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Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation


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.

Fig. 1: Optimization of α-CD3×CD28 CODV-Fab antibody and assessment of anti-CD28 contribution to T cell stimulation, survival and proliferation.
Fig. 2: CD38 trispecific antibody with minimal FcR binding reduced non-specific cytokine release by human PBMCs in vitro and lysed human multiple myeloma cells.
Fig. 3: Characterization of in vitro T cell subset expansion in response to CD38/CD28×CD3.
Fig. 4: CD28 expression on multiple myeloma cells increases susceptibility to cytolysis.
Fig. 5: In vitro activation of human PBMCs by α-CD28 superagonist requires bivalency of the antibody.
Fig. 6: Structural basis for CD3 and CD28 target recognition.
Fig. 7: Comparison of the effects of subcutaneous versus intravenous routes of administration on maximum concentration, inflammatory cytokine release and CD4/CD8 cell binding in vivo in NHPs.
Fig. 8: Vital microscopy analysis of myeloma cell cytolysis by the CD38 trispecific Ab in vitro in the presence of primary human T cells and protection against disseminated human multiple myeloma cell tumor growth in vivo.

Data availability

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. 14 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.


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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.

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Z.-y.Y., L.W., E.S., L.X., E.R., R.R.W., V.C.-R., F.S., P.P., N.E.M., S.R., K.R., P.K., J.F., R.V. and G.J.N. designed the research. Z.-y.Y., L.W., E.S., L.X., R.R.W., D.M.L., V.C.-R., B.C., C.P., B.Z., H.Q., P.K., J.F. and R.V. carried out the research. Z.-y.Y., L.W., E.S., L.X., D.M.L., V.P., B.O., G.U., E.F., C.B., T.D., T.B., S.P., C.L., A.P., G.D. and Z.S. performed the experiments. Z.-y.Y., L.W., E.S., L.X., R.R.W., D.M.L., V.C.-R., B.C., C.P., P.P., G.U., N.E.M., P.K., J.F., R.V., A.L. and G.J.N. analyzed the data. Z.-y.Y., L.W., E.S., D.M.L., R.R.W., G.U., P.P., T.B., N.E.M. and G.J.N. wrote the paper. All authors reviewed the paper.

Corresponding authors

Correspondence to Zhi-yong Yang or Gary J. Nabel.

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Competing interests

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.

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Extended data

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.

Extended Data Fig. 2 Binding of alternative human IgG4 Fc mutants to human Fc receptors.

(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. Source data

Extended Data Fig. 3 Crystal structure and modeling of the CD38 trispecific Ab.

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.

Extended Data Fig. 7 Cytolysis of the CD38 trispecific Ab of CD38+ hematological cancer cell lines.

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.

Supplementary information

Supplementary Information

Supplementary Tables 1–6.

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Supplementary Video 1

Time-lapse vital microscopy of myeloma cell cytolysis by the CD38 trispecific antibody in vitro in the presence of primary human T cells.

Source data

Source Data Fig. 1

Flow cytometry statistical source data

Source Data Fig. 2

Flow cytometry statistical source data

Source Data Fig. 3

Flow cytometry statistical source data

Source Data Fig. 4

Flow cytometry statistical source data

Source Data Extended Data Fig. 2

SPR and ELISA source data

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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).

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