Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy.
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Atomic coordinates and structure factors of the CD28 CODV-Fab and CD28:CODV-Fab complexes from Fig. 6 have been deposited in the PDB under accession codes 6O89 and 6O8D, respectively. Source data for Figs. 1–4 and Extended Data Fig. 2 are provided with the paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.
Bhoj, V. G. et al. Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy. Blood 128, 360–370 (2016).
Hui, R. et al. Pembrolizumab as first-line therapy for patients with PD-L1-positive advanced non-small cell lung cancer: a phase 1 trial. Ann. Oncol. 28, 874–881 (2017).
Leach, D. R., Krummel, M. F. & Allison, J. P. Enhancement of antitumor immunity by CTLA-4 blockade. Science 271, 1734–1736 (1996).
Zuniga, E. I., Macal, M., Lewis, G. M. & Harker, J. A. Innate and adaptive immune regulation during chronic viral infections. Annu. Rev. Virol. 2, 573–597 (2015).
Vesely, M. D., Kershaw, M. H., Schreiber, R. D. & Smyth, M. J. Natural innate and adaptive immunity to cancer. Annu. Rev. Immunol. 29, 235–271 (2011).
Sharma, P. & Allison, J. P. The future of immune checkpoint therapy. Science 348, 56–61 (2015).
Topalian, S. L. et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med. 366, 2443–2454 (2012).
Porter, D. L., Levine, B. L., Kalos, M., Bagg, A. & June, C. H. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N. Engl. J. Med. 365, 725–733 (2011).
Kochenderfer, J. N. et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood 116, 4099–4102 (2010).
Rosenberg, S. A. & Restifo, N. P. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348, 62–68 (2015).
Baeuerle, P. A. & Reinhardt, C. Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res. 69, 4941–4944 (2009).
Buie, L. W., Pecoraro, J. J., Horvat, T. Z. & Daley, R. J. Blinatumomab: a first-in-class bispecific T-cell engager for precursor B-cell acute lymphoblastic leukemia. Ann. Pharmacother. 49, 1057–1067 (2015).
Xu, L. et al. Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques. Science 358, 85–90 (2017).
Sun, L. L. et al. Anti-CD20/CD3 T cell-dependent bispecific antibody for the treatment of B cell malignancies. Sci. Transl. Med. 7, 287ra270 (2015).
Zugmaier, G. et al. Long-term survival and T-cell kinetics in relapsed/refractory ALL patients who achieved MRD response after blinatumomab treatment. Blood 126, 2578–2584 (2015).
Esensten, J. H., Helou, Y. A., Chopra, G., Weiss, A. & Bluestone, J. A. CD28 costimulation: from mechanism to therapy. Immunity 44, 973–988 (2016).
Hui, E. et al. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science 355, 1428–1433 (2017).
Chai, J. G. & Lechler, R. I. Immobilized anti-CD3 mAb induces anergy in murine naive and memory CD4+ T cells in vitro. Int. Immunol. 9, 935–944 (1997).
Steinmetz, A. et al. CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications. MAbs 8, 867–878 (2016).
Waibler, Z. et al. Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies. PLoS ONE 3, e1708 (2008).
Boise, L. H. et al. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Immunity 3, 87–98 (1995).
Alegre, M. L. et al. A non-activating “humanized” anti-CD3 monoclonal antibody retains immunosuppressive properties in vivo. Transplantation 57, 1537–1543 (1994).
McKeage, K. Daratumumab: first global approval. Drugs 76, 275–281 (2016).
Gratama, J. W. et al. Tetramer-based quantification of cytomegalovirus (CMV)-specific CD8+ T lymphocytes in T-cell-depleted stem cell grafts and after transplantation may identify patients at risk for progressive CMV infection. Blood 98, 1358–1364 (2001).
Halliley, J. L. et al. Long-lived plasma cells are contained within the CD19−CD38hiCD138+ subset in human bone marrow. Immunity 43, 132–145 (2015).
Robillard, N. et al. CD28, a marker associated with tumoral expansion in multiple myeloma. Clin. Cancer Res. 4, 1521–1526 (1998).
Nair, J. R. et al. CD28 expressed on malignant plasma cells induces a prosurvival and immunosuppressive microenvironment. J. Immunol. 187, 1243–1253 (2011).
Hunig, T. The rise and fall of the CD28 superagonist TGN1412 and its return as TAB08: a personal account. FEBS J. 283, 3325–3334 (2016).
Tyrsin, D. et al. From TGN1412 to TAB08: the return of CD28 superagonist therapy to clinical development for the treatment of rheumatoid arthritis. Clin. Exp. Rheumatol. 34, 45–48 (2016).
Findlay, L. et al. Improved in vitro methods to predict the in vivo toxicity in man of therapeutic monoclonal antibodies including TGN1412. J. Immunol. Methods 352, 1–12 (2010).
Evans, E. J. et al. Crystal structure of a soluble CD28-Fab complex. Nat. Immunol. 6, 271–279 (2005).
Kroschinsky, F. et al. New drugs, new toxicities: severe side effects of modern targeted and immunotherapy of cancer and their management. Crit. Care 21, 89 (2017).
Saber, H., Del Valle, P., Ricks, T. K. & Leighton, J. K. An FDA oncology analysis of CD3 bispecific constructs and first-in-human dose selection. Regul. Toxicol. Pharmacol. 90, 144–152 (2017).
Zuch de Zafra, C. L. et al. Targeting multiple myeloma with AMG 424, a novel anti-CD38/CD3 bispecific T-cell-recruiting antibody optimized for cytotoxicity and cytokine release. Clin. Cancer Res. 25, 3921–3933 (2019).
Brudno, J. N. & Kochenderfer, J. N. Toxicities of chimeric antigen receptor T cells: recognition and management. Blood 127, 3321–3330 (2016).
Ishiguro, T. et al. An anti-glypican 3/CD3 bispecific T cell-redirecting antibody for treatment of solid tumors. Sci. Transl. Med. 9, eaal4291 (2017).
June, C. H., O’Connor, R. S., Kawalekar, O. U., Ghassemi, S. & Milone, M. C. CAR T cell immunotherapy for human cancer. Science 359, 1361–1365 (2018).
Ribas, A. & Wolchok, J. D. Cancer immunotherapy using checkpoint blockade. Science 359, 1350–1355 (2018).
Wolchok, J. D. et al. Development of ipilimumab: a novel immunotherapeutic approach for the treatment of advanced melanoma. Ann. NY Acad. Sci. 1291, 1–13 (2013).
Przepiorka, D. et al. FDA approval: blinatumomab. Clin. Cancer Res. 21, 4035–4039 (2015).
Malavasi, F. et al. Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology. Physiol. Rev. 88, 841–886 (2008).
Wang, L. et al. CD38 expression predicts poor prognosis and might be a potential therapy target in extranodal NK/T cell lymphoma, nasal type. Ann. Hematol. 94, 1381–1388 (2015).
Burgler, S. Role of CD38 expression in diagnosis and pathogenesis of chronic lymphocytic leukemia and its potential as therapeutic target. Crit. Rev. Immunol. 35, 417–432 (2015).
Konopleva, M., Rissling, I. & Andreeff, M. CD38 in hematopoietic malignancies. Chem. Immunol. 75, 189–206 (2000).
Poret, N. et al. CD38 in hairy cell leukemia is a marker of poor prognosis and a new target for therapy. Cancer Res. 75, 3902–3911 (2015).
Bolzoni, M. et al. The link between bone microenvironment and immune cells in multiple myeloma: emerging role of CD38. Immunol. Lett. 205, 65–70 (2019).
Van de Donk, N., Richardson, P. G. & Malavasi, F. CD38 antibodies in multiple myeloma: back to the future. Blood 131, 13–29 (2018).
Van de Donk, N. W. et al. Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma. Blood 127, 681–695 (2016).
Lonial, S. et al. Daratumumab monotherapy in patients with treatment-refractory multiple myeloma (SIRIUS): an open-label, randomised, phase 2 trial. Lancet 387, 1551–1560 (2016).
Dimopoulos, M. A. et al. Daratumumab, lenalidomide, and dexamethasone for multiple myeloma. N. Engl. J. Med. 375, 1319–1331 (2016).
Palumbo, A. et al. Daratumumab, bortezomib, and dexamethasone for multiple myeloma. N. Engl. J. Med. 375, 754–766 (2016).
Mateos, M. V. et al. Daratumumab plus bortezomib, melphalan, and prednisone for untreated myeloma. N. Engl. J. Med. 378, 518–528 (2018).
Martin, T. et al. A phase 1b study of isatuximab plus lenalidomide and dexamethasone for relapsed/refractory multiple myeloma. Blood 129, 3294–3303 (2017).
Hoffmann, M. et al. Exhaustion of activated CD8 T cells predicts disease progression in primary HIV-1 infection. PLoS Pathog. 12, e1005661 (2016).
Rodriguez-Alba, J. C. et al. HIV disease progression: overexpression of the ectoenzyme CD38 as a contributory factor? Bioessays 41, e1800128 (2019).
Chen, L., Li, Y., Yi, X. & Gibbons, D. L. Targeting CD38 to improve anti-PD-1/CTLA-4 combination therapy in lung cancer. J. Clin. Oncol. 36, 144 (2018).
Tai, Y. T. & Anderson, K. C. Targeting CD38 alleviates tumor-induced immunosuppression. Oncotarget 8, 112166–112167 (2017).
DeFrancesco, L. CAR-T cell therapy seeks strategies to harness cytokine storm. Nat. Biotechnol. 32, 604 (2014).
Suntharalingam, G. et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N. Engl. J. Med. 355, 1018–1028 (2006).
Penaranda, C., Tang, Q. & Bluestone, J. A. Anti-CD3 therapy promotes tolerance by selectively depleting pathogenic cells while preserving regulatory T cells. J. Immunol. 187, 2015–2022 (2011).
Tabares, P. et al. Human regulatory T cells are selectively activated by low-dose application of the CD28 superagonist TGN1412/TAB08. Eur. J. Immunol. 44, 1225–1236 (2014).
Krejcik, J. et al. Monocytes and granulocytes reduce CD38 expression levels on myeloma cells in patients treated with daratumumab. Clin. Cancer Res. 23, 7498–7511 (2017).
Raghu, G. et al. SAR156597 in idiopathic pulmonary fibrosis: a phase 2 placebo-controlled study (DRI11772). Eur. Respir. J. 52, 1801130 (2018).
Merchant, A. M. et al. An efficient route to human bispecific IgG. Nat. Biotechnol. 16, 677–681 (1998).
Smith, K. B. & Ellis, S. A. Standardisation of a procedure for quantifying surface antigens by indirect immunofluorescence. J. Immunol. Methods 228, 29–36 (1999).
Cobbold, M. et al. Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers. J. Exp. Med. 202, 379–386 (2005).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013).
We thank R. Mashal and H. Van de Velde for critical comments on the manuscript, T. Majid and C. Lawendowski for excellent program management, A. E. Schroeer and B. DelGiudice for graphic arts support, and M. Sanicola-Nadel, T. Schmidt, T. Bouquin, D. Wiederschain, S. Sidhu, B. Thurberg, K. Klinger, J. Darbyshire, C. Dangler, Z. Jayyosi and C. J. Wei for organizational support. We also thank J. Kingsbury, S. Kathuria, L. Chen, N. Maestrali, S. Somarriba, E. Deschamps, N. Couteault and L. Maton for technical support.
All authors are or were employees of Sanofi while engaged in this research project. Sanofi develops and manufactures cancer treatment medicines. G.J.N. is the Chief Scientific Officer of Sanofi. G.J.N., Z.-y.Y., R.R.W., L.X., E.S., L.W., T.D., B.C., C.L. and C.P. are listed on intellectual property (WO2019074973A2) developed and owned by Sanofi related to the development of novel cancer treatments.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Expression of relevant cell surface antigens on an IL-2 promoter luciferase indicator line, as well as Bcl-xL kinetics and annexin V analysis in primary T cells.
(a) Cell surface expression of CD3, CD28, or CD38 respectively on GloResponse™ IL2-luc2P Jurkat Cells (Promega) was determined by flow cytometry as described in Methods in 1 independent experiment. (b) Induction of pro-survival protein Bcl-xL (left) and annexin V (right) after CD38 trispecific Ab, or indicated mutant Ab, treatment of primary CD3+ cells (n = 3 donors). Cells were isolated as described in Methods and analyzed by flow cytometry targeting CD3, Annexin V, and using a fixable viability dye. The data points and error bars were plotted with mean ± SD.
(a) Alternative mutations in the Fc region of IgG4 were prepared for analysis in Fc receptor binding assays. (b) Binding in the biacore assay was used to measure the affinity of the specified IgG4 Fc variants to indicated human Fc receptors immobilized on the chip. IgG4 Fc variants were used at 150 nM. The assay was repeated once with similar results. The binding to human FcRn was measured using ELISA by coating the human FcRn antigen on the plate in a single experiment.
The crystal structure of CD28: anti-CD28xCD3 CODV-Fab (a) and molecular model of anti-CD38 Fab. (b) were used to model the binary CD28: trispecific antibody model complex (Fig. 6b). The model is compatible with concurrent binding of the antibody with CD28 and CD38. The distance between the CDRH3s for anti-CD28/CD3 CODV-Fab measured ~60 Å, comparable to the anti-IL4/IL13 CODV-Fab and shorter than the anti-HIV CODV-Fab (exceeds 100 Å), possibly because different linkers were used in the CODV Fabs. The structure of CD3 in presence of CD3mid Fv has not been solved and thus not modeled.
Extended Data Fig. 4 Effect of trispecific Ab on immune cell distribution and binding of CD38 trispecific Ab to T cell subsets in the blood of NHPs.
Blood samples from animals treated with a single IV (30 ug/kg) or SC administration (100 ug/kg) were analyzed by flow cytometry (a) to quantitate T lymphocyte, B lymphocyte, NK cells and monocytes as indicated. T lymphocytes were selected using CD2 + /CD16-, B lymphocytes with CD2-/CD20 + /HLA-DR + , and NK cells with CD2 + / CD16 + , and (b) to quantitate cell-bound trispecific Ab levels on CD4+ T cells (CD2 + /CD4 + /CD8−) or CD8 cells (CD2 + /CD4-/CD8 + ). CD38 trispecific binding was determined using anti-human IgG4 (Southern Biotech) to determine percentage of cells bound with trispecific Ab on indicated subpopulations of T cells. The data were plotted as average of n = 3 animals ± SD.
Extended Data Fig. 5 Effect of CD38 trispecific Ab on T lymphocyte subset distribution in the blood of NHPs.
Blood samples from animals (n = 3) treated with a single SC administration (100 ug/kg) were analyzed by flow cytometry to quantitate CD4, CD8, CD8 effector memory and T regulatory cells as indicated. CD4+ T cells were selected using cell surface staining for CD2 + /CD4 + /CD8-, CD8 + T cells with CD2 + /CD4-/CD8 + ; CD8 + effector memory by CD2 + /CD4-/CD8 + /CD45RA + /CD197- and regulatory T cells with CD2 + /CD4 + /CD8-/CD127-/CD25hi. The data were plotted as individual animals.
Extended Data Fig. 6 Effect of intra-animal dose escalation on serum levels and inflammatory cytokine release in NHP.
Animals (n = 3) were injected IV with the CD38 trispecific Ab at 10 ug/kg on day 1, 30 ug/kg on day 4, and 100 ug/kg on day 7. Serum samples were measured for trispecific Ab serum concentration using a Gyrolab immunoassay (Gyros Protein Technologies). Plasma samples were analyzed for inflammatory cytokines IL-6 and IFN-γ as described in Methods. The data were plotted as individual animals.
Cytolytic activity of the CD38/CD28xCD3 trispecific FALA mutant Ab against indicated CD38+CD28- lines, including acute myelocytic leukemia (AML (KG-1)), a B cell lymphoma (OCI-Ly19), acute T lymphocytic leukemia (ALL (KOPN8)), and chronic lymphocytic lymphoma (CLL(Z-138)). The representative nonlinear regression graphs (ordinary fit) ± SD are from 2 PBMC donors performed in two separate experiments.
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Wu, L., Seung, E., Xu, L. et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation. Nat Cancer 1, 86–98 (2020). https://doi.org/10.1038/s43018-019-0004-z
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