancer response rate when used in combination. Currently therapeutics harnessing the innate immunity mainly involve bispecific antibodies with one arm targeting tumor antigen and one arm connecting to innate effector cells such as NK cells or macrophages, represented by CD30-CD16a bispecific and Her2-CD47 bispecific respectively. Considering that cancer patient’s immune status are heterogenous, varying from T cell exhaustion, macrophage disfunction (M2 bias), Treg cell presence to NK cell deficiencies, it would be advantageous to combine several innate immunity enhancers into one therapeutic molecule. Here we describe a long-acting tri-specific antibody termed IAMA-004 that targets Her2 as the primary target and CD47 and CD16a as effector cell engagers. CD47 is also a co-target on some tumor cells (CD47-Her2 double positive tumors). We have characterized this molecule both in vitro and in vivo. The in vitro experiments tested the molecule’s ability to kill Her2 high, Her2 low and Her2 negative tumor cell lines including BT-474, SK-OV-3, NCI-N87, OE19, HCC1954, MDA-MB-231, MDA-MB-468, in human PBMC ADCC assays with DS-8201 and Trastuzmab as the comparator molecules. In all of these ADCC experiments, IAMA-004 showed superior tumor cell killing activities to those mediated by DS-8201 or Trastuzmab. When combined with IAMA-005 (a Fc-IL2-IFN-α fusion cytokine), the tumor cell killing activities were dramatically increased. In addition, IAMA-004 was also tested in patient-derived Ex-Vivo fluids (one malignant pleural effusion and one malignant asities) and demonstrated tumor cell killing efficacies; and the killing activity could be attributed to the specific arms of the molecule. These results suggest that it is feasible to combine more than one innate engager in one molecule to carry out specific functions in the heterogeneous immune status for the treatment of advanced cancer patients. Citation Format: Liming Liu, Zhen Han, Yang Fan, Qi Pan. Pre-clinical development of IAMA-004, a tri-specific antibody targeting innate immune receptors and tumor targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1339.

Abstract 1328: AccuBaseTM (ceBE), a second-generation high-fidelity base editor, can proficiently modify genes in a human primary NK cells with minimizing off-target effect

Abstract Natural Killer (NK) cells hold great promise for cancer therapy owing to their innate ability to detect cells expressing signs of stress or infection. However, NK cell-based therapeutics face significant challenges: limited expansion and genetic modification resistance. In response to these challenges, we present our pioneering work in the development of Base Editing-Directed NK (BED-NK) cell products, referred to as NK510, which is an off-the-shelf allogeneic cell therapy product containing peripheral blood mononuclear cell (PBMC)-derived TIGIT knock-out NK cells. Through a meticulously planned optimization process, we have achieved over 80% editing efficiency in NK510s using our high-fidelity Base Editor, AccuBase™. By knocking out TIGIT (an inhibitory receptor in NK cells), we have prompted tumor cell-derived CD155 (an inhibitory ligands in tumor cells) to engage with CD226 (an activating receptor in NK cells), triggering activating signals in NK510s. Our results showed that CD155 positive tumor cells were more susceptible to NK510s than CD155 negative tumor cells. And target killing percentage is positively related to CD155 expression level in tumor cells. This transformation effectively converted an inhibitory ligand into an activating one, resulting in tumor regression in multiple mouse models, including non-small cell lung cancer (NSCLC) and sarcoma. We developed a GMP-compliant NKBoost+ expansion system to generate a millionfold expansion of NK510 within a 21-day expansion protocol. To investigate the safety of NK510 product, we conducted repeated dosing toxicity, tumorigenicity, and oncogenicity study in a GLP facility, and the results validated the safety of NK510. Additionally, we developed an analytical protocol based on Droplet Digital PCR (ddPCR) technology to investigate the pharmacokinetics (PK) and biodistribution of NK510s in NSCLC tumor-bearing mice. We observed an enrichment of NK510-derived genomic DNA (gDNA) in lung tissues of tumor-bearing mice at day 1. The concentration of NK510-derived gDNA was up to 2000 copies/ug DNA. The concentration of NK510-derived gDNA sharply decreased from day 2 to day 6, and then decreased to below the lower limit of quantification (LLOQ). Distinct from the PK data of NK510s, the concentration of NK510-derived gDNA started low at day 1 and reached peak concentration at day 2 (in blood samples) and at day 4 (in liver tissues), respectively. These results demonstrated the efficacy and safety of BED-NK product and underscore the feasibility of combining base editing with ex vivo expansion of NK cells, significantly enhancing their therapeutic potential in clinical settings. Citation Format: Han Ying, Chao Yao, Cheng Fang, Guanglei Li, Lixin Wang, Yufu Zhou, Chenyuan Gong, Juewan Li, Yiming Wang, Wenwen Fan, Fenghua Lv, Xiaohu Shao, Haiyang Zhao, Liling Wang, Jun Yan, Tianhong Xu, Shiguo Zhu. AccuBaseTM (ceBE), a second-generation high-fidelity base editor, can proficiently modify genes in a human primary NK cells with minimizing off-target effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1328.

The Combination of Radiation with PARP Inhibition Enhances Senescence and Sensitivity to the Senolytic, Navitoclax, in Triple Negative Breast Tumor Cells.

Despite significant advances in the treatment of triple-negative breast cancer, this disease continues to pose a clinical challenge, with many patients ultimately suffering from relapse. Tumor cells that recover after entering into a state of senescence after chemotherapy or radiation have been shown to develop a more aggressive phenotype, and to contribute to disease recurrence. By combining the PARP inhibitor (PARPi), talazoparib, with radiation, senescence was enhanced in 4T1 and MDA-MB-231 triple-negative breast cancer cell lines (based on SA-β-gal upregulation, increased expression of CDKN1A and the senescence-associated secretory phenotype (SASP) marker, IL6). Subsequent treatment of the radiation- and talazoparib-induced senescent 4T1 and MDA-MB231 cells with navitoclax (ABT-263) resulted in significant apoptotic cell death. In immunocompetent tumor-bearing mice, navitoclax exerted a modest growth inhibitory effect when used alone, but dramatically interfered with the recovery of 4T1-derived tumors induced into senescence with ionizing radiation and talazoparib. These findings support the potential utility of a senolytic strategy in combination with the radiotherapy/PARPi combination to mitigate the risk of disease recurrence in triple-negative breast cancer.

Combination of verteporfin-photodynamic therapy with 5-aza-2'-deoxycytidine enhances the anti-tumour immune response in triple negative breast cancer.

Triple negative breast cancer (TNBC) is a subtype of breast cancer characterised by its high tumourigenic, invasive, and immunosuppressive nature. Photodynamic therapy (PDT) is a focal therapy that uses light to activate a photosensitizing agent and induce a cytotoxic effect. 5-aza-2'-deoxycytidine (5-ADC) is a clinically approved immunomodulatory chemotherapy agent. The mechanism of the combination therapy using PDT and 5-ADC in evoking an anti-tumour response is not fully understood. The present study examined whether a single dose of 5-ADC enhances the cytotoxic and anti-tumour immune effect of low dose PDT with verteporfin as the photosensitiser in a TNBC orthotopic syngeneic murine model, using the triple negative murine mammary tumour cell line 4T1. Histopathology analysis, digital pathology and immunohistochemistry of treated tumours and distant sites were assessed. Flow cytometry of splenic and breast tissue was used to identify T cell populations. Bioinformatics were used to identify tumour immune microenvironments related to TNBC patients. Functional experiments showed that PDT was most effective when used in combination with 5-ADC to optimize its efficacy. 5-ADC/PDT combination therapy elicited a synergistic effect in vitro and was significantly more cytotoxic than monotherapies on 4T1 tumour cells. For tumour therapy, all types of treatments demonstrated histopathologically defined margins of necrosis, increased T cell expression in the spleen with absence of metastases or distant tissue destruction. Flow cytometry and digital pathology results showed significant increases in CD8 expressing cells with all treatments, whereas only the 5-ADC/PDT combination therapy showed increase in CD4 expression. Bioinformatics analysis of in silico publicly available TNBC data identified BCL3 and BCL2 as well as the following anti-tumour immune response biomarkers as significantly altered in TNBC compared to other breast cancer subtypes: GZMA, PRF1, CXCL1, CCL2, CCL4, and CCL5. Interestingly, molecular biomarker assays showed increase in anti-tumour response genes after treatment. The results showed concomitant increase in BCL3, with decrease in BCL2 expression in TNBC treatment. In addition, the treatments showed decrease in PRF1, CCL2, CCL4, and CCL5 genes with 5-ADC and 5-ADC/PDT treatment in both spleen and breast tissue, with the latter showing the most decrease. To our knowledge, this is the first study that shows which of the innate and adaptive immune biomarkers are activated during PDT related treatment of the TNBC 4T1 mouse models. The results also indicate that some of the immune response biomarkers can be used to monitor the effectiveness of PDT treatment in TNBC murine model warranting further investigation in human subjects.

Sequential T-Cell Engagement for Myeloma (“STEM”) Trial: A Phase 2 Study of Cevostamab Consolidation Following BCMA CAR T Cell Therapy

Background and significance: The BCMA-targeted CAR T products idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel) are currently approved for relapsed/refractory multiple myeloma (RRMM) patients with ≥4 prior lines of therapy, including an IMID, proteasome inhibitor, and CD38 antibody. However, despite unprecedented response rates, CAR T cells are not curative in these late-line patients, even for those in complete remission. Mechanisms of resistance may include lack of persistence or poor function of persisting CAR T cells, as well as BCMA-low or -negative residual tumor cells that serve as a reservoir for relapse. FcRH5 is another MM cell surface antigen, with expression independent of BCMA. Cevostamab is an FcRH5-targeted, T cell-engaging bispecific antibody (bsAb) with demonstrated activity in RRMM, including in patients with prior BCMA-directed therapies (Trudel et al, ASH 2021, #157). We hypothesize that consolidating BCMA CAR T cell therapy with a bsAb targeting a different antigen may re-invigorate persisting CAR+ T cells against residual BCMA+ tumor cells, while also activating endogenous T cells against FcRH5+, BCMA-low/negative tumor cells, ultimately improving rates of sustained minimal residual disease (MRD)-negativity and durability of response. Study design and methods: This is a single-institution, investigator-initiated study (NCT05801939) sponsored by the University of Pennsylvania, with funding support from Genentech. Targeted population are patients with RRMM who have received a commercially available CAR T cell product (ide-cel or cilta-cel) according to the FDA label, within the past 8 weeks, with stable disease or better. Major inclusion criteria include absolute neutrophil count ≥ 1, hemoglobin ≥ 7, platelets ≥ 50, and creatinine clearance ≥ 30 ml/min. Major exclusion criteria include prior cytokine release syndrome or ICANS ≥ grade 3, or any grade hemophagocytic lymphohistiocytosis (HLH) or Parkinsonism, or any active infection. The study schema is shown in Figure 1. Cevostamab is initiated 8-10 weeks after CAR T cell infusion. This timepoint was chosen to allow recovery from acute CAR T cell-related toxicities, but while CAR T cells may still be detectable in circulation. Cevostamab is given at a step-up dose of 3.6mg intravenously (IV) on Cycle 1, Day 1 (C1D1), followed by full dose of 160mg IV on C1D8. Subjects are hospitalized for 48 hours after each C1 dose to monitor for CRS and ICANS. They then continue cevostamab every 3 weeks for total of 8 cycles. If they are in an MRD-negative complete response (CR) after 8 cycles (Adaptive Clonoseq assay, at 10e-5 sensitivity), they stop therapy and are observed. If not, or if bone marrow results are indeterminate, they get another 8 cycles of cevostamab, then stop and are observed. The primary endpoint is frequency of MRD-negative CR at 12 months post-CAR T cell therapy. Assuming a roughly equal proportion of patients enrolling after ide-cel and cilta-cel, the null hypothesis is that the true MRD-negative CR rate at 12 months is 35%. Twenty-six evaluable subjects will be accrued in a single-stage design. The null hypothesis will be rejected if 14 or more subjects meet the primary endpoint. This design yields a one-sided type I error rate of 0.05 and power of 0.84 for an exact test when the true 12-month MRD-negative CR rate is 60%. Secondary endpoints include feasibility, safety/tolerability, and other efficacy measures (overall and CR rates, PFS, OS). Exploratory endpoints include 1) impact of cevostamab on pre- and post-therapy frequency and phenotype of both CAR+ and CAR-negative T cells in blood and marrow, assessed by multiparameter flow cytometry; 2) pre- and post-therapy expression of BCMA and FcRH5 on myeloma cells (when available), serum concentrations of soluble BCMA and FcRH5, and relationship of these factors to clinical outcome measures, and 3) pre- and post-therapy genotypic and phenotypic make-up of bone marrow microenvironment, assessed by single cell RNA sequencing (scRNAseq) and multiparameter flow cytometry, and relationship of these factors to clinical outcome measures. Conclusions: This phase 2 study is exploring the efficacy, safety, and feasibility of cevostamab consolidation following BCMA-directed CAR T cell therapy for RRMM, with the goal of sequential T cell engagement against 2 different antigens to eliminate residual disease. Accrual started in July 2023.

Early Intervention with Celmods, but Not Imids, Prevents Relapse to Forimtamig Driven By GPRC5D-Negative Myeloma Cells

Despite novel therapeutic options leading to a substantial increase in survival rates, multiple myeloma (MM) still represents an incurable disease. T cell bispecific antibodies (TCBs) have become a novel therapeutic option for relapsed refractory myeloma (RRMM) patients based on their promising objective response rates (ORR), favorable safety profile and off-the-shelf availability as compared to CAR-T cell therapies. Although BCMA- and GPRC5D-targeted TCBs have been reported to induce deep clinical responses, antigen drift represents a tumor intrinsic resistance mechanism limiting durability of responses 1. Here, we report that the combination of forimtamig, a 2:1 GPRC5D-targeted TCB, with CELMoDs, but not IMIDs, prevents tumor relapse driven by target-negative myeloma cells in a preclinical model of multiple myeloma. In order to mimic acquired TCB resistance in vivo, stem-cell humanized mice were engrafted with NCI-H929 tumors and treated once weekly (QW) with fixed duration forimtamig at 0.1 mg/kg for 6 cycles with a treatment-free follow up of more than 2 weeks. Single administration of forimtamig reduced tumor load by 80% at the end of cycle 1 (C1). At the end of cycle 4 (C4), all mice achieved a complete response (CR) with no detectable tumors. However, between C4 and cycle 6 (C6), tumor escape was observed in 47% of animals and progression free survival rate (PFS) at study termination was 53%. Relapsed tumors were subjected to quantitative immunohistochemistry and while prevalence for BCMA was comparable to baseline, GPRC5D expression was lost. To evaluate if cereblon modulation represents a strategy to improve PFS, we combined fixed-duration forimtamig with either pomalidomide (pom) or iberdomide (iber) at C1 day 1 (C1D1). Combination with pom and iber increased anti-tumor response compared to monotherapy during C1 deleting 88% and 92% of tumors, respectively. PFS for the combination with pom was not improved (~50%) as tumor escape was observed after treatment was stopped at C6. In contrast, no tumor relapse was observed for the combination with iber increasing PFS to 100%. Notably, as compared to forimtamig monotherapy, combination with pom and iber further increased serum levels of IFNg, IP-10, IL-2 and TNFa 48h after TCB dosing. We next explored if a sequential start of the combination would impact PFS and cytokine release and started co-administration at C3 instead of C1. Forimtamig induced strong tumor regressions in 90% of animals during C1-C4, but tumor escape was observed in 100% of animals starting after C4. When combined with pom or iber, PFS rates increased to 30% and 60%, respectively. Overall cytokine release was significantly lower compared to C1 and neither addition of pom nor iber led to an increase in cytokine production as compared to forimtamig monotherapy. To explore the potential of CELMoD combination at significantly lower TCB starting and target doses, we introduced step up dosing for forimtamig reflecting clinically relevant exposures and combined with thrice weekly (Q3W) or five time a week (Q5W) dosing of mezigdomide (mezi) starting at C1D1. Although forimtamig induced transient tumor regressions at C1D1 as well C1D15, mice showed progressive disease at the end of C1 and only 20% of mice achieved a CR at the end of the study. In contrast, combination with mezi led to rapid onset of tumor regression during C1 correlating with significantly improved PFS rates of 80% (Q3W) and 100% (Q5W). Cytokine release was measured after each step up and target dose and a maximal 2-fold increase in combination with mezi was detected. Interestingly, in contrast to IL-2 or IP-10, serum levels of IL-8 as well as MIP-1a decreased for the Q3W mezi schedule. Taken together, our data suggest that combination with CELMoDs but not IMIDs can prevent relapse to forimtamig driven by GPRC5D negative tumor cells. We confirm timing of intervention with CELMoDs to have an significant impact on PFS and cytokine release and suggest a broad therapeutic window using low dose forimtamig and intermittent dosing of iberdomide or mezigdomide.

Human Single Domain Antibody-Based CAR-T Cells Targeting BAFF-R Demonstrate Promising Preclinical Activity in B Cell Malignancies

Background: B-cell activating factor-receptor (BAFF-R) is a potential B-cell specific target, highly expressed in B cell malignancies and regulates B cell proliferation and survival. Single-chain antibody-based CAR-T cells targeting BAFF-R (PMB101) has demonstrated antitumor effects against human B cell malignancies and can overcome CD19 antigen loss (Qin et al. Sci Transl Med. 2019;11(511)). However, CARs built on antigen-specific single chain antibody variable fragment (scFv) may have some immunological risks. Recently, single variable domain of heavy chain antibodies are becoming an alternative to scFv to construct CARs. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain. Methods: A fully human antibody phage display library (IMARS, IASO Biotherapeutics) was used to select anti-BAFF-R clones using optimal protein/cell alternate panning. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry were applied to evaluate the specificity of clones. The screened clones were grafted into a second-generation CAR with CD8α hinge, 4-1BB costimulatory and CD3ζ intracellular signaling domains to generate CAR-T cells. PMB101 and CAR-T cells constructed with VAY736 were used as positive controls. Non-transduced T cells from the same donor were used as a negative control. Results: Four candidate clones including clone 1, 5, 77 and 80 were finally screened by in vitro functional evaluation, among which clone 5, 77 and 80 were variable domain heavy chain antibodies. The expression levels of the degranulation marker CD107a on CAR-T cells constructed with the four candidate clones were significantly upregulated after coculture with BAFF-R positive tumor cells, while BAFF-R negative tumor cells could not effectively activate CAR-T cells (data not shown). The four clones exhibited similar cytotoxicity in CAR-T cells compared with the PMB101 and VAY736 CAR-T cells using the luciferase-based cytotoxicity assay (data not shown). Xenografts were established in NPG mice following intravenous injection of 1 x 10 6 jeko-1 cells expressing the firefly luciferase gene on day - 5, and a single dose of 4 x 10 6 CAR T cells were infused IV on day 0. Compared to the other groups, no progressive tumor growth was discovered in mice treated with clone 5 CAR-T cells until day 49. Clone 5 CAR-T cells showed excellent in vivo antitumor activity to PMB101 and conferred long-term survival (data not shown). Conclusion: We developed a novel BAFF-R CAR-T cell product structured on single-domain antibody. This product demonstrated promising preclinical activity, and it may provide an alternative choice for patients with B cell malignancies.

Digital spatial profiling to predict recurrence in grade 3 stage I lung adenocarcinoma

ObjectiveEarly-stage lung adenocarcinoma is treated with local therapy alone, although patients with grade 3 stage I lung adenocarcinoma have a 50% 5-year recurrence rate. Our objective is to determine if analysis of the tumor microenvironment can create a predictive model for recurrence. MethodsThirty-four patients with grade 3 stage I lung adenocarcinoma underwent surgical resection. Digital spatial profiling was used to perform genomic (n = 31) and proteomic (n = 34) analyses of pancytokeratin positive and negative tumor cells. K-means clustering was performed on the top 50 differential genes and top 20 differential proteins, with Kaplan–Meier recurrence curves based on patient clustering. External validation of high-expression genes was performed with Kaplan–Meier plotter. ResultsThere were no significant clinicopathologic differences between patients who did (n = 14) and did not (n = 20) have recurrence. Median time to recurrence was 806 days; median follow-up with no recurrence was 2897 days. K-means clustering of pancytokeratin positive genes resulted in a model with a Kaplan–Meier curve with concordance index of 0.75. K-means clustering for pancytokeratin negative genes was less successful at differentiating recurrence (concordance index 0.6). Genes upregulated or downregulated for recurrence were externally validated using available public databases. Proteomic data did not reach statistical significance but did internally validate the genomic data described. ConclusionsGenomic difference in lung adenocarcinoma may be able to predict risk of recurrence. After further validation, stratifying patients by this risk may help guide who will benefit from adjuvant therapy.

Follicular lymphoma evolves with a surmountable dependency on acquired glycosylation motifs in the B-cell receptor

AbstractAn early event in the genesis of follicular lymphoma (FL) is the acquisition of new glycosylation motifs in the B-cell receptor (BCR) due to gene rearrangement and/or somatic hypermutation. These N-linked glycosylation motifs (N-motifs) contain mannose-terminated glycans and can interact with lectins in the tumor microenvironment, activating the tumor BCR pathway. N-motifs are stable during FL evolution, suggesting that FL tumor cells are dependent on them for their survival. Here, we investigated the dynamics and potential impact of N-motif prevalence in FL at the single-cell level across distinct tumor sites and over time in 17 patients. Although most patients had acquired at least 1 N-motif as an early event, we also found (1) cases without N-motifs in the heavy or light chains at any tumor site or time point and (2) cases with discordant N-motif patterns across different tumor sites. Inferring phylogenetic trees of the patients with discordant patterns, we observed that both N-motif–positive and N-motif–negative tumor subclones could be selected and expanded during tumor evolution. Comparing N-motif–positive with N-motif–negative tumor cells within a patient revealed higher expression of genes involved in the BCR pathway and inflammatory response, whereas tumor cells without N-motifs had higher activity of pathways involved in energy metabolism. In conclusion, although acquired N-motifs likely support FL pathogenesis through antigen-independent BCR signaling in most patients with FL, N-motif–negative tumor cells can also be selected and expanded and may depend more heavily on altered metabolism for competitive survival.

The search for therapeutic targets in lung cancer: Preclinical and human studies of carcinoembryonic antigen-related cell adhesion molecule 5 expression and its associated molecular landscape

ObjectivesCEACAM5 is a cell-surface glycoprotein expressed on epithelial cells of some solid tumors. Tusamitamab ravtansine (SAR408701), a humanized antibody-drug conjugate targeting CEACAM5, is in clinical development for nonsquamous non-small cell lung cancer (NSQ-NSCLC) with CEACAM5 high expression (HE), defined as membranous CEACAM5 immunohistochemistry staining at ≥ 2+ intensity in ≥ 50% of tumor cells. Materials and methodsWe investigated correlations between CEACAM5 expression by immunohistochemistry, CEACAM5 protein expression by ELISA, and CEACAM5 RNA expression by RNA-seq in NSQ-NSCLC patient-derived xenograft (PDX) models, and tumor responses to tusamitamab ravtansine in these models. We assessed prevalence of CEACAM5 HE, clinicopathologic characteristics and molecular markers in patients with NSQ-NSCLC in clinical cohorts. ResultsIn a lung PDX set of 10 NSQ-NSCLC specimens, correlations between CEACAM5 by IHC, ELISA and RNA-seq ranged from 0.72 to 0.88. In a larger lung PDX set, higher H-scores were present in NSQ- (n = 93) vs SQ-NSCLC (n = 128) models, and in 12 of these NSQ-NSCLC models, more tumor responses to tusamitamab ravtansine occurred in CEACAM5 HE (5/8; 62.5%) versus moderate or negative expression (1/4; 25%), including 3 with KRAS mutations among the 6 responders. In clinical NSQ-NSCLC samples, CEACAM5 HE prevalence was (52/214; 24.3%) in primary tumors and (6/17; 35.3%) in metastases. In NSQ-NSCLC primary tumors, CEACAM5 HE prevalence was significantly higher in KRAS-altered versus wild-type (35.0% vs 19.5%; P = 0.028) and in programmed cell death ligand 1 (PD-L1) negative (tumor cells 0%)/low (1–49%) versus high (≥50%) (33.3%, 26.1%, 5.0%; P = 0.031), but not significantly different in EGFR-mutated versus wild-type (20.0% vs 25.7%, P = 0.626). ConclusionsIn NSQ-NSCLC tumors, CEACAM5 HE prevalence was 24.3% overall and was higher with KRAS altered and with PD-L1 negative/low tumors but similar regardless of EGFR mutation status. These findings support targeting CEACAM5 and the clinical development of tusamitamab ravtansine for patients with NSQ-NSCLC with CEACAM5 HE.

Antibody-Drug Conjugates: A Review of Approved Drugs and Their Clinical Level of Evidence.

Antibody-drug conjugates (ADCs) are an innovative family of agents assembled through linking cytotoxic drugs (payloads) covalently to monoclonal antibodies (mAbs) to be delivered to tumor tissue that express their particular antigen, with the theoretical advantage of an augmented therapeutic ratio. As of June 2023, eleven ADCs have been approved by the Food and Drug Administration (FDA) and are on the market. These drugs have been added to the therapeutic armamentarium of acute myeloblastic and lymphoblastic leukemias, various types of lymphoma, breast, gastric or gastroesophageal junction, lung, urothelial, cervical, and ovarian cancers. They have proven to deliver more potent and effective anti-tumor activities than standard practice in a wide variety of indications. In addition to targeting antigen-expressing tumor cells, bystander effects have been engineered to extend cytotoxic killing to low-antigen-expressing or negative tumor cells in the heterogenous tumor milieu. Inevitably, myelosuppression is a common side effect with most of the ADCs due to the effects of the cytotoxic payload. Also, other unique side effects are specific to the tissue antigen that is targeted for, such as the cardiac toxicity with Her-2 targeting ADCs, and the hemorrhagic side effects with the tissue factor (TF) targeting Tisotumab vedotin. Further exciting developments are centered in the strategies to improve the tolerability and efficacy of the ADCs to improve the therapeutic window; as well as the development of novel payloads including (1) peptide-drug conjugates (PDCs), with the peptide replacing the monoclonal antibody, rendering greater tumor penetration; (2) immune-stimulating antibody conjugates (ISACs), which upon conjugation of the antigen, cause an influx of pro-inflammatory cytokines to activate dendritic cells and harness an anti-tumor T-cell response; and (3) the use of radioactive isotopes as a payload to enhance cytotoxic activity.

MRI appearances of stage IA ovarian carcinoma.

To analyze the MRI findings of stage IA ovarian cancer. The data on age distribution, clinical symptoms at onset, CA125 detection, MRI findings, including tumor volume, structure, diffusion-weighted imaging (DWI), apparent diffusion coefficient (ADC) and enhancement, etc., of the patients with stage IA ovarian cancer, who were admitted to Nantong tumor Hospital between 2013 and 2020 were analyzed retrospectively. Only 11 cases of stage IA ovarian cancer were recorded. The age of patients was 30-67 (average 52) years. The initial symptoms were mostly lower abdominal distension and abdominal pain. CA125 was 90% positive. MRI features 1. Large pelvic mass with a volume range of 23-2,009 cm3 (average 669 cm3). 2. Five cases of cyst type (with plaque-like, papillary, or mural nodule vegetations), two cases of cystic-solid mixed type (with thickened septum or wall), and four cases of solid type. 3. DWI diffusion was limited, and ADC was reduced on all solid portions (vegetation, septa, and cyst wall). 4. The solid parts were significantly enhanced on T1-enhanced MRI. 5. There was no metastasis in the pelvic cavity, and a few ascites (negative tumor cells) in three patients. MRI characteristics of stage IA ovarian carcinomas were large tumors, cystic, cystic-solid, or solid; solid portion limited diffusion on DWI and low ADC; enhancement of the cyst wall, vegetation, and septa; no pelvic metastasis.