Bispecific molecules

Abstract

Bispecific molecules that target cancer-associated fibroblasts are provided herein. Methods of treatment comprising administering a pharmaceutical composition comprising a bispecific molecule as described herein are further provided.

Description

[0001] BISPECIFIC MOLECULES CROSS-REFERENCE TO RELATED APPLICATIONS [1] This application claims the benefit of U.S. Provisional Patent Application No.63 / 596,211, filed November 3, 2023, which is hereby incorporated by reference in its entirety. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY [2] Incorporated by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: 139 bytes XML file named "BISPECIFIC MOLECULES Sequence Listing"; created on November 3, 2023. FIELD OF THE INVENTION [3] The present invention relates to bispecific molecules that target cancer-associated fibroblasts (CAFs) for the treatment of cancer. BACKGROUND [4] The Fibroblast-activation protein ^ (FAP or FAP ^), also known as Seprase, is a type II integral membrane serine peptidase. FAP is expressed selectively in reactive stromal fibroblasts of more than 90% of epithelial malignancies (primary and metastatic) examined, including lung, colorectal, bladder, ovarian and breast carcinomas, and in malignant mesenchymal cells of bone and soft tissue sarcomas, while it is generally absent from normal adult tissues (Brennen et al., Mol. Cancer Ther. 11(2): 257–266 (2012); Garin-Chesa et al., Proc Natl Acad Sci USA 87, 7235-7239 (1990); Rettig et al., Cancer Res.53:3327–3335 (1993); Rettig et al., Proc Natl Acad Sci USA 85, 3110-3114 (1988)). FAP is also expressed on certain malignant tumor cells. Due to its expression in many common cancers and its restricted expression in normal tissues, FAP has been considered a promising antigenic target for imaging, diagnosis and therapy of a variety of cancers. [5] CD40 is a member of the TNF receptor (TNFR) superfamily that is preferentially expressed by antigen presenting cells (APCs), such as dendritic cells, B cells and macrophages. Interaction with its trimeric ligand on activated T helper cells results in APC activation that includes upregulation of cytokines / chemokines (such as interleukin-12 (IL-12) and CXCL10), proteins involved in antigen- presentation (such as MHC class I and II ligands), T cell costimulatory ligands (such as CD80 and CD86), and array of other immune modulatory factors (i.e., adhesion molecules and other TNFRs). These “licensed” APCs can then trigger a cascade of events leading to induction of robust adaptive immune responses. [6] Therapeutics capable of activating CD40 signaling have the potential to inflame solid tumors through their ability to enhance generation of anti-tumor T cells and enhance T cell recruitment directly into the tumor lesion. Preclinical studies with anti-CD40 agonists suggest that triggering CD40   with crosslinking antibodies on APCs can substitute for CD4 T cell help to license APCs and facilitate the activation as well as expansion of CD8 effector T cells. In addition, CD40-activated macrophages may also exert direct tumoricidal functions. These anti-CD40 agonist antibodies have been demonstrated to be efficacious in multiple syngeneic tumor models alone or in combination with other therapies. Based on these pre-clinical studies several CD40 agonistic antibodies are under investigation in phase I / II clinical trials of solid tumor patients. To date, these monoclonal anti-CD40 antibodies have shown some signs of clinical activity, but are often associated with immune-related adverse effects, such as cytokine release syndrome and evidence of liver damage. These toxicities limit the dose of CD40 agonist that can be delivered, and thus may result in insufficient activation of the CD40 pathway in tumor-associated APC population, negatively impacting the efficacy of this therapeutic approach. Therapeutics capable of activation of CD40 signaling within tumor tissue only (while lacking CD40 agonist activities in normal tissues) may improve anti-tumor effects while reducing systemic toxicities. [7] The available pre-clinical and clinical data clearly demonstrate that there is a high clinical need for effective agonists of CD40 that are able to induce and enhance effective endogenous immune responses to cancer. SUMMARY [8] Based on the disclosure provided herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments (E). [9] The use of section headings herein is merely for the convenience of reading, and not intended to be limiting per se. The entire document is intended to be viewed as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated. E1. A bispecific molecule, comprising: (1) a Fab moiety that binds to FAP, a scFv moiety that binds to a second antigen, and a Fc region comprising CH2 and CH3 domains; or (2) a scFv moiety that binds to FAP, a Fab moiety that binds to a second antigen, and a Fc region comprising CH2 and CH3 domains; wherein: (i) said Fab moiety comprises two chains: a heavy chain comprising a heavy chain variable domain A (VHA) and a CH1 domain, and a light chain comprising a light chain variable domain A (VLA) and a CL domain; (ii) said scFv moiety comprises a heavy chain variable domain B (VHB) and a light chain variable domain B (VLB), wherein said VHB and VLB are connected via a first linker; (iii) said Fab is connected to said scFv moiety via a second linker; and (iv) said scFv moiety is connected to said CH2 domain via a third linker. E2. A bispecific molecule, comprising:   (i) two Fab moieties that bind to FAP, wherein each Fab moiety comprises two chains: a heavy chain comprising a heavy chain variable domain A (VHA) and a CH1 domain, and a light chain comprising a light chain variable domain A (VLA) and a CL domain; (ii) two scFv moieties that bind to CD40, wherein each scFv comprises a heavy chain variable domain B (VHB) and a light chain variable domain B (VLB), wherein said VHBand VLBare connected via a first linker; and (iii) one Fc region that comprises two chains, each chain comprising a monomeric CH2 domain and a monomeric CH3 domain; wherein a second linker connects the C-terminus of one CH1 domain to the N-terminus of one scFv, and a third linker connects the C-terminus of one scFv to the N-terminus of one chain of the Fc region. E3. A bispecific molecule, comprising: (i) two Fab moieties that bind to CD40, wherein each Fab moiety comprises two chains: a heavy chain comprising a heavy chain variable domain A (VHA) and a CH1 domain, and a light chain comprising a light chain variable domain A (VLA) and a CL domain; (ii) two scFv moieties that bind to FAP, wherein each scFv comprises a heavy chain variable domain B (VHB) and a light chain variable domain B (VLB), wherein said VHB and VLB are connected via a first linker; and (iii) one Fc region that comprises two chains, each chain comprising a monomeric CH2 domain and a monomeric CH3 domain; wherein a second linker connects the C-terminus of one CH1 domain to the N-terminus of one scFv, and a third linker connects the C-terminus of one scFv to the N-terminus of one chain of the Fc region. E4. A bispecific molecule, comprising: (i) two copies of a heavy chain that comprises, from N-terminus to C-terminus: VHA – CH1 – second linker – scFv – third linker – monomeric CH2 – monomeric CH3, wherein said scFv comprises a VHBand a VLB, and wherein said VHBand VLBare connected via a first linker; and (ii) two copies of a light chain that comprises, from N-terminus to C-terminus: VLA – CL; wherein said VHA-CH1 and said VLA-CL form a Fab that binds to CD40, said scFv binds FAP, and said two copies of monomeric CH2 – monomeric CH3 form a Fc region. E5. A bispecific molecule, comprising: (i) two copies of a heavy chain that comprises, from N-terminus to C-terminus: VHA – CH1 – second linker – scFv – third linker – monomeric CH2 – monomeric CH3, wherein said scFv comprises a VHBand a VLB, and wherein said VHBand VLBare connected via a first linker; and (ii) two copies of a light chain that comprises, from N-terminus to C-terminus: VLA – CL; wherein said VHA-CH1 and said VLA-CL form a Fab that binds to FAP, said scFv binds to CD40, and said two copies of monomeric CH2 – monomeric CH3 form a Fc region.   E6. The bispecific molecule of any one of E1-E5, wherein said first linker, second linker, and third linker each independently comprises any one of the amino acid sequences of SEQ ID NOs: 76-89. E7. The bispecific molecule of any one of E1-E6, wherein said second linker connects the C- terminus of said CH1 domain to the N-terminus of the scFv. E8. The bispecific molecule of any one of E1-E7, wherein said second linker comprises the amino acid sequence of (Gly-Gly-Gly-Gly-Ser)2 (SEQ ID NO:83). E9. The bispecific molecule of any one of E1-E8, wherein said third linker connects the C- terminus of the scFv to the N-terminus of the Fc region. E10. The bispecific molecule of any one of E1-E9, wherein said third linker comprises the amino acid sequence of GGGG (SEQ ID NO:76). E11. A bispecific molecule, comprising: (A) a Fab moiety that binds to CD40, a scFv moiety that binds to FAP, and a Fc region comprising CH2 and CH3 domains; or (B) a Fab moiety that binds to FAP, a scFv moiety that binds to CD40, and a Fc region comprising CH2 and CH3 domains. wherein: (i) said Fab moiety comprises two chains: a heavy chain comprising a heavy chain variable domain A (VHA) and a CH1 domain, and a light chain comprising a light chain variable domain A (VLA) and a CL domain; (ii) said scFv moiety comprises a heavy chain variable domain B (VHB) and a light chain variable domain B (VLB), wherein said VHBand VLBare connected via a first linker; (iii) said Fab is connected to said CH2 domain; and (iv) said scFv moiety is connected to said CH3 domain via a second linker. E12. A bispecific molecule, comprising: (i) two Fab moieties that bind to FAP, wherein each Fab moiety comprises two chains: a heavy chain comprising a heavy chain variable domain A (VHA) and a CH1 domain, and a light chain comprising a light chain variable domain A (VLA) and a CL domain; (ii) two scFv moieties that bind to CD40, wherein each scFv comprises a heavy chain variable domain B (VHB) and a light chain variable domain B (VLB), wherein said VHBand VLBare connected via a first linker; and (iii) one Fc region that comprises two chains, each chain comprising a monomeric CH2 domain and a monomeric CH3 domain; wherein the C-terminus of one CH1 domain is connected to the N-terminus of one chain of the Fc region, and wherein a second linker connects the C-terminus of one CH3 domain to the N-terminus of one scFv. E13. A bispecific molecule, comprising:   (i) two Fab moieties that bind to CD40, wherein each Fab moiety comprises two chains: a heavy chain comprising a heavy chain variable domain A (VHA) and a CH1 domain, and a light chain comprising a light chain variable domain A (VLA) and a CL domain; (ii) two scFv moieties that bind to FAP, wherein each scFv comprises a heavy chain variable domain B (VHB) and a light chain variable domain B (VLB), wherein said VHBand VLBare connected via a first linker; and (iii) one Fc region that comprises two chains, each chain comprising a monomeric CH2 domain and a monomeric CH3 domain; wherein the C-terminus of one CH1 domain is connected to the N-terminus of one chain of the Fc region, and wherein a second linker connects the C-terminus of one CH3 domain to the N-terminus of one scFv. E14. A bispecific molecule, comprising: (i) two copies of a heavy chain that comprises, from N-terminus to C-terminus: VHA– CH1 – monomeric CH2 – monomeric CH3 – second linker – scFv, wherein said scFv comprises a VHB and a VLB, and wherein said VHB and VLB are connected via a first linker; and (ii) two copies of a light chain that comprises, from N-terminus to C-terminus: VLA – CL; wherein said VHA-CH1 and said VLA-CL form a Fab that binds to CD40, said scFv binds FAP, and said two copies of monomeric CH2 – monomeric CH3 form a Fc region. E15. A bispecific molecule, comprising: (i) two copies of a heavy chain that comprises, from N-terminus to C-terminus: VHA– CH1 – monomeric CH2 – monomeric CH3 – second linker – scFv, wherein said scFv comprises a VHB and a VLB, and wherein said VHB and VLB are connected via a first linker; and (ii) two copies of a light chain that comprises, from N-terminus to C-terminus: VLA – CL; wherein said VHA-CH1 and said VLA-CL form a Fab that binds to FAP, said scFv binds to CD40, and said two copies of monomeric CH2 – monomeric CH3 form a Fc region. E16. The bispecific molecule of any one of E11-E15, wherein said second linker connects the C- terminus of the Fc region to the N-terminus of said scFv. E17 The bispecific molecule of any one of E11-E16, wherein said first linker and second linker each independently comprises any one of the amino acid sequences of SEQ ID NOs:76-89. E18. The bispecific molecule of any one of E11-E17, wherein said second linker comprises the amino acid sequence of (Gly-Gly-Gly-Gly-Ser)2(SEQ ID NO:83). E19. The bispecific molecule of any one of E11-E18, wherein said Fab is connected to said Fc region to form an IgG moiety.   E20. The bispecific molecule of any one of E1-E19, comprising a CD40 antigen-binding moiety that comprises: (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:31; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 32. E21. The bispecific molecule of any one of E1-E20, comprising a CD40 antigen-binding moiety that comprises: (i) a CDR-H1 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1; (ii) a CDR-H2 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2; (iii) a CDR-H3 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3; (iv) a CDR-L1 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4; (v) a CDR-L2 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5; and (vi) a CDR-L3 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6. E22. The bispecific molecule of any one of E1-E21, comprising a CD40 antigen-binding moiety that comprises: a CDR-H1 comprising SEQ ID NO:1, a CDR-H2 comprising SEQ ID NO:2, a CDR-H3 comprising SEQ ID NO:3, a CDR-L1 comprising SEQ ID NO:4, a CDR-L2 comprising SEQ ID NO:5, and a CDR-L3 comprising SEQ ID NO: 6. E23. The bispecific molecule of any one of E1-E22, comprising a CD40 antigen-binding moiety that comprises a VL framework derived from a human germline V ^ framework sequence, such as a V ^1 framework sequence or a V ^2 framework sequence. E24. The bispecific molecule of any one of E1-E22, comprising a CD40 antigen-binding moiety that comprises a VL framework derived from a human germline V ^ framework sequence, such as a V ^1 framework sequence, a V ^2 framework sequence, or V ^3 framework sequence. E25. The bispecific molecule of any one of E1-E23, comprising a CD40 antigen-binding moiety that comprises a VH framework derived from a human germline VH1, VH2, VH3, VH4, or VH5 framework sequence.   E26. The bispecific molecule of any one of E1-E25, comprising a CD40 antigen-binding moiety that comprises a VH framework derived from a human germline VH1 framework sequence. E27. The bispecific molecule of any one of E1-E25, comprising a CD40 antigen-binding moiety that comprises a VH framework derived from a human germline VH2 framework sequence. E28. The bispecific molecule of any one of E1-E25, comprising a CD40 antigen-binding moiety that comprises a VH framework derived from a human germline VH3 framework sequence. E29. The bispecific molecule of any one of E1-E25, comprising a CD40 antigen-binding moiety that comprises a VH framework derived from a human germline VH4 framework sequence. E30. The bispecific molecule of any one of E1-E25, comprising a CD40 antigen-binding moiety that comprises a VH framework derived from a human germline VH5 framework sequence. E31. The bispecific molecule of any one of E1-E30, comprising a CD40 antigen-binding moiety that comprise a VL framework sequence, wherein said VL framework sequence is at least 90% identical to the human germline framework sequence from which it is derived. E32. The bispecific molecule of any one of E1-E31, comprising a CD40 antigen-binding moiety that comprise a VH framework sequence, wherein said VH framework sequence is at least 90% identical to the human germline framework sequence from which it is derived. E33. The bispecific molecule of any one of E1-E32, comprising a CD40 antigen-binding moiety that comprises a VL framework sequence and a VH framework sequence, wherein said VL framework sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human germline framework sequence from which it is derived, and wherein said VH framework sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human germline framework sequence from which it is derived. E34. The bispecific molecule of any one of E1-E33, comprising a CD40 antigen-binding moiety that comprises a heavy chain variable region (VH) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 31. E35. The bispecific molecule of any one of E1-E34, comprising a CD40 antigen-binding moiety that comprises a light chain variable region (VL) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 32.   E36. The bispecific molecule of any one of E1-E36, comprising a CD40 antigen-binding moiety that is an scFv. E37. The bispecific molecule of any one of E1-E36, comprising a FAP antigen-binding moiety that comprises: (1) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 33, and the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 34; (2) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 35, and the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 36; (3) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 37, and the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 38; (4) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 39, and the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 40; or (5) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 41, and the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 42; E38. The bispecific molecule of any one of E1-E37, comprising a FAP antigen-binding moiety that comprises: (i) a CDR-H1 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7, 13, 19, or 25; (ii) a CDR-H2 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8, 14, 20, or 26; (iii) a CDR-H3 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9, 15, 21, or 27; (iv) a CDR-L1 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10, 16, 22, or 28; (v) a CDR-L2 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11, 17, 23, or 29; and (vi) a CDR-L3 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12, 18, 24, or 30. E39. The bispecific molecule of any one of E1-E38, comprising a FAP antigen-binding moiety that comprises: (1) a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 comprising SEQ ID NOs.6-12, respectively;   (2) a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 comprising SEQ ID NOs.13-18, respectively; (3) a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 comprising SEQ ID NOs.19-24, respectively; or (4) a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 comprising SEQ ID NOs.25-30, respectively. E40. The bispecific molecule of any one of E1-E39, comprising a FAP antigen-binding moiety that comprises a VL framework derived from a human germline V ^ framework sequence, such as a V ^1 framework sequence or a V ^2 framework sequence. E41. The bispecific molecule of any one of E1-E39, comprising a FAP antigen-binding moiety that comprises a VL framework derived from a human germline V ^ framework sequence, such as a V ^1 framework sequence, a V ^2 framework sequence, or V ^3 framework sequence. E42. The bispecific molecule of any one of E1-E41, comprising a FAP antigen-binding moiety that comprises a VH framework derived from a human germline VH1, VH2, VH3, VH4, or VH5 framework sequence. E43. The bispecific molecule of any one of E1-E42, comprising a FAP antigen-binding moiety that comprises a VH framework derived from a human germline VH1 framework sequence. E44. The bispecific molecule of any one of E1-E42, comprising a FAP antigen-binding moiety that comprises a VH framework derived from a human germline VH2 framework sequence. E45. The bispecific molecule of any one of E1-E42, comprising a FAP antigen-binding moiety that comprises a VH framework derived from a human germline VH3 framework sequence. E46. The bispecific molecule of any one of E1-E42, comprising a FAP antigen-binding moiety that comprises a VH framework derived from a human germline VH4 framework sequence. E47. The bispecific molecule of any one of E1-E42, comprising a FAP antigen-binding moiety that comprises a VH framework derived from a human germline VH5 framework sequence. E48. The bispecific molecule of any one of E1-E47, comprising a FAP antigen-binding moiety that comprise a VL framework sequence, wherein said VL framework sequence is at least 90% identical to the human germline framework sequence from which it is derived. E49. The bispecific molecule of any one of E1-E48, comprising a FAP antigen-binding moiety that comprise a VH framework sequence, wherein said VH framework sequence is at least 90% identical to the human germline framework sequence from which it is derived.   E50. The bispecific molecule of any one of E1-E49, comprising a FAP antigen-binding moiety that comprises a VL framework sequence and a VH framework sequence, wherein said VL framework sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human germline framework sequence from which it is derived, and wherein said VH framework sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human germline framework sequence from which it is derived. E51. The bispecific molecule of any one of E1-E50, comprising a FAP antigen-binding moiety that comprises: (1) a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 33, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 34; (2) a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 35, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 36; (3) a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 37, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 38; (4) a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 39, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 40; or (5) a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 41, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 42. E52. The bispecific molecule of any one of E1-E35 and E37-E51, comprising a FAP antigen- binding moiety that is an scFv.   E53. The bispecific molecule of any one of E1-E52, wherein said first linker comprises the amino acid sequence of (Gly-Gly-Gly-Gly-Ser)3(SEQ ID NO:84). E54. The bispecific molecule of any one of E1-E35 and E37-E53, comprising a scFv moiety that binds to FAP, wherein said scFv comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:93. E55. The bispecific molecule of any one of E1-E35 and E37-E53, comprising a scFv moiety that binds to FAP, wherein said scFv comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:94. E56. The bispecific molecule of any one of E1-E35 and E37-E53, comprising a scFv moiety that binds to FAP, wherein said scFv comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:95. E57. The bispecific molecule of any one of E1-E35 and E37-E53, comprising a scFv moiety that binds to FAP, wherein said scFv comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:96. E58. The bispecific molecule of any one of E1-E35 and E37-E53, comprising a scFv moiety that binds to FAP, wherein said scFv comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:97. E59. The bispecific molecule of any one of E1-E58, wherein said CH1 domain is the CH1 domain of an IgG (for example IgG1, lgG2, lgG3, or lgG4). E60. The bispecific molecule of any one of E1-E59, wherein said CH1 domain is the CH1 domain of a human IgG (for example, human IgG1, human IgG2, human IgG3, or human IgG4). E61. The bispecific molecule of any one of E1-E60, wherein said CH1 domain comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:47, 64, 68, or 72. E62. The bispecific molecule of any one of E1-E61, wherein said CH1 domain comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 90%, at least   91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:47. E63. The bispecific molecule of any one of E1-E62, wherein said CL domain is a kappa or lambda light chain CL. E64. The bispecific molecule of any one of E1-E63, wherein said CL domain is a human kappa or human lambda light chain CL. E65. The bispecific molecule of any one of E1-E64, wherein said CL domain comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:43 or 44. E66. The bispecific molecule of any one of E1-E64, wherein said CL domain comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:45 or 46. E67. The bispecific molecule of any one of E1-E66, wherein said Fc region is the Fc region of an IgA (for example IgA1 or lgA2), IgD, IgE, IgM, or IgG (for example IgG1, lgG2, lgG3, or lgG4). E68. The bispecific molecule of any one of E1-E67, wherein the Fc region is the Fc region of an IgG. E69. The bispecific molecule of E68, wherein the IgG is selected from the group consisting of IgG1, lgG2, lgG3, and lgG4. E70. The bispecific molecule of E69, wherein the IgG is IgG1, IgG2, or IgG4. E71. The bispecific molecule of any one of E1-E70, wherein said Fc region comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:48, 55, 56, 57, 58, 59, 60, 66, 70, or 74. E72. The bispecific molecule of any one of E1-E71, wherein said Fc region comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:48, 55, 56, 57, 58, 59, or 60.   E73. The bispecific molecule of any one of E1-E72, wherein said Fc region is derived from an IgG Fc, and further comprises one or more mutations selection from the group consisting of: L234A, L235A, L235E, G237A, and combination thereof (numbering according to the EU index). E74. The bispecific molecule of E73, comprising L234A and L235A mutations. E75. The bispecific molecule of any one of E1-E74, wherein said Fc region is derived from an IgG Fc, and further comprises one or more mutations selection from the group consisting of: V259C, A287C, R292C, V302C, L306C, V323C, I332C, and a combination thereof (numbering according to the EU index). E76. The bispecific molecule of any one of E1-E75, wherein said Fc region is derived from an IgG Fc, and further comprises one or more mutations selection from the group consisting of: L242C, A287C, R292C, N297G, V302C, L306C, K334C, and a combination thereof (numbering according to the EU index). E77. The bispecific molecule of E76, comprising a N297G mutation. E78. The bispecific molecule of E76, comprising A287C, N297G, and L306C mutations. E79. The bispecific molecule of E76, comprising R292C, N297G, and V302C mutations. E80. The bispecific molecule of any one of E1-E79, wherein said Fc region is derived from an IgG Fc, and further comprises one or more mutations selection from the group consisting of: M252Y, S254T, T256E, and a combination thereof. E81. The bispecific molecule of E80, comprising M252Y, S254T, T256E mutations. E82. The bispecific molecule of any one of E1-E81, wherein the lysine residue (K) at the C- terminus of the Fc region is deleted. E83. The bispecific molecule of any one of E1-E81, wherein the lysine residue (K) at the C- terminus of the Fc region is present. E84. The bispecific molecule of any one of E1-E81, wherein the glycine and lysine residues (GK) at the C-terminus of the Fc region are present. E85. The bispecific molecule of any one of E1-E81, wherein the glycine and lysine residues (GK) at the C-terminus of the Fc region are deleted. E86. A bispecific molecule comprising (i) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 99, and (ii) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at   least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 91. E87. A bispecific molecule comprising: two copies of a first polypeptide comprising SEQ ID NO: 99, and two copies of a second polypeptide comprising SEQ ID NO: 91. E88. A bispecific molecule comprising (i) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 101, and (ii) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 91. E89. A bispecific molecule comprising: two copies of a first polypeptide comprising SEQ ID NO: 101, and two copies of a second polypeptide comprising SEQ ID NO: 91. E90. A bispecific molecule comprising (i) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 103, and (ii) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 91. E91. A bispecific molecule comprising: two copies of a first polypeptide comprising SEQ ID NO: 103, and two copies of a second polypeptide comprising SEQ ID NO: 91. E92. A bispecific molecule comprising (i) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 105, and (ii) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 91. E93. A bispecific molecule comprising: two copies of a first polypeptide comprising SEQ ID NO: 105, and two copies of a second polypeptide comprising SEQ ID NO: 91. E94. A bispecific molecule comprising (i) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 107, and (ii) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 91.   E95. A bispecific molecule comprising: two copies of a first polypeptide comprising SEQ ID NO: 107, and two copies of a second polypeptide comprising SEQ ID NO: 91. E96. A bispecific molecule comprising (i) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 109, and (ii) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 91. E97. A bispecific molecule comprising: two copies of a first polypeptide comprising SEQ ID NO: 109, and two copies of a second polypeptide comprising SEQ ID NO: 91. E98. A bispecific molecule comprising (i) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 111, and (ii) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 91. E99. A bispecific molecule comprising: two copies of a first polypeptide comprising SEQ ID NO: 111, and two copies of a second polypeptide comprising SEQ ID NO: 91. E100 A bispecific molecule comprising (i) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 113, and (ii) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 91. E101. A bispecific molecule comprising: two copies of a first polypeptide comprising SEQ ID NO: 113, and two copies of a second polypeptide comprising SEQ ID NO: 91. E102. A bispecific molecule comprising (i) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115, and (ii) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 91. E103. A bispecific molecule comprising: two copies of a first polypeptide comprising SEQ ID NO: 115, and two copies of a second polypeptide comprising SEQ ID NO: 91.   E104. A bispecific molecule comprising (i) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 117, and (ii) a polypeptide comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 91. E105. A bispecific molecule comprising: two copies of a first polypeptide comprising SEQ ID NO: 117, and two copies of a second polypeptide comprising SEQ ID NO: 91. E106. A nucleic acid comprising a nucleotide sequence encoding the bispecific molecule of any one of E1-E105. E107. The nucleic acid of E106, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any one of SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, or 118, or a fragment of SEQ ID NOs.: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, or 118. E108. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:91. E109. The nucleic acid of E108, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:98. E110. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:99. E111. The nucleic acid of E110, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:100. E112. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:101. E113. The nucleic acid of E112, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:102. E114. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:103.   E115. The nucleic acid of E114, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:104. E116. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:105. E117. The nucleic acid of E116, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:106. E118. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:107. E119. The nucleic acid of E118, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:108. E120. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:109. E121. The nucleic acid of E120, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:110. E122. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:111. E123. The nucleic acid of E122, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:112. E124. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:113. E125. The nucleic acid of E124, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:114. E126. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:115. E127. The nucleic acid of E126, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%,   at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:116. E128. A nucleic acid encoding a polypeptide comprising the amino acid of SEQ ID NO:117. E129. The nucleic acid of E128, comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:118. E130. A vector comprising the nucleic acid of any one of E106-E129. E131. A host cell comprising the nucleic acid of any one of E106-E129, or the vector of E130. E132. The host cell of E131, wherein said host cell is a mammalian cell. E133. The host cell of E132, wherein said host cell is a CHO cell or a HEK-293 cell, or an Sp2.0 cell. E134. A kit comprising (i) a bispecific molecule of any one of E1-E105; a nucleic acid of any one of E106-E129, a vector of E130, a host cell of any one of E131-E133, or a combination thereof, and (ii) instructions for use. E135. A pharmaceutical composition comprising (i) a bispecific molecule of any one of E1-E105; a nucleic acid of any one of E106-E129, a vector of E130, a host cell of any one of E131-E133, or a combination thereof; and (ii) a pharmaceutically acceptable carrier, excipient, or diluent. E136. A method of making the bispecific molecule of any one of E1-E105, comprising culturing the host cell of any one of E131-E133, under a condition wherein the bispecific molecule is expressed. E137. The method of E136, further comprising harvesting the expressed bispecific molecule. E138. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific molecule of any one of E1-E105, or the pharmaceutical composition of E135. E139. The method of E138, wherein cancer is solid tumor. E140. The method of E138 or E139, wherein said cancer comprises stromal cells. E141. The method of any one of E138-E140, wherein said cancer comprises stromal cells that express FAP. E142. The method of any one of E138-E141, wherein said subject is a human.   E143. The method of any one of E138-E142, wherein the cancer is brain cancer, bladder cancer, breast cancer, clear cell kidney cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, gastric cancer, head / neck squamous cell carcinoma, lip cancer, oral cancer, liver cancer, lung squamous cell carcinoma, melanoma, mesothelioma, non-small-cell lung cancer (NSCLC), non- melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma, small-cell lung cancer (SCLC), Squamous Cell Carcinoma of the Head and Neck (SCCHN), triple negative breast cancer, renal cell carcinoma, or thyroid cancer. E144. The method of any one of E138-E142, wherein the cancer is adrenocortical tumor, alveolar soft part sarcoma, carcinoma, chondrosarcoma, desmoid tumors, desmoplastic small round cell tumor, endocrine tumors, endodermal sinus tumor, epithelioid hemangioendothelioma, Ewing sarcoma, germ cell tumor, hepatoblastoma, hepatocellular carcinoma, melanoma, nephroma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), osteosarcoma, paraspinal sarcoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, or Wilms tumor. E145. The method of any one of E138-E142, wherein the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML). E146. The method of any one of E138-E142, wherein the cancer is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or small lymphocytic lymphoma (SLL). E147. The method of any one of E138-E146, further comprising administering to said subject a therapeutically effective amount of a PD-1 inhibitor or PD-L1 inhibitor. E148. The method of E147, wherein said PD-1 inhibitor is an antigen-binding molecule that binds to PD-1. E149. The method of E147, wherein said PD-L1 inhibitor is an antigen-binding molecule that binds to PD-L1. E150. The method of any one of E147-E149, wherein said PD-1 or PD-L1 is human PD-1 or human PD-L1. E151. The method of any one of E147-E150, wherein said PD-1 inhibitor is nivolumab, pembrolizumab, cemiplimab, dostarlimab, or an antigen binding fragment thereof. E152. The method of any one of E147-E151, wherein said PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, or an antigen binding fragment thereof.   E153. The method of any one of E147-E152, wherein said bispecific molecule, or pharmaceutical composition, or PD-1 inhibitor, or PD-L1 inhibitor is administered intravenously. E154. The method of any one of E147-E153, wherein said bispecific molecule, or pharmaceutical composition, or PD-1 inhibitor, or PD-L1 inhibitor is administered subcutaneously. E155. The method of any one of E147-E154, wherein said bispecific molecule, pharmaceutical composition, or PD-1 or PD-L1 inhibitor is administered about twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months, once every three months, or once every four months. E156. The bispecific molecule of any one of E1-E105, or the pharmaceutical composition of E135 for use as a medicament. E157. The bispecific molecule of any one of E1-E105, or the pharmaceutical composition of E135 for use in treating cancer in a subject. E158. Use of the bispecific molecule of any one of E1-E105, or the pharmaceutical composition of E135 in the manufacture of a medicament for treating cancer in a subject. E159. Use of the bispecific molecule of any one of E1-E105, or the pharmaceutical composition of E135 for treating cancer in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG.1 is a schematic of the steps taken to generate bispecific molecules comprising a CD40 binding protein and FAP binding protein of the present disclosure. The bispecific molecule cartoon, in IgG-scFv format, is for illustration only. Not all bispecific molecules disclosed herein are in IgG-scFv format. Potential issues in selecting molecules include, but not limited to: purification, chain pairing, hotspot, viscosity, and aggregation issues.

[0011] FIG.2A is an illustration of a bispecific molecule comprising an IgG moiety and two scFv moieties (IgG-scFv). Figure 2B is an illustration of a bispecific molecule comprising two Fab moieties, two scFv moeities, and an Fc. A cysteine clamp may be introduced into scFv (represented as a horizontal line within scFv). The use (or non-use) of cysteine clamp may require evaluation of stability and biologically activities of the scFv.

[0012] FIG. 3 shows the tumor-localized immunostimulatory activity of the mouse surrogate FAP- targeted CD40 bispecific molecule. EMT6 breast preclinical tumor model was used. IHC analysis for FAP expression in EMT6 tumors showed that 10-20% FAP+ (figure not shown). EMT6 tumor-bearing animals were treated with either 30 mg / kg of control isotype mIgG1, 5 mg / kg dose of non-targeted   CD40 mAb or 30 mg / kg of CD40-FAP BsAb intra-peritoneally on study days 11 & 14 post implantation (study randomized at an average tumor volume of 110.48 mm3). PD evaluation was performed at 48 hours post second antibody treatment (day 16). Tumor weights were collected during harvest and used for normalization to determine absolute cell counts in tumors. Single cell suspensions of tumor, draining lymph node (dLN) and spleen were prepared for flow cytometry analysis of myeloid cell proportions and phenotypes. Activation of CD103+DCs is depicted by upregulation of CD86. Each dot represents data obtained from individual mouse. Statistical analysis was performed using one-way ANOVA comparing treatment to control group (*p ^ 0.05; ***p ^ 0.01, ****p ^ 0.001).

[0013] FIGs.4A-4B show that there is no systemic activation noted with the mouse surrogate FAP- targeted CD40 bispecific molecule. EMT6 tumor-bearing animals were treated with a single dose of either 25 mg / kg of control isotype mIgG1, 5 mg / kg of non-targeted CD40 mAb or 25 mg / kg of CD40- FAP bispecific molecule intra-peritoneally on study day 11 post implantation (average tumor volume of 118.11 mm3). Serum was collected at 72 hours post treatment and assessed for levels of pro- inflammatory cytokines (A) and liver enzymes (B). Each dot represents data obtained from individual mouse. Statistical analysis was performed using one-way ANOVA comparing treatment to control group (**p ^ 0.01).

[0014] FIGs.5A-5C show that FAP-targeted CD40 in combination with checkpoint blockade (anti-PD- 1) enhanced tumor growth inhibition in the EMT6 tumor model. Anti-tumor activity of CD40-FAP bispecific molecule in combination with anti-PD1 (clone 29F.1A12) in the EMT6 syngeneic tumor model. EMT6 tumor cells were implanted subcutaneously in the right flank of female wild type Balb / C animals on study day 0. Tumors were randomized and assigned on day 6 into three different treatment groups (n=10 / group) with an average tumor volume of 98.06 mm3. Grp1 - 25 mg / kg of control isotype mIgG1, Grp 2 – 5 mg / kg of non-targeted CD40 mAb + 5 mg / kg anti-PD-1 antibody and Grp 3 - 25 mg / kg of CD40-FAP bispecific + 5 mg / kg anti-PD-1 antibody. Animals were dosed intra- peritoneally on study days 7, 10, 13 and 17 (Q3D x 4). Tumor volume was measured twice per week. Individual tumor growth for the treatment groups is depicted as spider plots in panel (FIG.6A) and mean TV ^ SEM for each group until the last timepoint when all animals were on study (day 17) is depicted in panel (FIG.6B). Statistical analysis to evaluate effect of treatment on tumor size over time relative to control Grp1 was performed using Tukey’s all group comparison analysis. Animals with no measurable tumors defined as Complete Responders (CRs) have been assessed till end of study (day 47). Statistical analysis for survival was performed using the Log-rank (Mantel-Cox) test comparing treatment to control Grp1. FIG.6B: statistical analysis to evaluate effect of treatment on tumor size relative to control Grp1 up to day 17 was performed using 1 way ANOVA Tukey’s all group comparison – IVEA (**** p < 0.001). FIG.6C: treatment compared to control Grp1, using Kaplan- Meier estimator with Mantel-Cox logrank to compare curves (** P < 0.01).

[0015] FIG.6 is a schematic illustrating FAP x CD40 bispecific molecule-mediated clustering (crosslinking) of CD40 on immune cells in proximity to tumor cells, triggering an immune response. In the absence of the tumor antigen FAP (i.e., in normal, non-malignant cells), minimal clustering of   CD40 will occur due to lack of FAP binding, and immune activation will be limited. In contrast, in cancer-associated fibroblasts (“CAF”), FAP is highly expressed (shown as rectangle bars); therefore, through FAP-binding, the bispecific molecule promotes CD40 clustering and immune cell stimulation. DETAILED DESCRIPTION 1. Overview

[0016] Agonistic CD40 antibodies have shown some promising results in early clinical studies. However, common side effects include cytokine release syndromes and thromboembolic events. The early clinical results suggest a limited clinical efficacy and a local administration of the antibody (Vonderheide RH, Glennie M, Clin Cancer Res.2013, 19(5), 1035-1043).

[0017] Seeking to overcome this challenge, the inventors designed bispecific molecules to promote target-mediated clustering of CD40, so that activation of CD40 is localized to tumors. It is understood that upon binding of CD40 ligand, CD40 undergoes multimerization and clustering (sometime also referred to as “crosslinking”). Once the CD40 intracellular domains become clustered, it can initiate downstream responses. Therefore, the CD40 agonists disclosed herein were specifically selected for “crosslinking dependent” agonistic activity, meaning that the agonistic activity of the molecule is dependent upon the crosslinking of CD40. Without CD40 crosslinking, the binding of the molecule to CD40 leads to minimal agonist activity, thereby avoiding toxicities exhibited by previous generation of agonistic CD40 antibodies.

[0018] The activation of CD40 in tumor tissues is achieved through target-specific crosslinking, in particular through the binding of FAP antigen. FAP abundantly expressed in the stroma of many solid tumors by cancer-associated fibroblasts (CAFs). FAP is expressed selectively in reactive stromal fibroblasts of more than 90% of epithelial malignancies (primary and metastatic), including lung, colorectal, bladder, ovarian and breast carcinomas, and in malignant mesenchymal cells of bone and soft tissue sarcomas, while it is generally absent from normal adult tissues. FAP is also expressed on certain malignant tumor cells.

[0019] Although not wishing to be bound by a particular theory, FIG.6 illustrates the advantages of these target-specific, crosslinking dependent CD40 agonists. In the absence of the FAP (i.e., normal, non-malignant cells), minimal clustering of CD40 will occur, and immune activation will be limited. In contrast, in cancer-associated fibroblasts (CAFs), FAP is highly expressed; therefore, through FAP- binding, the FAP x CD40 bispecific molecule promotes CD40 clustering and immune cell activation. The advantages of this strategy are twofold: systemic toxicities should be limited because activation will be largely confined to tissue expressing FAP, and tumor-mediated CD40 crosslinking should drive potent agonism.

[0020] The inventors also discovered that, while FAP is a membrane-bound protein, its extracellular domain could be shed and circulate in peripheral blood. Indeed, soluble FAP (sFAP) was detected in   both healthy individuals as well as diseased subjects, including cancer patients. If a CD40 x FAP bispecific molecule can bind to sFAP and form a higher-order molecular complex (i.e., multiple sFAP molecules in complex with multiple copies of bispecific molecules), systemic toxicity remains to be a risk due to CD40 clustering and subsequent activation of immune cells in peripheral blood. Therefore, in some circumstances, it is desirable to use a bispecific molecule that does not trigger T-cell co- stimulatory molecule crosslinking through sFAP binding.

[0021] Accordingly, the present disclosure provides bispecific molecules that comprise a FAP binding moiety and a CD40 binding moiety; the bispecific molecules target fibroblasts through FAP binding and activate immune cells through CD40 activation. 2. Bispecific Molecules 2.1 Antigen-Binding Moiety or Antigen-Binding Protein Types

[0022] The bispecific molecules disclosed herein comprises two antigen-binding moieties, one being a CD40 antigen-binding moiety and one being a FAP antigen-binding moieties. These moieties sometimes also referred to as antigen-binding proteins. The antigen-binding moieties can take any one of many forms of antigen-binding proteins known in the art. In exemplary aspects, the antigen- binding moiety is an antibody or immunoglobulin, or an antigen binding fragment of an antibody or immunoglobulin.

[0023] Collectively, antibodies form a family of plasma proteins known as immunoglobulins and comprise of immunoglobulin domains. (Janeway et al., Immunobiology: The Immune System in Health and Disease, 4thed., Elsevier Science Ltd. / Garland Publishing, 1999). As used herein, the term “antibody” refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions. For example, an antibody may be an IgG which is a “Y-shaped” structure of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In IgG formats, the variable region is generally about 100-110 or more amino acids, comprises three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and substantially varies among other antibodies that bind to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region is made of the N-terminal regions of each light chain and heavy chain, while the constant region is made of the C- terminal portions of each of the heavy and light chains. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4thed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0024] Antibodies can comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has   several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. Accordingly, in exemplary embodiments, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3 or IgG4.

[0025] An antibody can be cleaved into fragments by enzymes, such as, e.g., papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab’)2fragment and a pFc’ fragment. In exemplary aspects of the present disclosure, the antigen binding moiety comprises an antigen binding fragment of an antibody. As used herein, “antigen binding fragment” of an antibody refers to a portion of an antibody molecule that retains the ability to specifically bind to an antigen (preferably with substantially the same binding affinity). Examples of an antigen-binding fragment include but not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain.

[0026] The architecture of antibodies has been exploited to create a growing range of alternative formats that span a molecular-weight range of at least about 12–150 kDa and has a valency (n) range from monomeric (n = 1), to dimeric (n = 2), to trimeric (n = 3), to tetrameric (n = 4), and potentially higher. The building block that is most frequently used to create novel antibody-based formats is the single-chain variable (V)-domain antibody fragment (scFv). Although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (see e.g., Bird et al. Science 242:423- 426 (1988) and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883).

[0027] Bispecific formats can generally be divided into five major classes: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A: 97-106 (2015). Exemplary bispecific molecules adopt IgG-scFv or Fab- scFv-Fc formats (see, e.g., Figs 2A-2B).

[0028] Bispecific molecules disclosed herein generally comprise two different chains, one derived from the heavy chain of an antibody, and one derived from the light chain of an antibody. Although the heavy / light chain has been modified and is no longer the classical immunoglobulin heavy / light chain, for convenience, it is still generally called “heavy chain” or “HC” if it is based on heavy chain backbone, and “light chain “ or “LC” if it is based on light chain backbone. For example, for tetravalent   bispecific molecule IgG-scFv, the “HC” comprises an IgG heavy chain fused to an scFv. It would be apparent to a skilled artisan whether HC is a traditional immunoglobulin heavy chain or a modified version based on immunoglobulin heavy chain backbone.

[0029] The antigen-binding moieties are sometimes characterized structurally by their Complementarity Determining Regions. As used herein, “Complementarity Determining Regions” (CDRs) can be identified according to the definitions of the Kabat, Chothia, the accumulation of both Kabat and Chothia, AbM, contact, North, and / or conformational definitions or any method of CDR determination well known in the art. See, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed. (hypervariable regions); Chothia et al., 1989, Nature 342:877-883 (structural loop structures). The identity of the amino acid residues in a particular antibody that make up a CDR can be determined using methods well known in the art. AbM definition of CDRs is a compromise between Kabat and Chothia and uses Oxford Molecular’s AbM antibody modeling software (Accelrys®). The “contact” definition of CDRs is based on observed antigen contacts, set forth in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. The “conformational” definition of CDRs is based on residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, J. Biol. Chem., 283:1156-1166). North has identified canonical CDR conformations using a different preferred set of CDR definitions (North et al., 2011, J. Mol. Biol.406: 228-256). In another approach, referred to herein as the “conformational definition” of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding (Makabe et al., 2008, J Biol. Chem.283:1156-1166). Martin definition (also called enhanced Chothia definition) combines the Kabat and Chothia definitions and differs from them only in the heavy chain, where CDR-H1 includes all residues of Kabat and Chothia while CDR-H2 is seven residues shorter than that defined by Kabat (Martin, Bioinformatics tools for antibody engineering. Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag GmbH; (2008). p.95–117; see also the database maintained by the Institute of Structural and Molecular Biology at the University College London, http: / / www.bioinf.org.uk / abs / #cdrid). Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. For example, “combined” CDRs may also be used. Therefore, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. For any given embodiment containing more than one CDR, the CDRs (or other residue of the antibody) may be defined in accordance with any of Kabat, Chothia, North, AbM, Contact, IMGT, Martin, combined Kabat and Chothia, and / or conformational definitions.

[0030] For example, the following Table shows several commonly used definitions of CDRs: Loop Kabat AbM Chothia1Contact2IMGT L1 L24L34 L24L34 L26L32 L30L36 L27L32   H1 H31-H35B H26-H35B H26-H32..34 H30-H35B H26-H35B (Kabat Numbering)3H1 H31-H35 H26-H35 H26-H32 H30-H35 H26-H33 2. Any of the numbering schemes can be used for these CDR definitions, except the contact definition uses the Chothia or Martin (Enhanced Chothia) definition. 3. The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. (This is because the Kabat numbering scheme places the insertions at H35A and H35B.)

[0031] The CDR sequences provided in the Sequence Tables are based on the Kabat definition. However, other definitions for CDRs may also be used. 2.2 Structure of FAP x CD40 Bispecific Molecules.

[0032] In various aspects, the invention provides bispecific molecules that bind to both FAP and CD40. In exemplary aspects, the bispecific molecule comprises 4 antigen binding sites, 2 of which bind to FAP protein and 2 of which bind to CD40. Optionally, each FAP binding site is identical to the other and / or each CD40 binding site is identical to the other.

[0033] In one particular example, the bispecific molecule comprises an antibody (IgG) moiety and an scFv moiety. As shown in FIG.2A, there are two Fab moieties that bind to one antigen (e.g., CD40). Each Fab moiety comprises two chains: a heavy chain comprising a heavy chain variable domain A (VHA) and a CH1 domain, and a light chain comprising a light chain variable domain A (VLA) and a CL domain. Each Fab is connected to one chain of Fc (CH2-CH3) to form an antibody (IgG). Because this part of the structure is essentially an IgG, there is no new linker between Fab and Fc (Fab and Fc are connected through “hinge” sequence just like a wildtype IgG). In addition, there are two scFv moieties that bind to the other antigen (e.g., FAP). Each scFv comprises a heavy chain variable domain B (VHB) and a light chain variable domain B (VLB); and the VHBand VLBare connected via a first linker. A second linker then connects the C-terminus of one CH3 domain to the N-terminus of one scFv. This particular structure is sometimes referred to as “IgG-scFv” format (one or more scFv moieties attached to an IgG molecule). Because each target (CD40, FAP) has two binding domains, the bispecific molecules exemplified in FIGs.2A-2B are often referred herein to as “bivalent” bispecific molecules; nonetheless, it should be noted that it is also acceptable in the art to refer to such kind of molecule as “tetravalent,” as altogether there are four binding domains.

[0034] In another particular example, the bispecific molecule comprises a scFv moiety that is inserted between the Fab and Fc region of an immunoglobulin (sometimes referred to as “Fab-scFv-Fc”). For example, as shown in FIG.2B, the bispecific molecule may comprises: (i) two Fab moieties that bind to one antigen (e.g., CD40), wherein each Fab moiety comprises two chains: a heavy chain comprising a heavy chain variable domain A (VHA) and a CH1 domain, and a light chain comprising a   light chain variable domain A (VLA) and a CL domain; (ii) two scFv moieties that bind to another antigen (e.g., FAP), wherein each scFv comprises a heavy chain variable domain B (VHB) and a light chain variable domain B (VLB), wherein the VHB and VLB are connected via a first linker; and (iii) one Fc region that comprises two chains, each chain comprising a monomeric CH2 domain and a monomeric CH3 domain. A second linker connects the C-terminus of one CH1 domain to the N- terminus of one scFv, and a third linker connects the C-terminus of one scFv to the N-terminus of one chain of the Fc region. Similar to the IgG-scFv structure, the molecule exemplified in FIG.2B is essentially a tetravalent molecule, with two binding moieties for CD40 and two binding moieties for FAP, but often called bivalent bispecific molecules.

[0035] It is believed that, as compared to monovalent binding of FAP (e.g., a hetero-Ig molecule in which one arm of the antibody binds to CD40 and one arm of the antibody binds to FAP), bivalent binding of FAP can enhance the crosslinking (clustering) of CD40 and potentially enhance the overall potency of the molecule: through bivalent binding, more CD40 molecules can be brought to the proximity of each other to promote crosslinking.

[0036] In some embodiments, the bispecific molecule comprises a FAP antigen-binding moiety, wherein said FAP antigen-binding moiety comprises: (i) the heavy chain CDR-H1, CDR-H2, and CDR- H3 of SEQ ID NO: 33, 35, 37, 39, or 41; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 34, 36, 38, 40, or 42. In some embodiments, the three heavy chain CDRs and three light chain CDRs come from the same clone as shown in Sequence Tables. In exemplary embodiments, the CDRs are defined according to Kabat, Chothia, AbM, contact, or IMGT.

[0037] In various embodiments, the FAP antigen-binding moiety comprises a VH comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% identical to the three heavy chain CDRs in SEQ ID NO:33; and a VL comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% to the three light chain CDRs in SEQ ID NO:34. The FAP antigen- binding moiety may further bind to its target (FAP) with a KD value of 100 nM or less, or a KD value of 50 nM or less.

[0038] In various embodiments, the FAP antigen-binding moiety comprises a VH comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% identical to the three heavy chain CDRs in SEQ ID NO:35; and a VL comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% to the three light chain CDRs in SEQ ID NO:36. The FAP antigen- binding moiety may further bind to its target (FAP) with a KD value of 100 nM or less, or a KD value of 50 nM or less.

[0039] In various embodiments, the FAP antigen-binding moiety comprises a VH comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% identical to the three heavy chain CDRs in SEQ ID NO:37; and a VL comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% to the three light chain CDRs in SEQ ID NO:38. The FAP antigen-   binding moiety may further bind to its target (FAP) with a KD value of 100 nM or less, or a KD value of 50 nM or less.

[0040] In various embodiments, the FAP antigen-binding moiety comprises a VH comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% identical to the three heavy chain CDRs in SEQ ID NO:39; and a VL comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% to the three light chain CDRs in SEQ ID NO:40. The FAP antigen- binding moiety may further bind to its target (FAP) with a KD value of 100 nM or less, or a KD value of 50 nM or less.

[0041] In various embodiments, the FAP antigen-binding moiety comprises a VH comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% identical to the three heavy chain CDRs in SEQ ID NO:41; and a VL comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% to the three light chain CDRs in SEQ ID NO:42. The FAP antigen- binding moiety may further bind to its target (FAP) with a KD value of 100 nM or less, or a KD value of 50 nM or less.

[0042] In some embodiments, the FAP antigen-binding moiety disclosed herein comprises: (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 33, and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 34.

[0043] In some embodiments, the FAP antigen-binding moiety disclosed herein comprises: (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 35, and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 36.

[0044] In some embodiments, the FAP antigen-binding moiety disclosed herein comprises: (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 37, and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 38.

[0045] In some embodiments, the FAP antigen-binding moiety disclosed herein comprises: (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 39, and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:40.

[0046] In some embodiments, the FAP antigen-binding moiety disclosed herein comprises: (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 41, and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 42.

[0047] In exemplary embodiments, the FAP antigen-binding moiety comprises: a VH comprises: (i) a CDR-H1 comprising any one of SEQ ID NOs: 7, 13, 19, and 25; (ii) a CDR-H2 comprising any one of SEQ ID NOs: 8, 14, 20, and 26; and (iii) a CDR-H3 comprising any one of SEQ ID NOs: 9, 15, 21, and 27; and a VL comprises: (i) a CDR-L1 comprising any one of SEQ ID NOs: 10, 16, 22, and 28; (ii) a CDR-L2 comprising any one of SEQ ID NOs: 11, 17, 23, and 29; and (iii) a CDR-L3 comprising any one of SEQ ID NOs: 12, 18, 24, and 30.

[0048] In exemplary embodiments, the FAP antigen-binding moiety comprises six CDR amino acid sequences selected from the group consisting of: (1) SEQ ID NOs: 7-12, (2) SEQ ID NOs: 13-18, (3) SEQ ID NOs: 19-24, and (4) SEQ ID NOs: 25-30.

[0049] In general, CDRs are separated by “framework” (FR) residues. A VH or VL domain framework comprises four framework sub-regions, FR1, FR2, FR3 and FR4, interspersed with CDRs in the following structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. Accordingly, the FAP antigen- binding moieties described herein may comprise a VH framework, such as a human germline VH framework sequence, and a VL framework, such as human germline VL framework sequences.

[0050] Preferred human germline light chain frameworks are frameworks derived from V ^ or V ^ germlines. It will be understood that if a sequence is “derived from” one or more germlines, what is referred to is a structural relationship, in which the features of a sequence correspond to the noted germline sequences, but may comprise somatic mutations or other amino acid differences relative to the noted germline sequence. For a sequence to be “derived from” a germline, an actual process of deriving that sequence from a germline sequence (either via molecular biology or computational analysis) is not necessarily required. For example, VL frameworks may be derived from one of the framework of the following germlines: DPK9 (IMGT name: IGKV1-39), DPK12 (IMGT name: IGKV2D- 29), DPK18 (IMGT name: IGKV2-30), DPK24 (IMGT name: IGKV4-1), HK102_V1 (IMGT name: IGKV1-5), DPK1 (IMGT name: IGKV1-33), DPK8 (IMGT name: IGKV1-9), DPK3 (IMGT name: IGKV1-6), DPK21 (IMGT name: IGKV3-15), Vg_38K (IMGT name: IGKV3-11 ), DPK22 (IMGT name: IGKV3-20), DPK15 (IMGT name: IGKV2-28), DPL16 (IMGT name: IGLV3-19), DPL8 (IMGT name: IGLV1-40), V1-22 (IMGT name: IGLV6-57). Alternatively, or in addition, the framework sequence may be derived from a human germline consensus framework sequence, such as the framework of human V ^1 consensus sequence, V ^3 consensus sequence, V ^1 consensus sequence, V ^2 consensus sequence, V ^3 consensus sequence. Sequences of human germline frameworks are available from various public databases, such as V-base, IMGT, NCBI, or Abysis.

[0051] The FAP antigen-binding moieties described herein may comprise a VL framework, wherein the framework may comprise one or more amino acid substitutions, additions, or deletions, while still retaining functional and structural similarity with the germline from which it was derived. In some aspects, the VL framework is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human germline VL framework sequence. In some aspects, the antigen-binding moiety comprises a VL framework comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions or deletions relative to the human germline VL framework sequence.

[0052] The VH framework sequence can be derived from a human VH3 germline, a VH1 germline, a VH5 germline, a human VH2 germline, or a VH4 germline. Preferred human germline heavy chain frameworks are frameworks derived from VH1, VH3, or VH5 germlines. For example, VH frameworks   may be derived from the framework of one of the following germlines: DP54 or IGHV3-7, DP47 or IGHV3-23, DP71 or IGHV4-59, DP75 or IGHV1-2_02, DP10 or IGHV1-69, DP7 or IGHV1-46, DP49 or IGHV3-30, DP51 or IGHV3-48, DP38 or IGHV3-15, DP79 or IGHV4-39, DP78 or IGHV4-30-4, DP73 or IGHV5-51, DP50 or IGHV3-33, DP46 or IGHV3-30-3, DP31 or IGHV3-9. Alternatively, or in addition, the framework sequence may be derived from the framework of a consensus sequence, such as: VH3 germline consensus sequence, VH1 germline consensus sequence, VH5 germline consensus sequence, VH2 germline consensus sequence, or VH4 germline consensus sequence.

[0053] The FAP antigen-binding moieties described herein may comprise a VH framework, wherein the framework may comprise one or more amino acid substitutions, additions, or deletions, while still retaining functional and structural similarity with the germline from which it was derived. In some aspects, the VH framework is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human germline VH framework sequence. In some aspects, the antigen-binding moiety comprises a VH framework comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions or deletions relative to the human germline VH framework sequence.

[0054] In exemplary embodiments, the FAP antigen-binding moiety comprises a VH that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 33, 35, 37, 29, or 41. In exemplary embodiments, the FAP antigen-binding moiety comprises a VL that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 34, 36, 38, 40, or 42. Preferably, the FAP antigen-binding moiety comprises a pair of VH and VL sequences listed under the same clone name in the Sequence Tables.

[0055] Also provided herein are bispecific molecules that comprises CD40 antigen-binding moieties. In some embodiments, the CD40 antigen-binding moiety comprises a VH comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% identical to the three heavy chain CDRs in SEQ ID NO:31; and a VL comprising three CDRs that in combination are at least 85%, at least 90%, or at least 95% to the three light chain CDRs in SEQ ID NO:32. The CD40 antigen-binding moiety may further bind to its target (CD40) with a KD value of 100 nM or less, or a KD value of 50 nM or less.

[0056] In some embodiments, the CD40 antigen-binding moiety comprises: (i) the heavy chain CDR- H1, CDR-H2, and CDR-H3 of SEQ ID NO:31; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 32.

[0057] In exemplary aspects, the CD40 antigen-binding moiety comprises: a CDR-H1 comprising SEQ ID NO:1, a CDR-H2 comprising SEQ ID NO:2, a CDR-H3 comprising SEQ ID NO:3, a CDR-L1   comprising SEQ ID NO:4, a CDR-L2 comprising SEQ ID NO:5, and a CDR-L3 comprising SEQ ID NO: 6.

[0058] Preferred VH and VL framework sequences for CD40 antigen-binding moieties disclosed herein are human framework sequences described in detail above, such as a VH framework that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human germline VH framework sequence, a VH framework comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions or deletions relative to the human germline VH framework sequence, a VL framework that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human germline VL framework sequence, or a VL framework comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions or deletions relative to the human germline VL framework sequence.

[0059] In exemplary embodiments, the CD40 antigen-binding moiety comprises a VH that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 31. In exemplary embodiments, the CD40 antigen- binding moiety comprises a VL that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 32.

[0060] The CD40 antigen-binding moiety and the FAP antigen-binding moiety may be linked by a linker. In addition, a linker may connect the VH and VL chains in an scFv.

[0061] A “linker” is a molecule or group of molecules that connects two separate entities (e.g., FAP- binding protein and CD40-binding protein) to one another and can provide spacing and flexibility between the two entities such that they are able to achieve a conformation in which they, e.g., specifically bind their respective targets (e.g., FAP and CD40). Protein linkers are particularly preferred, and they may be expressed as a component of the recombinant protein using standard recombinant DNA techniques well-known in the art. For recombinant proteins described herein comprising two or more linkers (for example IgG-scFv and Fab-scFv-Fc formats), the linkers may all be the same, or some or all of the linkers may be different from each other.

[0062] In some embodiments, the linker is a peptidyl linker. In some embodiments, the peptidyl linker comprises about 1 to 30 amino acid residues. Exemplary linkers include, e.g., a glycine rich peptide; a peptide comprising glycine and serine; a peptide having a sequence [Gly-Gly-Ser]n (SEQ ID NO:88), wherein n is 1, 2, 3, 4, 5, or 6; or a peptide having a sequence [Gly-Gly-Gly-Gly-Ser]n (SEQ ID NO: 87), wherein n is 1, 2, 3, 4, 5, or 6. A glycine rich peptide linker comprises a peptide linker, wherein at least 25% of the residues are glycine. Glycine rich peptide linkers are well known in the art (e.g.,   Chichili et al. Protein Sci.2013 February; 22(2): 153-167). The peptidyl linker may also be a proline- threonine rich peptide linker.

[0063] As shown in FIGs 2A-2B, when bispecific molecule comprises a scFv moiety, mutations may be introduced to scFv to further improve stability. For example, it has been reported that insufficient interface stability between the heavy and light chains of scFv fragments could be the main cause of irreversible scFv inactivation. Fv fragments have been reported to dissociate into heavy-chain variable domains (VH) and light-chain variable domains (VL) with KD values ranging from 10−9to 10−6M. An interdomain disulfide bond have been used to further improve scFv stability. For example, mutation to Cys at the site of H44 (Kabat numbering), and mutation to Cys at L100 (Kabat numbering) would not significantly affect the domain folding. The two cysteines can then form an intramolecular disulfide bond to further stabilize the scFv. Such mutation is sometimes referred to as “cysteine clamp.”

[0064] Specific examples of scFv comprising cysteine clamps are shown in Sequence Table C, where mutations at H44 (Kabat numbering) and at L100 (Kabat numbering) were used to create disulfide bonds (referred to as “C-C”).

[0065] Specific examples of scFv comprising cysteine clamp in a bispecific format are shown in Sequence Tables. In this particular example, a cysteine clamp is introduced in the scFv for all IgG- scFv bispecific molecules, and most of the Fab-scFv-Fc molecules, whereas no cysteine clamp is present in the scFv for one of the Fab-scFv-Fc bispecific molecules. Thus, the use (or non-use) of cysteine clamp may require evaluation of stability and biologically activities of the scFv. In general, it is believed that the removal of the constant domain (CH1 and C ^ or C ^) lowers the stability of the Fv domain. This may require the addition of a linker fusion between the VH and VL domains to avoid molecule dissociation. With the decreased interface between the HC and LC, some Fv domains may have an increased probability of being in a dissociated state, exposing their hydrophobic VH and VL interfaces. This could cause increased aggregation, and require additional stability using a disulfide bond or cysteine clamp that covalently links the VH to the VL. On the other hand, although cysteine clamp tends to create a stabilized product post purification, it could also lead to other issues. A poorly positioned cys-clamp can alter the orientation of the VH and VL domains such that it exposes new interfaces or cause a loss in antigen binding due to a new paratope interface. If possible, a scFv domain lacking a cys-clamp with good biophysical properties could be preferential. In IgG-scFv format, because scFv is located at the C-terminus and more exposed, it appears that a cys-clamp can improve the biophysical properties of scFv, whereas in the Fab-scFv-Fc format, it appears that the scFv without cysteine clamp may be feasible under some circumstances because the scFv is sandwiched between Fab and Fc and thus more protected.

[0066] In exemplary embodiments, the scFv moiety that binds to FAP and comprises an amino acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:93, 94, 95, 96, or 97.

[0067] In some embodiments, the bispecific molecule comprises a CH1 domain, preferably a human CH1 domain (such as a human IgG1 CH1, a human IgG2 CH1, a human IgG3 CH1, or a human IgG4 CH1). Non-limiting examples of human CH1 sequences are provided in the Sequence Tables. In some embodiments, the CH1 domain comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 47, 64, 68, or 72.

[0068] In some embodiments, the bispecific molecules described herein comprising a kappa or lambda light chain constant domain (CL). In some embodiments, the kappa light chain constant domain comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:43 or 44. In some embodiments, the lambda light chain constant domain comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:45 or 46.

[0069] The CH1 and CL domains, together with the VH and VL domain disclosed herein, may form a Fab moiety, such as the Fab moieties illustrated in Figs.2A-2B.

[0070] In certain embodiments, the bispecific molecules described herein comprises an Fc domain. The Fc domain can be derived from IgA (e.g., IgA1 or lgA2), IgG, IgE, or IgG (e.g., IgG1, lgG2, lgG3, or lgG4). In some embodiments, the Fc domain is a human IgG Fc (CH2-CH3). In some embodiments, the Fc domain comprises wild type sequence of an Fc domain. Non-limiting examples of human Fc sequences are provided in the Sequence Tables.

[0071] In some embodiments, the Fc domain comprises one or more mutations resulting in altered biological activity, such as to improve half-life / stability or to render the antibody more suitable for expression / manufacturability. For example, mutations may be introduced into the Fc domain to reduce the effector activity (e.g., WO 2005 / 063815), and / or to increase the homogeneity during the production of the recombinant protein.

[0072] In general, amino acid residues in the IgG heavy constant domain of an antibody are numbered according the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 as described in Kabat et al., 1991, referred to herein as the “EU index numbering.” Typically, the constant domain comprises from residue 118 to 447, and the Fc domain comprises from residue 236 to 447 of the human lgG1 constant domain. Comparison between EU numbering and other numbering systems can be found, e.g., at IGMT database.

[0073] Amino acid residues of the light chain constant domain are numbered according to Kabat et al., 1991, "Sequences of Proteins of Immunological Interest 5th Ed.", 1991, NATIONAL INSTITUTES OF HEALTH. Kappa light chain also has EU index numbering, and the EU index and Kabat numbering are identical. Lambda light chain does not have EU index numbering.

[0074] In some embodiments, the Fc domain is the Fc domain of human lgG1 and comprises one or more of the following effector-null mutations: L234A, L235A, and G237A (numbering according to the EU index), often referred as “LALA” mutations.

[0075] It has been reported that a single mutation of L235E was sufficient for knocking out binding to Fc receptors on U937 cells. Furthermore, the 100-fold reduction in binding to Fc ^R also resulted in lower T cell activation and proliferation in the presence of the L235E Fc mutant IgG1. Building upon this initial mutation it was found that the combination of L234A and L235A (commonly called LALA mutations) eliminated Fc ^RIIa binding. These two mutations were later shown to eliminate detectable binding to Fc ^RI, IIa, and IIIa for both IgG1 and IgG4. Other sites have been reported to knockout Fc receptor binding, such as Gly237Ala, Glu318Ala, Asp265Ala and Glu233Pro mutations.

[0076] In exemplary embodiments, the Fc region comprises a Stable Effector Functionless (SEFL) mutation to reduce the ability to interact with Fc ^ receptors. SEFL mutations are known in the art. See, e.g., Liu et al., J Biol Chem 292: 1876-1883 (2016); and Jacobsen et al., J. Biol. Chem.292: 1865- 1875 (2017). Further, US US9546203 discloses a Fc region comprising a N297G mutation, and one or more substitutions at position V259, A287, R292, V302, L306, V323, or I332, using EU numbering scheme, with a cysteine amino acid residue. In exemplary aspects, the SEFL mutation comprises one or more of the following mutations, numbered according to the EU system: L242C, A287C, R292C, N297G, V302C, L306C, and / or K334C. In exemplary aspects, the SEFL mutation comprises N297G. In exemplary aspects, the SEFL mutation comprises A287C, N297G, and L306C. In other exemplary aspects, the SEFL mutation comprises R292C, N297G, and V302C (i.e., SEFL2-2).

[0077] In exemplary embodiments, the Fc region comprises a YTE mutation. The M252Y / S254T / T256E (EU index numbering, referred to “YTE”) triple mutation have been shown to increase IgG half-life in cynomolgus monkeys by an approximate 4-fold increase.

[0078] C-terminal lysine clipping is a common phenomenon occurring during the bioproduction of monoclonal antibodies. Often, the lysine residue is removed via carboxypeptidase D (CpD), which results in generation of a mixture of antibody isoforms bearing zero or one C-terminal lysine residues on each heavy chain. Further, following C-terminal lysine cleavage, peptidylglycine ^-amidating monooxygenase (PAM) catalyzes the hydroxylation of glycine and removal of the glyoxylate from the glycine residue, leaving an amidated C-terminal proline. Therefore, during recombinant production of a monoclonal antibody, the product is often a mixture of C-terminal processing variants, with heavy chain C-terminus ends at (amidated) proline, glycine, or lysine. Sometimes, it may be desirable to   delete the C-terminal lysine of the Fc domain to increase the homogeneity during the production of the recombinant protein.

[0079] In some embodiments, the terminal lysine may be absent; in some embodiments, the terminal lysine may be present; in some embodiments, the terminal glycine-lysine may be absent; in some embodiments, the terminal glycine-lysine may be present.

[0080] In exemplary embodiments, the bispecific molecule described herein comprise Fc that is derived from an IgG1. In some embodiments, the Fc comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 48, 55, 56, 57, 58, 59, 60, 66, 70, or 74.

[0081] In exemplary embodiments, the bispecific molecules described herein comprise an IgG1 heavy chain constant domain (CH1-CH3). In some embodiments, the heavy chain constant domain comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:49, 50, 51, 52, 53, 54, 61, 62, 63, 67, 71, or 75. 2.3 Binding Characteristics

[0082] The bispecific molecules provided herein bind to their respective targets or antigens (CD40 and FAP) in a non-covalent and reversible manner. The use of “bind” here refers to characteristic of a molecule and should be interpreted as capable of binding to a target. It does not require that the molecule must exist in complex with its target.

[0083] In exemplary embodiments, the binding strength of the bispecific molecule to their targets or antigens (e.g., FAP or CD40) may be described in terms of its affinity, a measure of the strength of interaction between the binding site of the bispecific molecule and the target or antigen (e.g., FAP or CD40).

[0084] In exemplary embodiments, the binding strength of the bispecific molecule its target or antigen (e.g., FAP or CD40) may be described in terms of its sensitivity. KD is the equilibrium dissociation constant, a ratio of koff / kon, between the bispecific molecule and its target or antigen (e.g., FAP or CD40). KD and KA are inversely related. The KD value relates to the concentration of the bispecific molecule (the amount of bispecific molecule needed for a particular experiment) and so the lower the KD value (lower concentration needed) the higher the affinity of the bispecific molecule. In exemplary aspects, the binding strength of the bispecific molecule to its target (e.g., FAP or CD40) may be described in terms of KD. In exemplary aspects, the KD value of the bispecific molecules provided   herein is about 10-1M or less, about 10-2M or less, about 10-3M or less, about 10-4M or less, about 10-5M or less, about 10-6M or less, about 10-7M or less, about 10-8M or less, about 10-9M or less, about 10-10M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10-15M, from about 10-9M to about 10-15M, from about 10-10M to about 10-15M, from about 10-5M to about 10-14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10-14M, from about 10-9M to about 10-14M, from about 10-10M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10-13M, or from about 10-10M to about 10-13M.

[0085] In exemplary aspects, the KD of the bispecific molecules provided herein is micromolar, nanomolar, picomolar or femtomolar. In exemplary aspects, the KD of the bispecific molecules provided herein is within a range of about 10-4to 10-6M, or 10-7to 10-9M, or 10-10to 10-12M, or 10-13to 10-15M. In exemplary aspects, the bispecific molecule binds to the human FAP or human CD40 with a KD value that is from about 0.07 nM to about 4 nM. In exemplary aspects, the bispecific molecule binds to the human FAP or human CD40 with a KD of from about 0.01 nM to about 50 nM, from about 0.02 nM to about 50 nM, from about 0.05 nM to about 50 nM, from about 0.05 nM to about 45 nM, from 0.05 nM to about 40 nM, from about 0.05 nM to about 35 nM, from about 0.05 nM to about 30 nM, from about 0.05 nM to about 25 nM, from about 0.05 nM to about 20 nM, from about 0.05 nM to about 15 nM, or from about 0.05 nM to about 10 nM.

[0086] KDvalues can be determined using methods well established in the art. One exemplary method for measuring KD is surface plasmon resonance (SPR), a method well-known in the art (e.g., Nguyen et al. Sensors (Basel).2015 May 5; 15(5):10481-510). KD value may be measured by SPR using a biosensor system such as a BIACORE® system. BIAcore kinetic analysis comprises analyzing the binding and dissociation of an antigen from chips with immobilized molecules (e.g. molecules comprising epitope binding domains), on their surface. Another well-known method in the art for determining the KD of a protein is by using Bio-Layer Interferometry (e.g., Shah et al. J Vis Exp.2014; (84): 51383). KD value may be measured by Bio-Layer Interferometry using OCTET® technology (Octet QKe system, ForteBio). Alternatively or in addition, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used. Any method known in the art for assessing the binding affinity between two binding partners is encompassed herein. 3. Nucleic Acids, Vectors, and Host Cells 3.1 Nucleic acids

[0087] The present disclosure further provides nucleic acids comprising a nucleotide sequence encoding the bispecific molecules disclosed herein. The nucleic acid may comprise a single nucleic acid molecule, or two or more nucleic acid molecules (for example, a first nucleic acid molecule encoding a heavy chain amino acid sequence and a second nucleic acid molecule encoding a light   chain amino acid sequence). In some aspects, the nucleic acids of the present disclosure are recombinant.

[0088] In exemplary embodiments, the nucleic acid comprises a nucleotide sequence that has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) sequence identity to any one of SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, and 118. It would be immediately apparent to a skill artisan which part of SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, and 118 encode the amino acid sequences corresponding to the fragments listed in the sequence tables.

[0089] In addition, nucleic acid sequence that encoding a signal peptide may be added to the 5’ of SEQ ID NO: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, and 118. Recombinant expression of the bispecific molecules, as well as various antigen-binding moieties disclosed herein often require that the molecules be secreted. Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is an important step in protein secretion. It is understood that the signal peptide is present (and often critical) during the initial synthesis of a nascent protein, but then, signal peptide is cleaved during secretion process. Therefore, while the mature protein no longer has the signal peptide; having the signal peptide coding sequence in the nucleic acid is generally necessary to recombinantly express the protein.

[0090] The present disclosure further provides nucleic acids that are capable of hybridizing to any one of SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, and 118, or a complimentary sequence of any one of SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, and 118, under a moderately stringent condition, or under a highly stringent condition. A “moderately stringent condition” includes prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50 °C-65 °C, 5X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1 % SDS. A “highly stringent condition” includes, for example, (1) employ low ionic strength and high temperature for washing, for example 0.015M sodium chloride / 0.0015M sodium citrate / 0.5 % sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 °C; or (3) employ 50% formamide, 5XSSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 ^g / ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2X SSC (sodium chloride / sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1X SSC containing EDTA at 55°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.   3.2 Vectors

[0091] The nucleic acids of the present disclosure in some aspects are incorporated into a vector. In this regard, the present disclosure provides vectors comprising any of the presently disclosed nucleic acids. In exemplary aspects, the vector is a recombinant expression vector. For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the present disclosure are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The presently disclosed vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single- stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The vectors can comprise naturally-occurring or non-naturally-occurring internucleotide linkages, or both types of linkages. In some aspects, the altered nucleotides or non-naturally occurring internucleotide linkages do not hinder the transcription or replication of the vector.

[0092] The vector of the present disclosure can be any suitable vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJoIIa, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as ^GTIO, ^GTl 1, ^ZapII (Stratagene), ^EMBL4, and ^NMl 149, also can be used. Examples of plant expression vectors include pBI101, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). In some aspects, the vector is a viral vector, e.g., a retroviral vector.

[0093] The vectors of the present disclosure can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., infra, and Ausubel et al., infra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from CoIEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, and the like.

[0094] In some aspects, the vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.

[0095] The vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy   metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the presently disclosed expression vectors include, for instance, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0096] The vector can comprise a native or normative promoter operably linked to the nucleotide sequence encoding the polypeptide (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the bispecific molecule. The selection of promoters, e.g., strong, weak, inducible, tissue- specific and developmental- specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus. 3.3 Host cells

[0097] Provided herein are host cells comprising a nucleic acid or vector of the present disclosure. As used herein, the term "host cell" refers to any type of cell that can contain the presently disclosed vector and is capable of producing an expression product encoded by the nucleic acid (e.g., mRNA, protein). The host cell in some aspects is an adherent cell or a suspended cell, i.e., a cell that grows in suspension. The host cell in exemplary aspects is a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage.

[0098] In exemplary aspects, the cell is a eukaryotic cell, including, but not limited to, a yeast cell, filamentous fungi cell, protozoa cell, algae cell, insect cell, or mammalian cell. Such host cells are described in the art. See, e.g., Frenzel, et al., Front Immunol 4: 217 (2013). In exemplary aspects, the eukaryotic cells are mammalian cells. In exemplary aspects, the mammalian cells are non-human mammalian cells. In some aspects, the cells are Chinese Hamster Ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3, CS9), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to be deficient in dihydrofolatereductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), green African monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinomic human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryo fibroblast cells NIH 3T3, mouse fibroblast cells L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells Hep G2, mouse B myeloma cells J558L, or baby hamster kidney (BHK) cells (Gaillet et al.2007; Khan, Adv Pharm Bull 3(2): 257-263 (2013)). In a particular embodiment, the host cell is CS9 (a CHO cell line).

[0099] For purposes of amplifying or replicating the vector, the host cell is in some aspects is a prokaryotic cell, e.g., a bacterial cell.

[0100] Also provided by the present disclosure is a population of cells comprising at least one host cell described herein. The population of cells in some aspects is a heterogeneous population comprising the host cell comprising vectors described, in addition to at least one other cell, which does not comprise any of the vectors. Alternatively, in some aspects, the population of cells is a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the vector. The population in some aspects is a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a vector, such that all cells of the population comprise the vector. In exemplary embodiments of the present disclosure, the population of cells is a clonal population comprising host cells comprising a vector as described herein. 3.4. Methods of Manufacture

[0101] The bispecific molecules disclosed herein may be obtained by methods known in the art. Suitable methods of de novo synthesizing polypeptides are described in, for example, Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and U.S. Patent No. 5,449,752. Additional exemplary methods of making the peptides of the invention are set forth herein.

[0102] Also, in some aspects, the bispecific molecules disclosed herein are recombinantly produced using a nucleic acid encoding the amino acid sequence of the molecule using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual.3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

[0103] Methods of making bispecific molecules disclosed herein are further provided herein. In exemplary embodiments, the method comprises culturing a presently disclosed host cell so as to express the bispecific molecule and harvesting the expressed bispecific molecule. The host cell can be any of the host cells described herein. In exemplary aspects, the host cell is selected from the group consisting of: CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In exemplary aspects, the step of culturing a host cell comprises culturing the host cell in a growth medium to support the growth and expansion of the host cell. In exemplary aspects, the growth medium increases cell density, culture viability and productivity in a timely manner. In exemplary aspects, the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. In exemplary aspects, the growth medium is a fully chemically defined media consisting of amino acids, vitamins, trace elements, inorganic salts, lipids and insulin or insulin-like growth factors. In addition to nutrients, the growth medium also helps   maintain pH and osmolality. Several growth media are commercially available and are described in the art. See, e.g., Arora, “Cell Culture Media: A Review” MATER METHODS 3:175 (2013).

[0104] In exemplary aspects, the method of making bispecific molecules disclosed herein comprises culturing the host cell in a feed medium. In exemplary aspects, the method comprises culturing in a feed medium in a fed-batch mode. Methods of recombinant protein production are known in the art. See, e.g., Li et al., “Cell culture processes for monoclonal antibody production” MAbs 2(5): 466–477 (2010).

[0105] The method of making bispecific molecules disclosed herein can comprise one or more steps for purifying the molecule from a cell culture or the supernatant thereof and preferably recovering the purified protein. In exemplary aspects, the method comprises one or more chromatography steps, e.g., affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography. In exemplary aspects, the method comprises purifying the protein using a Protein A affinity chromatography resin.

[0106] In exemplary embodiments, the method further comprises steps for formulating the purified protein, etc., thereby obtaining a formulation comprising the purified protein. Such steps are described, for example, in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010. 4. Pharmaceutical Compositions and Method of Treatment 4.1 Pharmaceutical Compositions

[0107] Compositions comprising a bispecific molecule, a nucleic acid, a vector, a host cell, or a combination thereof, are provided herein. The compositions may comprise the bispecific molecule, nucleic acid, vector, or host cell, or a combination thereof, in isolated and / or purified form.

[0108] In exemplary aspects, the composition comprises agents which enhance the chemico-physico features of the antigen-binding molecule, nucleic acid, vector, or host cell, or a combination thereof, e.g., via stabilizing, for example, at certain temperatures (e.g., room temperature), increasing shelf life, reducing degradation, e.g., oxidation protease mediated degradation, increasing half-life of the bispecific molecule, etc.

[0109] In exemplary aspects of the present disclosure, the composition additionally comprises a pharmaceutically acceptable carrier, diluents, or excipient. The pharmaceutical composition can comprise any pharmaceutically acceptable ingredients, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring   agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing agents, solvents, stabilizing agents, suppository bases, surface active agents, surfactants, suspending agents, sweetening agents, therapeutic agents, thickening agents, tonicity agents, toxicity agents, viscosity- increasing agents, water-absorbing agents, water-miscible cosolvents, water softeners, or wetting agents. See, e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0110] In exemplary aspects, the pharmaceutical composition comprises formulation materials that are nontoxic to recipients at the dosages and concentrations employed. In specific embodiments, pharmaceutical compositions comprising an active agent and one or more pharmaceutically acceptable salts; polyols; surfactants; osmotic balancing agents; tonicity agents; anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; analgesics; or additional pharmaceutical agents. In exemplary aspects, the pharmaceutical composition comprises one or more polyols and / or one or more surfactants, optionally, in addition to one or more excipients, including but not limited to, pharmaceutically acceptable salts; osmotic balancing agents (tonicity agents); anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; and analgesics.

[0111] In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as   sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (A. R. Genrmo, ed.), 1990, Mack Publishing Company.

[0112] The pharmaceutical compositions can be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition can be for example between about 4 or about 5 and about 8.0 or about 4.5 and about 7.5 or about 5.0 to about 7.5. In exemplary embodiments, the pH of the pharmaceutical composition is between 5.5 and 7.5. 4.2 Methods of Treatment

[0113] Methods of treatment are additionally provided by the present disclosure. The method, in exemplary embodiments, is a method of treating a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition of the present disclosure in an amount effective to treat the subject.

[0114] The pharmaceutical compositions of the present disclosure are useful for activating CD40 signaling. Without being bound to a particular theory, the CD40 agonist activity of the compositions provided herein allow such entities to be useful in methods of enhancing T cell activity and enhancing an immune response, and, in particular, an immune response against a tumor or cancer.

[0115] Accordingly, provided herein are methods of enhancing T cell activity in a subject, enhancing T cell survival and effector function, restricting terminal differentiation and loss of replicative potential, promoting T cell longevity, and enhancing cytotoxicity against target (e.g., cancer) cells. In exemplary embodiments, the methods comprise administering to the subject the pharmaceutical composition of the present disclosure in an effective amount. In exemplary aspects, the T cell activity or immune response is directed against a cancer cell or cancer tissue or a tumor cell or tumor. In exemplary aspects, the immune response is a humoral immune response. In exemplary aspects, the immune response is an innate immune response. In exemplary aspects, the immune response which is enhanced is a T-cell mediated immune response.

[0116] As used herein, the term “enhance” and words stemming therefrom may not be a 100% or complete enhancement or increase. Rather, there are varying degrees of enhancement of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the pharmaceutical compositions of the present disclosure may enhance, e.g., T cell activity or enhance an immune response, to any amount or level. In exemplary embodiments, the enhancement provided by the methods of the present disclosure is at least or about a 10% enhancement (e.g., at least or about a 20% enhancement, at least or about a 30% enhancement, at least or about a 40% enhancement, at least or about a 50% enhancement, at least or about a 60% enhancement, at least or about a 70% enhancement, at least or about a 80% enhancement, at least or about a 90% enhancement, at least or about a 95% enhancement, at least or about a 98% enhancement).

[0117] Methods of measuring T cell activity and immune responses are known in the art. T cell activity can be measured by, for example, a cytotoxicity assay, such as those described in Fu et al., PLoS ONE 5(7): e11867 (2010). Other T cell activity assays are described in Bercovici et al., Clin Diagn Lab Immunol.7(6): 859–864 (2000). Methods of measuring immune responses are described in e.g., Macatangay et al., Clin Vaccine Immunol 17(9): 1452-1459 (2010), and Clay et al., Clin Cancer Res.7(5):1127-35 (2001).

[0118] Additionally provided herein are methods of treating a subject with cancer and methods of treating a subject with a solid tumor. In exemplary embodiments, the method comprises administering to the subject the pharmaceutical composition of the present disclosure in an amount effective for treating the cancer or the solid tumor in the subject.

[0119] The cancer treatable by the methods disclosed herein can be any cancer, e.g., any malignant growth or tumor caused by abnormal and uncontrolled cell division that may spread to other parts of the body through the lymphatic system or the blood stream. The cancer in some aspects is one selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer. In particular aspects, the cancer is selected from the group consisting of: head and neck, ovarian, cervical, bladder and oesophageal cancers, pancreatic, gastrointestinal cancer, gastric, breast, endometrial and colorectal cancers, hepatocellular carcinoma, glioblastoma, bladder, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma. In particular embodiments, the tumor is non-small cell lung cancer (NSCLC), head and neck cancer, renal cancer, triple negative breast cancer, and gastric cancer. In exemplary aspects, the subject has a tumor (e.g., a solid tumor, a hematological malignancy, or a lymphoid malignancy) and the pharmaceutical composition is administered to the subject in an amount effective to treat the tumor in the subject. In other exemplary aspects, the tumor is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, renal cancer, breast cancer, melanoma, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, prostate cancer, gastric cancer, lymphoma or leukemia, and the pharmaceutical composition is administered to the subject in an amount effective to treat the tumor in the subject.

[0120] As used herein, the term “treat,” as well as words related thereto, do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary   skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of treating cancer of the present disclosure can provide any amount or any level of treatment. Furthermore, the treatment provided by the method of the present disclosure can include treatment of one or more conditions or symptoms or signs of the cancer being treated. Also, the treatment provided by the methods of the present disclosure can encompass slowing the progression of the cancer. For example, the methods can treat cancer by virtue of enhancing the T cell activity or an immune response against the cancer, reducing tumor or cancer growth, reducing metastasis of tumor cells, increasing cell death of tumor or cancer cells, and the like. In exemplary aspects, the methods treat by way of delaying the onset or recurrence of the cancer by 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, two months, 4 months, 6 months, 1 year, 2 years, 4 years, or more. In exemplary aspects, the methods treat by way increasing the survival of the subject.

[0121] In particular, the bispecific molecules disclosed herein target cancer-associated fibroblasts that is present in tumor stroma. Tumor stroma, broadly defined as the non-cancer cell and non-immune cell components of tumors, is viewed traditionally as the structural components holding tumor tissues together. Tumor stroma is composed of extracellular matrix and specialized connective tissue cells, including fibroblasts and mesenchymal stromal cells. Tumors generally need stroma for nutritional support and the removal of waste products, but stromal content can vary markedly in different types of cancers. For example, many lymphomas have minimal stroma whereas the stroma may make up 90% of other solid tumors. The bispecific molecules disclosed herein in particular target FAP+ tumors. Fibroblasts are capable of infiltrating tumors and FAP+ cells can be easily identified by methods well known in the art, such as immunostaining. 4.3 Combination Therapy

[0122] Also provided herein are combination therapy. In some embodiments, the disclosure provides a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific molecule disclosed herein, and a therapeutically effective amount of PD-1 or PD-L1 pathway inhibitor.

[0123] Programmed cell death protein 1, also known as PD-1 or CD279 (cluster of differentiation 279), is a cell surface receptor that plays an important role in down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1 is an immune checkpoint and guards against autoimmunity through a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T-cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells). Through these mechanisms, PD-1 inhibits the immune system. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells.

[0124] A new class of drugs that block PD-1 pathway (PD-1 pathway inhibitors) can activate the immune system to attack tumors, and are therefore used with varying success to treat some types of cancer. The PD-1 protein in humans is encoded by the PDCD1 gene. PD-1 is a cell surface receptor   that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2.

[0125] Therapeutic approach of blocking the PD-1 / PD-L1 immune checkpoint proteins as anticancer agents has been successful in treating melanoma and NSCLC patients. However, overall more than 70% of cancer patients do not respond to PD-1 blockade. This may be because additional inhibitory mechanisms function to inhibit the activity of CD8+ T cells within the tumor microenvironment. In general, immune checkpoint inhibitors response rates are low in so-called “cold tumors.”

[0126] Based on the spatial distribution of CD8+ T lymphocytes in the tumor microenvironment (TME), a gradient of three immunophenotypes is observed: the immune-desert, immune-excluded and immune-inflamed phenotypes. In the immune-desert phenotype, immune cells are absent from the tumor and its periphery. In the immune-excluded phenotype, immune cells accumulate but do not efficiently infiltrate. In the immune-inflamed phenotype, immune cells infiltrate but their effects are inhibited. Notably, the three different phenotypes have different response rates to immune checkpoint inhibitors. Tumors with immune cell infiltration is often called “hot” tumors and typically respond to checkpoint inhibitor treatment well.

[0127] As discussed above, Cancer Associated Fibroblasts (CAFs) s are often key cellular components in the tumor stroma and can promote tumor growth. CAFs are predominantly located at the infiltrating edges of tumors, regulating tumor metastasis and influencing angiogenesis by synthesizing and remodeling the extracellular matrix (ECM) and producing cytokines, and transforming tumor margins into immune “cold” zones. CAFs led to immunosuppression and T-cell exclusion through several mechanisms. First, CAFs produce extracellular matrix that forms a physical barrier to prevent T-cell infiltration into the tumor area. Second, CXCL12 produced by CAFs has been shown to inhibit T-lymphocyte infiltration within tumors in a pancreatic cancer model. Third, CAFs can also reduce T-cell responses and exert immunosuppressive effects through the production of TGF ^ and IL-6.

[0128] The fibroblast taking molecules disclosed herein, especially the FAP x CD40 bispecific molecules, have the potential to inflame “cold” tumors to become “hot” tumors, and enhance the effectiveness of PD-1 / PD-L1 blockade. By targeting FAP on CAFs, the bispecific molecules activate CD40 pathway through a crosslinking dependent manner, thereby promoting T cell priming to tumor (neo)antigens, recruitment / infiltration into tumor, and activation / effector function. The increased infiltration of T cells means that “cold” tumors can become “hot” tumors.

[0129] PD-1 / PD-L1 pathway inhibitors disclosed herein include antigen-binding proteins, such as an antibody, or antigen binding fragment of an antibody, which specifically binds to PD-1 or PD-L1, in particular human PD-1 or human PD-L1. The antibody may be a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the constant region of the antibody comprises the constant region from human IgG1, IgG2, IgG3, or IgG4, as disclosed in detail above.   The antigen binding fragment maybe, for example, Fab, Fab’-SH, F(ab’)2, scFv and Fv fragments, as disclosed in detail above.

[0130] In certain embodiments, the antibody, or antigen-binding portion thereof, binds to PD-1, such as human PD-1. Examples of antibodies that bind to human PD-1, are described, e.g, in US7488802, US7521051, US8008449, US8354509, US8168757, WO2004 / 004771, WO2004 / 072286, WO2004 / 056875, and US2011 / 0271358. Specific anti-human PD-1 antibodies useful for the invention described herein include, for example: K-3945 (Pembrolizumab, Keytruda®; U.S. Patent No. 8,952,136); M-3475, a humanized IgG4 mAb with the structure described in WHO Drug Information, Vol.27, No.2, pages 161-162 (2013); nivoiumab (BMS-936558), a human IgG4 mAb with the structure described in WHO Drug Information, Vol.27, No.1, pages 68-69 (2013); the humanized antibodies h409A11, h409A16 and h409A17, which are described in WO2008 / 156712; AMP-514, which is being developed by Medlmmune; humanized antibody CT-011 (Pidilizumab) a monoclonal antibody being developed by Medivation, and anti-PD-1 antibodies disclosed in WO2015 / 119923 (the heavy and light chains comprise SEQ ID NO: 21 and SEQ ID NO: 22, respectively). Additional PD-1 inhibitors include Cemiplimab (Libtayo, approved for the treatment of cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for curative surgery or curative radiation); and Dostarlimab (Jemperli, approved for the treatment of mismatch repair deficient (dMMR) recurrent or advanced endometrial cancer and mismatch repair deficient (dMMR) recurrent or advanced solid tumors).

[0131] In certain embodiments, the antibody, or antigen-binding portion thereof, binds to PD-L1, such as human PD-L1. Examples of mAbs that bind to human PD-L1 are described, e.g., in WO2013 / 019906, W02010 / 077634 and US8383796. Specific anti-human PD-L1 antibodies useful for the invention described herein include, for example, MPDL3280A (Atezolizumab), MEDI4736 (Durvalumab), SB0010718C (Avelumab), BMS-936559, and an antibody which comprises the heavy chain and light chain variable regions of SEQ ID NO: 24 and SEQ ID NO: 21, respectively, of WO2013 / 019906.

[0132] In certain embodiments, the PD-1 / PD-L1 antibody, or antigen-binding portion thereof described herein inhibits the signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, or PD-L2. In certain embodiments, the antibody, or antigen-binding fragment thereof, inhibits at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the binding interactions between PD-1 and PD-L1 or PD-L2.

[0133] The PD-1 / PD-L1 inhibitor and the bispecific molecule disclosed herein may be administered concurrently or sequentially.

[0134] In certain embodiments, the PD-1 inhibitor is pembrolizumab. In certain embodiments, the PD-1 inhibitor is nivoiumab.   4.4 Subjects

[0135] In some embodiments of the present disclosure, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some aspects, the mammal is a human. 5. Kits

[0136] The present disclosure additionally provides kits comprising a bispecific molecule, nucleic acid, vector, or host cell of the present disclosure, or a combination thereof. In exemplary aspects, the bispecific molecule, nucleic acid, vector, or host cell is provided in the kit as a unit dose. For purposes herein “unit dose" refers to a discrete amount dispersed in a suitable carrier. In exemplary aspects, the unit dose is the amount sufficient to provide a subject with a desired effect, e.g., treatment of cancer. In exemplary aspects, the kit comprises several unit doses, e.g., a week or month supply of unit doses, optionally, each of which is individually packaged or otherwise separated from other unit doses. In some embodiments, the components of the kit / unit dose are packaged with instructions for administration to a patient. In some embodiments, the kit comprises one or more devices for administration to a patient, e.g., a needle and syringe, and the like. In some aspects, the bispecific molecule, nucleic acid, vector, host cell, or a combination thereof, is / are pre-packaged in a ready to use form, e.g., a syringe, an intravenous bag, etc. In exemplary aspects, the ready to use form is for a single use. In exemplary aspects, the kit comprises multiple single use, ready to use forms of the bispecific molecule, nucleic acid, vector, or host cell of the present disclosure. In some aspects, the kit further comprises other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein.

[0137] The following examples are given merely to illustrate the present invention and not in any way to limit its scope. EXAMPLES EXAMPLE 1. Anti-CD40 antibodies

[0138] Anti-CD40 antibodies were generated and characterized according to WO 2021 / 127528, pages 96-103. In particular, clone 30A12 and further optimized clones based on 30A12 (30A12.001, 30A12.002, and 30A12.003) were shown to have cross-linking dependent agonist activities, as shown in Table 1. Table 1. Functional activity of engineered variants of anti-CD40 antibodies   Antibody No Crosslinking No Crosslinking With Cros With Crosslinking Name EC50 (nM) % Max Activity slinking EC50 (nM) % Max Activity . nt- antbodes

[0139] Fully human antibodies to human FAP were generated by immunizing XENOMOUSE® transgenic mice (U.S. Pat. NOs.6,114,598; 6,162,963;6,833,268; 7,049,426; 7,064,244, which are incorporated herein by reference in their entirety; Green et al., 1994, Nature Genetics 7:13-21; Mendez et al., 1997, Nature Genetics 15:146-156; Green and Jakobovits, 1998, J. Ex. Med, 188:483- 495; Kellerman and Green, Current Opinion in Biotechnology 13, 593-597, 2002).4. Molecular Rescue and Sequencing of anti-FAP antibodies.

[0140] Through 3 arounds of harvesting, screening, and functional characterization, 82 unique heavy chains from 41 different VDJ recombination were resolved. Of these antibodies, light chain sequences for 2 antibodies from each VDJ group were resolved resulting in identification of 37 sequence- resolved unique antibodies identified from 29 different VDJ groups. Of these, 24 sequence-unique antibodies with 24 different CDR3s coming from 24 different VDJ groups were selected for bispecific molecule generation. EXAMPLE 3. Generation and characterization of bispecific molecules

[0141] This example describes the generation and characterization of FAP-targeted CD40 agonist bivalent bispecific molecules. A schematic of the steps carried out to produce bispecific molecules are shown in FIG.1. Details of steps are provided below. 1. Bispecific molecule production.

[0142] In order to generate a FAP-dependent CD40 agonist, bivalent bispecific molecules capable of binding to both human FAP and human CD40 were generated in an IgG-scFv format (FIG.2A) using the variable region binding domains of the anti-FAP and anti-CD40 antibodies described in Example 2. 2. Generation and evaluation of FAP x CD40 bispecific agonist antibodies in the IgG-scFv format.

[0143] A panel of FAP x CD40 bispecific molecules were generated in the IgG-scFv format (Table 2) and evaluated for binding to soluble forms of human and cynomolgus monkey FAP (Table 3) and human and cynomolgus monkey CD40 (Table 4) using the Octet assay. Association rate (Kon), disassociation rate (Kdis), and equilibrium binding constant (KD) were calculated. The majority of   bispecific molecules showed high affinity binding to both human and cynomolgus monkey FAP. These bispecific molecules were next evaluated for their ability to induce FAP-dependent activation of human B cells. CHO cells expressing human FAP, CHO cells expressing cynomolgus monkey FAP, or CHO cells without FAP expression were seeded into a 96 well plate. The next day varying concentrations of the FAP x CD40 IgG-scFv bispecific molecules were added to the wells along with purified human B cells or human PBMC and the plates were incubated for an additional 48 – 72 hours. Upregulation of CD86, a marker of CD40-mediated B cell activation, was quantified on B cells by flow cytometry by using CD20 as B cell marker. EC50 values for CD86 upregulation by CD20+ cells were calculated and demonstrate that the majority of FAP x CD40 IgG-scFv bispecific molecules were able to induce B cell activation in the presence of CHO cells expressing human FAP or cynomolgus monkey FAP while all but one showed no agonist activity in the absence of FAP expression (Table 5). Table 2. Anti-CD40xFAP IgG-scFv bispecific molecules Antibody ID IgG (CD40) scFv (FAP) 18799-1 30A12.001 35A10   Table 3. Anti-CD40xFAP IgG-scFv bispecific molecule binding to human and cynomolgus monkey FAP Human FAP Cyno FAPAntibody KD Kon D (nM)(1 / Ms)Kdis (1 / KD Kon Is)(nM)(1 / Ms)Kdis (1 / s)5 5 5 - 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 5 5

[0002] Table 4. Anti-CD40xFAP IgG-scFv bispecific molecule binding to human and cynomolgus monkey CD40 Human CD40 Cyno CD40Antibody KD KonKdis (1 / KD Kon ID M1Ms)M1MKdis (1 / s)3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Table 5. Anti-CD40xFAP IgG-scFv bispecific molecule functional activity on human B cells in the presence of CHO cells expressing human or cynomolgus monkey FAP Control Human FAPCHO C no FAPCHOCHO   18803-1 0.0029 0.0032 0.0055 0.0012 No 18804-1 0.0048 0.0029 0.0061 0.0016 No EXAMPLE 4. Molecular engineering of bispecific molecules

[0144] This example describes the further engineering of FAP x CD40 bispecific molecules in IgG-scFv format. 1. Sequence optimization of FAP x CD40 bispecific agonist antibodies in IgG-scFv format

[0145] Selected bispecific molecule sequences were analyzed specifically in the anti-FAP scFv domains for structural and chemical liabilities which could cause stability issues. Mutation sequences in the framework regions of the molecule were analyzed for potentially detrimental mutations occurring during somatic hypermutations. Fixes for these residues were to back mutate to the germline residue. Chemical liabilities were identified through sequence analysis for free cysteines, N-linked glycosylation, tryptophan oxidation, asparagine deamination, and aspartic acid isomerization. Replacement residues for those positions were selected after antibody homology modeling through MOE (Molecular Operating Environment by Chemical Computing Group) for best fit residues. Recombinant variants of the parental molecule were made with various combinations of the mentioned fixes (Table 6). These were produced   using standard cloning, expression, and purification technologies. Selected mutational variants were chosen based on purification quality, production yields, and functional activity. Table 6. Engineered anti-CD40xFAP IgG-scFv bispecific molecules Antibody IgG scFv Antibody IgG scFv Antibody IgG scFv ID (CD40) (FAP) ID (CD40) (FAP) ID (CD40) (FAP) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1   26046-1 30A12.001 35A10 26132-1 30A12.001 35D1 25997-1 30A12.001 35H1 26076-1 30A12.001 35A10 26133-1 30A12.001 35D1 25010-2 30A12.001 35H1 1 1 1 1 1 1 1 1 1 1 0 0 0 3

[0146] Table 7 summarizes the functional potency of engineered bispecific molecules. Assays were conducted following the protocol described in Example 3 using CHO cells stably expressing human FAP and examining CD86 upregulation by human B cells in the presence of the bispecific molecules. “EC50 transit” refers to the concentration of molecule that gives a 50% of average max activity for a given assay. Table 7. Functional potency of engineered anti-CD40xFAP IgG-scFv bispecific molecules on human B cells in the presence of CHO cells expressing human FAP Antibody N=1 N=2 Antibody N=1 N=2 Antibody N=1 N=2 ID EC50 EC50 ID EC50 EC50 ID EC50 EC50 7 1 5 1 1 8 2 9 0 4 4 0 2   26024-1 0.00178 0.00046 26047-1 0.00069 0.00034 26068-1 >2.813 >2.813 26025-1 0.00084 0.00045 26111-1 0.00036 0.00021 26069-1 >1.607 >1.607 2 9 7 9 6 1 8 7 4 9 2 8 5 5 5 8 2 1 2 0 9 4 7 3 2 6 9 9 1 3 2 1 5 2 6 3 EXAMPLE 5. FAP x CD40 bispecific molecules in IgG-scFv and Fab-scFv-Fc formats

[0147] This example demonstrates the generation and characterization of FAP x CD40 bispecific molecules in IgG-scFv and Fab-scFv-Fc formats. 2. Generation of sequence optimized FAP x CD40 bispecific agonist antibodies in IgG-scFv and Fab-scFv-Fc formats

[0148] Select bispecific molecules sequences from Example 4 were used to generate additional bispecific molecules in IgG-scFv and Fab-scFv-Fc formats. Selected mutational variants were chosen based on purification quality, production yields, and functional activity. Some of the Fab-scFv-Fc molecules were generated without cysteine-clamp. These molecules were produced using standard cloning, expression, and purification technologies. Molecules purified using Protein A column were tested for their functional activity following the protocol described in Example 3 using CHO cells stably expressing human FAP and examining CD86 upregulation by human B cells in the presence of the bispecific molecules. Table 8 shows the origin of the CD40 and FAP clones used to generate the bispecific molecules and their functional potency. Table 8. Sequence optimized anti-CD40 x FAP bispecific molecules in IgG-scFv and Fab-scFv-Fc with or without cysteine-clump (CC) Em Fabv EC ax Emax scF 50 (Fold EC50 (Fold e)   PUR129888-3 30A12.001 35F7 Fab-scFv-Fc 0.0057 5.50.0080 5.7PUR129889-3 30A12.001 6E1 Fab-scFv-Fc 0.0077 7.30.0100 8.03 . Selection of preferred molecules

[0149] Of these bispecific molecules, a number of molecules were not selected to move forward based on yield, purity, aggregation, FAP epitope, and functional activity. The final molecules included 4 IgG-scFv CC, 4 Fab-scFv-Fc CC, and one Fab-scFv-Fc, which were analyzed further. The sequences of these preferred bispecific molecules are shown in the Sequence Tables. Melting point (Tm), biophysical stability, thermostability, low molecular weight species, and high molecular weight species were assessed (data not shown). Functional activity of the molecules was assessed following the protocol described in Example 3 using CHO cells stably expressing human FAP and examining CD86 upregulation by human B cells in the presence of the bispecific molecules. In addition, the ability of the molecules to activate monocyte derived dendritic cells (MDDC) was assessed. Briefly, primary human monocytes were differentiated into MDDCs using standard protocol and the ability of the molecules to activate these cells in the presence of HEK293T cells expressing human FAP or human dermal fibroblast (HDF). MDDC activation was detected after 48 hours by measuring IL12p40 secretion by MSD. Based on data gathered from these analyses, three preferred molecules were selected: one IgG-scFv CC (26050-3) and two Fab- scFv-Fc CC (28906-3 and 29907-3). Table 9 and Table 10 summarize the results of these functional assays for select molecules. Table 9. Ability of select anti-CD40 x FAP bispecific molecules to activate human B cells in the presence of CHO cells expressing human FAP Emax Emax EC50 Emax w / o EC50 Emax w / o e) Table 10. Ability of select bispecific anti-CD40 x FAP bispecific molecules to activate human MDDC in the presence of 293 cells expressing human FAP or HDF   293-FAP HDF Antibod Fab scFv E E ) 4. Generation of half-life extended molecule

[0150] One of the bispecific molecules was also produced to incorporate YTE mutation in the Fc domain (M252Y / S254T / T256E) as half-life extension using standard cloning, expression, and purification technologies. Its activity in comparison to the non-YTE version of the molecule was examined by human B cell (Table 11) and human MDDC assays (Table 12). Table 11. Functional activity of half-life extended anti-CD40 x FAP bispecific molecules tested by primary human B cells and CHO cells expressing human FAP CHO-FAP CHO i Emax Emax Emax Emax e) Table 12. Functional activity of half-life extended anti-CD40 x FAP bispecific molecules tested by MDDC cells and 293T cells expressing human FAP Donor 1 Donor 2 )   40291 0.009 557 0.0080 546 0.017 192 0.016 130 EXAMPLE 6. Anti-tumor activity of stroma-targeted CD40 agonist bispecific molecule in mouse models

[0151] This example describes in vivo anti-tumor activity of the bispecific molecules disclosed herein. 1. Generation of a mouse surrogate FAP-targeted CD40 agonist bispecific molecule to test in preclinical tumor models with endogenous FAP expression

[0152] The anti-mouse CD40 agonist antibody (clone FGK45) and anti-mouse FAP antibody (clone 5A5) was used to generate a surrogate CD40 bispecific molecule in the IgG-scFv format with a mouse IgG1 N297G Fc domain (anti-muCD40 x muFAP bispecific molecule. The bispecific molecule contains the anti- CD40 moiety at the N-terminus of the molecule and the anti-FAP moiety as a scFv at the C-terminus of the molecule. This mouse surrogate was evaluated in the EMT6 breast tumor model with endogenous FAP content (10-20% endogenous FAP expression as detected using FAP IHC) (figure not shown). Furthermore, this tumor model is refractory to anti-PD-1 / PD-L1 checkpoint inhibitor treatment as single agent, enabling combination efficacy studies with CD40 monoclonal or FAP-CD40 bispecific molecule treatment. 2. In vivo tumor-localized immunostimulatory activity of the mouse surrogate FAP-targeted CD40 bispecific molecule, without systemic effects

[0153] To evaluate the pharmacodynamic (PD) effects of a stroma-targeted CD40 agonist bispecific molecule on immune cell activation in tumors versus periphery, EMT6 tumor cells were inoculated in the right flank of wild-type Balb / c mice at 3e5cells per implant on day 0. Tumors were randomized and assigned on day 11 into different treatment groups and dosed intra-peritoneally on days 11 and 14 with either isotype control mIgG1 (30 mg / kg), non-targeted anti-CD40 (5 mg / kg) or FAP-targeted CD40 bispecific moelcule (30 mg / kg). Tumors, tumor draining lymph nodes (dLNs), and spleen (Spl) were harvested at 48 hours post-treatment and single cell suspension prepared by enzymatic digestion of tissues. Flow cytometry analysis was performed to determine myeloid cell proportions and activation (up- regulation of CD86). As expected, non-targeted CD40 agonist antibody (anti-CD40) treatment increased and activated CD103+dendritic cells (DCs) located in tumor and in peripheral tissues including dLN and Spl. In contrast, the FAP-targeted CD40 bispecific molecule specifically activated and increased tumor infiltrating DCs in the EMT6 tumors (FIG.3). This shows that Surrogate CD40-FAP bispecific molecule activates tumor-resident APCs.

[0154] In a separate experiment, EMT6 tumor bearing animals were treated with a single dose of either 25 mg / kg of control isotype mIgG1, 5 mg / kg of non-targeted CD40 mAb or 25 mg / kg of CD40-FAP bispecific molecule intra-peritoneally on study day 11. Serum was collected at 72 hours post treatment and assessed for levels of pro-inflammatory cytokines and liver enzymes. Non-targeted anti-CD40 antibody increased concentrations of several pro-inflammatory cytokines (IL-12, TNFa, KC / CXCL1) in the serum of mice, consistent with systemic immune cell activation noted with CD40 agonists in the clinic. In contrast, the FAP-targeted CD40 bispecific molecule did not drive any systemic cytokine response (FIG.4A). We also did not detect any changes in liver enzymes (AST and ALT levels) in the bispecific molecule-treated group (FIG.4B). Taken together, these data indicate that a stroma-targeted CD40 agonist is capable of inducing localized immune cell activation in the tumor, without leading to systemic cytokine production or liver damage that has been associated with anti-CD40-mediated toxicity in the clinic. 3. Anti-tumor effects of FAP-targeted CD40 bispecific molecule in combination with immune checkpoint blockade

[0155] To evaluate the potential for this therapeutic approach to combine with blockade of an immune checkpoint inhibitor, we performed efficacy studies in the EMT6 tumor model that is refractory to single agent anti-PD1. Monotherapy with anti-CD40 monoclonal as well as FAP-CD40 bispecific also had minimal effect on EMT6 tumor growth (data not shown). To test the combination potential, mice bearing EMT6 tumors were treated with either an isotype control antibody (25 mg / kg), or the combination of non- targeted anti-CD40 agonist antibody (5 mg / kg) with anti-PD1 (5 mg / kg) or FAP-targeted CD40 bispecific (25 mg / kg) with anti-PD1 (5 mg / kg) (FIGs.5A-5C). Strikingly, both the combination groups demonstrated comparable and significant tumor growth inhibition in this model (FIGs.5A and 5B) and increased survival relative to isotype control group (FIG.5C). At the end of the study, there were 3 and 2 tumor-free animals (complete responders; CRs) in both groups 2 and 3, respectively.

[0156] Taken together, these data demonstrate that a stroma-targeted CD40 agonist therapy can enhance anti-tumor immune responses in combination with checkpoint blockade. Importantly, the stroma-targeted approach drives anti-tumor efficacy comparable to a non-targeted CD40 agonist but offers improved therapeutic index (tumor-localized CD40 activation with no systemic activation & liver tox). These datasets highlight the potential of the FAP-targeted CD40 bispecific molecule to delivery efficacy with fewer side-effects compared to non-targeted CD40 agonists. Sequence Table A: CD40 sequences SEQ Description sequence ID   2 CD40 CDR-H2 (30A12.001)ATGGSGISTYYADSVKG3 CD40 CDR-H3 G W E G G W P K G W P K P L N D E G D E Sequence Table B: 26050-3 sequences CD40_30A12.001 (IgG) / FAP_35F7 (scFv) C-C SEQ Description sequence ID   FAP CDR-L3 (35F7.23105A)ASFAGSYTWVFAP VH QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYGMHWVRQAPGKCLEWVAVIW G D G G W P K P L N D G D V N E E G D E C G T A G G T C G C T G A T T T G G G G   AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC C G G C A T C T C T G A C T A G T A T G G A A C G T A A G CD40_30A12.001 (Fab) / FAP_35F7 (scFv) / Fc SEQ Description Sequence ID   FAP VH QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYGMHWVRQAPGKCLEWVAVIW (35F7.23105A) YDGRNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGSGGG FDPWGQGTLVTVSS D G W G T L G W P K G T G M F E H Q K E G D E C G T A G G T C G C T G G G A T C G   CTTTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACT GTGCAAGAGATGGATCTGGTGGAGGATTTGATCCTTGGGGACAAGGAACACT TGTTACAGTTTCTTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGT G A G T A C C T G A A C C A G G T G C A T G G A A C G T A A G CD40_30A12.001 (IgG) / FAP_37B3 (scFv) C-C SEQ Description sequence ID   FAP CDR-L3 (37B3.23109A)ASYAGSSTWVFAP VH EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVIW D D G G W P K P L N D G D G D L E G D E C G T A G G T C G C T G A T T T G G G   CCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG C C G G C A T C T C C G C T C G C A A T G G A A C G T A A G CD40_30A12.001 (Fab) / FAP_37B3 (scFv) / Fc SEQ Description Sequence ID   FAP CDR-L3 (37B3.23109A)ASYAGSSTWVFAP VH EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVIW D D G W D I G G W P K G T G K S I V E K E G D E C G T A G G T C G C T G G G A T   GCCTGGAGTGGGTGGCAGTTATCTGGAATGATGGAAGTAATAAATACTATGC AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAAACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACT G C G A A A G T G G C C A C G A G A A G G C A T G G A A C G T A A G CD40_30A12.001 (IgG) / FAP_6G12 (scFv) C-C SEQ Description sequence ID   FAP CDR-L1 (6G12.23099A)SGSSSNIGSNTVNFAP CDR-L2 H G N G H G R G G W P K P L N D V S W S L E G D E C G T A G G T C G C T G A T T T G   CGAGGAGCAGTACGGCAGCACGTACCGTTGCGTCAGCGTCCTCACCGTCCTG CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAAG CCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG C G C C G G G T A C G G G G C C C G C A A T G G A A C G T A A G Sequence Table G: 28906-3 sequences CD40_30A12.001 (Fab) / FAP_6G12 (scFv) / Fc SEQ Description Sequence ID   FAP CDR-L2 (6G12.23099A)SNNQRPSFAP CDR-L3 H G N G H G R G G W P K G S G T S M S E S E G D E C G T A G G T C G C T G G C   TGTGCTGGTGAAACCCACAGAAACCCTCACGCTGACCTGCACCGTGTCTGGG TTCTCACTCTCTAATGCTAGAGTGGGTGTGAGCTGGATCCGTCAGCCCCCAG GGAAGTGCCTGGAGTGGCTTGCACACATTTTTTCGAATGACAAAAAATCCTA C T T C T G G G G C G C G G C C A C G C A C T T A T G G A A C G T A A G CD40_30A12.001 (Fab) / FAP_6G12 (scFv) / Fc SEQ Description Sequence ID   FAP CDR-L1 (6G12.23099A)SGSSSNIGSNTVNFAP CDR-L2 H G N G H G R G G W P K G S G T S Y S E S E G D E C G T A G G T C G C T G   CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGGTG GTGGCGGATCGGGAGGTGGCGGATCCCAGGTCACCTTGAAGGAGTCTGGTCC TGTGCTGGTGAAACCCACAGAAACCCTCACGCTGACCTGCACCGTGTCTGGG G A C T T C T G G G G C G C C G C C A C G C A C T T A T G G A A C G T A A G Sequence Table I: 28894-3 sequences CD40_30A12.001 (IgG) / FAP_6E1 (scFv) C-C SEQ Description sequence ID   FAP CDR-H2 (6E1.23098A)YISTSSTTIYYADSVKGFAP CDR-H3 S Y E G G G W P K P L N D G D Q V Q E G D E C G T A G G T C G C T G   CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAA CTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT T G G G G C G C C G G C A T C T C A T G T A T G T A T G G A A C G T A A G Sequence Table J: 28899-3 sequences CD40_30A12.001 (Fab) / FAP_6E1 (scFv) / Fc SEQ Description Sequence ID   FAP CDR-H3 (6E1.23098A)EPSYYYYYYGMDVFAP CDR-L1 S Y E G S Y T N G W P K G S G A S S L M L E G D E C G T C G T C C C T   CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC GTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACA AGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGG G G A C T A G G T T G A G A G G T C G C T A A G G C T T A T G G A A C G T A A G Sequence Table K: 28907-3 sequences CD40_30A12.001 (Fab) / FAP_6E1 (scFv) / Fc SEQ Description Sequence ID   FAP CDR-H1 (6E1.23098A)RYSMNFAP CDR-H2 S Y E G S Y T N G W P K G S G A S S L M L E G D E C G T A G G   (CD40_30A12.001 TGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTGTCCTCAGCCT / FAP CCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTC 6E1.23098A) TGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG C T G G G A T C A T G C T A C C T C G G C G C C T G G G C C A C A T G G A A C G T A A G eque ce a e . o s a eg o s a o e seque ces SEQ Name Sequence ID   Human LC TVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ (kappa) WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC E V S F ) P S I E F T N P E H F T N P E H F T N P E H F T N P E H F T N P   mutation and APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE terminal G SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG F T N P E H P S I E P S I E P S I E P S I E P S I E P S I E F T N P E H F T N P E H F T   domain with HTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFN SELF2 LALA YTE WYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALP mutation and APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE H F E I N F C V I G F D E I N F V S N   KVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0158] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.

[0159] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0160] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0161] Preferred embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be   practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED:

1. A bispecific molecule, comprising: (A) a Fab moiety that binds to CD40, a scFv moiety that binds to FAP, and a Fc region comprising CH2 and CH3 domains; or (B) a Fab moiety that binds to FAP, a scFv moiety that binds to CD40, and a Fc region comprising CH2 and CH3 domains; wherein: (i) said Fab moiety comprises two chains: a heavy chain comprising a heavy chain variable domain A (VHA) and a CH1 domain, and a light chain comprising a light chain variable domain A (VLA) and a CL domain; (ii) said scFv moiety comprises a heavy chain variable domain B (VHB) and a light chain variable domain B (VLB), wherein said VHB and VLB are connected via a first linker; (iii) said Fab is connected to said scFv moiety via a second linker; and (iv) said scFv moiety is connected to said CH2 domain via a third linker; wherein said CD40 binding moiety comprises: (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:31; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 32; and wherein said FAP binding moiety comprises: (1) (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:33; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 34; (2) (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:35; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 36; (3) (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:37; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 38; (4) (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:39; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 40; or (5) (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:41; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:

42.

2. A bispecific molecule, comprising: (A) a Fab moiety that binds to CD40, a scFv moiety that binds to FAP, and a Fc region comprising CH2 and CH3 domains; or (B) a Fab moiety that binds to FAP, a scFv moiety that binds to CD40, and a Fc region comprising CH2 and CH3 domains. wherein: (i) said Fab moiety comprises two chains: a heavy chain comprising a heavy chain variable domain A (VHA) and a CH1 domain, and a light chain comprising a light chain variable domain A (VLA) and a CL domain; (ii) said scFv moiety comprises a heavy chain variable domain B (VHB) and a light chain variable domain B (VLB), wherein said VHB and VLB are connected via a first linker; (iii) said Fab is connected to said Fc region; and (iv) said scFv moiety is connected to said Fc region via a second linker;  wherein said CD40 binding moiety comprises: (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:31; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 32; and wherein said FAP binding moiety comprises: (1) (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:33; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 34; (2) (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:35; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 36; (3) (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:37; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 38; (4) (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:39; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 40; or (5) (i) the heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:41; and (ii) the light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:

42.

3. The bispecific molecule of claims 1 or 2, comprising a CD40 antigen-binding moiety that comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 comprising SEQ ID Nos.1-6, respectively.

4. The bispecific molecule of any one of claims 1-3, comprising a FAP antigen-binding moiety that comprises: (1) a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 comprising SEQ ID Nos.7-12, respectively; (2) a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 comprising SEQ ID Nos.13-18, respectively; (3) a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 comprising SEQ ID Nos.19-24, respectively; or (4) a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 comprising SEQ ID Nos.25-30, respectively 5. The bispecific molecule of any one of claims 1-4, comprising a human VL framework and a human VH framework.

6. The bispecific molecule of any one of 1-5, comprising a FAP antigen-binding moiety that comprises: (1) a VH comprising a sequence that is at least 90 identical to SEQ ID NO: 33, and a VL comprising a sequence that is at least 90 identical to SEQ ID NO: 34; (2) a VH comprising a sequence that is at least 90 identical to SEQ ID NO: 35, and a VL comprising a sequence that is at least 90 identical to SEQ ID NO: 36;  (3) a VH comprising a sequence that is at least 90 identical to SEQ ID NO: 37, and a VL comprising a sequence that is at least 90 identical to SEQ ID NO: 38; (4) a VH comprising a sequence that is at least 90 identical to SEQ ID NO: 39, and a VL comprising a sequence that is at least 90 identical to SEQ ID NO: 40; (5) a VH comprising a sequence that is at least 90 identical to SEQ ID NO: 41, and a VL comprising a sequence that is at least 90 identical to SEQ ID NO:

42.

7. The bispecific molecule of any one of claims 1-6, comprising a FAP antigen-binding moiety that is an scFv.

8. The bispecific molecule of any one of claims 1-7, wherein said first linker, second linker, and third linker each independently comprises any one of the amino acid sequences of SEQ ID NOs: 76-89.

9. The bispecific molecule of any one of claims 1-8, comprising a scFv moiety that binds to FAP, wherein said scFv comprises an amino acid sequence at least 90 identical to SEQ ID NO:93, 94, 95, 96, or 97.

10. The bispecific molecule of any one of claims 1-9, comprising a FAP antigen-binding moiety that comprises a VH comprising a sequence that is at least 90 identical to SEQ ID NO: 31, and a VL comprising a sequence that is at least 90 identical to SEQ ID NO:

32.

11. The bispecific molecule of any one of claims 1-10, comprising a human IgG CH1 domain that is at least 70% identical to SEQ ID NO:47, 64, 68, or 72.

12. The bispecific molecule of any one of claims 1-11, comprising a human IgG Fc domain that is at least 70% identical to SEQ ID NO: 48, 55, 56, 57, 58, 59, 60, 66, 70, or 74.

13. The bispecific molecule of any one of claims 1-12, comprising a human kappa or lambda CL domain that is at least 70% identical to SEQ ID NO: 43, 44, 45, or 46.

14. A bispecific molecule comprising: (i) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 91, and (ii) a polypeptide comprising an amino acid sequence that is at least 90% identical SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, or 117.

15. The bispecific molecule of claim 14, comprising: (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 91, and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, or 117 16. A nucleic acid comprising a nucleotide sequence encoding the bispecific molecule of any one of claims 1-15.

17. A host cell comprising the nucleic acid of claim 16.

18. A pharmaceutical composition comprising the bispecific molecule of any one of claims 1-15.

19. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount the bispecific molecule of any one of claims 1-15, or the pharmaceutical composition of 18.

20. The method of claim 19, further comprising administering a PD-1 or PD-L1 inhibitor.

21. The method of claim 21, wherein said PD-1 inhibitor is pembrolizumab or nivolumab.

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