Supplementary Materials

The PDF file includes:

• Materials and Methods
• Fig. S1. Schematic of T cell activation at the immune synapse via signal 1 and signal 2.
• Fig. S2. Schematic of TSAxCD3 bispecific and mechanism for T cell activation at the immune synapse.
• Fig. S3. Schematic of TSAxCD3 and TSAxCD28 bispecifics and mechanism for T cell activation.
• Fig. S4. Introduction of co-stimulatory ligand expression on tumor cell lines inhibits tumor growth in vivo.
• Fig. S5. Biacore sensorgrams for binding of mIgG2a, hIgG4, and hIgG4s to mouse FcγRs.
• Fig. S6. PSMAxCD28 potentiates T cell activation only in the presence of TCR stimulation by CD20xCD3.
• Fig. S7. PSMAxCD28 and PSMAxCD3 bispecifics bind simultaneously to PSMA⁺ tumor cells.
• Fig. S8. TSA bispecifics potentiate T cell activation only in the presence of TSA expression on target cells.
• Fig. S9. PSMAxCD28 potentiates cynomolgus T cell activation only in the presence of TCR stimulation by PSMAxCD3.
• Fig. S10. MUC16xCD28 and MUC16xCD3 bispecifics bind simultaneously to MUC16⁺ tumor cells.
• Fig. S11. MUC16xCD28 potentiates cynomolgus T cell activation only in the presence of TCR stimulation by MUC16xCD3.
• Fig. S12. MUC16xCD28 potentiates purified T cell activation and cytotoxicity dependent on MUC16xCD3.
• Fig. S13. MUC16 bispecifics bind to MUC16-expressing cells in the presence of soluble.
• Fig. S14. MUC16xCD28 and MUC16xCD3 combination induces cytokines in humanized xenogenic mouse models.
• Fig. S15. MUC16 bispecifics bind to MUC16-expressing cells in the presence of soluble CA-125.
• Fig. S16. Targeting strategy for the generation of Cd3edg^hu/hu, Cd28^hu/hu, and Folh1^hu/hu mice.
• Fig. S17. Expression and functional validation of CD3 and CD28 in hPSMA/hCD3/hCD28 mice.
• Fig. S18. Validation of human PSMA expression in PSMA humanized mice.
• Fig. S19. Histology of PSMA⁺ tissue in hPSMA/hCD3/hCD28 mice treated with PSMA bispecifics.
• Fig. S20. Intratumoral T cells recognize p15E peptide.
• Fig. S21. PSMAxCD28 enhances anti-tumor immunity and T cell activation induced by PSMAxCD3.
• Fig. S22. CD28 superagonist induces systemic cytokine response in CD28 humanized mice.
• Table S1. Biacore kinetics of MUC16, PSMA, and CD28.
• Table S2. Biacore kinetics of mouse FcγRs.
• Table S3. Pharmacokinetic analysis of PSMA bispecifics in hPSMA/hCD3/hCD28 mice.
• Table S4. Summary of cynomolgus monkey toxicity study.

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Other Supplementary Material for this manuscript includes the following:

• Data file S1 (Microsoft Excel format). Primary data