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Cited by 200Pancreatic and Hepatic Oncology ResearchCancer Immunotherapy and BiomarkersImmunotherapy and Immune Responses

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Neoadjuvant Selicrelumab, an Agonist CD40 Antibody, Induces Changes in the Tumor Microenvironment in Patients with Resectable Pancreatic Cancer

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Byrne, Katelyn T., Betts, Courtney B., Mick, Rosemarie, Sivagnanam, Shamilene, Bajor, David L., Laheru, Daniel A., Chiorean, E. Gabriela, O'Hara, Mark H., Liudahl, Shannon M., Newcomb, Craig, Alanio, Cécile, Ferreira, Ana P., Park, Byung S., Ohtani, Takuya, Huffman, Austin P., Väyrynen, Sara A., Dias Costa, Andressa, Kaiser, Judith C., Lacroix, Andreanne M., Redlinger, Colleen, Stern, Martin, Nowak, Jonathan A., Wherry, E. John, Cheever, Martin A., Wolpin, Brian M., Furth, Emma E., Jaffee, Elizabeth M., Coussens, Lisa M., Vonderheide, Robert H.·2021·Clinical Cancer Research
DOI 10.1158/1078-0432.ccr-21-1047PMC PMC8667686

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Abstract

Purpose: CD40 activation is a novel clinical opportunity for cancer immunotherapy. Despite numerous active clinical trials with agonistic CD40 monoclonal antibodies (mAb), biological effects and treatment-related modulation of the tumor microenvironment (TME) remain poorly understood. Patients and Methods: Here, we performed a neoadjuvant clinical trial of agonistic CD40 mAb (selicrelumab) administered intravenously with or without chemotherapy to 16 patients with resectable pancreatic ductal adenocarcinoma (PDAC) before surgery followed by adjuvant chemotherapy and CD40 mAb. Results: The toxicity profile was acceptable, and overall survival was 23.4 months (95% confidence interval, 18.0–28.8 months). Based on a novel multiplexed immunohistochemistry platform, we report evidence that neoadjuvant selicrelumab leads to major differences in the TME compared with resection specimens from treatment-naïve PDAC patients or patients given neoadjuvant chemotherapy/chemoradiotherapy only. For selicrelumab-treated tumors, 82% were T-cell enriched, compared with 37% of untreated tumors (P = 0.004) and 23% of chemotherapy/chemoradiation-treated tumors (P = 0.012). T cells in both the TME and circulation were more active and proliferative after selicrelumab. Tumor fibrosis was reduced, M2-like tumor-associated macrophages were fewer, and intratumoral dendritic cells were more mature. Inflammatory cytokines/sec CXCL10 and CCL22 increased systemically after selicrelumab. Conclusions: This unparalleled examination of CD40 mAb therapeutic mechanisms in patients provides insights for design of subsequent clinical trials targeting CD40 in cancer.

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Figures

Figure 1. Survival estimates for patients who underwent surgical resection and eligible for adjuvant therapy. A, DFS from day of surgery by treatment arm and (B) OS from day of surgery by treatment arm and combined arms. C, Table comparing DFS and OS for the selicrelumab-treated cohort to two published cohorts: an untreated cohort and a chemo/chemoradiation-treated cohort, as indicated.
Figure 1. Survival estimates for patients who underwent surgical resection and eligible for adjuvant therapy. A, DFS from day of surgery by treatment arm and (B) OS from day of surgery by treatment arm and combined arms. C, Table comparing DFS and OS for the selicrelumab-treated cohort to two published cohorts: an untreated cohort and a chemo/chemoradiation-treated cohort, as indicated.CC BY-NC-ND · PubMed Central
Figure 2. Assessment of TME by histopathologic and IHC of the surgical resection specimen. Representative images of Mason's trichrome staining from (A) treatment-naïve cohort of UPenn patients or (B) patients treated with selicrelumab (arm I). Red lines indicate the border of tumor regions. C, Quantification of percent fibrosis in the TME for a set of untreated control PDAC resection samples and samples from arms I and II, as indicated. Each symbol represents a single patient, horizontal line indicates mean, and error bars indicate standard deviation from the mean (SD). Diamond symbols indicate representative image shown in A or B.
Figure 2. Assessment of TME by histopathologic and IHC of the surgical resection specimen. Representative images of Mason's trichrome staining from (A) treatment-naïve cohort of UPenn patients or (B) patients treated with selicrelumab (arm I). Red lines indicate the border of tumor regions. C, Quantification of percent fibrosis in the TME for a set of untreated control PDAC resection samples and samples from arms I and II, as indicated. Each symbol represents a single patient, horizontal line indicates mean, and error bars indicate standard deviation from the mean (SD). Diamond symbols indicate representative image shown in A or B.CC BY-NC-ND · PubMed Central
Figure 3. Immune infiltrates in the TME of patients. A, Density of CD45+ and pan cytokeratin+ (PanCK) cells in the TME of four treatment groups. B, Broad immune composition in treatment groups by spatial location. C, Broad immune composition in spatial locations as a function of treatment groups. For B and C, contribution of histopathologic region type is displayed in pie graphs above each stacked bar. For A–C, each “N” is an ROI from a total of 20 patients for treatment naïve, 13 patients for chemo/chemoradiation, 8 patients for selicrelumab, and 3 patients for selicrelumab/chemotherapy. For A left (CD45), B, and C, included ROIs were tumor, tumor-associated stroma, normal-adjacent pancreas, and tertiary lymphoid structures, treatment naïve, N = 295 ROIs; chemo/chemoradiation, N = 118 ROIs; selicrelumab, N = 87 ROIs; selicrelumab/chemotherapy, N = 33 ROIs. For A right (PanCK+), only tumor regions were analyzed: treatment naïve, N = 61 ROIs; chemo/chemoradiation, N = 40 ROIs; selicrelumab, N = 32 ROIs; selicrelumab/chemotherapy, N = 10 ROIs. Biostatistical comparisons for A–C are provided in Supplementary Table S3.D, Unsupervised hierarchical clustering of histopathologically identified invasive tumor regions. Input was patient summarized data for cell density for various immune cell types; each patient is a column. Treatment status of chemo/chemoradiation patients is shown as both black and green, as indicated. E, Broad immune composition for the cluster types identified in D. “a” is **** comparison cluster T cell to myeloid for CD4; “b” is **** cluster T-cell to granulocyte for neutrophils/eosinophils (neut/eos); “c” is **** cluster myeloid to granulocytic for neut/eos; “d” is *** cluster myeloid to granulocytic for DCs, “e” is **** cluster T cell to myeloid for DCs, “f” is * cluster T cell to granulocyte for monocytes/macrophages. F, Density of CD8+ T cells, CD4+ T helper (non-Treg), CD4+ T regulatory cells, and ratios of mature/immature DCs, CD163–/CD163+ monocytes/macrophages, and CD8+ T cells/CD68+ monocytes/macrophages. G, Percent cellular positivity for PD-1, granzyme B, and Ki67 on CD4+ and CD8+ T-cell subsets by treatment group. For A, F, G, patients who received gem + nab-paclitaxel in the neoadjuvant setting are colored black within the chemo/chemoradiation group, where all the other patients who received other chemo/chemoradiation combinations are green. For D–G, each “N” is a patient, N = 104 for treatment naïve, N = 13 for chemotherapy/chemoradiation, N = 8 for selicrelumab, N = 3 for selicrelumab/chemotherapy. For F–G, violin plots, all points are shown, lines at median and at 25th and 75th quartiles. Bar charts indicate mean ± SEM. Fisher exact test (2×3 contingency table) used to assess distributional differences (i.e., cluster skewing) among groups in D. For F–G, Kruskal–Wallis with Dunn multiple comparisons and correction. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Further experimental methods are provided in Supplementary Fig. S2 and Supplementary Table S4.
Figure 3. Immune infiltrates in the TME of patients. A, Density of CD45+ and pan cytokeratin+ (PanCK) cells in the TME of four treatment groups. B, Broad immune composition in treatment groups by spatial location. C, Broad immune composition in spatial locations as a function of treatment groups. For B and C, contribution of histopathologic region type is displayed in pie graphs above each stacked bar. For A–C, each “N” is an ROI from a total of 20 patients for treatment naïve, 13 patients for chemo/chemoradiation, 8 patients for selicrelumab, and 3 patients for selicrelumab/chemotherapy. For A left (CD45), B, and C, included ROIs were tumor, tumor-associated stroma, normal-adjacent pancreas, and tertiary lymphoid structures, treatment naïve, N = 295 ROIs; chemo/chemoradiation, N = 118 ROIs; selicrelumab, N = 87 ROIs; selicrelumab/chemotherapy, N = 33 ROIs. For A right (PanCK+), only tumor regions were analyzed: treatment naïve, N = 61 ROIs; chemo/chemoradiation, N = 40 ROIs; selicrelumab, N = 32 ROIs; selicrelumab/chemotherapy, N = 10 ROIs. Biostatistical comparisons for A–C are provided in Supplementary Table S3.D, Unsupervised hierarchical clustering of histopathologically identified invasive tumor regions. Input was patient summarized data for cell density for various immune cell types; each patient is a column. Treatment status of chemo/chemoradiation patients is shown as both black and green, as indicated. E, Broad immune composition for the cluster types identified in D. “a” is **** comparison cluster T cell to myeloid for CD4; “b” is **** cluster T-cell to granulocyte for neutrophils/eosinophils (neut/eos); “c” is **** cluster myeloid to granulocytic for neut/eos; “d” is *** cluster myeloid to granulocytic for DCs, “e” is **** cluster T cell to myeloid for DCs, “f” is * cluster T cell to granulocyte for monocytes/macrophages. F, Density of CD8+ T cells, CD4+ T helper (non-Treg), CD4+ T regulatory cells, and ratios of mature/immature DCs, CD163–/CD163+ monocytes/macrophages, and CD8+ T cells/CD68+ monocytes/macrophages. G, Percent cellular positivity for PD-1, granzyme B, and Ki67 on CD4+ and CD8+ T-cell subsets by treatment group. For A, F, G, patients who received gem + nab-paclitaxel in the neoadjuvant setting are colored black within the chemo/chemoradiation group, where all the other patients who received other chemo/chemoradiation combinations are green. For D–G, each “N” is a patient, N = 104 for treatment naïve, N = 13 for chemotherapy/chemoradiation, N = 8 for selicrelumab, N = 3 for selicrelumab/chemotherapy. For F–G, violin plots, all points are shown, lines at median and at 25th and 75th quartiles. Bar charts indicate mean ± SEM. Fisher exact test (2×3 contingency table) used to assess distributional differences (i.e., cluster skewing) among groups in D. For F–G, Kruskal–Wallis with Dunn multiple comparisons and correction. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Further experimental methods are provided in Supplementary Fig. S2 and Supplementary Table S4.CC BY-NC-ND · PubMed Central
Figure 4. Changes in serum cytokines between baseline and 5 days after selicrelumab. A, Upregulated or (B) downregulated fold changes in expression from baseline to 5 days after selicrelumab for each patient are displayed, in addition to mean ± SEM (gray bar and whisker plot), with dotted line indicating no change over baseline. For those with a statistically significant difference or a difference approaching significance, patient data points are color-coded (red, arm I; blue, arm II) with specific P values indicated as determined by Wilcoxon matched-pair signed rank test.
Figure 4. Changes in serum cytokines between baseline and 5 days after selicrelumab. A, Upregulated or (B) downregulated fold changes in expression from baseline to 5 days after selicrelumab for each patient are displayed, in addition to mean ± SEM (gray bar and whisker plot), with dotted line indicating no change over baseline. For those with a statistically significant difference or a difference approaching significance, patient data points are color-coded (red, arm I; blue, arm II) with specific P values indicated as determined by Wilcoxon matched-pair signed rank test.CC BY-NC-ND · PubMed Central
Figure 5. CyTOF analysis of antigen-presenting cells in PBMC. A, TSNE plot displaying the major cell populations within the CD45+ leukocyte population, gated as described in methods in Supplementary Fig. S4. B, Global MFI expression of MHC class II and costimulatory molecule expression. C–F, Changes in subsets or HLA-DR MFI expression for (C) B cells, (D) cDC1s, (E) M1-like myeloid cells, and (F) M2-like myeloid cells are shown. Red symbols indicate patients from arm I, blue indicates patients from arm II, circles denote baseline, squares indicate 5 days after selicrelumab (protocol day 8), and dotted line indicates mean values from normal donor controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as determined by Wilcoxon matched-pair signed rank test.
Figure 5. CyTOF analysis of antigen-presenting cells in PBMC. A, TSNE plot displaying the major cell populations within the CD45+ leukocyte population, gated as described in methods in Supplementary Fig. S4. B, Global MFI expression of MHC class II and costimulatory molecule expression. C–F, Changes in subsets or HLA-DR MFI expression for (C) B cells, (D) cDC1s, (E) M1-like myeloid cells, and (F) M2-like myeloid cells are shown. Red symbols indicate patients from arm I, blue indicates patients from arm II, circles denote baseline, squares indicate 5 days after selicrelumab (protocol day 8), and dotted line indicates mean values from normal donor controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as determined by Wilcoxon matched-pair signed rank test.CC BY-NC-ND · PubMed Central
Figure 6. CyTOF analysis of T cells in PBMC. A, TSNE plot displaying the major cell populations within the CD45+ leukocyte population, gated as described in methods in Supplementary Fig. S4. B, Heat map displaying the normalized proportions of the indicated markers on CD4+ effector T cells (CD4 Teff) or CD8+ effector T cells (CD8 Teff) from baseline to 5 days after selicrelumab (protocol day 8). C and D, Plotted changes in highlighted markers for CD4+ effector T cells (C) or CD8+ effector T cells (D). E, Changes in the proportion of circulating Tregs. Red symbols indicate patients from arm I, blue indicates patients from arm II, circles denote baseline, squares indicate 5 days after selicrelumab (protocol day 8), and dotted line indicates mean values from normal donor controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, as determined by Wilcoxon matched-pair signed rank test.
Figure 6. CyTOF analysis of T cells in PBMC. A, TSNE plot displaying the major cell populations within the CD45+ leukocyte population, gated as described in methods in Supplementary Fig. S4. B, Heat map displaying the normalized proportions of the indicated markers on CD4+ effector T cells (CD4 Teff) or CD8+ effector T cells (CD8 Teff) from baseline to 5 days after selicrelumab (protocol day 8). C and D, Plotted changes in highlighted markers for CD4+ effector T cells (C) or CD8+ effector T cells (D). E, Changes in the proportion of circulating Tregs. Red symbols indicate patients from arm I, blue indicates patients from arm II, circles denote baseline, squares indicate 5 days after selicrelumab (protocol day 8), and dotted line indicates mean values from normal donor controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, as determined by Wilcoxon matched-pair signed rank test.CC BY-NC-ND · PubMed Central
Figure 7. TCR deep sequencing and analysis of tumor-associated clones. To enrich for tumor-associated T-cell clonotypes, all CDR3 amino acid TCR template reads were first gated on positive detection in the tumor site. A, Total tumor T-cell clones detected in tumor and blood, as indicated. All clones reported are detected in the tumor; “Tumor only” clones are never detected in any blood sample; “Detected pre-surgery” indicates tumor-associated clones preexisting at baseline before neoadjuvant selicrelumab administration; “Detected pre- and post-surgery” indicates tumor-associated clones detected in the baseline sample and any blood sample after surgery; all “Post-surgery only” columns indicate tumor-associated clones found only in the tumor and post-surgery samples, and not detected in the baseline blood sample. B, Jaccard coefficient matrix, comparing the frequencies of shared TCR CDR3 sequences as indicated. Protocol time points indicated by cycle number (“C”) and day (“D”) represent treatment days for the adjuvant selicrelumab/chemotherapy phase of the study. C, Frequency of tumor clones detected in each subset of time points as indicated, grouped as described in A. Each symbol represents a single patient, with horizontal line indicating mean, and error bars indicating SEM (A and C), or each box indicates the mean value across all patients at indicated time points (B). For A and C, red symbols indicate patients from arm I, and blue indicates patients from arm II.
Figure 7. TCR deep sequencing and analysis of tumor-associated clones. To enrich for tumor-associated T-cell clonotypes, all CDR3 amino acid TCR template reads were first gated on positive detection in the tumor site. A, Total tumor T-cell clones detected in tumor and blood, as indicated. All clones reported are detected in the tumor; “Tumor only” clones are never detected in any blood sample; “Detected pre-surgery” indicates tumor-associated clones preexisting at baseline before neoadjuvant selicrelumab administration; “Detected pre- and post-surgery” indicates tumor-associated clones detected in the baseline sample and any blood sample after surgery; all “Post-surgery only” columns indicate tumor-associated clones found only in the tumor and post-surgery samples, and not detected in the baseline blood sample. B, Jaccard coefficient matrix, comparing the frequencies of shared TCR CDR3 sequences as indicated. Protocol time points indicated by cycle number (“C”) and day (“D”) represent treatment days for the adjuvant selicrelumab/chemotherapy phase of the study. C, Frequency of tumor clones detected in each subset of time points as indicated, grouped as described in A. Each symbol represents a single patient, with horizontal line indicating mean, and error bars indicating SEM (A and C), or each box indicates the mean value across all patients at indicated time points (B). For A and C, red symbols indicate patients from arm I, and blue indicates patients from arm II.CC BY-NC-ND · PubMed Central

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Authors
Byrne, K. T., Betts, C. B., Mick, R., Sivagnanam, S., Bajor, D. L., Laheru, D. A., Chiorean, E. G., O'Hara, M. H., Liudahl, S. M., Newcomb, C., Alanio, C., Ferreira, A. P., Park, B. S., Ohtani, T., Huffman, A. P., Väyrynen, S. A., Dias Costa, A., Kaiser, J. C., Lacroix, A. M., Redlinger, C., Stern, M., Nowak, J. A., Wherry, E. J., Cheever, M. A., Wolpin, B. M., Furth, E. E., Jaffee, E. M., Coussens, L. M., & Vonderheide, R. H.
Affiliations
  • University of Pennsylvania (US)
  • Portland State University (US)
  • Oregon Health & Science University (US)
  • Case Comprehensive Cancer Center (US)
  • Johns Hopkins University (US)
  • University of Washington (US)
  • 1Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
  • Harvard University (US)
  • 10Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.
  • Fred Hutch Cancer Center (US)
  • F. Hoffmann-La Roche AG (CH)
  • Brigham and Women's Hospital (US)
Corresponding author
Vonderheide, Robert H.
ORCID
Venue
Clinical Cancer Research
Publisher
American Association for Cancer Research (AACR)
Published
2021-06-10
Volume
27
Issue
16
Pages
4574-4586
ISSN
  • 1078-0432 (ISSN-L)
  • 1557-3265
DOI
10.1158/1078-0432.ccr-21-1047
PMC
PMC8667686
OpenAlex
W3171434019
Cited by
199
References
33
Citations by year
2026: 37 · 2025: 42 · 2024: 48 · 2023: 41 · 2022: 28 · 2021: 3
FWCI
10.91
Type
journal article
Language
en
Open access
Licence
Locations
  • Clinical Cancer Research (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • SU2C-Lustgarten Foundation (SU2C-AACR-DT14-14)
  • Pancreatic Cancer Action Network (Research Acceleration Network)
  • Lustgarten Foundation (NA)
  • National Cancer Institute (P30CA016520, R01CA229803, R01CA169123, U01CA224012, U2CCA233280, R01CA223150, R01CA226909, R21HD099367, UM1CA154967)
  • Parker Institute for Cancer Immunotherapy (NA)
  • Brenden-Colson Center for Pancreatic Care (NA)
  • Hale Center for Pancreatic Cancer Research (NA)
Topics
  • Health Sciences › Medicine › Oncology › Pancreatic and Hepatic Oncology Research
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
pancreatic ductal adenocarcinoma, neoadjuvant therapy, tumor microenvironment, T cell infiltration, tumor-associated macrophages, M2 macrophages, dendritic cell maturation, tumor fibrosis, cancer immunotherapy, overall survival
MeSH terms
Adult, Aged, Antibodies, Monoclonal, Female, Humans, Male, Middle Aged, Pancreatic Neoplasms, CD40 Antigens, Neoadjuvant Therapy, Tumor Microenvironment, Antibodies, Monoclonal, Humanized

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