Research ArticleCell biologyTherapeutics Open Access | 10.1172/JCI162148

Supreet Agarwal,1 Lei Fang,1 Kerry McGowen,1 JuanJuan Yin,1 Joel Bowman,1 Anson T. Ku,1 Aian Neil Alilin,1 Eva Corey,2 Martine P. Roudier,2 Lawrence D. True,3 Ruth Dumpit,4 Ilsa Coleman,4 John K. Lee,4 Peter S. Nelson,4 Brian J. Capaldo,1 Aida Mariani,5 Clare Hoover,5 Ilya S. Senatorov,1 Michael Beshiri,1 Adam G. Sowalsky,1 Elaine M. Hurt,5 and Kathleen Kelly1

1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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1Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

2Department of Urology and

3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.

4Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

5AstraZeneca, Gaithersburg, Maryland, USA.

Address correspondence to: Kathleen Kelly, Laboratory of Genitourinary Cancer Pathogenesis, Center for Cancer Research, National Cancer Institute, Building 37, Room 1068, Bethesda, Maryland 20892, USA. Phone: 240.760.6827; Email: kellyka@mail.nih.gov.

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Published September 19, 2023 - More info

Published in Volume 133, Issue 22 on November 15, 2023
J Clin Invest. 2023;133(22):e162148. https://doi.org/10.1172/JCI162148.
© 2023 Agarwal et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published September 19, 2023 - Version history
Received: May 26, 2022; Accepted: September 12, 2023 View PDF Abstract

Antibody-drug conjugates (ADCs) are a promising targeted cancer therapy; however, patient selection based solely on target antigen expression without consideration for cytotoxic payload vulnerabilities has plateaued clinical benefits. Biomarkers to capture patients who might benefit from specific ADCs have not been systematically determined for any cancer. We present a comprehensive therapeutic and biomarker analysis of a B7H3-ADC with pyrrolobenzodiazepine(PBD) payload in 26 treatment-resistant, metastatic prostate cancer (mPC) models. B7H3 is a tumor-specific surface protein widely expressed in mPC, and PBD is a DNA cross–linking agent. B7H3 expression was necessary but not sufficient for B7H3-PBD-ADC responsiveness. RB1 deficiency and/or replication stress, characteristics of poor prognosis, and conferred sensitivity were associated with complete tumor regression in both neuroendocrine (NEPC) and androgen receptor positive (ARPC) prostate cancer models, even with low B7H3 levels. Non-ARPC models, which are currently lacking efficacious treatment, demonstrated the highest replication stress and were most sensitive to treatment. In RB1 WT ARPC tumors, SLFN11 expression or select DNA repair mutations in SLFN11 nonexpressors governed response. Importantly, WT TP53 predicted nonresponsiveness (7 of 8 models). Overall, biomarker-focused selection of models led to high efficacy of in vivo treatment. These data enable a paradigm shift to biomarker-driven trial designs for maximizing clinical benefit of ADC therapies.

Graphical Abstract Introduction

Metastatic prostate cancer (mPC) remains a lethal disease accounting for more than 30,000 deaths annually in the United States (1). The use of androgen deprivation therapy (ADT) and androgen receptor (AR) signaling inhibitors (ARSI) have substantially prolonged the survival of patients with mPC in both pre and postchemotherapy settings. Unfortunately, durable complete responses are uncommon and mortality rates approach 100% with the development of castration resistant (CR) metastatic prostate cancer (mCRPC). Continuously evolving acquired resistance mechanisms include frequent AR mutations and structural genomic alterations that drive an AR-positive prostate cancer (ARPC) adenocarcinoma phenotype. Less common, but increasing in frequency, are resistance mechanisms that bypass an AR requirement through lineage plasticity with the emergence of phenotypes spanning neuroendocrine prostate cancer (NEPC) phenotypes and various other histologies (2). Across the landscape of genomic alterations in mCRPC, retinoblastoma (RB) transcriptional corepressor 1 (RB1) alteration is the only genomic factor strongly associated with poor survival (3), highlighting the need for potential therapeutic strategies targeting RB1-deficient tumors. Since the vast majority of mPC phenotypes eventually resist all currently approved therapeutics, new treatment strategies are essential.

A promising approach for developing effective and less toxic therapies for mPC involves selectively targeting tumor cells via tumor-specific cell surface proteins and cognate antigens. Exploiting prostate specific membrane antigen (PSMA) to deliver high dose radiation, PSMA-Lu177, to tumor cells overexpressing PSMA has recently gained FDA approval for the treatment of mPC (4). PSMA is expressed by the majority of, though not all, ARPCs, but emerging treatment-resistant phenotypes such as AR-negative and small cell neuroendocrine PC (SCNPC) generally do not express PSMA, prompting a search for alternate targets. CD276/B7H3 is a type I transmembrane protein overexpressed in several solid tumors and often correlated with poor survival and higher tumor grade (5, 6). B7H3 is overexpressed in prostate cancer compared with benign prostatic hyperplasia, and high B7H3 expression is positively correlated with adenocarcinoma aggressiveness, observed as overexpression in metastatic and castration-resistant disease (7–9). Further, B7H3 expression is not detected in human normal pancreas, lung, liver, kidney, colon, and heart (10). The preferential overexpression of B7H3 protein on the surface of cancer cells and the minimal expression on normal tissues makes it an ideal target for antibody-based therapeutics (11, 12), and targeting B7H3 is being widely pursued as more than 30 clinical trials are currently registered on clinicaltrials.gov.

One common strategy utilizing cell surface targets such as PSMA, B7H3, PSCA, TROP2, STEAP1, and CEACAM5 includes the development of antibody-drug conjugates (ADCs) (13, 14). ADCs combine the high target specificity of a monoclonal antibody with a cytotoxic agent for targeted killing of tumor cells. Several classes of cytotoxic drugs have been utilized in ADC designs, though most are either potent microtubule poisons or inducers of DNA damage (15). ADCs are rapidly internalized, releasing the antibody-linked payload to induce cell death. Several ADCs have been evaluated preclinically; however, only a few have been approved for clinical use due to either lack of efficacy or unacceptable toxicity, highlighting the need for strategies to define and use criteria for patient selection (16, 17). Generally, ADC-based trials have primarily focused on target antigen expression as a patient selection strategy, but absolute levels of target antigen have not been sufficient to predict response, suggesting that multiple factors be considered, including underlying mechanisms of vulnerability toward the cytotoxic payload (17). In fact, the FDA approved HER2 targeted ADC, Enhertu, has shown efficacy in the metastatic HER2-low breast cancer subtype (indication revised in August 2022) clearly suggesting a need for biomarkers other than the target antigen. Thus, a composite set of biomarkers, including target protein expression, are required to maximize clinical benefits of ADCs.

Pyrrolobenzodiazepines (PBDs) are DNA minor-groove crosslinking agents that have been used as payloads for several clinical grade ADCs (18). PBD dimers bind in a sequence-specific manner to form inter-strand crosslinks (ICLs) leading to double strand DNA breaks due to replication fork arrest (18). Determining the utility of PBD dimers for the treatment of cancers exhibiting replication stress and consequently increased potential vulnerability is a question of interest (19). Further, how tumor heterogeneity influences payload sensitivity and therapeutic efficacy has not been broadly investigated in preclinical cohorts of defined tumor types.

In this study, we evaluated a therapeutic strategy targeting B7H3 for the treatment of mPC. We profiled a spectrum of mPC tumors to assess the heterogeneity of B7H3 expression with respect to metastatic site and tumor phenotype. We tested a humanized B7H3-ADC armed with PBD payload across a range of molecularly characterized, clinically relevant mPC PDX and organoid models that reflect the diversity of human mPCs. Although we anticipated that DNA-double strand break repair defects and levels of B7H3 expression would drive B7H3-PBD-ADC responses, we observed high efficacy in (a) select B7H3-low expressing models with no apparent mutations in DNA repair pathway genes, and (b) no response in a group of B7H3+ adenocarcinomas. By integrating genomic and transcriptomic characteristics with B7H3-PBD-ADC response data, we uncovered additional biomarkers that represent vulnerabilities derived from more than one sensitivity or resistance pathway. These analyses demonstrate how a diverse cohort of mPCs distribute into distinct biomarker classes that reflect ADC mechanisms of action. Collectively, the results have the potential to inform patient selection for prospective trials and contribute to the interpretation of patient response and resistance outcomes.

Results

B7H3 is expressed across a range of mPC phenotypes and diverse metastatic sites. To assess the potential clinical utility of targeting B7H3 as a treatment strategy for advanced prostate cancer, we evaluated the transcript abundance of B7H3 and other previously studied cell surface targets — PSMA, PSCA, TROP2, STEAP1, and CEACAM5 — in 185 tumors from 98 patients with treatment refractory mPC and across a panel of 26 mPC PDX and organoid models representing the genomic and phenotypic heterogeneity of patient tumors. The 26 preclinical models tested in this study comprise tumors of ARPC phenotype with AR signaling (intact: n = 13, experimentally CR: n = 6) as well as ARNEG/VERYLow non-NEPC (n = 2), denoted as DNPC, (2) and SCNPC (n = 5), denoted at SCNPC. The latter 2 groups were collectively categorized as non-ARPC (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI162148DS1). The models included were primarily from the LuCaP PDX series (20) and also included 2 NCI mPC patient biopsy-derived organoids (PDOs) of the ARPC phenotype (21). We also categorized each patient tumor and mPC model into phenotypic categories based on gene expression signatures reflecting AR signaling and neuroendocrine (NE) pathway activity. We first quantified CD276(B7H3) transcript abundance in patient samples (185 tumors from 98 patients with mPC) and the above described 26 mPC models using RNA-Seq measurements. Overall, the vast majority of samples expressed CD276 transcripts, and there was limited variation within or between mPC phenotypes compared with other targets (Figure 1, A and B, and Supplemental Figure 1B). CD276 was also the most consistently expressed target across different mCRPC phenotypes. In contrast, other markers such as FOLH1(PSMA) expression varied substantially both within a phenotype and between phenotypes (P = 1 × 10–9 for the mean Log2 FKPM values between ARPC versus NEPC) (Figure 1B: FOLH1 (blue), CD276(green)). We also evaluated the intraindividual heterogeneity of CD276 transcript levels in multiple tumors acquired from the same patient. With few exceptions, there was a tight distribution of CD276 expression within individuals (Supplemental Figure 1C).

Figure 1

CD276/B7H3 expression in samples from patients with mPC and mPC PDX/organoid models. (A) CD276/B7H3, FOLH1/PSMA, PSCA, TACSTD2/TROP2, STEAP1, and CEACAM5 transcript abundance determined by RNA-Seq analysis of 185 metastatic prostate tumors from 98 patients. Transcript levels are shown as Log2 FPKM. (B) Comparisons of CD276/B7H3 (green dots) and FOLH1/PSMA (blue dots) expression by phenotypes of metastatic tumors. Groups were compared using 2-sided Wilcoxon rank tests with Benjamini-Hochberg multiple-testing correction. (C) IHC assessments of B7H3 protein expression. Representative staining of tumors with low, medium, and high B7H3 expression in AR+/NE– and AR–/NE+ phenotypes. (D) Distribution of B7H3 protein expression in 181 metastatic tumors within and between 58 patients. (E) Distribution of B7H3 protein expression in mPCs categorized by phenotype (AR+/NE–; n = 146, AR+/NE+; n = 10, AR–/NE–; n = 3, AR–/NE+; n = 18, Cases not evaluated n = 4), **P ≤ 0.01, ***P ≤ 0.001. Wilcoxon test. (F) Western blot quantification of B7H3 protein expression in PDX tissue samples and 2 PDOs (NCI-PC44, NCI-PC155) by Simple Western. ARPC samples with high B7H3 expression are categorized separately in the B7H3HI group. Y-axis represents CD276/B7H3 protein quantification scaled by a factor of 10. For pairwise comparison between groups, Wilcoxon test was used with P value adjusted using the Holm method. P < 0.05 was considered significant. (G and H) Flow cytometry analysis for B7H3 cell–surface expression from organoids dissociated into single cells. P < 0.05; significant, Wilcoxon test. (G) Median Fluorescence Intensity (MFI) and (H) Percentage positive cells are shown for 9 analyzed models.

We next evaluated B7H3 protein expression across a cohort of PC metastases using a tissue microarray (TMA) comprised of 181 tumors from 58 patients, with a range of 1 to 4 tumors per patient. A total of 3 tumors were not analyzed due to insufficient tumor content, leaving 178 evaluable tumors. Overall, B7H3 protein exhibited more variation compared with transcript expression: of 178 tumors evaluated, 149 expressed B7H3 (H-score > 20) and 29 lacked expression (Figure 1, C and D). B7H3 was detected across diverse metastatic sites with bone metastases exhibiting the highest levels (Supplemental Figure 1D). Tumors categorized as AR+/NE– ARPC generally expressed higher B7H3 levels compared with other phenotypes, but a subset of AR–/NE+ SCNPC and AR–/NE– tumors also expressed B7H3 (Figure 1E). Collectively, these results indicate that B7H3 may represent a target for antigen-directed therapeutics across a range of clinical mPC phenotypes.

Additionally, we used a quantitative immunoblot technique to determine the relative amount of total B7H3 protein expressed in mPC preclinical models. Like patient samples, we observed wide variation (more than 30-fold) in B7H3 protein levels (Figure 1F and Supplemental Figure 1E). ARPC models demonstrated a range of expression clustering as a high group (B7H3Hi) and an intermediate-to-low group, the latter of which overlapped in median level with the non-ARPC group (Figure 1F). There was no apparent common genotypic or phenotypic feature in B7H3HI ARPC group. Consistent with patient data, B7H3 mRNA levels were not strongly correlated with B7H3 protein levels in models of either phenotype (Supplemental Figure 1F), emphasizing the minimal utility in transcriptional based assays for quantitative analyses (12). Importantly, in FACS analysis, despite variability in the median fluorescence intensity across the models tested (n = 9), EpCam+ tumor cells homogenously expressed B7H3 (80%–100% cells) at the cell surface, which makes B7H3 an ideal target for ADC based therapy (Figure 1, G and H, and Supplemental Figure 2A). B7H3 cell surface level was well correlated with total B7H3 protein (Supplemental Figure 2B).

As AR signaling is a major determinant of ARPC phenotypic subclasses, we determined the relationship of B7H3 RNA and protein to AR target gene output. However, consistent with the analyses of human mPC tumors, there was no correlation of B7H3 protein levels with AR signature scores (Supplemental Figure 2C).

B7H3-PBD-ADC is cytotoxic for defined subclasses of prostate cancer. We next sought to determine the efficacy of a B7H3 targeted ADC directing the genotoxic PBD (B7H3-PBD-ADC) to mPC cells. We compared the targeted delivery of PBD via B7H3-PBD-ADC, relative to the nontargeted control R347-PBD-ADC, across a panel of mPC organoids where phenotype, genotype, and B7H3 levels were established (Figure 2, A and B, Supplemental Figure 3A, and Supplemental Data File 1). All non-ARPC models were highly sensitive to the B7H3-PBD-ADC with normalized AUC (nAUC) ranging from 0.2–0.5 and IC50 from 0.03–2.08 ng/mL (Figure 2C and Table 1). In contrast, the ARPC models displayed a broad range of responses, with nAUC ranging from 0.3–1 and displaying less steep dose response slopes in responders compared with the non-ARPC models (Figure 2, B and C). The relative dose required for cytotoxicity in comparing targeted B7H3-PBD-ADCs and control R347-PBD-ADCs was a minimum of 1,000-fold for the most sensitive models, while the majority of models were unaffected by even the highest concentration of R347-PBD (4 mg/mL) (Figure 2B and Supplemental Figure 3, B and C).

Figure 2

B7H3-PBD-ADC activity requires, but is not correlated with, B7H3 protein levels. (A) Schematic of the ex vivo drug assay. (B) Representative drug response curves for B7H3-PBD-ADC and R347-PBD-ADC (control ADC) in PDX-derived organoids (PDXOs) of SCNPC and ARPC phenotypes. Percentage viability was plotted relative to the control. (C) Comparison of B7H3-PBD-ADC response and B7H3 protein expression across the models; n = 26. (D) nAUC values for B7H3-PBD-ADC in ARPC (n = 19) and non-ARPC models (n = 7). ARPC models are categorized into 3 groups: high B7H3 expressors (B7H3HI) n = 4, responder (R) n = 7, and nonresponder (NR) n = 8. Red line indicates median nAUC for the groups. Wilcoxon test was used for pairwise comparison between groups with P value adjusted using the Holm method. P < 0.05 was considered significant. (E) FACS sorting strategy for selecting B7H3-KO 145.2 cells. (F) Western blot for FACS sorted 145.2 B7H3+ and B7H3– (B7H3 KO) cells grown as organoids. (G) Dose response curves for 145.2 presorted and sorted B7H3NEG and B7H3+ organoids treated with ADC for 10 days. (H) 145.2 B7H3+, B7H3NEG, and admix (mix of B7H3+ and B7H3NEG cells in approximately equal proportion) ODXs treated with ADCs or vehicle, once weekly for 2 weeks, as indicated by arrows; n = 8/group, except B7H3NEG (2 mice with necrotic tumors at Day 14 excluded from B7H3-PBD group), B7H3NEG and admix (Vehicle group; n = 2 each), B7H3+ (Vehicle group; n = 4), Admix (B7H3-PBD and R347-PBD; n = 5 each). Average tumor volume is plotted from the day of first treatment indicated as Day 0. Top panel comparing average tumor volumes for R347-PBD and vehicle treated mice. Bottom panel comparing average tumor volumes for B7H3-PBD treated B7H3+, B7H3NEG, and admix xenografts. Wilcoxon test, *P < 0.05. (I) Western blot for B7H3 knockdown in NCI-PC155 organoids. (J) Dose response curves for NCI-PC155 organoids after B7H3 knockdown (sgB7H3 group). Error bars indicate the SEM.