Solid tumor t cell engagers and uses thereof

Abstract

Disclosed herein is a platform technology for designing T cell engagers that provide both Signal 1 and Signal 2 to T cells and target tumor-associated antigens on the surface of cancer cells. Non-limiting examples of such T cell engagers and nucleic acids encoding the same, as well as lipid nanoparticles comprising mRNAs encoding the T cell engagers are provided. Such T cell engagers can be used to treat cancers, including solid tumors such as ovarian cancers.

Description

[0001] Attorney Docket No.: 45817-0187WO1

[0002] SOLID TUMOR T CELL ENGAGERS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of priority of U. S. Provisional Appl. No.

[0003] 63 / 728,873, filed December 6, 2024, the contents of which are incorporated by reference herein in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 21, 2025, is named 45817-0187WOl_SL.xml and is 257,376 bytes in size.

[0006] BACKGROUND

[0007] Bispecific antibodies are of increasing relevance for therapeutic use. The conventional means of producing bispecific antibodies which are monovalent against CD3, i.e., include only one anti-CD3 binding moiety, is to produce them recombinantly to remove impurities that are bivalent against CD3. However, this is a costly and laborious process. Further, conventional means are limited by their inability to target more than a single tumor-associated antigen (TAA). Tumor heterogeneity7, immune escape, and diverse patient TAA expression profiles are common reasons for limited efficacy of antibodies targeting a single TAA.

[0008] The ability to bind to two different targets with the same antibody defines bispecific antibody function. To achieve this desired outcome, bispecific antibodies are generally designed based on a heteromeric Fc antibody format using various strategies such as knobs-in-holes, electrostatic steering, or a common light chain to generate an antibody capable of binding two different targets. However, when T cell binding specificity is included in the bispecific antibody, a functional Fc can drive crosslinking of activated T cells via the anti-CD3 binding and Fc mediated complement dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) resulting in the potential depletion of activated T cells. This results in the undesirable depletion of the activated T cells that are required for Attorney Docket No.: 45817-0187WO1

[0009] elimination of the targeted cancer cells. An antibody-like format that ensures targeting of CD3 is monovalent, i.e.. with no risk of cross-linking CD3, and which has a halflife adequate for therapy is needed in the art.

[0010] The instant disclosure advances the art by providing such antibody-like formats especially suited for treatment of solid tumors. As described in more detail below, these novel antibody-like formats permit mRNA multiplexing as well as the ability to provide both Signal 1 and Signal 2 to T cells whilst also binding to one or more TAAs that are expressed in solid tumors.

[0011] SUMMARY

[0012] Provided herein is a platform technology for designing and using multispecific binding molecules to treat disease (e.g., cancer such as solid tumors (e.g., MUC16+and / or CLDN6+and / or TROP2+solid tumors). The multi-specific binding molecules of this disclosure are T cell engagers. Using mRNA multiplexing, mRNAs encoding these T cell engagers can be used to bind to the T cell receptor (TCR) complex on a T cell (e.g., CD3) to provide Signal I, and / or to a costimulatory receptor on aT cell (e.g., 4-1BB) to provide Signal 2, as well as to several (e.g., 1, 2, 3, 4, 5) tumor-associated antigens (TAAs). A Signal 1 T cell engager molecule (TCE1) is comprised of a binding moiety (e.g., a Fab) that specifically binds to a T cell surface antigen in the TCR complex (e.g., CD3). The binding moiety that specifically binds to the T cell surface antigen (e.g., CD3) is chosen so as to not lead to nonspecific T cell activation. In some instances, the binding moiety' that specifically binds to CD3 is a Fab comprising a VH / CH1 domain and a VL / CL domain. Appended to the C-terminal of the CH I of the VH / CH1 domain of the Fab are at least two VHHs that specifically bind to the same TAA. In some cases, the TAA is a solid tumor-associated antigen (e.g., MUC16, CLDN6, TROP2). The VL / CL domain of the Fab is modified by attaching to the C-terminal of the CL, a VHH that binds to human serum albumin (HSA) so as to enhance the circulating half-life of the multi-specific binding molecule. In some cases, more than one (e.g., 2, 3, 4) Signal 1 T cell engager molecules are employed. If two Signal 1 T cell engager molecules are employed, they comprise binding moieties to different TAAs. For Attorney Docket No.: 45817-0187WO1

[0013] example, one Signal 1 T cell engager molecule comprises a binding moiety to MUC16, and the other Signal 1 T cell engager molecule comprises a binding moiety to CLDN6. Tn the context of the Fab-VHH-VHH format, a common light chain is employed to multiplex several T cell engager molecules such that they can be delivered in a single formulation. This approach is not possible with conventional protein delivery; however, it is achievable utilizing the mRNA delivery system described herein. mRNA multiplexing of the multi-specific binding molecules described herein facilitates proper assembly and expression of multiple therapeutic anti-CD3 multi-specific molecules in vivo. This disclosure demonstrates that it is possible to express multiple TAA-targeted anti-CD3 multi-specific binding molecules by co-formulating strands of mRNA encoding several heavy chains and one strand of mRNA encoding a common light chain with an anti-HSA binding domain. The Fab-VHH-VHH format overcomes the toxicity' concerns observed with bivalent anti-CD3, the efficacy shortcomings of targeting only one TAA, and the short half-life of proteins. The constructs described herein enable the targeting of multiple (e.g., 2. 3, 4, 5) TAAs and T cells to thereby bridge cancer cells, which can be diverse in terms of their antigen expression, and immune cells to facilitate elimination of the cancer cells. In addition to the Signal 1 T cell engager molecule(s), the disclosure also features a Signal 2 T cell engager (TCE2) that binds to a costimulatory receptor on a T cell (e.g., 4-1BB). Such a T cell engager is comprised of a binding moiety7(e.g., a VHH) that specifically7binds to a TAA (e.g., a TAA expressed in solid tumors) linked to a binding moiety (e.g., a VHH) that specifically binds to a costimulatory receptor on the T cell (e.g.,4-lBB). In some instances, the TAA targeted by the Signal 2 TCE is TROP2. In other instances, TAA targeted by the Signal 2 TCE is MUC16. If TCE1 and TCE2 comprise binding moieties that binds to MUC16, then the binding moiety that binds to MUC16 in the TCE2 molecule is a different binding moiety7than those used in TCE1. mRNAs encoding these TCE molecules can be administered to human subjects using a delivery vehicle such as a nanoparticle (e.g., a lipid nanoparticle). One, two, three, four, or more mRNAs can be formulated in one or more LNPs. In some cases, mRNAs encoding a T cell engager(s) that provide Signal 1 are formulated in one nanoparticle and mRNAs encoding a T cell engager that provide Signal 2 are Attorney Docket No.: 45817-0187WO1

[0014] formulated in a separate nanoparticle. In certain cases, these nanoparticles are mixed together for administration to a patient or are administered separately (e.g., concurrently or sequentially). In other cases, mRNAs encoding a T cell engager(s) that provide Signal 1 and mRNAs encoding a T cell engager that provide Signal 2 are formulated in the same nanoparticle. In yet another instance, a LNP comprising the Signal 2 engager can be administered alone. In some cases, the nanoparticle is formulated with one mRNA. In some cases, the nanoparticle is formulated with two mRNAs. In some cases, the nanoparticle is formulated with three mRNAs. In some cases, the nanoparticle is formulated with four mRNAs.

[0015] The present disclosure provides, among other things, a first lipid nanoparticle (LNP) and a second LNP, wherein the first LNP comprises a first mRNA molecule, a second mRNA molecule, and a third mRNA molecule. The first mRNA molecule comprises a first ORF encoding a first polypeptide comprising from the N-terminal to C -terminal a first VH and a first CH I linked via the C-terminal of the first CH I directly or via a first linker to the N-terminal of a first VHH that binds to a first tumor-associated antigen, the C-terminal of the first VHH linked directly or via a second linker to the N-terminal of a second VHH that binds to the first tumor-associated antigen. In some cases, the first VHH and the second VHH have the same amino acid sequence. The second mRNA molecule comprises a second ORF encoding a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a third linker to the N-terminal of a third VHH that binds to a second tumor-associated antigen, the C-terminal of the third VHH linked directly or via a fourth linker to the N-terminal of a fourth VHH that binds to the second tumor-associated antigen. In some cases, the third VHH and the fourth VHH have the same amino acid sequence. The third mRNA molecule comprises a third ORF encoding a third polypeptide comprising from the N-terminal to C-terminal a first VL and a first CL linked via the C-terminal of the CL directly or via a fifth linker to the N-terminal of a fifth VHH that binds to human serum albumin. The first VH and first CHI of the first polypeptide associates with the first VL and first CL of the third polypeptide to form a Fab and the second VH and second CHI of the second polypeptide associates with the first VL Attorney Docket No.: 45817-0187WO1

[0016] and first CL of the third polypeptide to form a Fab, wherein the Fabs specifically bind to human CD3 (e.g., human CD3E). The second LNP comprises the first mRNA molecule, the second mRNA molecule, the third mRNA molecule, and a fourth mRNA molecule. The fourth mRNA molecule comprises a fourth ORF encoding a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that binds to a third tumor-associated antigen linked directly or via a sixth linker to the N-terminal of an immunologically silent human Ig Fc domain comprising from N terminal to C-terminal a hinge, a CH2 domain, and a CH3 domain of an immunologically silent human Ig Fc domain. In some cases, the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain from a human IgG4PAA polypeptide. The C-terminal of the immunologically silent human Ig Fc domain linked directly or via a seventh linker to the N-terminal of a seventh VHH that binds to a costimulatory molecule on the surface of an activated T cell. The fourth polypeptide associates with itself to form a homodimer. In some cases, all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0017] In another aspect, the disclosure provides a lipid nanoparticle comprising a first mRNA molecule, a second mRNA molecule, a third mRNA molecule, and a fourth mRNA molecule. The first mRNA molecule comprises a first ORF encoding a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a first linker to the N-terminal of a first VHH that binds to a first tumor-associated antigen, the C-terminal of the first VHH linked directly or via a second linker to the N-terminal of a second VHH that binds to the first tumor-associated antigen. In some cases, the first VHH and the second VHH have the same amino acid sequence. The second mRNA molecule comprises a second ORF encoding a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a third linker to the N-terminal of a third VHH that binds to a second tumor-associated antigen, the C-terminal of the third VHH linked directly or via a fourth linker to the N-terminal of a fourth VHH that binds to the second tumor-associated antigen. In some cases, the third VHH and the Attorney Docket No.: 45817-0187WO1

[0018] fourth VHH have the same amino acid sequence. The third mRNA molecule comprises a third ORF encoding a third polypeptide comprising from the N-terminal to C-terminal a first VL and a first CL linked via the C-terminal of the CL directly or via a fifth linker to the N-terminal of a fifth VHH that binds to human serum albumin. The fourth mRNA molecule comprises a fourth ORF encoding a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that binds to a third tumor-associated antigen linked directly or via a sixth linker to the N-terminal of an immunologically silent human Ig Fc domain comprising from N terminal to C-terminal a hinge, a CH2 domain, and a CH3 domain of an immunologically silent human Ig Fc domain. In certain cases, the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain from an IgG4PAA polypeptide. The C-terminal of the immunologically silent human Ig Fc domain linked directly or via a seventh linker to the N-terminal of a seventh VHH that binds to a costimulatory molecule on the surface of an activated T cell. The first VH and first CHI of the first polypeptide associates with the first VL and first CL of the third polypeptide to form a Fab and the second VH and second CHI of the second polypeptide associates with the first VL and first CL of the third polypeptide to form a Fab, wherein the Fabs specifically bind to human CD3 (e g., human CD3E). The fourth polypeptide associates with itself to form a homodimer. In some cases, all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0019] In some instances, the first, second, and third tumor-associated antigens are solid tumor-associated antigens. In certain cases, the first tumor-associated antigen is MUC16, the second tumor-associated antigen is CLDN6, the third tumor-associated antigen is TROP2; and the coslimulatory molecule is 4- IBB. In other cases, the first tumor-associated antigen is MUC16, the second tumor-associated antigen is CLDN6, the third tumor-associated antigen is MUC16; and the costimulatory molecule is 4-1BB.

[0020] In certain instances, the C-terminal of the first CHI is linked via the first linker to the N-terminal of the first VHH, optionally wherein the first linker is G4S (SEQ ID NO:4); the C-terminal of the first VHH is linked via the second linker to the Attorney Docket No.: 45817-0187WO1

[0021] N-terminal of the second VHH, optionally wherein the second linker is (G4S)3 (SEQ ID NO:5); the C-terminal of the second CEI1 is linked via the third linker to the N-terminal of the third VHH, optionally wherein the third linker is G4S (SEQ ID NO:4); the C-terminal of the third VHH is linked via the fourth linker to the N-terminal of the fourth VHH, optionally wherein the fourth linker is (G4S)3 (SEQ ID NO: 5); the C-terminal of the first CL is linked via the fifth linker to the N-terminal of the fifth VHH, optionally wherein the fifth linker is G4S (SEQ ID NO:4); and the C-terminal of the immunologically silent human Ig Fc domain is linked via the seventh linker to the N-terminal of the sixth VHH, optionally wherein the seventh linker is G4S (SEQ ID NO:4).

[0022] In some instances, the first CHI and the second CHI have the identical amino acid sequence. In some cases, the first CHI and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:28. In some instances, the first CL and the second CL have the identical amino acid sequence. In some cases, the first CL and the second CL comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:30.

[0023] In certain instances, the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain of a human IgG4PAA polypeptide which comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:33.

[0024] In some instances, the first VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40. In some cases, the first VHH binds to the 5-SEA domain of human MUC16. In certain instances, the second VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40. In some cases, the second VHH binds to the 5-SEA domain of human MUC16. In some instances, the third VHH comprises a VHH-CDR1, a VHH-CDR2, Attorney Docket No.: 45817-0187WO1

[0025] and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43. In certain cases, the third VHH binds specifically to human CLDN6 relative to CLDN3, CLDN4. and CLDN9. In some instances, the fourth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43. In certain cases, the fourth VHH binds specifically to human CLDN6 relative to human CLDN3, human CLDN4, and human CLDN9. In some instances, the fifth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:208. In certain instances, the sixth VHH comprises aVHH-CDRl, aVHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:206. In some cases, the sixth VHH binds specifically to human TROP2 relative to human EpCAM. In some instances, the seventh VHH comprises a VHH-CDR1. a VHH-CDR2. and a VHH-CDR3 of the VHH set forth in SEQ ID NO: 37. In some instances, the first VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40; the second VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40; the third VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 43; the fourth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43; the fifth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:208; the sixth VHH comprises an amino acid sequence that is 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% Attorney Docket No.: 45817-0187WO1

[0026] identical to the sequence set forth in SEQ ID NO: 206; and the seventh VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:37.

[0027] In some instances, the first VH and second VH each comprise a CDR1, a CDR2, and a CDR3 of the VH set forth in SEQ ID NO: 26, and the first VL and second VL each comprise a CDR1, a CDR2, and a CDR3 of the VL set forth in SEQ ID NO:27. In some cases, the first VH and second VH each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:26, and the first VL and second VL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:27. In certain cases, the first VH and the first CHI and the second VH and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 31, and the first VL and first CL and second VL and second CL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:32.

[0028] In some instances, the first polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:55. In certain instances, the second polypeptide comprises an amino acid sequence that is 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 the Attorney Docket No.: 45817-0187WO1

[0029] sequence set forth in SEQ ID NO:56. In some instances, the third polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 57. In certain instances, the fourth polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:58.

[0030] In certain instances, the first ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO: 60 or 230. In some instances, the second ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO: 61 or 231. In some instances, the third ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:62 or 232. In certain instances, the fourth ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:63 or 233.

[0031] In certain instances, the mRNA molecules further comprise one or more of: a 5’ terminal cap (optionally comprising m7GpppGm); a 5’UTR (optionally comprising the sequence of SEQ ID NO: 64); if a signal sequence is not included as part of the ORF, a signal sequence (optionally comprising the sequence of any one of SEQ ID NOs:225. 226, 68, or 227); a 3'UTR (optionally wherein the 3’UTR comprises one or more miR binding sites and / or an IDR, and further optionally comprising the sequence of any one of SEQ ID NOs:234, 235, 130, or 236); and a poly A tail (optionally wherein the poly A tail is about 100 nucleotides in length, optionally Attorney Docket No.: 45817-0187WO1

[0032] comprising the sequence of SEQ ID NO: 132). In some cases, all the uracils of the mRNA molecules are N1 -methylpseudouracils, or the uridines of the mRNA are Nl-methy 1 ps eudouri di nes.

[0033] In certain instances, the lipid nanoparticle or lipid nanoparticles comprise an ionizable amino lipid, a structural lipid, a phospholipid, and a polyethylene glycol (PEG)-modified lipid. In some cases, the ionizable amino lipid is present at about 40 to 50 mole ratio %, the structural lipid is present at 35 to 45 mole ratio %, the phospholipid is present at 5 to 15 mole ratio %, and the PEG-modified lipid is present at 1.5 to 5 mole ratio %. In other cases, the ionizable amino lipid is present at about 47.5 mole ratio %, the structural lipid is present at about 39 mole ratio %. the phospholipid is present at about 10.5 mole ratio %, and the PEG-modified lipid is present at about 3 mole ratio %. In yet other cases, the ionizable amino lipid is present at 47.5 mole ratio %, the structural lipid is present at 39 mole ratio %, the phospholipid is present at 10.5 mole ratio %, and the PEG-modified lipid is present at 3 mole ratio %. In some cases, the ionizable amino lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate and the PEG-modified lipid is 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78.81,84,87,90,93, 96, 99, 102, 105, 108, 111,114, 117,120, 123,126, 129, 132-tetratetracontaoxatetratriacontahectyl stearate. In certain cases, the ionizable amino lipid comprises Compound 2 and the PEG-modified lipid comprises Compound I.

[0034] In certain instances, the ionizable amino lipid is Compound 2 and is present at about 47.5 mole ratio %, the structural lipid is cholesterol and is present at about 39 mole ratio %, the phospholipid is DSPC and is present at about 10.5 mole ratio %, and the PEG-modified lipid is Compound I and is present at about 3 mole ratio %. In some cases, the ionizable amino lipid is Compound 2 and is present at 47.5 mole ratio %, the structural lipid is cholesterol and is present at 39 mole ratio %, the phospholipid is DSPC and is present at 10.5 mole ratio %, and the PEG-modified lipid is Compound I and is present at 3 mole ratio %. Attorney Docket No.: 45817-0187WO1

[0035] In certain aspects, the disclosure features a pharmaceutical composition comprising the first LNP and second LNP or the LNP described above, and a pharmaceutically acceptable carrier.

[0036] In another aspect, the disclosure provides a method of treating a solid tumor expressing one or more of MUC16, CLDN6, or TROP2 in a human subject in need thereof. In some cases, the solid tumor is an epithelial cancer such as ovarian cancer. The method involves administering to the human subject a therapeutically effective amount of the first LNP and the second LNP or the LNP or the pharmaceutical composition described herein. In some cases, the ovarian cancer is an epithelial ovarian cancer, fallopian tube cancer, or primary’ peritoneal cancer. In certain cases, the treatment is a second, third, or later line treatment. In cases where the method comprises administering a first LNP and a second LNP, these LNPs can be administered at substantially the same time or sequentially. In certain cases, administration is by intravenous administration. In some cases, the first and second LNPs are mixed together and administered intravenously to the human subject.

[0037] In another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid nanoparticle comprising a first mRNA molecule, a second mRNA molecule, and a third mRNA molecule. The first mRNA molecule comprises a first ORF encoding a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a first linker to the N-terminal of a first VHH that binds to a first tumor-associated antigen, the C-terminal of the first VHH linked directly or via a second linker to the N-terminal of a second VHH that binds to the first tumor-associated antigen. In some cases, the first VHH and the second VHH have the same amino acid sequence. The second mRNA molecule comprises a second ORF encoding a second polypeptide comprising from the N-terminal to C-terminal a first VL and a first CL linked via the C-terminal of the CL directly or via a third linker to the N-terminal of a third VHH that binds to human serum albumin. The third mRNA molecule comprises a third ORF encoding a third polypeptide comprising from the N-terminal to C-terminal a fourth VHH that binds to a second tumor-associated antigen linked directly or via a fourth linker to the N-terminal of an Attorney Docket No.: 45817-0187WO1

[0038] immunologically silent human Ig Fc domain comprising from N terminal to C-terminal a hinge, a CH2 domain, and a CH3 domain of an immunologically silent human Ig Fc domain. In some cases, the immunologically silent human Ig Fc domain is from an IgG4PAA polypeptide. The C-terminal of the immunologically silent human Ig Fc domain linked directly or via a fifth linker to the N-terminal of a fifth VHH that binds to a costimulatory molecule on the surface of an activated T cell. The first VH and first CHI of the first polypeptide associates with the first VL and first CL of the second polypeptide to form a Fab wherein the Fab specifically bind to human CD3. In some cases, the human CD3 is human CD3E. The third polypeptide associates with itself to form a homodimer. In some cases, all the uracils in the first, second, and third mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are N 1 -methylpseudouridines.

[0039] In some instances, the pharmaceutical composition further comprises a fourth mRNA molecule. The fourth mRNA molecule comprises a fourth ORF encoding a fourth polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a sixth linker to the N-terminal of a sixth VHH that binds to a third tumor-associated antigen, the C-terminal of the sixth VHH linked directly or via a seventh linker to the N-terminal of a seventh VHH that binds to the third tumor-associated antigen. In some cases, the sixth VHH and the seventh VHH have the same amino acid sequence. The second VH and second CHI of the fourth polypeptide associates with the first VL and first CL of the second polypeptide to form a Fab and wherein the Fab specifically bind to human CD3 (e g., human CD3E). In some cases, all the uracils in the fourth mRNAs are N1-methylpseudouracils. or the uridines of the mRNA are N1 -methylpseudouridines.

[0040] In some instances, the first, second, and third tumor-associated antigens are solid tumor-associated antigens. In certain instances, the first tumor-associated antigen is MUC16, the second tumor-associated antigen is TROP2, the third tumor-associated antigen is CLDN6; and the costimulatory molecule is 4-1BB. In other instances, the first tumor-associated antigen is MUC16, the second tumor-associated antigen is MUC16, the third tumor-associated antigen is CLDN6; and the costimulatory molecule is 4- IBB. Attorney Docket No.: 45817-0187WO1

[0041] In certain instances, the C-terminal of the first CHI is linked via a first linker to the N-terminal of the first VHH, optionally wherein the first linker is G4S (SEQ ID NO:4); the C-terminal of the first VHH is linked via the second linker to the N-terminal of the second VHH, optionally wherein the second linker is (G4S)3 (SEQ ID NO: 5); the C-terminal of the first CL is linked via the third linker to the N-terminal of the third VHH, optionally wherein the third linker is G4S (SEQ ID NO:4); the C-terminal of the immunologically silent human Ig Fc domain is linked via the fifth linker to the N-terminal of the fifth VHH, optionally wherein the fifth linker is G4S (SEQ ID NO:4); the C-terminal of the second CHI is linked via the sixth linker to the N-terminal of the sixth VHH, optionally wherein the sixth linker is G4S (SEQ ID NO:4); and the C-terminal of the sixth VHH is linked via the seventh linker to the N-terminal of the seventh VHH, optionally wherein the seventh linker is (G4S)3 (SEQ ID NO:4).

[0042] In some instances, the first CHI and the second CHI have the identical amino acid sequence, optionally wherein the first CHI and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:28; and the first CL and the second CL have the identical amino acid sequence, optionally wherein the first CL and the second CL comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 30.

[0043] In certain instances, the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain of an IgG4PAA polypeptide which comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:33.

[0044] In some instances, the first VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40; the second VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID Attorney Docket No.: 45817-0187WO1

[0045] NO:40; the third VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:208; the fourth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:206 or SEQ ID NO:237; the fifth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:37; the sixth VHH comprises a VHH-CDR1, a VHH-CDR2. and a VHH-CDR3 of the VHH set forth in SEQ ID NO: 43; and the seventh VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43.

[0046] In certain instances, the first VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 40; the second VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40; the third VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:208; the fourth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:206 or SEQ ID NO:237; the fifth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:37; the sixth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43; and the seventh VHH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least Attorney Docket No.: 45817-0187WO1

[0047] 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 sequence set forth in SEQ ID NO:43.

[0048] In some instances, the first VH and second VH each comprise a CDR1, a CDR2, and a CDR3 of the VH set forth in SEQ ID NO:26, and the first VL and second VL each comprise a CDR1, a CDR2, and a CDR3 of the VL set forth in SEQ ID NO:27. In certain cases, the first VH and second VH each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:26, and

[0049] the first VL and second VL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:27. In some cases, the first VH and the first CHI and the second VH and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:31, and the first VL and first CL and second VL and second CL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 32.

[0050] In certain instances, the first polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:55, the second polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:57, the third polypeptide comprises an amino acid sequence that is 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 Attorney Docket No.: 45817-0187WO1

[0051] to the sequence set forth in SEQ ID NO:58 or 59, and the fourth polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:56.

[0052] In some instances, the first ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO: 60, the second ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:62, the third ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:63 or 223, and the fourth ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:61.

[0053] In another aspect, the disclosure encompasses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid nanoparticle (LNP) comprising a first mRNA molecule, a second mRNA molecule, and a third mRNA molecule. The first mRNA molecule comprises a first ORF encoding a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a first linker to the N-terminal of a first VHH that binds to a first tumor-associated antigen, the C-terminal of the first VHH linked directly or via a second linker to the N-terminal of a second VHH that binds to the first tumor-associated antigen. In some cases, the first VHH and the second VHH are identical. The second mRNA molecule comprises a second ORF encoding a second polypeptide comprising from the N-terminal to C-terminal a first VL and a first CL linked via the C-terminal of the CL directly or via a third linker Attorney Docket No.: 45817-0187WO1

[0054] to the N-terminal of a third VHH that binds to human serum albumin, the C-terminal of the third VHH linked directly or via a fourth linker to the N-terminal of a fourth VHH that binds to a costimulatory molecule on the surface of activated T cells. The third mRNA molecule comprises a third ORF encoding a third polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a fifth linker to the N-terminal of a fifth VHH that binds to a second tumor-associated antigen, the C-terminal of the fifth VHH linked directly or via a sixth linker to the N-terminal of a sixth VHH that binds to the second tumor-associated antigen. In some cases, the fifth VHH and the sixth VHH are identical. The first VH and first CHI of the first polypeptide associates with the first VL and the first CL of the second polypeptide to form a first Fab, and the second VH and second CHI of the third polypeptide associates with the first VL and the first CL of the second polypeptide to form a second Fab, wherein the first Fab and the second Fab each specifically bind to human CD3 (e g., human CD3E). In some cases, all the uracils in the first, second, and third mRNAs are N1-methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0055] In some instances, the first, second, and third tumor-associated antigens are solid tumor-associated antigens. In certain cases, the first tumor-associated antigen is MUC16, the second tumor-associated antigen is CLDN6; and the costimulatory molecule on the surface of activated T cells is 4-1BB.

[0056] In certain instances, the C-terminal of the first CHI is linked via a first linker to the N-terminal of the first VHH, optionally wherein the first linker is G4S (SEQ ID NO:4); the C-terminal of the first VHH is linked via the second linker to the N-terminal of the second VHH, optionally wherein the second linker is (G4S)3 (SEQ ID NO:5); the C-terminal of the first CL is linked via the third linker to the N-terminal of the third VHH, optionally wherein the third linker is G4S (SEQ ID NO:4); the C-terminal of the third VHH is linked via the fourth linker to the N-terminal of the fourth VHH, optionally wherein the fourth linker is (G4S) (SEQ ID NO:4); the C-terminal of the second CHI is linked via the fifth linker to the N-terminal of the fifth VHH, optionally wherein the fifth linker is G4S (SEQ ID NO:4); and the C-terminal Attorney Docket No.: 45817-0187WO1

[0057] of the fifth VHH is linked via the sixth linker to the N-terminal of the sixth VHH, optionally wherein the sixth linker is (G4S)3 (SEQ ID NO:5).

[0058] In some instances, the first CHI and the second CHI have the identical amino acid sequence, optionally wherein the first CHI and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:28; and the first CL and the second CL have the identical amino acid sequence, optionally wherein the first CL and the second CL comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 30.

[0059] In certain instances, the first VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40; the second VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40; the third VHH comprises a VHH-CDR 1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:208; the fourth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:37; the fifth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43; and the sixth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43.

[0060] In some instances, the first VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 40; the second VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40; the third VHH comprises an amino acid sequence that is 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 the sequence set Attorney Docket No.: 45817-0187WO1

[0061] forth in SEQ ID NO:208; the fourth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: or SEQ ID NO: 37; the fifth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43; and the sixth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43.

[0062] In some instances, the first VH and second VH each comprise a CDR1, a CDR2, and a CDR3 of the VH set forth in SEQ ID NO:26, and the first VL and second VL each comprise a CDR1, a CDR2, and a CDR3 of the VL set forth in SEQ ID NO:27. In certain cases, the first VH and second VH each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:26, and the first VL and second VL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:27. In some cases, the first VH and the first CHI and the second VH and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 31, and the first VL and first CL and second VL and second CL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:32. Attorney Docket No.: 45817-0187WO1

[0063] In certain instances, the first polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:55, the second polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:222, and the third polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 56.

[0064] In some instances, the first ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO: 60; the second ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:224, and the third ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:61.

[0065] In another aspect, the disclosure features a pharmaceutical composition compnsing a pharmaceutically acceptable carrier and a lipid nanoparticle. The LNP comprises means for encoding a human CD3E binding agent linked to (i) means for encoding a human MUC 16 binding agent; and (ii) means for encoding a HSA binding agent; and a means for encoding a human TROP2 binding agent linked via an immunologically silent human Ig Fc domain to means for encoding a human 4- IBB binding agent. In some cases, the LNP further comprises a means for encoding a human CD3E binding agent linked to (i) means for encoding a human CLDN6 binding agent; and (ii) means for encoding a HSA binding agent. Attorney Docket No.: 45817-0187WO1

[0066] In a different aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid nanoparticle. The LNP comprises means for encoding a human CD3E binding agent linked to (i) means for encoding a human MUC 16 binding agent; and (ii) means for encoding a HSA binding agent linked to means for encoding a human 4- IBB binding agent; and a means for encoding a human CD3E binding agent linked to (i) means for encoding a human CLDN6 binding agent; and (ii) means for encoding a HSA binding agent linked to means for encoding a human 4- IBB binding agent.

[0067] In another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid nanoparticle. The LNP comprises means for encoding a human CD3E binding agent linked to (i) means for encoding a human MUC16 binding agent; and (ii) means for encoding a HSA binding agent; and means for encoding a human MUC16 binding agent linked via an immunologically silent human Ig Fc domain to means for encoding a human 4-1BB binding agent. In some cases, the LNP further comprises means for encoding a human CD3E binding agent linked to (i) means for encoding a human CLDN6 binding agent; and (ii) means for encoding a HSA binding agent.

[0068] In yet another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a first lipid nanoparticle and a second lipid nanoparticle. The first lipid nanoparticle comprises means for encoding a human CD3E binding agent linked to (i) means for encoding a human MUC 16 binding agent; and (ii) means for encoding a HSA binding agent. In some cases, the first LNP further comprises a means for encoding a human CD3E binding agent linked to (i) means for encoding a human CLDN6 binding agent; and (ii) means for encoding a HSA binding agent. The second lipid nanoparticle comprises means for encoding a human CD3E binding agent linked to (i) means for encoding a human MUC 16 binding agent; and (ii) means for encoding a HSA binding agent; and a means for encoding a human TROP2 binding agent linked via an immunologically silent human Ig Fc domain to means for encoding a human 4- IBB binding agent. In some cases, the second LNP further comprises a means for encoding a human CD3E Attorney Docket No.: 45817-0187WO1

[0069] binding agent linked to (i) means for encoding a human CLDN6 binding agent; and (ii) means for encoding a HS A binding agent.

[0070] In another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid nanoparticle comprising a first mRNA molecule, optionally a second mRNA molecule, a third mRNA molecule, and a fourth mRNA molecule. The first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen. In some cases, the first and second VHH are identical. In certain cases, the first tumor-associated antigen is human MUC16. The second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen. In some cases, the third and fourth VHH are identical. In certain cases, the second tumor-associated antigen is human CLDN6. The third mRNA molecule encodes a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin. The fourth mRNA molecule encodes a fourth polypeptide compnsing from the N-terminal to C-terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells. In some cases, the third tumor-associated antigen is human TROP2 and the costimulatory molecule on the surface of activated T cells is human 4-1 BB. The first and second polypeptides each separately pair with the third polypeptide and the fourth Attorney Docket No.: 45817-0187WO1

[0071] polypeptide self-associates. In some cases, the first and second CHI are identical. In certain cases, the human CD3 is human CD3E. In some cases, all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0072] In another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid nanoparticle comprising a first mRNA molecule, optionally a second mRNA molecule, a third mRNA molecule, and a fourth mRNA molecule. The first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen. In some cases, the first and second VHH are identical. In certain cases, the first tumor-associated antigen is human MUC16. The second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen. In some cases, the third and fourth VHH are identical. In certain cases, the second tumor-associated antigen is human CLDN6. The third mRNA molecule encodes a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin. The fourth mRNA molecule encodes a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that specifically binds to a third tumor- associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells. In some cases, the third tumor-associated antigen is human TROP2 and the Attorney Docket No.: 45817-0187WO1

[0073] costimulatory molecule on the surface of activated T cells is human 4-1BB. The first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide self-associates. In some cases, the first and second CHI are identical. In certain cases, the human CD3 is human CD3E. In some cases, all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0074] In another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a first LNP and a second LNP. The first LNP comprises a first mRNA molecule, optionally a second mRNA molecule, and a third mRNA molecule. The first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen. In some cases, the first and second VHH are identical. In certain cases, the first tumor-associated antigen is human MUC16. The second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen. In some cases, the third and fourth VHH are identical. In certain cases, the second tumor-associated antigen is human CLDN6. The third mRNA molecule encodes a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin. The second LNP comprises the first mRNA molecule, the optional second mRNA molecule, third mRNA molecule, and a fourth mRNA molecule. The fourth mRNA molecule encodes a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N- Attorney Docket No.: 45817-0187WO1

[0075] terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells. In some cases, the third tumor-associated antigen is human TROP2 and the costimulatory molecule on the surface of activated T cells is human 4-1BB. The first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide selfassociates. In some cases, the first and second CHI are identical. In certain cases, the human CD3 is human CD3E. In some cases, all the uracils in the first, second, third, and fourth mRNAs are N 1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0076] In another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a first LNP and a second LNP. The first LNP comprises a first mRNA molecule, optionally a second mRNA molecule, and a third mRNA molecule. The first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen. In some cases, the first and second VHH are identical. In certain cases, the first tumor-associated antigen is human MUC16. The second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen. In some cases, the third and fourth VHH are identical. In certain cases, the second tumor-associated antigen is human CLDN6. The third mRNA molecule encodes a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin. The second LNP comprises the first mRNA molecule, the Attorney Docket No.: 45817-0187WO1

[0077] optional second mRNA molecule, third mRNA molecule, and a fourth mRNA molecule. The fourth mRNA molecule encodes a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells. In some cases, the third tumor-associated antigen is human MUC16 and the costimulatory molecule on the surface of activated T cells is human 4-1BB. The sixth VHH is different in sequence from the first and second VHH. The first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide self-associates. In some cases, the first and second CHI are identical. In certain cases, the human CD3 is human CD3E. In some cases, all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are N1-methylpseudouridines.

[0078] In another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a first LNP and a second LNP. The first LNP comprises a first mRNA molecule, optionally a second mRNA molecule, and a third mRNA molecule. The first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen. In some cases, the first and second VHH are identical. In certain cases, the first tumor-associated antigen is human MUC16. The second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen. In some cases, the third Attorney Docket No.: 45817-0187WO1

[0079] and fourth VHH are identical. In certain cases, the second tumor-associated antigen is human CLDN6. The third mRNA molecule encodes a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin. The second LNP comprises a fourth mRNA molecule. The fourth mRNA molecule encodes a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells. In some cases, the third tumor-associated antigen is human TROP2 and the costimulatory molecule on the surface of activated T cells is human 4-1BB. The first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide self-associates. In some cases, the first and second CHI are identical. In certain cases, the human CD3 is human CD3E. In some cases, all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are N1-methylpseudouridines.

[0080] In another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a first LNP and a second LNP. The first LNP comprises a first mRNA molecule, optionally a second mRNA molecule, and a third mRNA molecule. The first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CH1 linked via the C-terminal of the first CH1 directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen. In some cases, the first and second VHH are identical. In certain cases, the first tumor-associated antigen is human MUC16. The second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CH1 linked via the C-terminal of the second CH1 directly or via a linker to the N-terminal of a Attorney Docket No.: 45817-0187WO1

[0081] third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen. In some cases, the third and fourth VHH are identical. In certain cases, the second tumor-associated antigen is human CLDN6. The third mRNA molecule encoding a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin. The second LNP comprises a fourth mRNA molecule. The fourth mRNA molecule encoding a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells. In some cases, the third tumor-associated antigen is human MUC16 and the costimulatory molecule on the surface of activated T cells is human 4-1 BB. The first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide self-associates. In some cases, the first and second CHI are identical. In certain cases, the human CD3 is human CD3E. In some cases, all the uracils in the first, second, third, and fourth mRNA molecules are N1 -methylpseudouracils, or the uridines of the mRNA molecules are N1 -methylpseudouridines.

[0082] In certain instances, the first mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:60. In some instances, the second mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:61. In some instances, the third mRNA molecule comprises a sequence that is 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 Attorney Docket No.: 45817-0187WO1

[0083] 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO:62. In certain instances, the fourth mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:63 or 223. In some cases, all the uracils in the four mRNA molecules are N1 -methylpseudouracils, or the uridines of the mRNA molecules are N1 -methylpseudouridines.

[0084] In some instances, the first mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:230; the second mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:231; the third mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:232; and the fourth mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:233 or 223. In some cases, all the uracils in the four mRNA molecules are N1-methylpseudouracils, or the uridines of the mRNA molecules are N1-methylpseudouridines.

[0085] In another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a LNP comprising a first mRNA molecule and a second mRNA molecule. The first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N- Attorney Docket No.: 45817-0187WO1

[0086] terminal of a first VHH that specifically binds to a tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen. In some cases, the first and second VHH are identical. In certain cases, the tumor-associated antigen is human MUC16. The second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a third VHH that specifically binds to human serum albumin linked via the C-terminal of the third VHH directly or via a linker to the N-terminal of a fourth VHH that specifically binds to a costimulatory molecule on the surface of activated T cells. In some cases, the costimulatory molecule on the surface of activated T cells is 4-1BB. The first and second polypeptides associate with each other. In some cases, the human CD3 is human CD3E. In certain cases, all the uracils in the first and second mRNA molecules are N1 -methylpseudouracils, or the uridines of the first and second mRNA molecules are N1 -methylpseudouridines.

[0087] In some instances, the first mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:60 or 230. In certain instances, the second mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:224.

[0088] In another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a LNP comprising a first mRNA molecule, optionally a second mRNA molecule, and a third mRNA molecule. The first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor- Attorney Docket No.: 45817-0187WO1

[0089] associated antigen. In some cases, the first and second VHH are identical. In certain cases, the first tumor-associated antigen is human MUC16. The second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen. In some cases, the third and fourth VHH are identical. In certain cases, the second tumor-associated antigen is human CLDN6. The third mRNA molecule encodes a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin linked via the C-terminal of the fifth VHH directly or via a linker to the N-terminal of a sixth VHH that specifically binds to a costimulatory molecule on the surface of activated T cells. In some cases, the costimulatory molecule on the surface of activated T cells is 4-1BB. The first and second polypeptides associate with each other and the third and second polypeptides associate with each other. In some cases, the human CD3 is human CD3E. In certain cases, all the uracils in the first, second, and third mRNA molecules are N1 -methylpseudouracils, or the uridines of the first, second, and third mRNA molecules are N1 -methylpseudouridines.

[0090] In some instances, the first mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:60 or 230. In certain instances, the second mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:61 or 231. In some instances, the third mRNA molecule comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at Attorney Docket No.: 45817-0187WO1

[0091] least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO:224.

[0092] In yet another aspect, the disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a LNP comprising an mRNA molecule encoding a polypeptide comprising from the N-terminal to C-terminal a first VHH that specifically binds to a tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to a costimulatory molecule on the surface of activated T cells. In one case, the tumor-associated antigen is human TROP2. In another case, the tumor-associated antigen is human MUC16. In certain cases, the costimulatory molecule on the surface of activated T cells is human 4-1 BB. The polypeptide self-associates. In some cases, all the uracils in the mRNA are N1-methylpseudouracils. or the uridines of the mRNA are N1 -methylpseudouridines. This pharmaceutical composition can be administered together with a separate pharmaceutical composition comprising a pharmaceutically acceptable carrier and a LNP comprising mRNA molecules encoding one or more T cell engagers that bind to both CD3 and a TAA(s) of interest (e.g., MUC16, CLDN6). To be clear the pharmaceutical composition comprising the LNP of this aspect can be used in combination with a pharmaceutical composition comprising a separate LNP(s) containing one or more mRNAs encoding proteins that target TAAs of other cancers of interest (i. e., not just MUC16 and / or CLDN6).

[0093] In some instances, the mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:63 or 223.

[0094] In certain instances, the above-described mRNA molecules further comprise one or more of: (i) a 5’ terminal cap (optionally comprising m7GpppGm); (ii) a 5’UTR (optionally comprising the sequence of SEQ ID NO:64); (iii) if a signal sequence is not included in the ORF, a signal sequence (optionally comprising the sequence of any one of SEQ ID NOs:225, 226, 68, or 227); (iv) a 3’UTR (optionally Attorney Docket No.: 45817-0187WO1

[0095] wherein the 3’UTR comprises one or more miR binding sites and / or an IDR, and further optionally comprising the sequence of any one of SEQ ID NOs:234, 235, 130, or 236); and (v) a poly A tail (optionally wherein the poly A tail is about 100 nucleotides in length, optionally comprising the sequence of SEQ ID NO: 132).

[0096] In some instances, all the uracils in the mRNA molecules described above are N1 -methylpseudouracils, or the uridines of the mRNA molecules described above are N 1 -methy Ips eudouri dines.

[0097] In certain instances, the lipid nanoparticles comprise an ionizable amino lipid, a structural lipid, a phospholipid, and a polyethylene glycol (PEG)-modified lipid. In some cases, the ionizable amino lipid is present at about 40 to 50 mole ratio %, the structural lipid is present at 35 to 45 mole ratio %, the phospholipid is present at 5 to 15 mole ratio %, and the PEG-modified lipid is present at 1.5 to 5 mole ratio %. In certain cases, the ionizable amino lipid is present at about 47.5 mole ratio %, the structural lipid is present at about 39 mole ratio %, the phospholipid is present at about 10.5 mole ratio %, and the PEG-modified lipid is present at about 3 mole ratio %. In one case, the ionizable amino lipid is present at 47.5 mole ratio %, the structural lipid is present at 39 mole ratio %, the phospholipid is present at 10.5 mole ratio %, and the PEG-modified lipid is present at 3 mole ratio %. In some cases, the ionizable amino lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate and the PEG-modified lipid is 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate. In one case, the ionizable amino lipid composes Compound 2 and the PEG-modified lipid comprises Compound I. In some cases, the structural lipid is cholesterol. In certain cases, the phospholipid is DSPC. In one case, the ionizable amino lipid is Compound 2, the structural lipid is cholesterol, the phospholipid is DSPC, and the PEG-modified lipid is Compound I. In a certain case, the ionizable amino lipid is Compound 2 and is present at about 47.5 mole ratio %, the structural lipid is cholesterol and is present at about 39 mole ratio %, the phospholipid is DSPC and is present at about 10.5 mole ratio %, and the PEG-modified lipid is Compound I and is present at about 3 mole ratio %. In a specific Attorney Docket No.: 45817-0187WO1

[0098] case, the ionizable amino lipid is Compound 2 and is present at 47.5 mole ratio %, the structural lipid is cholesterol and is present at 39 mole ratio %, the phospholipid is DSPC and is present at 10.5 mole ratio %, and the PEG-modified lipid is Compound I and is present at 3 mole ratio %.

[0099] In another aspect, the disclosure features a method of treating a solid tumor expressing one or more of MUC16, CLDN6, or TROP2 in a human subject in need thereof. The method involves administering to the human subject a therapeutically effective amount of the pharmaceutical composition described above. In some cases, the solid tumor is an epithelial cancer. In one case, the solid tumor is ovarian cancer. In certain cases, the ovarian cancer is an epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer. In certain cases, the ovarian cancer is a relapsing or remitting ovarian cancer. In some cases, the treatment is a second, third, or later line treatment. In some cases, the human subject is diagnosed with early stage ovarian cancer. In another case, the human subject has been diagnosed with late stage ovarian cancer. In another embodiment, the subject has failed prior cancer therapies. In certain cases, the human subject has a platinum-resistant ovarian cancer. In other cases, the human subject has a platinum-sensitive ovarian cancer. In some cases where two or more different LNP compositions are to be administered, they may be administered substantially at the same time or sequentially. In some cases, the two or more different LNPs are mixed together and administered. In some cases, administration is performed intravenously. In other cases, administration is performed subcutaneously.

[0100] The mRNA molecules that encode the Signal 1 TCE(s) and Signal 2 TCE can be formulated together in a single delivery vehicle such as a nanoparticle, e.g., a lipid nanoparticle, or formulated in two or more delivery vehicles (e.g., lipid nanoparticles). For illustrative purposes, different options for mRNA multiplexing are provided below. In these different options. Ml = Signal 1 TCE “Heavy Chain’" comprising MUC16 binders (e.g., comprising SEQ ID NO: 60 or 230); Cl = Signal 1 TCE “Heavy Chain” comprising CLDN6 binders (e.g., comprising SEQ ID NO: 61 or 231); T2 = Signal 2 TCE comprising a TROP2 binder and a 4-1BB binder (e.g., comprising SEQ ID NO: 63 or 233); M2 = Signal 2 TCE comprising a MUC16 binder Attorney Docket No.: 45817-0187WO1

[0101] and a4-lBB binder (e.g., comprising SEQ ID NO: 223); CLC = Signal 1 TCE “Common Light Chain7’ (e.g., comprising SEQ ID NO: 62 or 232). The MUC16 binder in M2 is different from the MUC16 binders in Ml.

[0102] Option A '.

[0103] LNP1: Ml + C1 + CLC

[0104] LNP2: Ml + CI + CLC + T2

[0105] Option B

[0106] LNP1: Ml + C1 + CLC + T2

[0107] Option C'.

[0108] LNP1: Ml + C1 + CLC

[0109] LNP2: T2

[0110] Option D

[0111] LNP1: Ml + C1 + CLC

[0112] Option E:

[0113] LNP1: T2

[0114] Options A, B, C, and D can exclude CL In addition. Options A. B, C. and E can employ M2 instead of T2. In Option D, the CLC can also comprise a 4-1BB binder. Each of these options are encompassed by this disclosure and can be used to treat a cancer such as a solid tumors (e.g., a MUC16+solid tumor; a CLDN6+solid tumor; or a TROP2+solid tumor; a MUC16+TROP2+solid tumor; TROP2+CLDN6+solid tumor; or a MUC16+CLDN6+solid tumor). The skilled person will readily recognize that the TAAs used in these constructs can be replaced by other TAAs that are expressed on the cancer being targeted for treatment.

[0115] In some instances, a single drug product is administered to a human subject in need thereof, wherein the drug product comprises the 4 mRNAs (see, Fig. IB) at a defined ratio (Ml: Cl: CLC: T2 = about 39.6: about 39.6: about 15.8: about 5). In certain cases, the defined ratio of Ml: Cl: CLC: T2 = 39.6: 39.6: 15.8: 5. The defined ratio can be adjusted as needed for improving therapeutic goals. For example, the percentage of T2 may be increased. In some instances, the drug product is prepared by formulating the three Signal 1 mRNAs in one LNP, and the Signal 2 mRNA in a second LNP, which are then mixed to create a single drug product. That single drug product is then administered (e.g., intravenously) to a human subject in Attorney Docket No.: 45817-0187WO1

[0116] need thereof (e g., one having ovarian cancer). In some instances, the drug product is prepared by formulating all four mRNAs in a single LNP. That drug product is then administered (e.g., intravenously) to a human subject in need thereof (e g., one having ovarian cancer).

[0117] It is to be understood that this disclosure also encompasses all of the abovedescribed compositions and methods wherein the mRNAs are replaced by polypeptides for the Signal 1 TCE(s) and / or Signal 2 TCE(s). In some such instances, the polypeptides can be administered without a delivery vehicle(s).

[0118] Enumerated Embodiments:

[0119] Embodiment 1. A first lipid nanoparticle (LNP) and a second LNP, wherein:

[0120] (1) the first LNP comprises a first mRNA molecule, a second mRNA molecule, and a third mRNA molecule, wherein:

[0121] (i) the first mRNA molecule comprises a first ORF encoding a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CH 1 directly or via a first linker to the N-terminal of a first VHH that binds to a first tumor-associated antigen, the C-terminal of the first VHH linked directly or via a second linker to the N-terminal of a second VHH that binds to the first tumor-associated antigen, optionally wherein the first VHH and the second VHH have the same amino acid sequence;

[0122] (ii) the second mRNA molecule comprises a second ORF encoding a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a third linker to the N-terminal of a third VHH that binds to a second tumor-associated antigen, the C-terminal of the third VHH linked directly or via a fourth linker to the N-terminal of a fourth VHH that binds to the second tumor-associated antigen, optionally wherein the third VHH and the fourth VHH have the same amino acid sequence,

[0123] (iii) the third mRNA molecule comprises a third ORF encoding a third polypeptide comprising from the N-terminal to C-terminal a first VL and a first CL linked via the C-terminal of the CL directly or via a fifth linker to the N-terminal of a fifth VHH that binds to human serum albumin; Attorney Docket No.: 45817-0187WO1

[0124] wherein the first VH and first CHI of the first polypeptide associates with the first VL and first CL of the third polypeptide to form a Fab and wherein the Fab specifically bind to human CD3, optionally wherein the human CD3 is human CD3E, wherein the second VH and second CHI of the second polypeptide associates with the first VL and first CL of the third polypeptide to form a Fab and wherein the Fab specifically bind to human CD3, optionally wherein the human CD3 is human CD3E; and

[0125] (2) the second LNP comprises the first mRNA molecule, the second mRNA molecule, the third mRNA molecule, and a fourth mRNA molecule, wherein:

[0126] (iv) the fourth mRNA molecule comprises a fourth ORF encoding a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that binds to a third tumor-associated antigen linked directly or via a sixth linker to the N-terminal of an immunologically silent human Ig Fc domain comprising from N terminal to C-terminal a hinge, a CH2 domain, and a CH3 domain of an immunologically silent human Ig Fc domain, optionally wherein the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain from a human IgG4P AA polypeptide, the C-terminal of the immunologically silent human Ig Fc domain linked directly or via a seventh linker to the N-terminal of a seventh VHH that binds to a costimulatory molecule on the surface of an activated T cell,

[0127] wherein the fourth polypeptide associates with itself to form a homodimer; and optionally wherein all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0128] Embodiment 2. A lipid nanoparticle comprising a first mRNA molecule, a second mRNA molecule, a third mRNA molecule, and a fourth mRNA molecule, wherein:

[0129] (i) the first mRNA molecule comprises a first ORF encoding a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a first linker to the N-terminal of a first VHH that binds to a first tumor-associated antigen, the C-terminal of the first VHH linked directly or via a second linker to the N-terminal of a second Attorney Docket No.: 45817-0187WO1

[0130] VHH that binds to the first tumor-associated antigen, optionally wherein the first VHH and the second VHH have the same amino acid sequence;

[0131] (ii) the second mRNA molecule comprises a second ORF encoding a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a third linker to the N-terminal of a third VHH that binds to a second tumor-associated antigen, the C-terminal of the third VHH linked directly or via a fourth linker to the N-terminal of a fourth VHH that binds to the second tumor-associated antigen, optionally wherein the third VHH and the fourth VHH have the same amino acid sequence,

[0132] (iii) the third mRNA molecule comprises a third ORF encoding a third polypeptide comprising from the N-terminal to C-terminal a first VL and a first CL linked via the C-terminal of the CL directly or via a fifth linker to the N-terminal of a fifth VHH that binds to human serum albumin;

[0133] (iv) the fourth mRNA molecule comprises a fourth ORF encoding a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that binds to a third tumor-associated antigen linked directly or via a sixth linker to the N-terminal of an immunologically silent human Ig Fc domain comprising from N terminal to C-terminal a hinge, a CH2 domain, and a CH3 domain of an immunologically silent human Ig Fc domain, optionally wherein the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain from a human IgG4PAA polypeptide, the C-terminal of the immunologically silent human Ig Fc domain linked directly or via a seventh linker to the N-terminal of a seventh VHH that binds to a costimulatory molecule on the surface of an activated T cell,

[0134] wherein the first VH and first CHI of the first polypeptide associates with the first VL and first CL of the third polypeptide to form a Fab and wherein the Fab specifically bind to human CD3, optionally wherein the human CD3 is human CD3E, wherein the second VH and second CHI of the second polypeptide associates with the first VL and first CL of the third polypeptide to form a Fab and wherein the Fab Attorney Docket No.: 45817-0187WO1

[0135] specifically bind to human CD3, optionally wherein the human CD3 is human CD3E; and

[0136] wherein the fourth polypeptide associates with itself to form a homodimer; and optionally wherein all the uracils in the first, second, third, and fourth mRNAs are N 1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0137] Embodiment 3. The first LNP and second LNP of Embodiment 1 or the LNP of Embodiment 2. wherein the first, second, and third tumor-associated antigens are solid tumor-associated antigens.

[0138] Embodiment 4. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 3, wherein:

[0139] the first tumor-associated antigen is MUC16,

[0140] the second tumor-associated antigen is CLDN6,

[0141] the third tumor-associated antigen is TROP2; and

[0142] the costimulatory molecule is 4-1 BB.

[0143] Embodiment 5. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 4, wherein:

[0144] the first tumor-associated antigen is MUC16,

[0145] the second tumor-associated antigen is CLDN6,

[0146] the third tumor-associated antigen is MUC16; and

[0147] the costimulatory molecule is 4- IBB.

[0148] Embodiment 6. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 5, wherein:

[0149] the C-terminal of the first CHI is linked via the first linker to the N-terminal of the first VHH, optionally wherein the first linker is G4S (SEQ ID NO:4); Attorney Docket No.: 45817-0187WO1

[0150] the C-terminal of the first VHH is linked via the second linker to the N-terminal of the second VHH. optionally wherein the second linker is (G4S)3 (SEQ ID NO:5);

[0151] the C-terminal of the second CHI is linked via the third linker to the N-terminal of the third VHH, optionally wherein the third linker is G4S (SEQ ID NO:4);

[0152] the C-terminal of the third VHH is linked via the fourth linker to the N-terminal of the fourth VHH, optionally wherein the fourth linker is (G4S)3 (SEQ ID NO:5);

[0153] the C-terminal of the first CL is linked via the fifth linker to the N-terminal of the fifth VHH, optionally wherein the fifth linker is G4S (SEQ ID NO:4); and the C-terminal of the immunologically silent human Ig Fc domain is linked via the seventh linker to the N-terminal of the sixth VHH, optionally wherein the seventh linker is G4S (SEQ ID NO:4).

[0154] Embodiment 7. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 6, wherein:

[0155] the first CHI and the second CHI have the identical amino acid sequence, optionally wherein the first CHI and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:28; and

[0156] the first CL and the second CL have the identical amino acid sequence, optionally wherein the first CL and the second CL comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:30.

[0157] Embodiment 8. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 7, wherein the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain of a human IgG4PAA polypeptide that comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least Attorney Docket No.: 45817-0187WO1

[0158] 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 sequence set forth in SEQ ID NO:33.

[0159] Embodiment 9. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 8. wherein:

[0160] the first VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40, optionally wherein the first VHH binds to the 5-SEA domain of human MUC16;

[0161] the second VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40, optionally wherein the second VHH binds to the 5-SEA domain of human MUC16;

[0162] the third VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43, optionally wherein the third VHH binds specifically to human CLDN6 relative to CLDN3, CLDN4, and CLDN9;

[0163] the fourth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43, optionally wherein the fourth VHH binds specifically to human CLDN6 relative to CLDN3, CLDN4, and CLDN9;

[0164] the fifth VHH comprises a VHH-CDR1, a VHH-CDR2. and a VHH-CDR3 of the VHH set forth in SEQ ID NO:208;

[0165] the sixth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:206, optionally wherein the sixth VHH binds specifically to human TROP2 relative to EpCAM; and

[0166] the seventh VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:37.

[0167] Embodiment 10. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 9. wherein:

[0168] the first VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40; Attorney Docket No.: 45817-0187WO1

[0169] the second VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40;

[0170] the third VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43;

[0171] the fourth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43;

[0172] the fifth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:208;

[0173] the sixth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:206; and

[0174] the seventh VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 37.

[0175] Embodiment 11. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 10, wherein:

[0176] the first VH and second VH each comprise a CDR1. a CDR2, and a CDR3 of the VH set forth in SEQ ID NO:26, and

[0177] the first VL and second VL each comprise a CDR1, a CDR2, and a CDR3 of the VL set forth in SEQ ID NO:27. Attorney Docket No.: 45817-0187WO1

[0178] Embodiment 12. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 11, wherein:

[0179] the first VH and second VH each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 26, and

[0180] the first VL and second VL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 27.

[0181] Embodiment 13. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 12, wherein:

[0182] the first VH and the first CHI and the second VH and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:31, and

[0183] the first VL and first CL and second VL and second CL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:32.

[0184] Embodiment 14. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 13, wherein:

[0185] the first polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:55,

[0186] the second polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at Attorney Docket No.: 45817-0187WO1

[0187] least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:56,

[0188] the third polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 57, and

[0189] the fourth polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:58.

[0190] Embodiment 15. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 14, wherein:

[0191] the first ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:60 or 230,

[0192] the second ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:61 or 231,

[0193] the third ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:62 or 232, and

[0194] the fourth ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:63 or 233. Attorney Docket No.: 45817-0187WO1

[0195] Embodiment 16. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 15, wherein the mRNA molecules further comprise:

[0196] (i) a 5’ terminal cap, optionally comprising m7GpppGm;

[0197] (ii) a 5’UTR, optionally comprising the sequence of SEQ ID NO:64;

[0198] (iii) if a signal sequence is not included as part of the ORF, a signal sequence, optionally comprising the sequence of any one of SEQ ID NOs:225, 226. 68. or 227;

[0199] (iv) a 3’UTR, optionally wherein the 3’UTR comprises one or more miR binding sites and / or an IDR, and further optionally comprising the sequence of any one of SEQ ID NOs:234, 235, 130, or 236; and

[0200] (v) a poly A tail, optionally wherein the poly A tail is about 100 nucleotides in length, optionally comprising the sequence of SEQ ID NO: 132,

[0201] optionally wherein all the uracils are N1 -methylpseudouracils, or the uridines of the mRNA are Nl-methylpseudouridines.

[0202] Embodiment 17. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 16, wherein the lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and a polyethylene glycol (PEG)-modified lipid, optionally wherein the ionizable amino lipid is present at about 40 to 50 mole ratio %, the structural lipid is present at 35 to 45 mole ratio %, the phospholipid is present at 5 to 15 mole ratio %, and the PEG-modified lipid is present at 1.5 to 5 mole ratio %, and further optionally wherein the ionizable amino lipid is present at about 47.5 mole ratio %, the structural lipid is present at about 39 mole ratio %, the phospholipid is present at about 10.5 mole ratio %, and the PEG-modified lipid is present at about 3 mole ratio %, and also optionally wherein the ionizable amino lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate and the PEG-modified lipid is 134-hydroxy-3.6,9,12,15,18,21,24.27,30,33,36,39,42,45,48,51,54,57,60,63.66,69,72,75,78,81,84,87,90.93,96,99,102,105,108,111.114,117,120,123.126,129,132-tetratetracontaoxatetratriacontahectyl stearate. Attorney Docket No.: 45817-0187WO1

[0203] Embodiment 18. The first LNP and second LNP or the LNP of any one of Embodiments 1 to 17, wherein the ionizable amino lipid is Compound 2 and is present at about 47.5 mole ratio %, the structural lipid is cholesterol and is present at about 39 mole ratio %, the phospholipid is DSPC and is present at about 10.5 mole ratio %, and the PEG-modified lipid is Compound I and is present at about 3 mole ratio %.

[0204] Embodiment 19. A pharmaceutical composition comprising the first LNP and second LNP or the LNP of any one of Embodiments 1 to 18, and a pharmaceutically acceptable carrier.

[0205] Embodiment 20. A method of treating a solid tumor expressing one or more of MUC16, CLDN6, or TROP2 in a human subject in need thereof, optionally wherein the solid tumor is an epithelial cancer such as ovarian cancer, the method comprising administering to the human subject a therapeutically effective amount of the first LNP and the second LNP or the LNP of any one of Embodiments 1 to 18, or the pharmaceutical composition of claim 19, optionally wherein the ovarian cancer is an epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer, further optionally wherein the treatment is a second, third, or later line treatment, and also optionally wherein if the method comprises administering the first LNP and the second LNP, these LNPs are administered at substantially the same time or sequentially.

[0206] Embodiment 21. A composition comprising a plurality of lipid nanoparticles (LNPs) comprising a first mRNA molecule, a second mRNA molecule, and a third mRNA molecule, wherein:

[0207] (i) the first mRNA molecule comprises a first ORF encoding a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a first linker to the N-terminal of a first VHH that binds to a first tumor-associated antigen, the C-terminal of the first VHH linked directly or via a second linker to the N-terminal of a second Attorney Docket No.: 45817-0187WO1

[0208] VHH that binds to the first tumor-associated antigen, optionally wherein the first VHH and the second VHH have the same amino acid sequence;

[0209] (ii) the second mRNA molecule comprises a second ORF encoding a second polypeptide comprising from the N-terminal to C-terminal a first VL and a first CL linked via the C-terminal of the CL directly or via a third linker to the N-terminal of a third VHH that binds to human serum albumin;

[0210] (iii) the third mRNA molecule comprises a third ORF encoding a third polypeptide comprising from the N-terminal to C-terminal a fourth VHH that binds to a second tumor-associated antigen linked directly or via a fourth linker to the N-terminal of an immunologically silent human Ig Fc domain comprising from N terminal to C-terminal a hinge, a CH2 domain, and a CH3 domain of an immunologically silent human Ig Fc domain, optionally wherein the immunologically silent human Ig Fc domain is from an IgG4PAA polypeptide, the C-terminal of the immunologically silent human Ig Fc domain linked directly or via a fifth linker to the N-terminal of a fifth VHH that binds to a costimulatory molecule on the surface of an activated T cell,

[0211] wherein the first VH and first CHI of the first polypeptide associates with the first VL and first CL of the second polypeptide to form a Fab and wherein the Fab specifically bind to human CD3, optionally wherein the human CD3 is human CD3E, wherein the third polypeptide associates with itself to form a homodimer; and optionally wherein all the uracils in the first, second, and third mRNAs are N1-methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0212] Embodiment 22. The composition of Embodiment 21. further comprising a fourth mRNA molecule, wherein:

[0213] (iv) the fourth mRNA molecule comprises a fourth ORF encoding a fourth polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a sixth linker to the N-terminal of a sixth VHH that binds to a third tumor-associated antigen, the C-terminal of the sixth VHH linked directly or via a seventh linker to the N-terminal of a Attorney Docket No.: 45817-0187WO1

[0214] seventh VHH that binds to the third tumor-associated antigen, optionally wherein the sixth VHH and the seventh VHH have the same amino acid sequence,

[0215] wherein the second VH and second CHI of the fourth polypeptide associates with the first VL and first CL of the second polypeptide to form a Fab and wherein the Fab specifically bind to human CD3, optionally wherein the human CD3 is human CD3E, and

[0216] optionally wherein all the uracils in the fourth mRNAs are N1-methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0217] Embodiment 23. The composition of Embodiment 21 or 22. wherein the first, second, and third tumor-associated antigens are solid tumor-associated antigens.

[0218] Embodiment 24. The composition of Embodiment 21 or 22, wherein:

[0219] the first tumor-associated antigen is MUC16,

[0220] the second tumor-associated antigen is TROP2,

[0221] the third tumor-associated antigen is CLDN6; and

[0222] the costimulatory molecule is 4-1BB.

[0223] Embodiment 25. The composition of Embodiment 21 or 22. wherein:

[0224] the first tumor-associated antigen is MUC16,

[0225] the second tumor-associated antigen is MUC16,

[0226] the third tumor-associated antigen is CLDN6; and

[0227] the costimulatory molecule is 4- IBB.

[0228] Embodiment 26. The composition of any one of Embodiments 21 to 25, wherein: the C-terminal of the first CHI is linked via a first linker to the N-terminal of the first VHH, optionally wherein the first linker is G4S (SEQ ID NO:4);

[0229] the C-terminal of the first VHH is linked via the second linker to the N-terminal of the second VHH, optionally wherein the second linker is (G4S)3 (SEQ ID NO:5); Attorney Docket No.: 45817-0187WO1

[0230] the C-terminal of the first CL is linked via the third linker to the N-terminal of the third VHH. optionally wherein the third linker is G4S (SEQ ID NO:4);

[0231] the C-terminal of the immunologically silent human Ig Fc domain is linked via the fifth linker to the N-terminal of the fifth VHH, optionally wherein the fifth linker is G4S (SEQ ID NO:4);

[0232] the C-terminal of the second CHI is linked via the sixth linker to the N-terminal of the sixth VHH, optionally wherein the sixth linker is G4S (SEQ ID NO:4); and

[0233] the C-terminal of the sixth VHH is linked via the seventh linker to the N-terminal of the seventh VHH, optionally wherein the seventh linker is (G4S)3 (SEQ ID NO:5).

[0234] Embodiment 27. The composition of any one of Embodiments 21 to 26, wherein: the first CHI and the second CHI have the identical amino acid sequence, optionally wherein the first CHI and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:28; and

[0235] the first CL and the second CL have the identical amino acid sequence, optionally wherein the first CL and the second CL comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:30.

[0236] Embodiment 28. The composition of any one of Embodiments 21 to 27, wherein the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain of an IgG4PAA polypeptide that comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:33. Attorney Docket No.: 45817-0187WO1

[0237] Embodiment 29. The composition of any one of Embodiments 21 to 28, wherein: the first VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40;

[0238] the second VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40;

[0239] the third VHH comprises a VHH-CDR1. a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:208;

[0240] the fourth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:206 or SEQ ID NO:237;

[0241] the fifth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:37;

[0242] the sixth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43; and

[0243] the seventh VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43.

[0244] Embodiment 30. The composition of any one of Embodiments 21 to 29, wherein: the first VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40;

[0245] the second VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40;

[0246] the third VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:208;

[0247] the fourth VHH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, Attorney Docket No.: 45817-0187WO1

[0248] at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:206 or SEQ ID NO:237;

[0249] the fifth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:37;

[0250] the sixth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43; and

[0251] the seventh VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43.

[0252] Embodiment 31. The composition of any one of Embodiments 21 to 30, wherein: the first VH and second VH each comprise a CDR1, a CDR2, and a CDR3 of the VH set forth in SEQ ID NO:26, and

[0253] the first VL and second VL each comprise a CDR1, a CDR2, and a CDR3 of the VL set forth in SEQ ID NO:27.

[0254] Embodiment 32. The composition of any one of Embodiments 21 to 31, wherein: the first VH and second VH each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 26, and

[0255] the first VL and second VL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 27. Attorney Docket No.: 45817-0187WO1

[0256] Embodiment 33. The composition of any one of Embodiments 21 to 32, wherein: the first VH and the first CHI and the second VH and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:31, and

[0257] the first VL and first CL and second VL and second CL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:32.

[0258] Embodiment 34. The composition of any one of Embodiments 21 to 33, wherein: the first polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:55,

[0259] the second polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:57,

[0260] the third polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:58 or 59, and

[0261] the fourth polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:56.

[0262] Embodiment 35. The composition of any one of Embodiments 21 to 34, wherein: Attorney Docket No.: 45817-0187WO1

[0263] the first ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:60,

[0264] the second ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO: 62,

[0265] the third ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:63 or 223, and

[0266] the fourth ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:61.

[0267] Embodiment 36. A composition comprising a plurality' of lipid nanoparticles (LNPs) comprising a first mRNA molecule, a second mRNA molecule, and a third mRNA molecule, wherein:

[0268] (i) the first mRNA molecule comprises a first ORF encoding a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a first linker to the N-terminal of a first VHH that binds to a first tumor-associated antigen, the C-terminal of the first VHH linked directly or via a second linker to the N-terminal of a second VHH that binds to the first tumor-associated antigen, optionally wherein the first VHH and the second VHH are identical;

[0269] (ii) the second mRNA molecule comprises a second ORF encoding a second polypeptide comprising from the N-terminal to C-terminal a first VL and a first CL linked via the C-terminal of the CL directly or via a third linker to the N-terminal of a third VHH that binds to human serum albumin, the C-terminal of the third VHH Attorney Docket No.: 45817-0187WO1

[0270] linked directly or via a fourth linker to the N-terminal of a fourth VHH that binds to a costimulatory molecule on the surface of activated T cells; and

[0271] (iii) the third mRNA molecule comprises a third ORF encoding a third polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a fifth linker to the N-terminal of a fifth VHH that binds to a second tumor-associated antigen, the C-terminal of the fifth VHH linked directly or via a sixth linker to the N-terminal of a sixth VHH that binds to the second tumor-associated antigen, optionally wherein the fifth VHH and the sixth VHH are identical;

[0272] wherein the first VH and first CHI of the first polypeptide associates with the first VL and the first CL of the second polypeptide to form a first Fab,

[0273] wherein the second VH and second CHI of the third polypeptide associates with the first VL and the first CL of the second polypeptide to form a second Fab, wherein the first Fab and the second Fab each specifically bind to human CD3, optionally wherein the human CD3 is human CD3E, and

[0274] optionally wherein all the uracils in the first, second, and third mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0275] Embodiment 37. The composition of Embodiment 36. wherein the first, second, and third tumor-associated antigens are solid tumor-associated antigens.

[0276] Embodiment 38. The composition of Embodiment 36 or 37, wherein:

[0277] the first tumor-associated antigen is MUC16,

[0278] the second tumor-associated antigen is CLDN6; and

[0279] the costimulatory molecule on the surface of activated T cells is 4- IBB.

[0280] Embodiment 39. The composition of any one of Embodiments 36 to 38, wherein: the C-terminal of the first CHI is linked via a first linker to the N-terminal of the first VHH, optionally wherein the first linker is G4S (SEQ ID NO:4); Attorney Docket No.: 45817-0187WO1

[0281] the C-terminal of the first VHH is linked via the second linker to the N-terminal of the second VHH. optionally wherein the second linker is (G4S)3 (SEQ ID NO:5);

[0282] the C-terminal of the first CL is linked via the third linker to the N-terminal of the third VHH, optionally wherein the third linker is G4S (SEQ ID NO:4);

[0283] the C-terminal of the third VHH is linked via the fourth linker to the N-terminal of the fourth VHH, optionally wherein the fourth linker is (G4S) (SEQ ID NO:4);

[0284] the C-terminal of the second CHI is linked via the fifth linker to the N-terminal of the fifth VHH, optionally wherein the fifth linker is G4S (SEQ ID NO:4); and

[0285] the C-terminal of the fifth VHH is linked via the sixth linker to the N-terminal of the sixth VHH, optionally wherein the sixth linker is (G4S)3 (SEQ ID NO:5).

[0286] Embodiment 40. The composition of any one of Embodiments 36 to 39, wherein: the first CHI and the second CHI have the identical amino acid sequence, optionally wherein the first CHI and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:28; and

[0287] the first CL and the second CL have the identical amino acid sequence, optionally wherein the first CL and the second CL comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:30.

[0288] Embodiment 41. The composition of any one of Embodiments 36 to 40, wherein: the first VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40;

[0289] the second VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40; Attorney Docket No.: 45817-0187WO1

[0290] the third VHH comprises a VHH-CDR1. a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:208;

[0291] the fourth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:37;

[0292] the fifth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43; and

[0293] the sixth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43.

[0294] Embodiment 42. The composition of any one of Embodiments 36 to 41, wherein: the first VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40;

[0295] the second VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40;

[0296] the third VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:208;

[0297] the fourth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: or SEQ ID NO: 37;

[0298] the fifth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43; and Attorney Docket No.: 45817-0187WO1

[0299] the sixth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43.

[0300] Embodiment 43. The composition of any one of Embodiments 36 to 42, wherein: the first VH and second VH each comprise a CDR1, a CDR2, and a CDR3 of the VH set forth in SEQ ID NO:26, and

[0301] the first VL and second VL each comprise a CDR1, a CDR2, and a CDR3 of the VL set forth in SEQ ID NO:27.

[0302] Embodiment 44. The composition of any one of Embodiments 36 to 43, wherein: the first VH and second VH each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 26, and

[0303] the first VL and second VL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 27.

[0304] Embodiment 45. The composition of any one of Embodiments 36 to 44, wherein: the first VH and the first CHI and the second VH and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:31, and

[0305] the first VL and first CL and second VL and second CL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:32. Attorney Docket No.: 45817-0187WO1

[0306] Embodiment 46. The composition of any one of Embodiments 36 to 45, wherein: the first polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:55,

[0307] the second polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:222,

[0308] the third polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:56.

[0309] Embodiment 47. The composition of any one of Embodiments 36 to 46, wherein: the first ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:60,

[0310] the second ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:224, and

[0311] the third ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:61.

[0312] Embodiment 48. A composition comprising a plurality of lipid nanoparticles compnsing: Attorney Docket No.: 45817-0187WO1

[0313] means for encoding a human CD3E binding agent linked to (i) means for encoding a human MUC16 binding agent; and (ii) means for encoding a HSA binding agent; and

[0314] means for encoding a human TROP2 binding agent linked via an immunologically silent human Ig Fc domain to means for encoding a human 4-1BB binding agent.

[0315] Embodiment 49. The composition of Embodiment 48, further comprising:

[0316] means for encoding a human CD3E binding agent linked to (i) means for encoding a human CLDN6 binding agent; and (ii) means for encoding a HSA binding agent.

[0317] Embodiment 50. A composition comprising a plurality7of lipid nanoparticles comprising:

[0318] means for encoding a human CD3E binding agent linked to (i) means for encoding a human MUC 16 binding agent; and (ii) means for encoding a HSA binding agent linked to means for encoding a human 4-1BB binding agent; and

[0319] means for encoding a human CD3E binding agent linked to (i) means for encoding a human CLDN6 binding agent; and (ii) means for encoding a HSA binding agent linked to means for encoding a human 4-1BB binding agent.

[0320] Embodiment 51. A composition comprising a plurality' of lipid nanoparticles comprising:

[0321] means for encoding a human CD3E binding agent linked to (i) means for encoding a human MUC 16 binding agent; and (ii) means for encoding a HSA binding agent; and

[0322] means for encoding a human MUC 16 binding agent linked via an immunologically silent human Ig Fc domain to means for encoding a human 4-1BB binding agent.

[0323] Embodiment 52. The composition of Embodiment 51, further comprising: Attorney Docket No.: 45817-0187WO1

[0324] means for encoding a human CD3E binding agent linked to (i) means for encoding a human CLDN6 binding agent; and (ii) means for encoding a HSA binding agent.

[0325] Embodiment 53. A composition comprising a plurality' of lipid nanoparticles comprising a first mRNA molecule, optionally a second mRNA molecule, a third mRNA molecule, and a fourth mRNA molecule, wherein:

[0326] the first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen, optionally wherein the first and second VHH are identical, and further optionally wherein the first tumor-associated antigen is human MUC16; and the second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen, optionally wherein the third and fourth VHH are identical, and further optionally wherein the second tumor-associated antigen is human CLDN6;

[0327] the third mRNA molecule encoding a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin; and

[0328] the fourth mRNA molecule encoding a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically Attorney Docket No.: 45817-0187WO1

[0329] binds to a costimulatory molecule on the surface of activated T cells, optionally wherein the third tumor-associated antigen is human TROP2 and the costimulatory molecule on the surface of activated T cells is human 4- IBB,

[0330] wherein the first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide self-associates, optionally wherein the first and second CHI are identical, and further optionally wherein the human CD3 is human CD3E, and

[0331] optionally wherein all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are Nl-methylpseudouri dines.

[0332] Embodiment 54. A composition comprising a lipid nanoparticle comprising a first mRNA molecule, optionally a second mRNA molecule, a third mRNA molecule, and a fourth mRNA molecule, wherein:

[0333] the first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen, optionally wherein the first and second VHH are identical, and further optionally wherein the first tumor-associated antigen is human MUC16; and the second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CH I directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen, optionally wherein the third and fourth VHH are identical, and further optionally wherein the second tumor-associated antigen is human CLDN6;

[0334] the third mRNA molecule encoding a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly Attorney Docket No.: 45817-0187WO1

[0335] or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin; and

[0336] the fourth mRNA molecule encoding a fourth polypeptide comprising from the N-terminal to C -terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells, optionally wherein the third tumor-associated antigen is human TROP2 and the costimulatory molecule on the surface of activated T cells is human 4-1BB,

[0337] wherein the first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide self-associates, optionally wherein the first and second CHI are identical, and further optionally wherein the human CD3 is human CD3E, and

[0338] optionally wherein all the uracils in the first, second, third, and fourth mRNAs are N 1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0339] Embodiment 55. A composition comprising a plurality of lipid nanoparticles comprising a first LNP and a second LNP, wherein

[0340] (i) the first LNP comprises a first mRNA molecule, optionally a second mRNA molecule, and a third mRNA molecule, wherein:

[0341] the first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CH I linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen, optionally wherein the first and second VHH are identical, and further optionally wherein the first tumor-associated antigen is human MUC16; and the second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C- Attorney Docket No.: 45817-0187WO1

[0342] terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen, optionally wherein the third and fourth VHH are identical, and further optionally wherein the second tumor-associated antigen is human CLDN6;

[0343] the third mRNA molecule encoding a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin; and

[0344] (ii) the second LNP comprises the first mRNA molecule, the optional second mRNA molecule, third mRNA molecule, and a fourth mRNA molecule, wherein: the fourth mRNA molecule encoding a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells, optionally wherein the third tumor-associated antigen is human TROP2 and the costimulatory molecule on the surface of activated T cells is human 4- IBB,

[0345] wherein the first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide self-associates, optionally wherein the first and second CHI are identical, and further optionally wherein the human CD3 is human CD3E, and

[0346] optionally wherein all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are Nl-methylpseudouri dines.

[0347] Embodiment 56. A composition comprising a plurality of lipid nanoparticles comprising a first LNP and a second LNP, wherein Attorney Docket No.: 45817-0187WO1

[0348] (i) the first LNP comprises a first mRNA molecule, optionally a second mRNA molecule, and a third mRNA molecule, wherein:

[0349] the first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen, optionally wherein the first and second VHH are identical, and further optionally wherein the first tumor-associated antigen is human MUC16; and the second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen, optionally wherein the third and fourth VHH are identical, and further optionally wherein the second tumor-associated antigen is human CLDN6;

[0350] the third mRNA molecule encoding a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin; and

[0351] (ii) the second LNP comprises the first mRNA molecule, the optional second mRNA molecule, third mRNA molecule, and a fourth mRNA molecule, wherein: the fourth mRNA molecule encoding a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells, optionally wherein the third tumor-associated antigen is human MUC16 and the costimulatory Attorney Docket No.: 45817-0187WO1

[0352] molecule on the surface of activated T cells is human 4-1BB, wherein the sixth VHH is different in sequence from the first and second VHH,

[0353] wherein the first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide self-associates, optionally wherein the first and second CHI are identical, and further optionally wherein the human CD3 is human CD3E, and

[0354] optionally wherein all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are Nl-methylpseudouri dines.

[0355] Embodiment 57. A composition comprising a plurality of lipid nanoparticles comprising a first LNP and a second LNP, wherein

[0356] (i) the first LNP comprises a first mRNA molecule, optionally a second mRNA molecule, and a third mRNA molecule, wherein:

[0357] the first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen, optionally wherein the first and second VHH are identical, and further optionally wherein the first tumor-associated antigen is human MUC16; and the second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CH I directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen, optionally wherein the third and fourth VHH are identical, and further optionally wherein the second tumor-associated antigen is human CLDN6; and

[0358] the third mRNA molecule encoding a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly Attorney Docket No.: 45817-0187WO1

[0359] or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin; and

[0360] (ii) the second LNP comprises a fourth mRNA molecule, wherein:

[0361] the fourth mRNA molecule encoding a fourth polypeptide comprising from the N-terminal to C -terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells, optionally wherein the third tumor-associated antigen is human TROP2 and the costimulatory molecule on the surface of activated T cells is human 4- IBB,

[0362] wherein the first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide self-associates, optionally wherein the first and second CHI are identical, and further optionally wherein the human CD3 is human CD3E, and

[0363] optionally wherein all the uracils in the first, second, third, and fourth mRNAs are N1 -methylpseudouracils, or the uridines of the mRNA are Nl-methylpseudouri dines.

[0364] Embodiment 58. A composition comprising a plurality of lipid nanoparticles comprising a first LNP and a second LNP wherein:

[0365] (i) the first LNP comprises a first mRNA molecule, optionally a second mRNA molecule, and a third mRNA molecule, wherein:

[0366] the first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen, optionally wherein the first and second VHH are identical, and further optionally wherein the first tumor-associated antigen is human MUC16; and Attorney Docket No.: 45817-0187WO1

[0367] the second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen, optionally wherein the third and fourth VHH are identical, and further optionally wherein the second tumor-associated antigen is human CLDN6; and

[0368] the third mRNA molecule encoding a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum albumin; and

[0369] (ii) the second LNP comprises a fourth mRNA molecule, wherein:

[0370] the fourth mRNA molecule encoding a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that specifically binds to a third tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked directly or via a linker to the N-terminal of a seventh VHH that specifically binds to a costimulatory molecule on the surface of activated T cells, optionally wherein the third tumor-associated antigen is human MUC16 and the costimulatory molecule on the surface of activated T cells is human 4-1BB,

[0371] wherein the first and second polypeptides each separately pair with the third polypeptide and the fourth polypeptide self-associates, optionally wherein the first and second CHI are identical, and further optionally wherein the human CD3 is human CD3E and

[0372] optionally wherein all the uracils in the first, second, third, and fourth mRNA molecules are N1 -methylpseudouracils, or the uridines of the mRNA molecules are N1 -methylpseudouridines.

[0373] Embodiment 59. The composition of any one of Embodiments 53 to 58, wherein: Attorney Docket No.: 45817-0187WO1

[0374] the first mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:60;

[0375] the second mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:61;

[0376] the third mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:62; and

[0377] the fourth mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:63 or 223,

[0378] optionally wherein all the uracils in the four mRNA molecules are N1-methylpseudouracils, or the uridines of the mRNA molecules are N1-methylpseudouridines.

[0379] Embodiment 60. The composition of any one of Embodiments 53 to 59, wherein: the first mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:230;

[0380] the second mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:231;

[0381] the third mRNA molecule comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, Attorney Docket No.: 45817-0187WO1

[0382] at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO:232; and

[0383] the fourth mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:233 or 223,

[0384] optionally wherein all the uracils in the four mRNA molecules are N1-methylpseudouracils, or the uridines of the mRNA molecules are N1-methylpseudouridines.

[0385] Embodiment 61. A composition comprising a plurality of lipid nanoparticles comprising a first mRNA molecule and a second mRNA molecule, wherein:

[0386] the first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a linker to the N-terminal of a first VHH that specifically binds to a tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen, optionally wherein the first and second VHH are identical, and further optionally wherein the tumor-associated antigen is human MUC16;

[0387] the second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a third VHH that specifically binds to human serum albumin linked via the C-terminal of the third VHH directly or via a linker to the N-terminal of a fourth VHH that specifically binds to a costimulatory molecule on the surface of activated T cells, optionally wherein the costimulatory molecule on the surface of activated T cells is 4-1BB,

[0388] wherein the first and second polypeptides associate with each other, optionally wherein the human CD3 is human CD3E and

[0389] further optionally wherein all the uracils in the first and second mRNA molecules are N1 -methylpseudouracils, or the uridines of the mRNA are Nl-methylpseudouri dines. Attorney Docket No.: 45817-0187WO1

[0390] Embodiment 62. The composition of Embodiment 6E wherein:

[0391] the first mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:60 or 230; and

[0392] the second mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:224.

[0393] Embodiment 63. A composition comprising a plurality of lipid nanoparticles comprising a first mRNA molecule, optionally a second mRNA molecule, and a third mRNA molecule, wherein:

[0394] the first mRNA molecule encodes a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CH 1 directly or via a linker to the N-terminal of a first VHH that specifically binds to a first tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a second VHH that specifically binds to the first tumor-associated antigen, optionally wherein the first and second VHH are identical, and further optionally wherein the first tumor-associated antigen is human MUC16; and the second mRNA molecule encodes a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a linker to the N-terminal of a third VHH that specifically binds to a second tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of a fourth VHH that specifically binds to the second tumor-associated antigen, optionally wherein the third and fourth VHH are identical, and further optionally wherein the second tumor-associated antigen is human CLDN6;

[0395] the third mRNA molecule encoding a third polypeptide comprising from the N-terminal to C-terminal a VL and a CL linked via the C-terminal of the CL directly or via a linker to the N-terminal of a fifth VHH that specifically binds to human serum Attorney Docket No.: 45817-0187WO1

[0396] albumin linked via the C-terminal of the fifth VHH directly or via a linker to the N-terminal of a sixth VHH that specifically binds to a costimulatory molecule on the surface of activated T cells, optionally wherein the costimulatory molecule on the surface of activated T cells is 4-1BB; and

[0397] wherein the first and second polypeptides associate with each other and the third and second polypeptides associate with each other;

[0398] optionally wherein the human CD3 is human CD3E and

[0399] further optionally wherein all the uracils in the first, second, and third mRNA molecules are N1 -methylpseudouracils, or the uridines of the mRNA are Nl-methylpseudouri dines.

[0400] Embodiment 64. The composition of Embodiment 63, wherein:

[0401] the first mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:60 or 230;

[0402] the second mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:61 or 231; and

[0403] the third mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:224.

[0404] Embodiment 65. A composition comprising a plurality of lipid nanoparticles comprising:

[0405] an mRNA molecule encoding a polypeptide comprising from the N-terminal to C-terminal a first VHH that specifically binds to a tumor-associated antigen, the C-terminal of which is linked directly or via a linker to the N-terminal of an immunologically silent human Ig Fc domain, the C-terminal of which is linked Attorney Docket No.: 45817-0187WO1

[0406] directly or via a linker to the N-terminal of a second VHH that specifically binds to a costimulatory molecule on the surface of activated T cells, optionally wherein the tumor-associated antigen is human TROP2 or human MUC 16 and the costimulatory molecule on the surface of activated T cells is human 4-1BB,

[0407] wherein the polypeptide self-associates,

[0408] optionally wherein all the uracils in the mRNA are N1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

[0409] Embodiment 66. The composition of Embodiment 65, wherein:

[0410] the mRNA molecule comprises a sequence that is 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 the nucleic acid sequence set forth in SEQ ID NO:63 or 223.

[0411] Embodiment 67. The composition of any one of Embodiments 21 to 66, further comprising:

[0412] (i) a 5’ terminal cap, optionally comprising m7GpppGm;

[0413] (ii) a 5’UTR, optionally comprising the sequence of SEQ ID NO:64;

[0414] (iii) if a signal sequence is not included in the ORF, a signal sequence, optionally comprising the sequence of any one of SEQ ID NOs:225, 226, 68, or 227;

[0415] (iv) a 3’UTR, optionally wherein the 3’UTR comprises one or more miR binding sites and / or an IDR, and further optionally comprising the sequence of any one of SEQ ID NOs:234. 235, 130, or 236; and

[0416] (v) a poly A tail, optionally wherein the poly A tail is about 100 nucleotides in length, optionally comprising the sequence of SEQ ID NO: 132.

[0417] Embodiment 68. The composition of any one of Embodiments 21 to 67, wherein all the uracils are N 1 -methylpseudouracils, or the uridines of the mRNA are N I -methylpseudouridines. Attorney Docket No.: 45817-0187WO1

[0418] Embodiment 69. The composition of any one of Embodiments 21 to 68, wherein the lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and a polyethylene glycol (PEG)-modified lipid, optionally wherein the ionizable amino lipid is present at about 40 to 50 mole ratio %, the structural lipid is present at 35 to 45 mole ratio %, the phospholipid is present at 5 to 15 mole ratio %, and the PEG-modified lipid is present at 1.5 to 5 mole ratio %, and further optionally wherein the ionizable amino lipid is present at about 47.5 mole ratio %, the structural lipid is present at about 39 mole ratio %, the phospholipid is present at about 10.5 mole ratio %, and the PEG-modified lipid is present at about 3 mole ratio %, and also optionally wherein the ionizable amino lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate and the PEG-modified lipid is 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate.

[0419] Embodiment 70. The composition of Embodiment 69, wherein the ionizable amino lipid is Compound 2.

[0420] Embodiment 71. The composition of Embodiment 69 or 70, wherein the structural lipid is cholesterol.

[0421] Embodiment 72. The composition of any one of Embodiments 69 to 71, wherein the phospholipid is DSPC.

[0422] Embodiment 73. The composition of any one of Embodiments 69 to 72, wherein the PEG-modified lipid is Compound I.

[0423] Embodiment 74. The composition of any one of Embodiments 69 to 73, wherein the ionizable amino lipid is Compound 2, the structural lipid is cholesterol, the phospholipid is DSPC, and the PEG-modified lipid is Compound I. Attorney Docket No.: 45817-0187WO1

[0424] Embodiment 75. The composition of any one of Embodiments 69 to 74, wherein the ionizable amino lipid is Compound 2 and is present at about 47.5 mole ratio %, the structural lipid is cholesterol and is present at about 39 mole ratio %, the phospholipid is DSPC and is present at about 10.5 mole ratio %, and the PEG-modified lipid is Compound I and is present at about 3 mole ratio %.

[0425] Embodiment 76. The composition of any one of Embodiments 21 to 75, wherein the composition is a pharmaceutical composition and comprises a pharmaceutically acceptable carrier.

[0426] Embodiment 77. A method of treating a solid tumor expressing one or more of MUC16, CLDN6, or TROP2 in a human subject in need thereof, optionally wherein the solid tumor is an epithelial cancer such as ovarian cancer, the method comprising administering to the human subject a therapeutically effective amount of the composition of any one of claims 21 to 76, optionally wherein the ovarian cancer is an epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer, further optionally wherein the treatment is a second, third, or later line treatment. In some cases, the human subject is diagnosed with early stage ovarian cancer. In another case, the human subject has been diagnosed with late stage ovarian cancer. In another embodiment, the subject has failed prior cancer therapies. In certain cases, the human subject has a platinum-resistant ovarian cancer. In other cases, the human subject has a platinum-sensitive ovarian cancer.

[0427] BRIEF DESCRIPTION OF DRAWINGS FIG. 1A is a schematic representation showing delivery of multiple mRNA strands that encode for distinct secreted engager proteins. In this instance, four different mRNAs encode four polypeptides - two “heavy chains’", one “common light chain’", and one homodimerizing Fc polypeptide - that assemble to form three distinct TCE proteins that bind to three different TAAs. Two TCEs share a common light chain and bind to CD3 in a monovalent fashion. The common light chain (CLC) comprises a VHH that specifically binds to HSA. The homodimerizing Fc polypeptide Attorney Docket No.: 45817-0187WO1

[0428] comprises the hinge-CH2-CH3 domain of human IgG4PAA. The four mRNA strands can be formulated in a single delivery vehicle (such as a nanoparticle, e.g., LNP) or can be formulated in two or three LNPs.

[0429] FIG. IB is a diagram depicting the assembled engager proteins where the Signal 2 TCE binds to the same TAA as a Signal 1 TCEs, to force a cis-signaling TCE2. In this instance, there are two Signal 1 TCEs that bind to CD3 on T cells and to MUC 16 ('‘Ml”) or CLDN6 ('‘Cl”) on tumor cells, and a Signal 2 TCE that binds to 4-1BB on T cells and MUC 16 (“M2”) on tumor cells. The dashed line around the CLDN6 TCE protein is to indicate this component can be optionally included.

[0430] FIG. 1C is a diagram depicting the assembled engager proteins where each TCE binds to a different TAA and provides both Signal 1 and Signal 2 to the T cell. In this instance, there are two Signal 1+2 TCEs that bind to both CD3 and 4-1BB on T cells and to MUC 16 or CLDN6 on tumor cells. The dashed line around the CLDN6 TCE protein is to indicate that this component can be optionally included.

[0431] FIG. ID is a diagram depicting the assembled engager proteins where each TCE binds to a different TAA. In this instance, there are two Signal 1 TCEs that bind to CD3 on T cells and to MUC16 (“Ml”) or CLDN6 (“Cl”) on tumor cells, and a Signal 2 TCE that binds to 4- IBB on T cells and to TROP2 (“T2”) on tumor cells. The dashed line around the CLDN6 TCE protein is to indicate that this component can be optionally included.

[0432] FIG. 2 graphically demonstrates the cytotoxicity' of an anti-MUC16 VHH in the Signal 1 T cell engager format in vitro. Several anti-MUC16 clones were tested in bivalent format in media alone, or in the presence of 1.5 mg / mL HSA or 200 ng / mL CA125. The cytotoxicity EC50 of each protein in each media condition is shown in the table. Clones 15 and 41 bind to the most membrane-proximal region of the MUC 16 extracellular domain.

[0433] FIG. 3 graphically demonstrates the cytotoxicity' of various MUC 16 TCE1 proteins in vitro. The top four anti-MUC16 VHH clones - 8, 15, 24, and 41 - were tested in bivalent (x2) or biparatopic TCE1 format in the presence of 1.5mg / mL HSA and 200 ng / mL CA125. Each biparatopic construct included either clone 15 or clone 41, both of which bind to the most membrane-proximal region of the MUC 16 Attorney Docket No.: 45817-0187WO1

[0434] extracellular domain. The graph titles refer to the N-terminal VHH in the TCE1 protein. The cytotoxicity EC50 of each protein is shown in the table.

[0435] FIG. 4 graphically demonstrates cytotoxicity of humanized anti-MUC16 VHH proteins in the bivalent TCE1 format in vitro. Two humanized variants of clone 15 were tested in comparison to the parental clone in the presence of 1.5 mg / mL HSA and 200 ng / mL CA125. The cytotoxicity EC50 of each protein is shown in the table.

[0436] FIG. 5 graphically demonstrates the cytotoxicity of an anti-CLDN6 VHH in the Signal 1 T cell engager format in vitro. Several anti-CLDN6 clones were tested in bivalent format in the absence or presence of 1.5 mg / mL HSA. The cytotoxicity' EC50 of each protein in each media condition is shown in the table.

[0437] FIG. 6 graphically demonstrates cytotoxicity of humanized anti-CLDN6 VHH proteins in the bivalent TCE1 format in vitro. Humanized variants of clones 18 and 51 were tested in comparison to the parental clone in the presence of 1.5 mg / mL HSA, against two different cell lines. The cytotoxicity EC50 of each protein against each cell line is shown in the table.

[0438] FIG. 7 graphically demonstrates the TAA expression-dependent cytotoxicity of selected TCE1 proteins in vitro. The selected MUC16 and CLDN6 TCE1 proteins were tested in the presence of 1.5 mg / mL HSA and 200 ng / mL CA125 against a cell line that does not express these TAAs. A third TCE1 protein targeting a TAA that is expressed on this cell line was also tested as a positive control. The cytotoxicity EC50 of each protein is shown in the table.

[0439] FIG. 8 demonstrates the in vitro binding specificity7of humanized anti-CLDN6 VHH proteins in the bivalent TCE1 format across human and cynomolgus claudin family members. Humanized clones 51h5 and 18h5 were tested across a range of concentrations for binding to Expi293 cells that had been transiently transfected with plasmids encoding the indicated human or cynomolgus claudin family protein. Binding was assessed by flow cytometry; MFI = mean fluorescence intensity.

[0440] FIG. 9 graphically demonstrates the 4- IBB agonism of parental and humanized TROP2 TCE2 proteins in the presence of TROP2+target cells in vitro using a 4-1BB luciferase reporter Jurkat cell line. The activation EC50 is shown in the table. Attorney Docket No.: 45817-0187WO1

[0441] FIG. 10 graphically demonstrates cytotoxicity of aMUC16 TCE1 protein alone and in combination with 10 nM of a MUC16 TCE2 protein targeting a different MUC16 epitope in vitro. The cytotoxicity EC50 of the TCE1 protein in the presence or absence of the TCE2 is shown in the table.

[0442] FIG. 11 graphically demonstrates cytotoxicity of a MUC16 VHH in either the TCE1 format or the trispecific TCE1+2 format in vitro. The cytotoxicity EC50 of each protein is shown in the table.

[0443] FIG. 12 graphically demonstrates cytotoxicity of aMUC16 TCE1 protein alone and in combination with 10 nM of a TROP2 TCE2 protein in vitro. The cytotoxicity EC50 of the TCE1 protein in the presence or absence of the TCE2 is shown in the table.

[0444] FIG. 13 demonstrates that TROP2 TCE2 protein can enhance in vitro cytotoxicity7of MUC16 TCE1 protein in both cis and trans. The diagrams at the top depict that in the cis-signaling condition, the TAAs targeted by TCE1 and TCE2 are expressed on the same cell, whereas in the trans-signaling condition, the TAAs targeted by TCE1 and TCE2 are not co-expressed, so each engager binds to a different target cell. The graph demonstrates that the cytotoxicity EC50 of the MUC16 TCE1 protein is reduced in the presence of 10 nM TROP2 TCE2 in both target cell conditions.

[0445] FIG. 14A graphically demonstrates the in vitro cytotoxicity of MUC16 TCE1 and CLDN6 TCE1 proteins alone or combined 1:1, and TROP2 TCE2 protein, against different target cell lines expressing varying levels of the target TAAs. TAA expression of the cell lines decreases from left to right.

[0446] FIG. 14B graphically demonstrates the enhancement by TROP2 TCE2 of the in vitro cytotoxicity potency ofthe 1:1 mixture of MUC16 and CLDN6 TCE1 proteins, against different target cell lines expressing vary ing levels of the target TAAs. The dashed line indicates the cytotoxicity EC50 of the TCE1 combo alone. The TCE2 EC50 of cytotoxicity enhancement is indicated on each graph. TAA expression of the cell lines decreases from left to right.

[0447] FIG. 15A graphically depicts the level of engager protein secreted into the supernatant by cells transfected in vitro with mRNAs encoding each of the listed Attorney Docket No.: 45817-0187WO1

[0448] TCEs. For TCE1 transfections, TCE1 heavy chain mRNA (MUC16, CLDN6, or 1:1 ratio of MUC16 and CLDN6) and TCE1 common light chain mRNA were cotransfected at a 5:1 mass ratio. Control refers to an mRNA with anon-translatable ORF.

[0449] FIG. 15B graphically demonstrates in vitro cytotoxicity of secreted engager proteins in the mRNA-transfected supernatants from FIG. 14A. Transfected supernatants from TCE1 conditions were tested alone or in combination with TROP2 TCE2 transfected supernatants. The TCE1 cytotoxicity EC50 of each condition is show n in the table.

[0450] FIG. 16 demonstrates that recombinant proteins encoded by mRNA-2151 elicit potent, TCE1 dose-dependent cytotoxic activity in vitro when applied to PBMCs derived from ovarian cancer patients spanning treatment histories from 1+ to 5+ lines of standard-of-care therapy. The Hl 975 tumor cell line was selected as the target due to its clinically relevant expression levels of MUC16, CLDN6, and TROP2, which reflect antigen profiles observed in ovarian cancer patient tumors.

[0451] FIG. 17A graphically demonstrates the kinetics of MUC16 TCE1 protein expression in vivo in the plasma of naive wildtype mice following a single intravenous administration of mRNAs formulated in LNPs. MUC16 TCE1 heavy chain mRNA and TCE1 common light chain mRNA were co-administered at a 3:2 mass ratio at the total dose level indicated. Pharmacokinetic parameters are shown in the table. Cmax = maximum concentration, tmax = time point at which Cmax is reached; AUC = area under curve; ti / 2 = half-life; LLOQ = lower limit of quantitation of the assay.

[0452] FIG. 17B graphically demonstrates the kinetics of CLDN6 TCE1 protein expression in vivo in the plasma of naive wildtype mice following a single intravenous administration of mRNAs formulated in LNPs. CLDN6 TCE1 heavy chain mRNA and TCE1 common light chain mRNA were co-administered at a 3:2 mass ratio at the total dose level indicated. Pharmacokinetic parameters are shown in the table. Cmax = maximum concentration, tmax = time point at which Cmax is reached; AUC = area under curve; ti / 2 = half-life; LLOQ = lower limit of quantitation of the assay. Attorney Docket No.: 45817-0187WO1

[0453] FIG. 17C graphically demonstrates the kinetics of TROP2 TCE2 protein expression in vivo in the plasma of naive wildtype mice following a single intravenous administration of mRNA formulated in LNPs. Pharmacokinetic parameters are shown in the table. Cmax= maximum concentration; tmax= time point at which Cmaxis reached; AUC = area under curve; t1 / 2= half-life; LLOQ = lower limit of quantitation of the assay.

[0454] FIG. 18A graphically demonstrates the kinetics of MUC16 TCE1 protein expression in vivo in the plasma of naive wildtype cynomolgus macaques following a single intravenous administration of mRNAs formulated in LNPs. MUC16 TCE1 heavy chain mRNA, CLDN6 TCE1 heavy chain mRNA, TCE1 common light chain mRNA, and TROP2 TCE2 mRNA were co-administered at a 26.7:26.7:10.6:36 mass ratio (equivalent to 32% MUC16 TCE1, 32% CLDN6 TCE1, and 36% TROP2 TCE2) at the total dose levels indicated. Pharmacokinetic parameters are shown in the table. Cmax= maximum concentration; AUC = area under curve.

[0455] FIG. 18B graphically demonstrates the kinetics of CLDN6 TCE1 protein expression in vivo in the plasma of nai ve wildtype cynomolgus macaques following a single intravenous administration of mRNAs formulated in LNPs. MUC16 TCE1 heavy chain mRNA, CLDN6 TCE1 heavy chain mRNA, TCE1 common light chain mRNA, and TROP2 TCE2 mRNA were co-administered at a 26.7:26.7:10.6:36 mass ratio (equivalent to 32% MUC16 TCE1, 32% CLDN6 TCE1, and 36% TROP2 TCE2) at the total dose levels indicated. Pharmacokinetic parameters are shown in the table. Cmax= maximum concentration; AUC = area under curve.

[0456] FIG. 18C graphically demonstrates the kinetics of TROP2 TCE2 protein expression in vivo in the plasma of naive wildtype cynomolgus macaques following a single intravenous administration of mRNAs formulated in LNPs. MUC16 TCE1 heavy chain mRNA, CLDN6 TCE1 heavy chain mRNA, TCE1 common light chain mRNA, and TROP2 TCE2 mRNA were co-administered at a 26.7:26.7: 10.6:36 mass ratio (equivalent to 32% MUC16 TCE1, 32% CLDN6 TCE1, and 36% TROP2 TCE2) at the total dose levels indicated. Pharmacokinetic parameters are shown in the table. Cmax= maximum concentration; AUC = area under curve. Attorney Docket No.: 45817-0187WO1

[0457] FIG. 19 demonstrates the anti-tumor efficacy of MUC16 TCE1, CLDN6 TCE1, and TROP2 TCE2 mRNAs in an in vivo humanized xenograft model. Human ovarian cancer cell line OVCAR3 was implanted subcutaneously into immunocompromised NSG mice. Tumor-bearing mice were intraperitoneally implanted with activated human T cells (ATCs) and then received four weekly intravenous administrations at the indicated dose levels of the indicated mRNA(s) formulated in LNPs. MUC16 TCE1 refers to a 3:2 mass ratio of individually formulated MUC16 TCE1 heavy chain mRNA and TCE1 common light chain mRNA. CLDN6 TCE1 refers to a 3:2 mass ratio of individually formulated CLDN6 TCE1 heavy chain mRNA and TCE1 common light chain mRNA. MUC16 TCE1 + CLDN6 TCE1 (1:1) refers to a 1.5:1.5:2 mass ratio of individually formulated MUC16 TCE1 heavy chain mRNA, CLDN6 TCE1 heavy chain mRNA, and TCE1 common light chain mRNA. Control TCE1 refers to a 3:2 mass ratio of individually formulated negative control TCE1 heavy chain mRNA (anti-CD3E Fab with no anti-TAA VHH domains) and TCE1 common light chain mRNA. Solid line indicates ATC administration. Dashed lines indicate LNP dosing. CRs = complete responders, which are defined as mice with no measurable tumor, or with 3 consecutive tumor measurements < 30 mm3.

[0458] FIG. 20 demonstrates the anti-tumor efficacy of MUC16 TCE1, CLDN6 TCE1, and TROP2 TCE2 mRNAs in an in vivo humanized xenograft model. Human ovarian cancer cell line OVCAR3 was implanted subcutaneously into immunocompromised NSG mice. Tumor-bearing mice were intraperitoneally implanted with activated human T cells (ATCs) and then received four weekly intravenous administrations at the indicated dose levels of the indicated mRNA(s) formulated in LNPs. TCE1 refers to a 2.5:2.5:1 mass ratio of individually formulated MUC16 TCE1 heavy chain mRNA, CLDN6 TCE1 heavy chain mRNA, and TCE1 common light chain mRNA. TCE2 refers to TROP2 TCE2 mRNA. Solid line indicates ATC administration. Dashed lines indicate LNP dosing. CRs = complete responders, which are defined as mice with no measurable tumor, or with three consecutive tumor measurements < 30 mm3. Attorney Docket No.: 45817-0187WO1

[0459] DETAILED DESCRIPTION

[0460] This disclosure features a platform technology for secreted multi-specific binding molecules - both polypeptides and nucleic acids that encode these polypeptides (e.g., mRNAs) - that have been designed to provide both Signal 1 and Signal 2. Signaling from the TCR / CD3 complex, so-called “Signal 1,” initiates T cell activation. Subsequently, costimulatory receptors such as 4- IBB or CD28 can provide “Signal 2” to enhance T cell activation. Encompassed herein are polypeptides and nucleic acids that encode these polypeptides (e.g., mRNAs) that have been designed to (i) simultaneously engage Signal 1 ( e.g., human CD3) and one or more tumor-associated antigens (TAAs) on a cancer cell to induce T cell-mediated cytotoxicity against the targeted cancer cell (e.g., in a solid tumor) as well as (ii) simultaneously engage Signal 2 ( e.g., human 4-1BB) and a tumor-associated antigen (TAA) on a cancer cell to enhance T cell-mediated cytotoxicity against the targeted cancer cell (e.g., in a solid tumor). Such T cell engagers, through multiplexing, minimize antigen escape, enhance potency, and broaden tumor and patient coverage as well as providing convenience and manufacturing advantages. The secreted multi-specific binding molecules that simultaneously engage Signal 1 on T cells and a tumor-associated antigen (TAA) are referred to herein as Signal 1 T cell engagers (TCE1). One or more (e.g., 1, 2, 3) TCEls may be employed. The secreted multi-specific binding molecules that simultaneously engage a Signal 2 on T cells and a tumor-associated antigen (TAA) are referred to herein as Signal 2 T cell engagers (TCE2).

[0461] The CD3 targeting moiety7of TCEls must be monovalent because crosslinking of CD3 can cause severe toxicity and death. The conventional means of producing these antibodies is to produce them recombinantly and purify to remove impurities that are bivalent against CD3. However, this is a costly and laborious process. Further, conventional means are limited to targeting only a single TAA. Tumor heterogeneity, immune escape, and patient TAA expression profiles are common reasons for limited efficacy of antibodies targeting a single TAA. The anti-CD3 bispecific binding molecules of this disclosure, which are produced in vivo by administration of mRNA molecules (e.g., formulated in a delivery vehicle such as a nanoparticle), are designed to bind to one or more tumor-associated antigens and to Attorney Docket No.: 45817-0187WO1

[0462] only induce a cytotoxic CD8+ T cell response after complexing to a tumor-associated antigen, but not induce nonspecific cytotoxic T cell activation. To meet these design goals, the bispecific binding molecules of this disclosure have several therapeutic design attributes. The binding molecules of this disclosure lack Fc gamma receptor binding and so do not deplete the activated T cells that are required for elimination of the targeted cancer cells. In addition, the binding molecules have an antibody-like format that ensures targeting of CD3 is monovalent, i.e., with no risk of cross-linking CD3, and have the desired half-life for a therapeutic.

[0463] The format of the binding molecules of this disclosure comprises a “heavy chain” comprising an anti-CD3 binding VH / CH1 domain of a Fab and TAA-targeting VHHs that are appended to the C-terminal of the CHI of the VH / CH1 domain of the Fab. Surprisingly, this format was found to yield superior results as compared to other formats tested. A “common light chain” is used comprising the VL / CL domain of the Fab that binds to CD3, wherein the C-terminal of the CL of the Fab is linked to a VHH that binds to HSA so as to enhance the circulating half-life of the binding molecule. With the VH / CH1 Fab-VHH-VHH “heavy chain” format, a “common light chain” is used to multiplex mRNAs allowing the multiplexed products to target multiple TAAs at the same time using a single formulation. This approach is not possible with conventional protein delivery but is possible when delivering this format as mRNA that encodes for the described protein components that assemble and express the desired therapeutic anti-CD3 binding molecules in vivo. This disclosure demonstrates that with a combination of up to 4 mRNAs, it is possible to express 4 polypeptides that bind 3 TAAs by delivering 2 strands of mRNA encoding “heavy chains” targeting different TAAs (e.g., human TROP2, human MUC16, human CLDN6), 1 strand of mRNA encoding a “common light chain” that comprises an anti-HSA binding domain, and 1 strand of mRNA comprising a binding molecule that specifically binds to a TAA (e.g., human TROP2, human MUC16, human CLDN6) which is linked (e.g.. via an immunologically silent human Ig Fc domain such as human IgG4PAA) to a binding moiety that specifically binds to a Signal 2 receptor on a T cell (e.g., human 4-1BB). This proof of concept enables formulations comprising vary ing numbers of mRNAs encoding vary ing numbers of anti-TAA polypeptides to Attorney Docket No.: 45817-0187WO1

[0464] arrive at the desired protein therapeutics in vivo. Such mRNA molecules can be delivered in the same or in separate (e.g., two. three) delivery vehicles (e.g., lipid nanoparticles (LNPs)). In some cases, the mRNAs are delivered in the same LNP. In some instances, a single drug product is administered to a human subject in need thereof, wherein the drug product comprises the 4 mRNAs (see, Fig. IB) at a defined ratio (Ml: Cl: CLC: T2 = about 39.6: about 39.6: about 15.8: about 5). In certain cases, the defined ratio of Ml: Cl: CLC: T2 = 39.6: 39.6: 15.8: 5. The defined ratio can be adjusted as needed for improving therapeutic goals. For example, the percentage of T2 may be increased. The multiplexed Fab-VHH-VHH format overcomes toxicity concerns of bivalent anti-CD3, efficacy shortcomings of targeting only one TAA, and short half-life of proteins.

[0465] In one aspect, the disclosure provides a platform technology that features a delivery vehicle composition (e.g., a nanoparticle composition such as a LNP composition) comprising multiple mRNAs that encode T cell engagers. The multiple mRNAs are formulated in one or more (e.g.. 1, 2, 3) delivery vehicles (e.g.. LNPs). In some cases, the mRNAs are formulated in a single delivery vehicle (e.g., LNP). In certain cases, the mRNAs are formulated in two delivery vehicles (e.g., LNPs). The composition comprises (i) mRNAs that encode multi-specific binding molecules that are T cell engagers that provide Signal 1 (e.g., by binding to human CD3) (TCE1) and (ii) mRNAs that encode multi-specific binding molecules that are T cell engagers that provide a Signal 2 (e.g., by binding to human 4- IBB) (TCE2). TCE1 and TCE2 also bind to TAAs (e.g., human solid tumor TAAs). The TCE1 molecules comprise a first polypeptide comprising from the N-terminal to C-terminal a VH / CH1 domain of an Fab that specifically binds to human CD3 linked directly or via a linker (e.g., G4S (SEQ ID NO:4) to a VHH that binds to a tumor-associated antigen; and a second polypeptide comprising from the N-terminal to C-terminal a VL / CL domain of a Fab that specifically binds to human CD3 linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that binds to an agent for the purpose of halflife extension (e.g., HSA), wherein the VH and the VL from the first and second polypeptides when associated form a binding site that specifically binds to human CD3. In certain cases, the C-terminal of the VHH that binds to HSA is linked via a Attorney Docket No.: 45817-0187WO1

[0466] linker (e.g., G4S (SEQ ID NO:4) or (G4S)3 (SEQ ID NO:5)) to the N-terminal of a VHH that specifically binds to human 4- IBB. In some cases, the C-terminal of the CH 1 is linked directly or via a linker to two or at least two VHHs that bind to a tumor-associated antigen, wherein the C-terminal of the CHI is linked to the N-terminal of a first VHH and the C-terminal of the first VHH is linked (e.g., via (G4S)3 (SEQ ID NO: 5)) to the N-terminal of the second VHH. In some cases, the two VHHs of TCE1 are human MUC16 binders, e.g., that bind to the same or different epitopes of human MUC16. In other cases, the two VHHs of TCE1 are human CLDN6 binders, e.g., that bind to the same or different epitopes of CLDN6. In certain cases, the two VHHs of TCE1 are identical.

[0467] As set forth above, the VH and CHI associates with the VL and CL to form a Fab, and the Fab specifically binds to human CD3 (e g., human CD3E). In some cases, at least two TCEls are employed, wherein the first TCE1 specifically binds to both human CD3 and human MUC16 and the second TCE1 binds to both human CD3 and human CLDN6. Also featured are mRNAs that encode a TCE2. TCE2 comprises from N-terminal to C-terminal a VHH that specifically binds to either TROP2 or MUC16, an immunologically silent human Ig Fc domain such as the human IgG4PAA hinge-Fc domain, a G4S (SEQ ID NO: 4) linker, and a VHH that specifically binds to human 4-1BB. The C-terminal of the VHH that specifically binds to either TROP2 or MUC16 is linked to the N-terminal of the immunologically silent human Ig Fc domain (e.g., human IgG4PAA hinge-Fc domain), and the C-terminal of the immunologically silent human Ig Fc domain (e.g., human IgG4PAA hinge-Fc domain) is connected to the N-terminal of the VHH that specifically binds to human 4-1BB through the G4S (SEQ ID NO:4) linker.

[0468] In some aspects, the disclosure provides a composition comprising the mRNAs that encode TCE1 and / or TCE2 formulated in LNPs. In some cases, mRNA encoding a single TCE polypeptide is formulated in a single LNP. In some cases, mRNAs encoding two TCE polypeptides are formulated in a single LNP. In certain cases, mRNAs encoding three TCE polypeptides are formulated in a single LNP. In other cases, mRNAs encoding four TCE polypeptides are formulated in a single LNP. In some cases, the LNP composition comprises mRNAs that encodes a TCE2 Attorney Docket No.: 45817-0187WO1

[0469] polypeptide formulated in a single LNP. In other cases, the LNP composition comprises mRNAs that encode TCE1 polypeptides formulated in a single LNP. In yet other cases, the LNP composition comprises mRNAs that encode TCE1 and TCE2 polypeptides formulated in a single LNP.

[0470] In other aspects, the disclosure provides a composition comprising a mixture of LNPs comprising mRNAs that encode a TCE1 or two TCEls along with LNPs comprising mRNAs that encode TCE2. In some cases, two LNPs are used, the first comprising two or three mRNAs encoding TCE1 polypeptides and the second LNP comprising four mRNAs encoding TCE1 polypeptides and TCE2 polypeptides.

[0471] In some cases, the lipid nanoparticle comprises a structural lipid, a phospholipid, a PEG-lipid and an ionizable amino lipid. In one case, the ionizable amino lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate. In one case, the PEG-lipid is 134-hydroxy-3.6,9,12,15,18,21,24.27,30,33,36,39,42,45,48,51,54,57,60,63.66,69,72,75,78,81,84,87,90.93,96,99.102, 105, 108, 111.114, 117, 120, 123.126, 129, 132-tetratetracontaoxatetratriacontahectyl stearate. In one case, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one case, the structural lipid is cholesterol. In some cases, the lipid nanoparticle comprises Compound 2, DSPC, cholesterol, and Compound I. In some cases, the LNP comprises about 47.5 mole ratio % Compound 2, about 10.5 mole ratio % DSPC, about 39 mole ratio % cholesterol, and about 3 mole ratio % Compound I.

[0472] The disclosure features delivery vehicle compositions comprising nucleic acids (e.g., mRNAs) encoding T cell engagers comprising a means for binding human CD3 (e.g., human CD3E) linked to (i) one, two, or more means for binding a solid tumor TAA (e.g., MUC16, CLDN6); and (ii) means for binding an agent for the purpose of half-life extension (e.g., HSA). Nucleic acid molecules (e.g., mRNA molecules) encoding such engagers allow for targeting multiple TAAs at the same time, thereby reducing the possibility of antigenic escape by tumor cells using one drug product. More specifically, the data in the instant Examples demonstrate that the messenger RNA (mRNA) platform enables multiplexing of three or more (e.g., 3, 4, 5) mRNA sequences to encode two or more T cell engagers (TCEs) in a single drug Attorney Docket No.: 45817-0187WO1

[0473] product. Targeting multiple tumor-associated antigens (TAAs) simultaneously using this platform can circumvent target-mediated resistance and achieve outcomes superior to those seen with single TAA targeting. Furthermore, targeting both Signal 1 and Signal 2 enhances cytotoxicity and efficacy and allows for treatment of solid tumors.

[0474] In some instances, the disclosure features a first LNP (LNP1) comprising an mRNA encoding a heavy chain comprising MUC16 binders (MUC HC), an mRNA encoding a heavy chain comprising CLDN6 binders (CLDN HC), and an mRNA encoding a light chain comprising a HSA binder (LC); and a second LNP (LNP2) comprising an mRNA encoding a TROP2 binder linked to a 4- IBB binder (TROP2 Fc). In some cases, LNP1 and LNP2 are administered (e.g., intravenously) separately to a human subject in need thereof (e.g., one with ovarian cancer). In other cases, LNP1 and LNP2 are mixed together to create a single drug product and administered (e.g., intravenously) to a human subject in need thereof (e.g., one with ovarian cancer). In certain instances, the disclosure features a LNP comprising each of MUC HC + CLDN HC + LC + TROP2 Fc. A pharmaceutical composition comprising a population of this single LNP is administered (e.g., intravenously) to a human subject in need thereof (e.g., one with ovarian cancer).

[0475] In some instances, the disclosure provides a pharmaceutical composition comprising population of LNPs containing MUC HC + LC. In other instances, the disclosure features a pharmaceutical composition comprising a population of LNPs containing CLDN6 HC + LC. These two instances are examples of standalone / single targeted TCEs. These pharmaceutical compositions can be used alone or multiplexed with mRNAs encoding proteins binding other targets.

[0476] Definitions

[0477] As used herein, the term “about’' refers to a stated numerical term and a value that is no more than 10% above or below the value being described. For example, the term “about 5 nM” indicates disclosure of both the stated value of 5 nM and a range of from 4.5 nM to 5.5 nM. When used with respect to time, e.g., “about” 1 week, the Attorney Docket No.: 45817-0187WO1

[0478] term ‘‘about” means + / - 3 days, so about 1 week refers to 1 week and a range of 4 days to 10 days.

[0479] As used herein, the terms "conservative mutation,” “conservative substitution,” “conservative amino acid substitution,” and the like refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and / or steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in Table I below.

[0480] Table I - Representative Physicochemical Properties of Naturally-Occurring Amino Acids

[0481] Electrostatic

[0482] 3 1 Side- character at Steric Amino Acid Letter Letter chain

[0483] physiological pH Volume^ Code Code Polarity

[0484] (7.4)

[0485] Alanine Ala A nonpolar neutral small Arginine Arg R polar cationic large Asparagine Asn N polar neutral intermediate Aspartic acid Asp D polar anionic intermediate Cysteine Cys C nonpolar neutral intermediate Glutamic acid Glu E polar anionic intermediate Glutamine Gin Q polar neutral intermediate Glycine Gly G nonpolar neutral small Both neutral and

[0486] cationic forms in

[0487] Histidine His H polar large equilibrium at pH

[0488] 7.4

[0489] Isoleucine He I nonpolar neutral large Leucine Leu L nonpolar neutral large Lysine Lys K polar cationic large Methionine Met M nonpolar neutral large Phenylalanine Phe F nonpolar neutral large Proline ProPnonneutral intermediate polar

[0490] Serine Ser S polar neutral small Threonine Thr T polar neutral intermediate Tryptophan Trp W nonpolar neutral bulky Tyrosine Tyr Y polar neutral large Valine Vai V nonpolar neutral intermediate ^based on volume in A3: 50-100 is small, 100-150 is intermediate,

[0491] 150-200 is large, and >200 is

[0492]

[0493] bulky Attorney Docket No.: 45817-0187WO1

[0494] From this table it is appreciated that the conservative amino acid families include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg).

[0495] As used herein, the term “fusion protein” refers to a protein that is joined via a covalent bond to another molecule.

[0496] As used herein, the term “lipid nanoparticle” refers to a transfer vehicle including one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids). Examples of lipid nanoparticles are formulated to deliver one or more mRNA molecules to one or more target cells in vivo. Examples of suitable lipids include, for example, ionizable amino lipids, phospholipids, cholesterol lipids, and PEG lipids. Other formulations will be known to those of skill in the art and such lipids can be used to formulate LNPs to enhance the delivery of mRNA into the target cells.

[0497] As used herein, the terms “percent (%) sequence identity,” “percent (%) identity,” and the like, with respect to a reference polynucleotide or polypeptide sequence, is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity' of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid Attorney Docket No.: 45817-0187WO1

[0498] sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence. B) is calculated as: 100 multiplied by (the fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0499] As used herein, the phrase ‘‘specifically binds” refers to a binding reaction which is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological molecules that is recognized, e.g., by an antibody or antigen-binding fragment thereof, with particularity, i.e., at levels above background binding. In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen via the antigen binding domain will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD of greater than 100 nM (e.g.. greater than 500 nm. 1 pM, 100 pM, 500 pM, or 1 mM) for that particular antigen or epitope thereof. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0500] As used herein, the terms “treat” or “treatment” refer to therapeutic treatment, in which the object is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as a cancer. Beneficial or desired clinical results of treatment Attorney Docket No.: 45817-0187WO1

[0501] include, without limitation, alleviation of symptoms, diminishment of extent of disease, stabilized (i. e.. not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Those in need of treatment include those already having the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be inhibited.

[0502] As used herein, the term “alkyl,” “alkyl group,” or “alkylene” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted. The notation “Ci-i4 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1-14 carbon atoms. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups.

[0503] As used herein, the term “alkenyl,” “alkenyl group,” or “alkenylene” means a linear or branched hydrocarbon including two or more carbon atoms (e.g, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted. The notation “C2-14 alkenyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. For example, Cis alkenyl may include one or more double bonds. A Cis alkenyl group including two double bonds may be a linoleyl group. Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.

[0504] As used herein, the term “alkynyl,” “alkynyl group,” or “alkynylene” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted. The notation “C2-14 alky nyl” means an optionally substituted linear or branched hydrocarbon including 2- Attorney Docket No.: 45817-0187WO1

[0505] 14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may include one, two. three, four, or more carbon-carbon triple bonds. For example. Cis alkynyl may include one or more carbon-carbon triple bonds. Unless otherwise specified, an alky nyl group described herein refers to both unsubstituted and substituted alky nyl groups.

[0506] As used herein, the term “carbocycle” or “carbocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings. The notation “C3-6 carbocycle” means a carbocycle including a single ring having 3-6 carbon atoms. Carbocycles may include one or more carboncarbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or ary l groups). Examples of carbocycles include cyclopropyl, cyclopentyd, cyclohexy l, phenyl, naphthyl, and 1,2 dihydronaphthyl groups. The term “cycloalkyd” as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond. Unless otherwise specified, carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles.

[0507] As used herein, the term “heterocycle” or “heterocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom. Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms. Rings may be three, four, five, six, seven, eight, nine, ten. eleven, twelve, thirteen, or fourteen membered rings. Heterocycles may include one or more double or triple bonds and may be non-aromatic or aromatic (e.g., heterocycloalkyd or heteroaryd groups). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidiny 1, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl. morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. The term “heterocycloalkyl” as used herein means a non-aromatic heterocycle and may or may not include any double or triple bond. Unless otherwise specified, heterocycles described herein refers to both Attorney Docket No.: 45817-0187WO1

[0508] unsubstituted and substituted heterocycle groups, i.e., optionally substituted heterocycles.

[0509] As used herein, the term “heteroalkyl,’’ “heteroalkenyl,” or “heteroalkynyl” refers respectively to an alkyl, alkenyl, alkynyl group, as defined herein, which further comprises one or more (e.g., 1. 2, 3, or 4) heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus) wherein the one or more heteroatoms is inserted between adjacent carbon atoms within the parent carbon chain and / or one or more heteroatoms is inserted between a carbon atom and the parent molecule, i.e., between the point of attachment. Unless otherwise specified, heteroalkyls, heteroalkenyls, or heteroalkynyls described herein refers to both unsubstituted and substituted heteroalkyls, heteroalkenyls, or heteroalkynyls, i.e., optionally substituted heteroalkyls, heteroalkenyls, or heteroalkynyls.

[0510] As used herein, a “biodegradable group” is a group that may facilitate faster metabolism of a lipid in a mammalian entity. A biodegradable group may be selected from the group consisting of. but is not limited to. -C(O)O-. -OC(O)-. -C(O)N(R')-. -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, an aryl group, and a heteroar l group. As used herein, an “aryl group” is an optionally substituted carbocyclic group including one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, a “heteroaryl group” is an optionally substituted heterocyclic group including one or more aromatic rings.

[0511] Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both ary l and heteroaryl groups may be optionally substituted. For example, M and M' can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the Formulas herein, M and M' can be independently selected from the list of biodegradable groups above. Unless otherwise specified, ary l or heteroaryl groups described herein refers to both unsubstituted and substituted groups, i. e.. optionally substituted aryl or heteroaryl groups.

[0512] Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified. Optional substituents may be selected from the group consisting of, but are not limited to, a halogen atom (e.g., a Attorney Docket No.: 45817-0187WO1

[0513] chloride, bromide, fluoride, or iodide group), a carboxylic acid (e.g., C(O)OH), an alcohol (e.g.. a hydroxyl. OH), an ester (e.g., C(O)OR OC(O)R), an aldehyde (e.g. C(O)H), a carbonyl (e g., C(O)R, alternatively represented by C=O), an acyl halide (e.g., C(O)X, in which X is a halide selected from bromide, fluoride, chloride, and iodide), a carbonate (e.g., OC(O)OR), an alkoxy (e.g., OR), an acetal (e.g., C(OR)2R"", in which each OR are alkoxy groups that can be the same or different and R"" is an alkyl or alkenyl group), a phosphate (e.g, P(O)43'), a thiol (e.g, SH), a sulfoxide (e.g., S(O)R), a sulfinic acid (e.g., S(O)OH), a sulfonic acid (e.g., S(O)2OH), athial (e.g., C(S)H), a sulfate (e.g., S(O)42'), a sulfonyl (e.g., S(O)2 ), an amide (e.g, C(O)NR2, or N(R)C(O)R), an azido (e.g., N3), a nitro (e.g. NO2), a cyano (e.g., CN), an isocyano (e.g., NC), an acyloxy (e.g., OC(O)R), an amino (e.g., NR2, NRH, or NH2), a carbamoyl (e g., OC(O)NR2, OC(O)NRH, or OC(O)NH2), a sulfonamide (e.g., S(O)2NR2, S(O)2NRH, S(O)2NH2, N(R)S(O)2R, N(H)S(O)2R, N(R)S(O)2H, or N(H)S(O)2H), an alkyl group, an alkenyl group, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any of the preceding, R is an alkyl or alkenyl group, as defined herein. In some embodiments, the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein. For example, a C1-6 alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.

[0514] Compounds of the disclosure that contain nitrogens can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and / or hydrogen peroxides) to afford other compounds of the disclosure. Thus, all shown and claimed nitrogen-containing compounds are considered, when allowed by valency and structure, to include both the compound as shown and its N-oxide derivative (which can be designated as N- 0 orN+-O). Furthermore, in other instances, the nitrogens in the compounds of the disclosure can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as m CPBA. All shown and claimed nitrogen-containing compounds are also considered, when allowed by valency and structure, to cover both the compound as show n and its N-hydroxy (i.e., N-OH) and N-alkoxy (i.e., N-OR, wherein R is substituted or Attorney Docket No.: 45817-0187WO1

[0515] unsubstituted Ci-Ce alkyl, Ci-Ce alkenyl, Ci-Cg alkynyl, 3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

[0516] T Cell Engager Structure

[0517] This disclosure encompasses polypeptides and nucleic acids that encode T cell engagers (TCEs) that provide Signal 1 (TCE1) and that bind to one or more TAAs on a cancer cell (e.g., in a solid tumor) as well as T cell engagers that provide Signal 2 (TCE2) and that bind to one or more TAAs on a cancer cell. In some cases, the TAAs are expressed on the cell surface of a solid tumor (e.g., ovarian cancer). Non-limiting examples of TCEs are provided below.

[0518] The TCEls of this disclosure comprise a binding moiety that binds specifically to human CD3 (e.g., human CD3E) linked to a binding moiety or binding moieties that bind to a tumor-associated antigen(s) (TAA(s)). In certain instances, the TAA is present on a solid tumor, such as an epithelial cell tumor, e.g., an ovarian tumor. For example, in one embodiment, the TAA is human MUC16 or human CLDN6. The TCE1 also comprises a binding moiety that specifically binds to an agent for the purpose of half-life extension (e.g.. HSA). In some instances, the binding moiety that binds specifically to human CD3 (e.g., human CD3E) is a Fab comprising a VH / CH1 domain and a VL / CL domain. In certain instances, the binding moieties that bind to a TAA and HSA are VHHs. In one case, the C-terminal of the CHI of the Fab that binds specifically to human CD3 (e.g., human CD3E) is linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to a TAA (e g., human MUC16 or human CLDN6). In certain cases, the C-terminal of that VHH is linked directly or via a linker (e.g., (G4S)3 (SEQ ID NO: 5)) to the N-terminal of another VHH that binds to the same TAA. The two VHHs may be identical in sequence or may have different sequences (e.g., they may bind different epitopes of the same TAA). In some cases, the C-terminal of the CL of the VL / CL of the Fab that binds specifically to human CD3 (e.g., human CD3E) is linked Attorney Docket No.: 45817-0187WO1

[0519] directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that binds to an agent for the purpose of half-life extension (e.g.. HSA).

[0520] In some instances, a single TCE1 is employed. In such cases, the single TCE1 comprises a VH / CH1 domain and a VL / CL domain of a Fab that specifically binds to human CD3 (e.g., human CD3E) wherein the C-terminal of the CHI is linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to MUC16, and the C-terminal of the VHH that specifically binds to MUC16 is linked directly or via a linker (e.g., (G4S)3 (SEQ ID NO: 5)) to the N-terminal of another VHH that specifically binds to MUC16. In some cases, both VHHs that specifically binds to MUC16 are identical in sequence. In certain cases, the VHHs that specifically bind to MUC16 are not identical in sequence. TCE1 also comprises a “common light chain” which comprises a VL / CL domain of a Fab that specifically binds to human CD3 (e.g., human CD3E) wherein the C-terminal of the CL is linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to an agent for the purpose of half-life extension (e.g., HSA).

[0521] In other cases, two or more TCEls are employed. In some such cases, a first TCE1 comprises a VH / CH1 domain and a VL / CL domain of a Fab that specifically binds to human CD3 (e.g., human CD3E) wherein the C-terminal of the CHI is linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to human MUC16, and the C-terminal of the VHH that specifically binds to human MUC16 is linked directly or via a linker (e.g., (G4S)3 (SEQ ID NO: 5)) to the N-terminal of another VHH that specifically binds to human MUC16. In some cases, both VHHs that specifically bind to human MUC16 are identical in sequence. In other cases, the VHHs that specifically binds to human MUC16 are not identical in sequence. TCE1 also comprises a “common light chain” which comprises a VL / CL domain of a Fab that specifically binds to human CD3 (e.g., human CD3E) wherein the C-terminal of the CL is linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to an agent for the purpose of half-life extension (e.g., HSA). In some instances, a second TCE1 is also employed. In some such cases, the second TCE1 comprises a VH / CH1 domain and a Attorney Docket No.: 45817-0187WO1

[0522] VL / CL domain of a Fab that specifically binds to human CD3 (e.g., human CD3E) wherein the C-terminal of the CHI is linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to human CLDN6, and the C-terminal of the VHH that specifically binds to human CLDN6 is linked directly or via a linker (e.g., (G4S)3 (SEQ ID NO: 5)) to the N-terminal of another VHH that specifically binds to human CLDN6. In some cases, both VHHs that specifically bind to human CLDN6 are identical in sequence. The VH / CH1 domain of the second TCE1 pairs with the VL / CL domain of the “common light chain.”

[0523] The disclosure also features a TCE2. In some instances, the TCE2 comprises a VHH that specifically binds to TROP2 linked via a hinge-CH2-CH3 domain of an immunologically silent human Ig (e.g., IgG), the C-terminal of which is attached to a linker (e g., G4S (SEQ ID NO:4)), the C-terminal of which in turn is attached to the N-terminal of a VHH that specifically binds to 4- IBB. In some cases, the hinge-CH2-CH3 domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of human IgG4PAA (SEQ ID NO:33). IgG4PAA is human IgG4 with mutations S228P / F234A / L235A and has reduced binding to FcyR. The amino acid sequence of human IgG4PAA is provided below (the hinge is shown in bold; the CH2 domain is italicized; and the CH3 domain is underlined; the CH2 and CH3 domains together form the Fc):

[0524] ESKYGPPCPPCPAPP. AAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPE VQFNWYVDG VEVHNAKTKPREEQFNSTYR WSVL TVLHQD WLNGKEYKCKVSNK

[0525] GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:33)

[0526] In some cases, the hinge and Fc domain comprises an amino acid sequence set forth in SEQ ID NO:33 except for 1, 2, 3 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. These substitutions can be made to e.g., alter effector function (e.g., increasing or decreasing effector function), or to promote heterodimerization. Such substitutions made as part of Fc domain engineering are well known in the art. See e.g., 1,.i u H, Saxena A, Sidhu SS and Wu D (2017) Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds. From. Immunol. 8:38. doi: Attorney Docket No.: 45817-0187WO1

[0527] 10.3389 / fimmu.2017.00038; Wilkinson I, Anderson S, Fry J, Julien LA, Neville D, Qureshi O, et al. (2021) Fc-engineered antibodies with immune effector functions completely abolished. PLoS ONE 16(12): e0260954. doi.org / 10.1371 / joumal.pone.0260954; Delidakis et al., Wilkinson I, Anderson S, Fry J, Julien LA, Neville D, Qureshi O, et al. (2021) Fc-engineered antibodies with immune effector functions completely abolished. PLoS ONE 16(12): e0260954. / / doi.org / 10.1371 / joumal.pone.0260954, all of which are incorporated by reference herein.

[0528] In a different aspect, the disclosure features T cell engagers that employ the two TCEls described above but uses a different TCE2. The TCE2 in this case comprises a VHH that specifically binds to human MUC16 linked via a hinge-CH2-CH3 domain of an immunologically silent human Ig (e.g., IgG), the C-terminal of which is attached to a linker (e.g., G4S (SEQ ID NO:4)), wherein the C-terminal of the linker is attached to the N-terminal of a VHH that specifically binds to human 4-1BB. In some cases, the VHH that binds to human MUC16 in TCE2 has a different amino acid sequence than the VHHs that bind human MUC 1 in TCE1 (e g., it binds a different MUC 16 epitope). In certain cases, the VHH that binds to human MUC 16 in TCE2 is non-competing with the TCE1 MUC 16 VHH. In some cases, the hinge-CH2-CH3 domain comprises an amino acid sequence that is 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 the hinge-Fc domain amino acid sequence of human IgG4PAA (SEQ ID NO:33). In other cases, the hinge-CH2-CH3 domain comprises the hinge-Fc domain amino acid sequence of human IgG4PAA set forth in SEQ ID NO:33 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0529] In yet another aspect, the disclosure encompasses T cell engagers wherein both Signal 1 and Signal 2 are provided by the same target cancer cell (in cis). In such instances, a TCE1 comprises a VH / CH1 domain and a VL / CL domain of a Fab that specifically binds to human CD3 (e.g., human CD3E) wherein the C-terminal of the CHI is linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to MUC16, and the C-terminal of the VHH that Attorney Docket No.: 45817-0187WO1

[0530] specifically binds to MUC16 is linked directly or via a linker (e.g., (G4S)3 (SEQ ID NO: 5)) to the N-terminal of another VHH that specifically binds to MUC16. In some cases, both VHHs that specifically bind to MUC16 are identical in sequence. TCE1 also comprises a ''common light chain” which comprises a VL / CL domain of a Fab that specifically binds to human CD3 (e.g., human CD3E) wherein the C-terminal of the CL is linked directly or via a linker (e.g.. G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to an agent for the purpose of half-life extension (e.g., HSA), the C-terminal of which is linked to the N-terminal of a VHH that specifically binds to human 4-1BB. In some instances, a second TCE1 is also employed. In some such cases, the second TCE1 comprises a VH / CH1 domain and a VL / CL domain of a Fab that specifically binds to human CD3 (e.g., human CD3E) wherein the C-terminal of the CHI is linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to CLDN6, and the C-terminal of the VHH that specifically binds to CLDN6 is linked directly or via a linker (e.g.. (G4S)3 (SEQ ID NO: 5)) to the N-terminal of another VHH that specifically binds to CLDN6. In some cases, both VHHs that specifically bind to CLDN6 are identical in sequence. The VH / CH1 of the second TCE1 pairs with the VL / CL of the “common light chain” which comprises a VL / CL of a Fab that specifically binds to human CD3 (e.g.. human CD3E) wherein the C-terminal of the CL is linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to an agent for the purpose of half-life extension (e.g., HSA), the C-terminal of which is linked to the N-terminal of a VHH that specifically binds to human 4-1BB.

[0531] The individual components of these T cell engagers are discussed in more detail below.

[0532] Anti-CD3 Binding Moiety (Signal 1 TCE)

[0533] Cluster of Differentiation 3 (CD3) is a protein complex and T cell co-receptor that is involved in activating both cytotoxic T cells (CD8+T cells) and T helper cells (CD4+T cells). It is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD35 chain, and two CD3s chains. These chains associate Attorney Docket No.: 45817-0187WO1

[0534] with the T cell receptor (TCR) and the CD3 zeta (ζ chain to generate an activation signal in T lymphocytes. The TCR, CD3 zeta, and the other CD3 molecules together constitute the TCR complex.

[0535] As part of the TCR-CD3 complex, CD3 epsilon is present on the surface of T-lymphocyte and plays an essential role in the adaptive immune response. When antigen presenting cells (APCs) activate T cell receptor (TCR), TCR-mediated signals are transmitted across the cell membrane by the CD3 chains - i.e., CD3 delta (CD3D), CD3 epsilon (CD3E), CD3 gamma (CD3G) and CD3 zeta (CD3Z). All the CD3 chains contain immunoreceptor tyrosine-based activation motifs (IT AMs) in their cytoplasmic domain. Upon TCR engagement, these motifs become phosphorylated by Src family protein tyrosine kinases LCK and FYN, resulting in the activation of downstream signaling pathways. In addition to this role in signal transduction in T cell activation, CD3E plays an essential role in T cell development. It initiates the TCR-CD3 complex assembly by forming the two heterodimers CD3D / CD3E and CD3G / CD3E. It also participates in internalization and cell surface down-regulation of TCR-CD3 complexes via endocytosis sequences present in CD3E cytosolic region.

[0536] The amino acid sequence of mature full length human CD3E is provided below”

[0537] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKN IGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDA NFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYY WSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPI RKGQRDLYSGLNQRRI (SEQ ID NO 1)

[0538] The amino acid sequence of mature full length cynomolgus CD3E is provided below:

[0539] QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRL FLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATI VIVDICTTLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPN PDYEPIRKGQQDLYSGLNQRRI (SEQ ID NO:2)

[0540] The amino acid sequence of the extracellular domain of mature human CD3E is provided below: Attorney Docket No.: 45817-0187WO1

[0541] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNI GSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0542] (SEQ ID NO:3)

[0543] The T cell Signal 1 targeting component of the T cell engagers of this disclosure specifically bind to CD3. In certain instances, the T cell Signal 1 targeting component of the T cell engagers of this disclosure specifically bind to human CD3E.

[0544] In some instances, the T cell Signal 1 targeting component of the T cell engagers of this disclosure specifically bind to human and cynomolgus CD3E. In certain cases, the T cell Signal 1 targeting component of the T cell engagers of this disclosure

[0545] specifically bind to the extracellular domain of human CD3E. In some cases, the T cell Signal 1 targeting component of the T cell engagers of this disclosure specifically bind to the extracellular domain of human and cynomolgus CD3E.

[0546] Provided herein are binding polypeptides (e.g., Fab) that specifically bind to CD3 (e g., human CD3E) and comprise the six CDRs of an anti-CD3 antibody. Table II provides the six CDR sequences of a non-limiting example of an anti-CD3 antibody (humanized SP34) based on common CDR definitions known in the art.

[0547] Table II: CDR Sequences of Humanized SP34

[0548] Kabat Chothia Enhanced Chothia Contact IMGT VHCDR1 TYAMN GFTFNTY GFTFNTYAMN NTYAMN GFTFNTYA (SEQ ID NO:6) (SEQ ID NO: 12) (SEQ ID NO: 14) (SEQ ID NO: 16) (SEQ ID NO:22) VHCDR2 RIRSKYNNYATY RSKYNNYA RIRSKYNNYATY WVARIRSKYN IRSKYNNYAT YADSVKD (SEQ ID NO: 13) (SEQ ID NO: 15) NYATY (SEQ ID NO:23) (SEQ ID NOY) (SEQ ID NO: 17)

[0549] VHCDR3 HGNFGNSYVSW HGNFGNSYVS HGNFGNSYVSW ARHGNFGNSY ARHGNFGNSY FAY WFAY FAY VSWFA VSWFAY

[0550] (SEQ IDNO:8) (SEQ IDNO:8) (SEQ IDNO:8) (SEQ ID NO: 18) (SEQ ID NO:24)

[0551] VLCDR1 RSSTGAVTTSNY RSSTGAVTTSN RSSTGAVTTSNY VTTSNYANWV TGAVTTSNY AN (SEQ ID NO:9) YAN AN (SEQ ID NO: 19) (SEQ ID NO:25)

[0552] (SEQ IDNO:9) (SEQ ID NO: 9)

[0553] VLCDR2 GTNKRAP GTNKRAP GTNKRAP GLIGGTNKRA GTN

[0554] (SEQ ID NO: 10) (SEQ ID NO: 10) (SEQ ID NO: 10) (SEQ ID NO:20)

[0555]

[0556] Attorney Docket No.: 45817-0187WO1

[0557] VLCDR3 ALWYSNLWV ALWYSNLWV ALWYSNLWV ALWYSNLW ALWYSNLWV (SEQ ID NO: 11) (SEQ IDNO:11) (SEQ ID NO: 11) (SEQ IDNO:21) (SEQ ID NO: 11)

[0558]

[0559] This disclosure relates in part to a binding molecule (e.g., Fab) that

[0560] specifically binds to CD3 (e.g., human and / or cyno CD3) comprising the six CDRs of an anti-CD3 antibody described herein. In some cases, these binding molecules bind CD3E (e.g., human and / or cyno CD3E). In some instances, these binding molecules comprise the six CDRs of an anti-CD3 antibody based on the Kabat definition. In some instances, these binding molecules comprise the six CDRs of an anti-CD3 antibody based on the Chothia definition. In some instances, these binding molecules comprise the six CDRs of an anti-CD3 antibody based on the enhanced Chothia definition. In some instances, these binding molecules comprise the six CDRs of an anti-CD3 antibody based on the contact definition. In some instances, these binding molecules comprise the six CDRs of an anti-CD3 antibody based on the IMGT definition.

[0561] In some cases, the VH and VL CDRs in Table II can include any of the amino acid substitutions of any one or more of SEQ ID NOs.: 54 or 72 to 83 listed in Table 1 of US 2017 / 0355767, which is incorporated by reference herein.

[0562] This disclosure also relates to binding polypeptides (e.g., Fab) that bind CD3 (e.g., human and / or cyno CD3) comprising the VH and VL of a humanized SP34 described herein. In some cases, these binding polypeptides bind CD3E (e.g., human and / or cyno CD3E). Table III provides the amino acid sequences of an exemplary humanized VH and VL of humanized SP34.

[0563] Table III; Humanized SP34 VH and VL Amino Acid Sequences _

[0564] SEQ VH or VL Amino acid sequence

[0565] ID NO: sequence

[0566] 26 SP34-VH- EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQ hl APGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKS ILYLQMNNLRAEDTAVYYCARHGNFGNSYVSWFAYWGQ GTLVTVSA

[0567]

[0568] Attorney Docket No.: 45817-0187WO1

[0569] 27 SP34-VL- QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQ hl QKPGQAFRGL1GGTNKRAPGVPARFSGSLIGDKAALT1TGA

[0570] QPEDEAEYFCALWYSNLWVFGGGTKLTVL

[0571]

[0572] In some cases, the VH comprises an amino acid sequence that in association with a VL binds human CD3E and is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence set forth in SEQ ID NO:26. In some cases, the VH comprises an amino acid sequence set forth in SEQ ID NO:26, except for 0, 1, 2. 3, 4, 5. 6, 7, 8, 9, 10. 11. 12. 13. 14, or 15 amino acid substitutions, wherein the VH in association with a VL binds human CD3E. In some cases, the VL comprises an amino acid sequence that in association with a VH binds human CD3E and is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence set forth in SEQ ID NO:27. In some cases, the VL comprises an amino acid sequence set forth in SEQ ID NOs:27, except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, wherein the VL in association with a VH binds human CD3E. In some instances, the variability in the VH and VL, when present, is in the framework regions and not in the CDRs. In some cases, the binding molecule comprises an anti-CD3E binding VH and VL described in WO 2007 / 042261, US 2017 / 0355767, US 10407501, US 9587021. or US 8236308 In other cases, the VH and VL are not any one of the anti-CD3E binding VH or VL described in WO 2007 / 042261, US 2017 / 0355767, US 10407501, US 9587021, or US 8236308. For example, in some instances, this disclosure does not encompass binding molecules comprising the VHs set forth in SEQ ID NOs: 6-9 or the VLs set forth in SEQ ID NOs: 10-12 of US 2017 / 0355767. In one instance, the binding polypeptide that binds CD3 (e.g., human CD3E) in the T cell engager 1 is a Fab and comprises a VH comprising the sequence set forth in SEQ ID NO:26 and a VL comprising the sequence set forth in SEQ ID NO: 27.

[0573] In some instances, the C-terminal of the VH is linked directly or via a linker (e.g., peptide, chemical) to a CHI of a human immunoglobulin (e.g., IgGl, IgG2, IgG3. IgG4). In some cases, the CHI comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence: Attorney Docket No.: 45817-0187WO1

[0574] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:28).

[0575] In some cases, the CHI comprises an amino acid sequence that differs from SEQ ID NO:28 at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions a result of amino acid substitutions and / or deletions.

[0576] In some cases, the C-terminal of the CHI is linked to an upper hinge region of a human immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4). In some cases, the upper hinge region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence: EPKSCDKTHT (SEQ ID NO:29). In some cases, the upper hinge comprises an amino acid sequence that differs from SEQ ID NO:29 at 0, 1, 2, 3, or 4 positions a result of amino acid substitutions and / or deletions.

[0577] In some instances, the VL is linked directly or via a linker (e.g., peptide, chemical) to a CL of a human immunoglobulin (human lambda or human kappa). In some cases, the CL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence:

[0578] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE

[0579] C (SEQ ID NQ:30).

[0580] In some cases, the CL comprises an amino acid sequence that differs from SEQ ID NO:30 at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions a result of amino acid substitutions and / or deletions. In some cases, the CL comprises an S or SG at the C-terminal of SEQ ID NO: 30

[0581] Non-limiting examples of CD3E binding polypeptides are provided in Table IV below.

[0582] Table IV; Humanized Anti-CD3 Fab sequences _

[0583] SEQ VH: CH1: Amino acid sequence

[0584] ID NO: Upper

[0585] Hinge or

[0586]

[0587] Attorney Docket No.: 45817-0187WO1

[0588] VL: CL

[0589] sequence

[0590] 31 ModSP34- EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVR VHCHl-hl QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDS KSILYLQMNNLRAEDTAVYYCARHGNFGNSYVSWFAYW GQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VP S S SLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHT

[0591] 32 ModSP34- QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQ VLClam-hl QKPGQAFRGLIGGTNKRAPGVPARFSGSLIGDKAALTITG AQPEDEAEYFCALWYSNLWVFGGGTKLTVLGQPKAAPS VTLFPP S SEELQ ANKATLVCLISDFYPGAVTV AWKADS SP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTEC

[0592]

[0593] In some cases, the VH: CHl:upper hinge construct in association with a VL binds human CD3E and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence set forth in SEQ ID NO:31. In some cases, the VH: CHl:upper hinge construct comprises an amino acid sequence set forth in SEQ ID NO:31, except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, wherein the VH: CHI: upper hinge construct in association with a VL binds human CD3E. In some cases, the VL: CL construct in association with a VH binds human CD3E and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence set forth in SEQ ID NO:32. In some cases, the VL: CL construct comprises an amino acid sequence set forth in SEQ ID NO:32, except for 0, 1.2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15. 16. 17. 18, 19, or 20 amino acid substitutions, wherein the VL: CL construct in association with a VH binds human CD3E. The variability in the VH: CH1: upper hinge and VL when present is generally in the framework regions, constant region and / or hinge region and not in the CDRs. In some instances, the VH and VL of these constructs are one of the anti-CD3E binding Attorney Docket No.: 45817-0187WO1

[0594] VH or VL described in WO 2007 / 042261 or US 2017 / 0355767. In other instances, the VH and VL of these constructs are not any one of the anti-CD3E binding VH or VL described in WO 2007 / 042261 or US 2017 / 0355767.

[0595] In one instance, the binding polypeptide that binds CD3 (e.g., human CD3E) in the T cell engager 1 is a Fab and comprises the sequences set forth in SEQ ID NO:31 and SEQ ID NO:32.

[0596] Anti-4-lBB Binding Moiety (Signal 2 TCE)

[0597] 4-1BB is a costimulatory glycoprotein receptor that is part of the tumor necrosis factor superfamily (TNFRSF) and was initially identified as a receptor protein expressed on activated cytotoxic and helper T lymphocytes. The 4- IBB protein is an inducible cell surface receptor that is expressed in the presence of activating stimuli and functions in cell signaling during T cell activation and proliferation. The endogenous ligand for 4-1BB is 4-1BBL (CD137L / TNFSF9). Interaction of the receptor with its ligand results in thymocyte and splenic T cell proliferation, thereby playing a role in the body’s immune response. Antibodies directed against 4-1BB are also capable of providing costimulatory signals to the T cell, reproducing the agonism provided by the endogenous 4-1 BBL.

[0598] The amino acid sequence of human 4-1BB (Uniprot: Q07011) is provided below:

[0599] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFS SAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDC KQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVV CGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVV KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:34) The amino acid sequence of cynomolgus 4- IBB (Uniprot: A9YYE7) is provided below:

[0600] MGNSCYNIVATLLLVLNFERTRSLQDLCSNCPAGTFCDNNRSQICSPCPPNSFS SAGGQRTCDICRQCKGVFKTRKECSSTSNAECDCISGYHCLGAECSMCEQDC KQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVV CGPSPADLSPGASSATPPAPAREPGHSPQIIFFLALTSTVVLFLLFFLVLRFSVV KRSRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:35) Attorney Docket No.: 45817-0187WO1

[0601] The amino acid sequence of mouse 4-1BB (Uniprot: P20334) is provided

[0602] below:

[0603] MGNNCYNVVVIVLLLVGCEKVGAVQNSCDNCQPGTFCRKYNPVCKSCPPST FSSIGGQPNCNICRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCTRCEKDC RPGQELTKQGCKTCSLGTFNDQNGTGVCRPWTNCSLDGRSVLKTGTTEKDV VCGPPVVSFSPSTTISVTPEGGPGGHSLQVLTLFLALTSALLLALIFITLLFSVLK WIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQEEEGGGGGYEL (SEQ ID NO:36)

[0604] The disclosure features a binding molecule or polypeptide comprising a VHH that specifically binds to 4- IBB (human or human and cyno 4- IBB). In some

[0605] instances, the VHH comprises an amino acid sequence that is 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 the

[0606] sequence set forth below:

[0607] QVQLVESGGGLVQPGGSLRLSCAASGFSRNDYAIGWFRQAPGKEREGVSCIG RGDGSTSYEESVEGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCATDRSHRC PIWEGYVEVWGQGTLVTVSS (SEQ ID NO:37)

[0608] In some cases, the disclosure features a binding molecule or polypeptide comprising a VHH that specifically binds to 4-1BB (human or human and cyno 4-IBB), wherein the VHH comprises the sequence of SEQ ID NO:37 except for 1, 2. 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some instances, the substitutions are in the framework regions. In some cases, the substitutions are conservative amino acid substitutions.

[0609] Also encompassed by this disclosure are binding molecules or polypeptides compnsing a VHH that specifically binds to 4- IBB (human or human and cyno 4-1BB), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 of the VHH of SEQ ID NO:37. The VHH CDRs of the VHH can be based on any CDR definition. In one case, a VHH that specifically binds to 4-1BB (human or human and cyno 4-1BB) comprises the three CDRs according to the IMGT definition provided below:

[0610] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0611]

[0612] Attorney Docket No.: 45817-0187WO1

[0613] VHHlh3 / B8h3 GFSRNDYA IGRGDGST ATDRSHRCPIWEGYVEV (SEQ ID NO:147) (SEQ ID NO 148) (SEQ ID NO: 149)

[0614]

[0615] In another case, a VHH that specifically binds to 4- IBB (human or human and cyno 4-1BB) comprises the three CDRs according to the Kabat definition provided below:

[0616] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH5 / B8h3 DYAIG CIGRGDGSTSYEESVEG DRSHRCPIWEGYVEV (SEQ ID NO: 150) (SEQ ID NO 151) (SEQ ID NO 152)

[0617]

[0618] In another case, a VHH that specifically binds to 4-1BB (human or human and cyno 4-1BB) comprises the three CDRs according to the Chothia definition provided below:

[0619] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH5 / B8h3 GFSRNDY GRGDGS DRSHRCPIWEGYVEV (SEQ ID NO 153) (SEQ ID NO 154) (SEQ ID NO 152)

[0620]

[0621] In yet another case, a VHH that specifically binds to 4- IBB (human or human and cyno 4-1BB) comprises the three CDRs according to the enhanced Chothia definition provided below:

[0622] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH5 / B8h3 GFSRNDYAIG CIGRGDGSTS DRSHRCPIWEGYVEV (SEQ ID NO 155) (SEQ ID NO 156) (SEQ ID NO 152)

[0623]

[0624] In a further case, a VHH that specifically binds to 4-1BB (human or human and cyno 4- IBB) comprises the three CDRs according to the Contact definition provided below:

[0625] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH5 / B8h3 NDYAIG GVSCIGRGDGSTS ATDRSHRCPIWEGYVE (SEQ ID NO 157) (SEQ ID NO: 158) (SEQ ID NO 178)

[0626]

[0627] Attorney Docket No.: 45817-0187WO1

[0628] In certain instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds 4-1BB (human or human and cyno 4-1BB), wherein the VHH comprises:

[0629] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 147, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 148, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 149;

[0630] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 150, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 151, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 152;

[0631] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 152;

[0632] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 155, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 156, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 152; or (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 157, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 158, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 178.

[0633] In certain instances, the N-terminal of the VHH that specifically binds to 4-1BB is linked via a linker (e.g., G4S (SEQ ID NO:4)) to the C-terminal of a hinge-CH2-CH3 domain of a human Ig (e.g., IgG). In some cases, the N-terminal of the hinge-CH2-CH3 domain of the human Ig is connected to the C-terminal of a VHH that specifically binds either human TROP2 or human MUC16. In some cases, the hinge-CH2-CH3 domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the hinge-Fc domain of human IgG4PAA (SEQ ID NO:33)

[0634] Exemplary Anti-TAA Binding Moieties Attorney Docket No.: 45817-0187WO1

[0635] The T cell engager molecules of this disclosure also comprise a binding moiety or binding moieties that specifically bind to a TAA. In some instances, the TAA is human MUC 16. In other instances, the TAA is human CLDN6. In yet other instances, the TAA is human TROP2. In some instances, the T cell engager comprises at least two binding moieties that specifically bind to a TAA. In some cases, the at least two binding moieties have the same amino acid sequence. In other instances, the at least two binding moieties have different amino acid sequences. In some cases, the at least two binding moieties that have different amino acid sequences bind to different epitopes of the same TAA. In certain cases, the binding moieties that specifically bind to a TAA described above are VHHs. In some cases, the C-terminal of the CHI of the VH / CH1 domain of the Fab that binds to CD3 is linked directly or via a linker (e.g., G4S (SEQ ID NO:4)) to the N-terminal of a VHH that specifically binds to a TAA. In certain cases, when there are two or more VHHs that bind to a TAA, the VHHs are linked via a linker (e.g.. (G4S)3 (SEQ ID NO:5)). In one instance, the Fab polypeptide comprising the VH / CH1 domain of a Fab that binds human CD3 is linked via the C-terminal of the CHI to a linker (e.g., SEQ ID NO:4), the C-terminal of which is linked to the N-terminal of a first VHH that specifically binds to a TAA and the C-terminal of the first VHH is linked via a linker (e.g., SEQ ID NO:5) to the N-terminal of a second VHH that specifically binds to a TAA. In some cases, the first and second VHH are identical. In some cases, the first and second VHH specifically bind to human MUC 16. In other cases, the first and second VHH specifically bind to human CLDN6. In other instances, the first and second VHH are not identical. In some cases, the first and second VHH bind the same TAA but bind different epitopes. In some instances, the VL / CL domain of the Fab that binds human CD3 is linked via the C-terminal of the CL to a linker (e.g., SEQ ID NO:4), the C-terminal of which is linked to the N-terminal of a first VHH that specifically binds to an agent for the purpose of half-life extension (e.g., HSA). In some cases, the C-terminal of the VHH that specifically binds to an agent for the purpose of half-life extension (e.g., HSA) is linked via a linker (e.g., SEQ ID NO:5) to the N-terminal of a VHH that binds to human 4-1 BB. Attorney Docket No.: 45817-0187WO1

[0636] MUC16

[0637] Mucin 16 (MUC16) is a type I transmembrane protein that includes a single membrane-spanning domain, a cytoplasmic tail and a highly glycosylated N-terminal domain consisting of tandem repeat sequences. The N-terminal region of MUC16 is composed of approximately 12,000 amino acids. The tandem repeat region is composed of about 60 repeats wherein each repeat is 156 amino acids in length. The tandem repeat regions are frequently rich in serine and threonine residues that are potential sites for O-linked glycosylation. Glycosylation at these sites provides clusters of carbohydrate moieties that can act as ligands for binding other molecules and are essential to MUC16 structure and function. The tandem repeat domain is interspersed with sea urchin sperm protein, enterokinase, and agrin (SEA) domains, 14-leucine-rich repeats, and two Ankyrin domains. Of the several SEA domains present in MUC16, the 5-SEA domain is the most membrane proximal region. The carboxy -terminal of MUC16 is divided into three major regions, the juxtamembrane domain, the transmembrane domain, and the cytoplasmic tail (CT). The CT domain is composed of 32 amino acids and contains one serine, two threonine, and three ty rosine residues that can be potential phosphory lation sites. Studies show that phosphory lation of the CT domain leads to cleavage of extracellular portion of MUC16. The MUC16 CT domains also contain a polybasic sequence of amino acids (RRRKK (SEQ ID NO: 254)) predicted to bind to the ezrin / radixin / moesin (ERM) family of proteins, and that can facilitate interaction of MUC16 with numerous membrane- associated proteins and with actin-cytoskeleton.

[0638] The aberrant expression of MUC16 within tumor cells is closely associated with oncogenesis, proliferation, and metastasis. This association involves various mechanisms, including cellular proliferation, viability, apoptosis resistance, chemotherapeutic resilience, metabolic shifts, and immune surveillance evasion, and appears to play an important role in promoting inflammatory signaling in cancer. MUC16 is expressed in a wide variety of cancers (e.g., epithelial cancers) such as, but not limited to, breast cancer, endometrial cancer, ovarian cancer, and pancreatic cancer. Attorney Docket No.: 45817-0187WO1

[0639] The amino acid sequence of the 5-SEA domain of human MUC16 (Uniprot: Q8WXI7) is provided below:

[0640] AASHLLrLFTLNFTlTNLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPL YSGCRLTLLRPEKDGEATGVDAICTHRPDPTGPGLDREQLYLELSQLTHSITEL GPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTINNLRYMADMGQPG SLKFNITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRVDLLCTY LQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGPDEPPTT PKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGV LQHLLRPLFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLD IQQLYWELSQLTHGVTQLGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNW NLSNPDPTSSEYITLLRDIQDKVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTV KALFSSNLDPSLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSS SSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFS DCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFLRMTRNGTQLQN FTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGVITCLICGVLVT TRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ (SEQ ID NO:38)

[0641] The amino acid sequence of the 5-SEA domain of cynomolgus MUC16 is provided below:

[0642] PGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGCRLTLLRPEKDGEATGVDVIC THRPDPTGPGLDREQLYLELSQLTHSITELGPYTLDNDSLFVNGFTHRSSVPTT STGMVSEEPFTLNFTINNLRYMADMGQPGSLKFNITDNVMQHLLSPLFQRSSV GARYTGCRVIALRSVKNGAKTQVDILCTYLQPLSGPGLPIKQVFHELSQQTHG ITRLGPYFLDKDSLYLNGYNEPGPDEPPTTPKPATTFLPPLSDATTAMGYHLK TLTLNFTISNIQYSPDMGNGSAAFNSTERVLKHLLRALFQKSSMGPFYLGCQLI SLRPEKNGAATGVDTTCTYHTDPVGPGLDIQQLYWELSQLTHGVTQLGFYVL DRDSLFINGYAPQNLSIQGKYQIHFHIVNWNLSNPDPTSSEYIALLRDIQDKVT TLYKGSRLHDTFCYCLVTNLTMDSMLVTVKALFSSDLDPSLVEQVFLDKTLN ASSHWLGSTYQLVDIHVTEMEPSVYQPTKPTSSSSTQHFYLNFTITNLPYSQDI SQPSTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNSHHTGVDSL CNFSPLARRVDRVAIYEEFLRMTRNGSQLQNFTLDRSSVLVDGYSPNRNEPLT GNSDLP (SEQ ID NO:39)

[0643] The disclosure features a binding molecule or polypeptide comprising a VHH that specifically binds to MUC16 (human or human and cyno MUC16). In some instances, the VHH specifically binds to the 5-SEA domain of human MUC16. In certain instances, the VHH specifically binds to the most membrane proximal region up to the 2nd SEA domain of human MUC16. In some instances, the VHH comprises an amino acid sequence that is 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 the sequence set forth below: Attorney Docket No.: 45817-0187WO1

[0644] QVQLVESGGGVVQPGGSLRLSCALSGRTIGGVTMGWFRQAPGKEREFVADIR WADTRTSYADPVKGRFTTSGDNAKNTVYLQMNSLRAEDTAVYFCAALWKG GSKDNYKYWGQGTLVTVSS (SEQ ID NO:40). In other instances, the VHH comprises an amino acid sequence that is 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 the sequence set forth below: QVQLVESGGGLVQPGGSLRLSCSASGSTFKVSVMTWYRQAPGKPRELVSSISS NGDGTAYADFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNFLHRVKS PDSWGQGTLVIVSS (SEQ ID NO:237)

[0645] In some cases, the disclosure features a binding molecule or polypeptide comprising a VHH that specifically binds to MUC16 (human or human and cyno MUC16), wherein the VHH comprises the sequence of SEQ ID NO:40 or 237 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some instances, the substitutions are in the framework regions. In some cases, the substitutions are conservative amino acid substitutions.

[0646] Also encompassed by this disclosure are binding molecules or polypeptides comprising a VHH that specifically binds to MUC16 (human or human and cyno MUC16), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 of the VHH of SEQ ID NO:40 or 237. The VHH CDRs of the VHH can be based on any CDR definition.

[0647] In one case, a VHH that specifically binds to MUC 16 (human or human and cyno MUC 16) comprises the three CDRs according to the IMGT definition provided below:

[0648] VHH-ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0649] GRTIGGVT IRWADTRT AALWKGGSKDNYKY VHH15h9 / 15h9 (SEQ ID NO: 179) (SEQ ID NO: 180) (SEQ ID NO: 181) GSTFKVSV ISSNGDGT NFLHRVKSPDS

[0650]

[0651] VHH24hl / 24hl (SEQ ID NO:238) (SEQ ID NO:239) (SEQ ID NO:240)

[0652] In another case, a VHH that specifically binds to MUC 16 (human or human and cyno MUC 16) comprises the three CDRs according to the Kabat definition provided below: Attorney Docket No.: 45817-0187WO1

[0653] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH15h9 / 15h9 GVTMG DIRWADTRTSYADPVKG LWKGGSKDNYKY (SEQ ID NO: 182) (SEQ ID NO: 183) (SEQ ID NO: 184) VHH24hl / 24hl VSVMT SISSNGDGTAYADFVKG LHRVKSPDS (SEQ ID NO:241) (SEQ ID NO: 242) (SEQ ID NO:243)

[0654]

[0655] In another case, a VHH that specifically binds to MUC16 (human or human and cyno MUC16) comprises the three CDRs according to the Chothia definition provided below:

[0656] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH15h9 / 15h9 GRTIGGV RWADTR LWKGGSKDNYKY (SEQ ID NO: 185) (SEQ ID NO: 186) (SEQ ID NO: 184) VHH24hl / 24hl GSTFKVS SSNGDG LHRVKSPDS

[0657] (SEQ ID NO: 244) (SEQ ID NO:245) (SEQ ID NO:243)

[0658]

[0659] In yet another case, a VHH that specifically binds to MUC16 (human or human and cyno MUC16) comprises the three CDRs according to the enhanced Chothia definition provided below:

[0660] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH15h9 / 15h9 GRTIGGVTMG DIRWADTRTS LWKGGSKDNYKY (SEQ ID NO: 187) (SEQ ID NO: 188) (SEQ ID NO: 184) VHH24hl / 24hl GSTFKVSVMT SISSNGDGTA LHRVKSPDS

[0661] (SEQ ID NO:246) (SEQ ID NO:247) (SEQ ID NO:243)

[0662]

[0663] In a further case, a VHH that specifically binds to MUC16 (human or human and cyno MUC16) comprises the three CDRs according to the Contact definition provided below:

[0664] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH15h9 / 15h9 GGVTMG FVADIRWADTRTS AALWKGGSKDNYK (SEQ ID NO: 189) (SEQ ID NO: 190) (SEQ ID NO: 191)

[0665]

[0666] Attorney Docket No.: 45817-0187WO1

[0667] VHH24hl / 24hl KVSVMT LVASISSNGDGTA NFLHRVKSPD (SEQ ID NO:248) (SEQ ID NO:249) (SEQ ID NO:250)

[0668]

[0669] In certain instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds MUC16 (human or human and cyno MUC16). wherein the VHH comprises:

[0670] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 179, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 180, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 181;

[0671] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 182, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 183, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 184;

[0672] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 185, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 186. and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 184;

[0673] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 187, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 188, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 184;

[0674] (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191;

[0675] (1) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:238, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:239, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:240;

[0676] (g) VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:241, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:242, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:243; Attorney Docket No.: 45817-0187WO1

[0677] (h) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 244, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:245, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:243;

[0678] (i) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 246, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:247, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:243; or (j) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:248, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:249, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:250.

[0679] In some instances, the VHH specifically binds to the 5-SEA domain of human MUC16. In certain instances, the VHH specifically binds to the most membrane proximal region up to the 2nd SEA domain of human MUC16.

[0680] Claudin 6 (CLDN6)

[0681] Claudin 6 (CLDN6) is one of the many members of the CLDN family. It has four transmembrane domains and a PDZ -binding region at the carboxyl end of the cytoplasm. CLDN6 is the only CLDN to potentially show specificity, as it can activate cell adhesion signals and regulate the activity of nuclear receptors. CLDN6 is expressed in a variety of embryonic epithelia, induces epithelial cell junction formation and polari ty, and participates in the differentiation of stem cells into epithelial cells. It serves a major role in maintaining the function of Tight Junctions (TJs). Changes in CLDN expression cause TJ disorders and leakage. The decrease in cell polarity increases the supply of nutrition and growth factors for tumor cells. Furthermore, the decrease in intercellular adhesion may increase metastatic potential of tumor cells. CLDN6 is expressed in several tumor types such as breast cancer, ovarian cancer, endometrial cancer, testicular cancer, gastric cancer, liver cancer, cervical cancer, lung cancer, esophageal cancer, and atypical teratoid / rhabdoid tumors.

[0682] The amino acid sequence of human Claudin 6 (Uniprot: P56747) is provided below: Attorney Docket No.: 45817-0187WO1

[0683] MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIVVAQVVWEGL WMSCVVQSTGQMQCKVYDSLLALPQDLQAARAECVIALLVALFGLLVYLAG AKCTrCVEEKDSKARLVLTSGIVFVISGVLTLIPVCWTAHAIIRDFYNPLVAEA QKRELGASLYLGWAASGLLLLGGGLLCCTCPSGGSQGPSHYMARYSTSAPAI SRGPSEYPTKNYV (SEQ ID NO:41)

[0684] The amino acid sequence of cynomolgus CLDN6 (Uniprot: F7AWZ8) is provided below:

[0685] MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIVVAQVVWEGL WMSCVVQSTGQMQCKVYDSLLALPQDLQAARALCVIALLVALFGLLVYLAG AKCTTCVEEKDSKARLVLTSGIVFVISGVLTLIPVCWTAHAIIRDFYNPLVAEA QKRELGASLYLGWAASGLLLLGGGLLCCTCPSGGSRGPSHYMARYSTSAPAI SRGPSEYPTKNYV (SEQ ID NO:42)

[0686] The disclosure features a binding molecule or polypeptide comprising a VHH that specifically binds to CLDN6 (human or human and cyno CLDN6). In some cases, the VHH does not bind to closely related family CLDN members (e.g., CLDN3, CLDN4, CLDN9). In one case, the VHH does not bind to CLDN9. In some instances, the VHH comprises an amino acid sequence that is 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 the sequence set forth below:

[0687] QVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMAWFRQAPGKEREFVAGTS WSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADTIWTT GWSSAANFGSWGQGTQVTVSS (SEQ ID NO:43)

[0688] In some cases, the disclosure features a binding molecule or polypeptide comprising a VHH that specifically binds to CLDN6 (human or human and cyno CLDN6), wherein the VHH comprises the sequence of SEQ ID NO:43 except for 1, 2. 3, 4, 5. 6, 7, 8. 9, or 10 amino acid substitutions. In some instances, the substitutions are in the framework regions. In some cases, the substitutions are conservative amino acid substitutions.

[0689] Also encompassed by this disclosure are binding molecules or polypeptides comprising a VHH that specifically binds to CLDN6 (human or human and cyno CLDN6), wherein the VHH compnses the VHH CDR1, VHH CDR2, and VHH CDR3 of the VHH of SEQ ID NO: 43. The VHH CDRs of the VHH can be based on Attorney Docket No.: 45817-0187WO1

[0690] any CDR definition. In one case, a VHH that specifically binds to CLDN6 (human or human and cyno CLDN6) comprises the three CDRs according to the IMGT

[0691] definition provided below:

[0692] VHH-ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0693] VHH22h5 / GRTFSRSA TSWSGSST AADTIWTTGWSSAANFGS

[0694]

[0695] CSV51h5 (SEQ ID NO:192) (SEQ ID NO: 193) (SEQ ID NO: 194)

[0696] In another case, a VHH that specifically binds to CLDN6 (human or human and cyno CLDN6) comprises the three CDRs according to the Kabat definition provided below:

[0697] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH22h5 / RS AMA GTSWSGSSTYYADSVKG DTIWTTGWSSAANFGS CSV51h5 (SEQ ID NO:195) (SEQ ID NO: 196) (SEQ ID NO:197)

[0698]

[0699] In another case, a VHH that specifically binds to CLDN6 (human or human and cyno CLDN6) comprises the three CDRs according to the Chothia definition provided below:

[0700] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH22h5 / GRTFSRS SWSGSS DTIWTTGWS S AANFGS CSV51h5 (SEQ ID NO: 198) (SEQ ID NO: 199) (SEQ ID NO:197)

[0701]

[0702] In yet another case, a VHH that specifically binds to CLDN6 (human or

[0703] human and cyno CLDN6) comprises the three CDRs according to the enhanced Chothia definition provided below:

[0704] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH22h5 / GRTFSRSAMA GTSWSGSSTY DTIWTTGWS S AANFGS CSV51h5 (SEQ ID NO:200) (SEQ ID NO:201) (SEQ ID NO:197)

[0705]

[0706] In a further case, a VHH that specifically binds to CLDN6 (human or human and cyno CLDN6) comprises the three CDRs according to the Contact definition provided below: Attorney Docket No.: 45817-0187WO1

[0707] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH22h5 / SRSAMA FVAGTSWSGSSTY AADTIWTTGWSSAANFG CSV51h5 (SEQ ID NO:202) (SEQ ID NO:203) (SEQ ID NO:204)

[0708]

[0709] In certain instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds CLDN6 (human or human and cyno CLDN6), wherein the VHH comprises:

[0710] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 192, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 193. and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 194;

[0711] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197;

[0712] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 199, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197;

[0713] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 200, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:201, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; or

[0714] (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:202, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:204.

[0715] TROP2

[0716] The Trophoblast cell surface antigen 2 (Trop2) protein is 323 ammo acids in length and includes a signal peptide (SP) (ammo acids 1-26), an extracellular domain (ECD) (aa 27-274). a single transmembrane helix (TMD) (aa 275-297) and a cytoplasmic tail (ICD) (aa 298-323). The Trop2-ECD (H27-T274) is composed of three subdomains: a cysteine-rich domain (CRD), a thyroglobulin type-1 domain (TY), and a cysteine-poor domain (CPD). The ECD of Trop2 undergoes post- Attorney Docket No.: 45817-0187WO1

[0717] translational modification at four N-glycosylation sites: N33, N120, N168, and N208. The ECD of Trop2 shares 47.6% sequence identity with the ECD of epithelial cell adhesion molecule (EpCAM).

[0718] The amino acid sequence of human TROP2 (Uniprot: P09758) is provided below:

[0719] MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGR CQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGL YDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHI LIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQ NTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIY YLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEI KELGELRKEPSL (SEQ ID NO:44)

[0720] The amino acid sequence of cynomolgus TROP2 (NCBI Reference Sequence: XP 001114599.1) is provided below:

[0721] MARGPGLAPPPLRLPLLLLLLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGR CQCRALGSGVAVDCSTLTSKCLLLKARMSAPKNARTLVRPNEHALVDNDGL YDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHI LIDLRHRPTASAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQN TSQKAAGDVDIGDAAYYFERDVKGESLFQGRGGLDLRVRGEPLQVERTLIYY LDEIPPKFSMKRLTAGLIAVIVVVVVALVAGVAVLVISNRRKSGKYKKVEIKE LGELRKEPSL (SEQ ID NO:205)

[0722] Trop2 exhibits limited expression in normal tissues but is over-expressed across various solid tumors including breast cancer, non-small-cell lung cancer (NSCLC), oral squamous cell carcinoma (OSCC), salivary gland carcinomas (SGC), thyroid cancer, gastric cancer, pancreatic cancer, gallbladder cancer, colorectal cancer, prostate cancer, ovarian cancer, cervical cancer, and urothelial cancer.

[0723] Overexpression of Trop2 is correlated with tumor invasion, metastasis, and poor prognosis.

[0724] The disclosure features a binding molecule or polypeptide comprising a VHH that specifically binds to TROP2 (human or human and cyno TROP2). In some instances, the VHH comprises an amino acid sequence that is 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 the sequence set forth below: Attorney Docket No.: 45817-0187WO1

[0725] QVQLVESGGGLVQPGGSLRLSCAASGRIWSQYNIAWIRQAPGKEREFVALIN HNGGVTRYGDSVKGRFTISRDNSKNTVYLQMNSLRAEDTAIYYCAAGRLWT SGVDDGYDYWGQGTQVTVSS (SEQ ID NO:206)

[0726] In some cases, the disclosure features a binding molecule or polypeptide comprising a VHH that specifically binds to TROP2 (human or human and cyno TROP2), wherein the VHH comprises the sequence of SEQ ID NO:206 except for 1.

[0727] 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some instances, the

[0728] substitutions are in the framework regions. In some cases, the substitutions are conservative amino acid substitutions.

[0729] Also encompassed by this disclosure are binding molecules or polypeptides compnsing a VHH that specifically binds to TROP2 (human or human and cyno TROP2), wherein the VHH compnses the VHH CDR1, VHH CDR2, and VHH

[0730] CDR3 of the VHH of SEQ ID NO:206. The VHH CDRs of the VHH can be based on any CDR definition. In one case, a VHH that specifically binds to TROP2 (human or human and cyno TROP2) comprises the three CDRs according to the IMGT definition provided below:

[0731] VHH-ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0732] GRIWSQYN VHH7h7 / LTT51h7 INHNGGVT AAGRLWTSGVDDGYDY (SEQ ID NO:209)

[0733]

[0734] (SEQ ID NO:210) (SEQ ID NO:211)

[0735] In another case, a VHH that specifically binds to TROP2 (human or human and cyno TROP2) comprises the three CDRs according to the Kabat definition

[0736] provided below:

[0737] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH7h7 / QYNIA LINHNGGVTRYGDSVKG GRLWTSGVDDGYDY LTT51h7 (SEQ ID NO:212) (SEQ ID NO:213) (SEQ ID NO:214)

[0738]

[0739] In another case, a VHH that specifically binds to TROP2 (human or human and cyno TROP2) comprises the three CDRs according to the Chothia definition provided below: Attorney Docket No.: 45817-0187WO1

[0740] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH7h7 / GRIWSQY NHNGGV GRLWTSGVDDGYDY LTT51h7 (SEQ ID NO:215) (SEQ ID NO:216) (SEQ ID NO:214)

[0741] In yet anot ler case, a VHH that specifically binds to TROP2 (human or human and cyno TROP2) comprises the three CDRs according to the enhanced Chothia definition provide! I below:

[0742] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH7h7 / GRIWSQYNIA LINHNGGVTR GRLWTSGVDDGYDY LTT51h7 (SEQ ID NO:217) (SEQ ID NO:218) (SEQ ID NO:214)

[0743]

[0744] In a further case, a VHH that specifically binds to TR0P2 (human or human and cyno TR0P2) comprises the three CDRs according to the Contact definition provided below:

[0745] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH7h7 / SQYNIA FVALINHNGGVTR AAGRLWTSGVDDGYD LTT51h7 (SEQ ID NO:219) (SEQ ID NO:220) (SEQ ID NO: 221)

[0746]

[0747] In certain instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds TROP2 (human or human and cyno TROP2), wherein the VHH comprises:

[0748] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:209, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:210, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:211;

[0749] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:212, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:213, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:214;

[0750] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:215, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:216, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:214; Attorney Docket No.: 45817-0187WO1

[0751] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:217, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:218. and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:214; or (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:219, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:220, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:221.

[0752] Anti-HSA Binding Moiety

[0753] The Signal 1 T cell engagers (TCEls) of this disclosure also comprise a binding moiety that specifically binds to human serum albumin. Such binding moieties are useful to extend the half-life of the T cell engager. In certain instances, the binding moiety that specifically binds to human serum albumin is linked via a linker (e.g., SEQ ID NO:4) to the VL / CL domain of the Fab that specifically binds to human CD3 (e.g., human CD3E). In one instance, the C-terminal of the CL is linked to linker (e.g., SEQ ID NO:4), the C-terminal of which is linked to the N-terminal of the binding moiety that specifically binds to human serum albumin. In certain cases, the binding moiety that specifically binds to human serum albumin is a VHH.

[0754] The amino acid sequence of HSA is provided below:

[0755] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQ YLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLR ETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEE TFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDE LRDEGKASSAKQGLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLV TDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVGSKDVCKNYAEAKDVFLGMFLYEYARRHP DYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQN CELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDCLSVFLNQLCVLHEKTPVSDRVTKCCTESLVNGRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO:207) Attorney Docket No.: 45817-0187WO1

[0756] In some instances, the binding moiety that specifically binds to HSA is a

[0757] VHH. The Table below provides the three VHH CDR sequences of a non-limiting example of an anti-HSA VHH (VHSA1) based on common CDR definitions known in the art.

[0758] Table V: CDR Sequences of a VHH that Specifically Binds to HSA (VHSA1)

[0759] Kabat Chothia Enhanced Chothia Contact IMGT VHHCDR1 GFTFSSF SSFGMS GFTFSSFG SFGMS (SEQ ID GFTFSSFGMS (SEQ ID (SEQ ID (SEQ ID NO: 159 ) NO: 162) (SEQ ID NO: 164) NO: 166) NO: 169) VHHCDR2 WVSSI SGSGSD SISGSGSDTLYA SGSGSD LL ISGSGSDT DSVKG (SEQ ID SISGSGSDTL (SEQ ID (SEQ ID (SEQ ID NO: 160) NO: 163) (SEQ ID NO: 165) NO: 167) NO: 170) VHHCDR3 GGSLSR TIGGSLS TIGGSLSR GGSLSR (SEQ ID GGSLSR (SEQ ID (SEQ ID (SEQ ID NO: 161) NO: 161) (SEQ ID NO: 161) NO: 168) NO: 171)

[0760]

[0761] This disclosure relates in part to a binding molecule that specifically binds to HSA comprising the three VHH CDRs of an anti-HSA antibody described herein. In some instances, these binding molecules comprise the three VHH CDRs of an anti-HSA antibody based on the Kabat definition. In some instances, these binding molecules comprise the three VHH CDRs of an anti-HSA antibody based on the Chothia definition. In some instances, these binding molecules comprise the three

[0762] VHH CDRs of an anti-HSA antibody based on the enhanced Chothia definition. In some instances, these binding molecules comprise the three VHH CDRs of an anti-HSA antibody based on the contact definition. In some instances, these binding molecules comprise the three VHH CDRs of an anti-HSA antibody based on the

[0763] IMGT definition. In some cases, the T cell engager comprises a VHH that binds to HSA which has three CDRs based on any one CDR definition set out in Table V.

[0764] In one instance, the HSA-binding VHH comprises the amino acid sequence provided below (the CDRs based on IMGT definition are underlined): Attorney Docket No.: 45817-0187WO1

[0765] EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS GSGSDTLYADSVKGRFTISRDNAKTTLYLOMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSS (SEQ ID NO:208)

[0766] In some cases, the VHH specifically binds to HSA and comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:208. In some cases, VHH specifically binds to HSA and comprises an amino acid sequence set forth in SEQ ID NO:208, except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. In some instances, the variability in the VHH when present is in the framework regions and not in the CDRs.

[0767] In some cases, the Signal 1 T cell engagers (TCEls) comprise a VHH that binds to HSA. The VHH of such a T cell engager comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%. at least 95%, or 100% identical to the sequence set forth in SEQ ID NO:208. The C-terminal of the CL of the Fab is linked to the N-terminal of a linker (e.g., SEQ ID NO:4). In some cases, the C-terminal of the linker (e.g., SEQ ID NO:4) is connected to the N-terminal of the VHH. In certain cases, the VHH is 100% identical to SEQ ID NO:208. In some cases, the C-terminal of the VHH that binds to HSA is linked via a linker (e.g., SEQ ID NO:5) to the N-terminal of a VHH that binds to human 4-1BB. In certain cases, the VHH that binds to human 4-1BB comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence set forth in SEQ ID NO:37. In some cases, the C-terminal of the linker (e.g., SEQ ID NO:5) is connected to the N-terminal of the VHH that binds to human 4-1BB. In certain cases, the VHH is 100% identical to SEQ ID NO: 37.

[0768] Linkers

[0769] The disclosed T cell engagers can contain a linker or linkers, which connect two domains of the T cell engager. In some embodiments, a linker or linkers connect the different binding domains of a T cell engager. In some embodiments, a linker connects a binding domain such as a Fab with a VHH. In some embodiments, a linker Attorney Docket No.: 45817-0187WO1

[0770] connects the C-terminal of a Fab with the N-terminal of a VHH. In some embodiments, a linker connects the C-terminal of a CHI domain of a Fab with the N-terminal of a VHH. In other embodiments, a linker connects the C-terminal of a CL domain of a Fab with the N-terminal of a VHH. In some embodiments, a T cell engager may comprise two separate linkers, each of which connect different domains of the T cell engager. For example, a first linker connects a Fab and a VHH and a second linker connects the VHHs (e.g., when there are more than one VHH).

[0771] The linkers used in the disclosed T cell engagers can be encoded in vivo via expression of an mRNA. The linker may be flexible. The linker may comprise 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17. 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39. 40. 41. 42. 43. 44. 45, 46, 47, 48, 49, or 50 or more amino acids. A linker may comprise glycine (G), serine (S), or various repeats or combinations thereof.

[0772] Suitable linkers can include, but are not limited to GGGGS (SEQ ID NO: 4); i.e.. a -G4S linker”), GSGG (SEQ ID NO:45), GSGGSG (SEQ ID NO:46), GSGGGG (SEQ ID NO:47), GSG, GSGGSGGSGGSGGG (SEQ ID NO:48), GGGSGGGSGG (SEQ ID NO:49), GGGG (SEQ ID NO:50), GGG, SGG, GGGSGGSG (SEQ ID NO:51), GGSGGSGGGS (SEQ ID NO:52), and GGGGGS (SEQ ID NO:53). In some embodiments, the linker comprises GGGGS (SEQ ID NO:4). In other embodiments, the linker comprises (GGGGS)3 (SEQ ID NO:5). In some cases, the linker comprises (GGGGS )n, wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO:54)

[0773] Exemplary T Cell Engager Polypeptides

[0774] The disclosure features several non-limiting examples of T cell engager polypeptides.

[0775] One of the TCE integrated products of this disclosure comprises the Signal 1 and Signal 2 TCEs described below. In one case, the Signal 1 TCEs comprise the polypeptides of SEQ ID NO:55 and SEQ ID NO:57. In another case, the Signal 1 TCEs comprise the polypeptides of SEQ ID NO:55 and SEQ ID NO:57 (which associate together) as well as the polypeptides of SEQ ID NO: 56 and SEQ ID NO: 57 Attorney Docket No.: 45817-0187WO1

[0776] (which associate together). The Signal 2 TCE comprises the polypeptides of SEQ ID NO:58 (which associates with itself).

[0777] Signal 1 TCEs

[0778] Below is the amino acid sequence of a VH / CH1 domain of a Fab that binds human CD3 (e.g. human CD3E) and which includes two VHHs that bind human MUC16 (the linkers are underlined).

[0779] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNNLRAEDTAVYYCARHG NFGNSYVSWFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTGGGGSQVQLVESGGGVVQPGGSLRLS CALSGRTIGGVTMGWFRQAPGKEREFVADIRWADTRTSYADPVKGRFTTSG DNAKNTVYLQMNSLRAEDTAVYFCAALWKGGSKDNYKYWGQGTLVTVSS GGGGSGGGGSGGGGSQVQLVESGGGVVQPGGSLRLSCALSGRTIGGVTMGW FRQAPGKEREFVADIRWADTRTSYADPVKGRFTTSGDNAKNTVYLQMNSLR AEDTAVYFCAALWKGGSKDNYKYWGQGTLVTVSS (SEQ ID NO:55)

[0780] Below is the amino acid sequence of a VH / CH1 domain of a Fab that binds human CD3 (e.g. human CD3E) and that includes two VHHs that bind human CLDN6 (the linkers are underlined).

[0781] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNNLRAEDTAVYYCARHG NFGNSYVSWFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTGGGGSQVQLVESGGGLVQPGGSLRLS CAASGRTFSRSAMAWFRQAPGKEREFVAGTSWSGSSTYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCAADTIWTTGWSSAANFGSWGQGTQVTV SSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAM Attorney Docket No.: 45817-0187WO1

[0782] AWFRQAPGKEREFVAGTSWSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCAADTIWTTGWSSAANFGSWGQGTQVTVSS (SEQ ID NO:56)

[0783] Common Light Chain

[0784] Below is the amino acid sequence of a VL / CL domain of a Fab that binds human CD3 (e.g. human CD3E) and includes a VHH that binds to HSA (the linker is underlined).

[0785] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAFRGLIGG TNKRAPGVPARFSGSLIGDKAALTITGAQPEDEAEYFCALWYSNLWVFGGGT KLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPG KGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY YCTIGGSLSRSSQGTLVTVSS (SEQ ID NO:57)

[0786] Signal 2 TCE

[0787] Below is the amino acid sequence of a VHH that binds to human TROP2 linked via its C-terminal to a silent hinge-Fc domain, which in turn is linked via its C-terminal to the N-terminal of a VHH that binds to human 4-1BB (the immunologically silent human Ig hinge-Fc domain is underlined):

[0788] QVQLVESGGGLVQPGGSLRLSCAASGRIWSQYNIAWIRQAPGKEREFVALIN HNGGVTRYGDSVKGRFTISRDNSKNTVYLQMNSLRAEDTAIYYCAAGRLWT SGVDDGYDYWGQGTQVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSOEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGOPREPQVYTLPPS OEEMTKNOVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSOVQLV ESGGGLVQPGGSLRLSCAASGFSRNDYAIGWFRQAPGKEREGVSCIGRGDGS Attorney Docket No.: 45817-0187WO1

[0789] TSYEESVEGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCATDRSHRCPIWEG YVEVWGQGTLVTVSS (SEQ ID NO: 58)

[0790] Alternative Design 1

[0791] The alternative design comprises the same Signal 1 molecules as described above, but a different Signal 2 TCE. The TCE integrated products of this alternative design comprises the Signal 1 and Signal 2 TCEs described below. In one case, the Signal 1 TCEs comprise the polypeptides of SEQ ID NO:55 and SEQ ID NO:57. In another case, the Signal 1 TCEs comprise the polypeptides of SEQ ID NO:55 and SEQ ID NO:57 (which associate together) as well as the polypeptides of SEQ ID NO:56 and SEQ ID NO:57 (which associate together). The Signal 2 TCE comprises the polypeptides of SEQ ID NO:59 (which associates with itself).

[0792] Alternative Signal 2 TCE (A Cis Signaling Option)

[0793] Below is the amino acid sequence of a VHH that binds to human MUC16 linked via its C-terminal to a silent hinge-Fc domain, which in turn is linked via its C-terminal to the N-terminal of a VHH that binds to human 4- IBB (the immunologically silent human Ig hinge-Fc domain is underlined):

[0794] QVQLVESGGGLVQPGGSLRLSCSASGSTFKVSVMTWYRQAPGKPRELVSSISS NGDGTAYADFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNFLHRVKS PDSWGOGTLVIVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSOEDPEVOFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HODWLNGKEYKCKVSNKGLPSSIEKTISKAKGOPREPQVYTLPPSQEEMTKN OVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWOEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSOVOLVESGGGLVQ PGGSLRLSCAASGFSRNDYAIGWFRQAPGKEREGVSCIGRGDGSTSYEESVEG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCATDRSHRCPIWEGYVEVWGQG TLVTVSS (SEQ ID NO:59) Attorney Docket No.: 45817-0187WO1

[0795] Another exemplary amino acid sequence of an alternative Signal 2 TCE is provided below:

[0796] QVQLVESGGGLVQAGGSLRLSCLASGSTFKVSVMTWYRQAPGKPRDLVASIS SNGDGTAYADFVKGRFTISRDNAKNTVYLQMDSLRPEDTAVYYCNFLHRVK SPDSWGQGTQVIVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSOEDPEVOFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHODWLNGKEYKCKVSNKGLPSSIEKTISKAKGOPREPOVYTLPPSOEEMTK NOVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWOEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSOVOLVESGGGLV QPGGSLRLSCAASGFSRNDYAIGWFRQAPGKEREGVSCIGRGDGSTSYEESVE GRFTISRDNAKNTVYLQMNSLRAEDTAVYYCATDRSHRCPIWEGYVEVWGQ GTLVTVSS (SEQ ID NO:251)

[0797] T

[0798]

[0799] rispecific Signal 1 2 (Another Cis Signaling Option)

[0800] The cis signaling option comprises the polypeptides of SEQ ID NO:55 and SEQ ID NO:222 (which associate together). In another case, this option further comprises the polypeptides of SEQ ID NO:56 and SEQ ID NO:222 (which associate together)

[0801] Below is the amino acid sequence of a VL / CL domain of a Fab that binds human CD3 (e.g. human CD3E) and includes a VHH that binds to HSA linked to a VHH that binds to 4-1BB (e.g., human 4-1BB).

[0802] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAFRGLIGG TNKRAPGVPARFSGSLIGDKAALTITGAQPEDEAEYFCALWYSNLWVFGGGT KLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPG KGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY YCTIGGSLSRSSQGTLVTVSSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGF SRNDYAIGWFRQAPGKEREGVSCIGRGDGSTSYEESVEGRFTISRDNAKNTVY LQMNSLRAEDTAVYYCATDRSHRCPIWEGYVEVWGQGTLVTVSS (SEQ ID NO:222) Attorney Docket No.: 45817-0187WO1

[0803] The disclosure encompasses T cell engager polypeptides that provide T cell Signal 1, which specifically bind to CD3 (e.g.. human CD3E), MUC16 (e.g., human MUC16), and HSA, and that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequences set forth in SEQ ID NOs: 55 and 57, respectively. In one instance the Signal 1 T cell engager component comprises the sequences set forth in SEQ ID NOs: 55 and 57, respectively. Nucleic acid molecules (e.g., mRNAs) encoding these Signal 1 T cell engager molecules are also encompassed by this disclosure.

[0804] The disclosure also encompasses T cell engager polypeptides that provide T cell Signal I, which specifically bind to CD3 (e.g., human CD3E), CLDN6 (e.g., human CLDN6), and HSA, and that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequences set forth in SEQ ID NOs: 56 and 57, respectively. In one instance the Signal 1 T cell engager component comprises the sequences set forth in SEQ ID NOs: 56 and 57, respectively. Nucleic acid molecules (e.g., mRNAs) encoding these Signal 1 T cell engager molecules are also encompassed by this disclosure.

[0805] The disclosure further encompasses T cell engager polypeptides that provide T cell Signal 2 and specifically bind to TROP2 (e.g., human TROP2) and 4-1BB (e.g., human 4-1BB), and that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequences set forth in SEQ ID NO: 58. In one instance the Signal 2 T cell engager component comprises the sequences set forth in SEQ ID NOs: 58. Nucleic acid molecules (e.g., mRNAs) encoding these Signal 2 T cell engager molecules are also encompassed by this disclosure.

[0806] The disclosure also features T cell engager polypeptides that provide T cell Signal 2 and specifically bind to MUC16 (e.g., human MUC16) and 4-1BB (e.g., human 4-1BB), and that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequences set forth in SEQ ID NO: 59. In one instance the Signal 2 T cell engager component comprises the sequences set forth in SEQ ID NO: 59. Nucleic acid molecules (e.g., Attorney Docket No.: 45817-0187WO1

[0807] mRNAs) encoding these Signal 2 T cell engager molecules are also encompassed by this disclosure.

[0808] In another aspect, the disclosure features T cell engager polypeptides that provide both T cell Signal 1 and Signal 2 and specifically bind to CD3 (e.g., human CD3E), MUC16 (e.g., human MUC16), HSA, and 4-1BB (e.g., human 4-1BB), and that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequences set forth in SEQ ID NO: 55 and 222, respectively. In yet another aspect, the disclosure features T cell engager polypeptides that provide both T cell Signal 1 and Signal 2 and specifically bind to CD3 (e.g., human CD3E), CLDN6 (e.g.. human CLDN6). HSA, and 4-1BB (e.g., human 4- IBB), and that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequences set forth in SEQ ID NO: 56 and 222, respectively. In one instance the Signal 1 + 2 T cell engager component comprises the sequences set forth in SEQ ID NOs: 55 and 222, respectively. In another instance the Signal 1 + 2 T cell engager component comprises the sequences set forth in SEQ ID NOs: 56 and 222, respectively. Nucleic acid molecules (e.g., mRNAs) encoding these Signal 1 + 2 T cell engager molecules are also encompassed by this disclosure.

[0809] In all of the above cases, variability can be achieved by altering the sequence of framework regions of the VH, VL, and VHH domains. In addition, variability can be affected by altering the sequence of the CHI and / or CL domains. Furthermore, variability of the above-described molecules can be the result of changing one or more of the linker sequences.

[0810] Nucleic Acids Encoding T Cell Engagers

[0811] The T cell engager compositions of the disclosure can be administered in protein form, but preferably in the form of nucleic acids which are expressed in vivo. The nucleic acids described herein may be used to encode any one or more (e.g., 1, 2, 3) of the disclosed T cell engagers in a subject. In some cases, nucleic acids (e.g., mRNA) encoding one or more (e.g., 1. 2, 3) of the T cell engagers are provided in the same delivery’ vehicle (e.g., LNP). In other cases, nucleic acids (e.g., mRNA) Attorney Docket No.: 45817-0187WO1

[0812] encoding one or more (e.g., 1, 2, 3) of the T cell engagers are provided in separate delivery vehicles (e.g., LNP). These nucleic acids (e.g., RNAs, such as mRNAs) may be used as therapeutic agents to express the T cell engagers of the disclosure as a therapy to treat a target disease (e.g., solid cancers).

[0813] The present disclosure provides nucleic acid, such as mRNA, encoding the disclosed T cell engagers. The nucleic acids, such as mRNA (e.g., modified mRNA), can be used as therapeutics, which can be administered to a subject in order to treat a disease. In some cases, the disease is cancer. In one instance, the cancer is a solid tumor. In some cases, the disease is an epithelial cancer. In one instance, the cancer is ovarian cancer.

[0814] The present disclosure also provides methods of expressing at least one or multiple (e.g., 2, 3) of the disclosed T cell engagers in vivo in a subject by administering to the subject one or more nucleic acids (e.g., mRNA) that encode the various T cell engagers. The mRNAs encoding one or more of the T cell engagers can be delivered in a nanoparticle (e.g., a lipid nanoparticle). In some cases, mRNAs encoding one, two, or three T cell engagers are administered in a delivery vehicle such as a nanoparticle, e.g., a lipid nanoparticle (LNP). In some cases, the lipid nanoparticle comprises an ionizable amino lipid (e.g., Compound 2); a structural lipid (e.g., cholesterol), a phospholipid (e.g., DSPC); and a PEG-lipid (e.g., Compound 1). In certain cases, the nucleic acid(s) (e.g., mRNAs) are formulated in the delivery vehicle (e.g., a nanoparticle such as a LNP).

[0815] For example, two or more (e.g., 2, 3, 4, 5) different T cell engagers that provide Signal 1 (e.g., by binding to CD3 on a T cell) can be concurrently expressed in a subject by administering to the subject three or more different mRNAs, e.g.,: a first mRNA encoding a VH / CH1 domain of a Fab that (when paired with the appropriate VL / CL domain of the Fab) binds to human CD3 (e.g., human CD3E), the C -terminal of which is linked to one or two or more VHHs that bind to a first TAA (e.g., human MUC16); a second mRNA encoding a VL / CL domain of a Fab that (when paired with the appropriate VH / CH1 domain of the Fab) binds to human CD3 Attorney Docket No.: 45817-0187WO1

[0816] (e.g., human CD3E), the C-terminal of which is linked to a binding molecule that binds to an agent for the purpose of half-life extension (e.g., HSA); and a third mRNA encoding a VH / CH1 domain of a Fab that (when paired with the appropriate VL / CL domain of the Fab) binds to human CD3 (e.g., human CD3E), the C-terminal of which is linked to one or two VHHs that bind to a second TAA (e.g., human CLDN6). Each of the polypeptides encoded by the mRNAs coding for the VH / CH1 domain of a Fab associate independently with the “common” VL / CL domain of the Fab to form two separate T cell engagers: one of which binds to human CD3, human MUC16, and HSA, and the second which binds to human CD3, human CLDN6, and HSA. In some cases, the mRNA encoding the CD3XCLDN6 T cell engager is not included. In certain cases, instead of the mRNA encoding the CD3XCLDN6 T cell engager, mRNA encoding a T cell engager that binds to human CD3 (e.g., CD3E) and a TAA other than human CLDN6 is employed. As will be apparent to those of skill in the art, additional or different mRNAs can be added to change the TAA(s) targeted or increase the number of TAAs targeted by the encoded T cell engagers.

[0817] In some cases, the nucleic acids (e.g., mRNAs) encode polypeptides comprising an amino acid sequence that is 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 the sequence of SEQ ID NO:55 and 57, respectively. In other cases, the nucleic acids (e.g., mRNAs) encode polypeptides comprising an amino acid sequence that is 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 the sequence of SEQ ID NO:56 and 57, respectively. In such cases, two nucleic acids (e.g., mRNAs) are used. In some other cases, the nucleic acids (e.g., mRNAs) encode the polypeptides comprising an amino acid sequence that is 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 the sequence of SEQ ID NO:55 and 57, respectively, as well as the polypeptides comprising an amino acid sequence that is 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 the sequence of SEQ ID Attorney Docket No.: 45817-0187WO1

[0818] NO:56 and 57, respectively. In such cases, three nucleic acid molecules (e.g., mRNAs) are employed. The mRNAs can be formulated in a delivery vehicle (e.g., a nanoparticle such as a LNP).

[0819] In addition to nucleic acids encoding the Signal 1 TCE, the disclosure also features nucleic acids encoding Signal 2 TCE. In some instances, mRNA encoding the Signal 2 TCE comprises from 5’ to 3’: an mRNA encoding a polypeptide comprising a VHH that binds to human TROP2 connected to an immunologically silent human Ig hinge-Fc domain (such as SEQ ID NO:33 or a variant thereof), linked via a linker (e.g., SEQ ID NO: 4) to a VHH that binds to human 4-1BB. In some cases, the nucleic acids (e.g., mRNAs) encode a polypeptide comprising an amino acid sequence that is 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 the sequence of SEQ ID NO:58. In other instances, mRNA encoding the Signal 2 TCE comprises from 5‘ to 3’: an mRNA encoding a polypeptide comprising a VHH that binds to human MUC16 connected to an immunologically silent human Ig silent hinge-Fc domain (such as SEQ ID NO:33 or a variant thereof), linked via a linker (e.g., SEQ ID NO: 4) to a VHH that binds to human 4-1BB. In some cases, the nucleic acids (e.g., mRNAs) encode a polypeptide comprising an amino acid sequence that is 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 the sequence of SEQ ID NO:59.

[0820] In certain cases, the nucleic acids (e.g.. mRNAs) that encode TCEl(s) (e.g.. the polypeptides of SEQ ID NOs.: 55 and 57 and the polypeptides of SEQ ID NOs.: 56 and 57) are formulated together in one delivery’ vehicle (e.g., LNP) and the nucleic acid (e.g., mRNA) that encodes the TCE2 (e.g., the polypeptide of SEQ ID NO: 58 or SEQ ID NO: 59) is formulated in a separate delivery’ vehicle (e.g., LNP). In other cases, the nucleic acids (e.g.. mRNAs) that encode the TCEl(s) and the nucleic acid (e.g., mRNA) that encodes the TCE2 are formulated in the same delivery vehicle (e.g., LNP). In some cases, the delivery’ vehicle is a lipid nanoparticle that comprises an ionizable amino lipid (e.g., Compound 2); a structural lipid (e.g.. cholesterol), a phospholipid (e.g., DSPC): and a PEG-lipid (e.g., Compound 1). Attorney Docket No.: 45817-0187WO1

[0821] In other cases, the mRNAs encode TCEs where Signal 1 and Signal 2 are provided in a cis signaling format. For example, the mRNAs encode a first mRNA encoding a VH / CH1 of a Fab that (when paired with the appropriate VL / CL) binds to human CD3 (e.g., human CD3E), the C-terminal of which is linked to one or two or more VHHs that bind to a first TAA (e.g., human MUC16); a second mRNA encoding a VL / CL of a Fab that (when paired with the appropriate VH / CH1 domain of the Fab) binds to human CD3 (e.g., human CD3E), the C-terminal of which is linked to a binding molecule that binds to an agent for the purpose of half-life extension (e.g., HSA), the C-terminal of which is linked to a VHH that binds to human 4- IBB; and a third mRNA encoding a VH / CH1 domain of a Fab that (when paired with the appropriate VL / CL domain of the Fab) binds to human CD3 (e.g., human CD3E), the C-terminal of which is linked to one or two VHHs that bind to a second TAA (e.g....

Claims

1. Attorney Docket No.: 45817-0187WO12.WHAT IS CLAIMED IS:

1. A composition comprising four mRNA molecules, wherein:4.(i) the first mRNA molecule comprises a first ORF encoding a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a first linker to the N-terminal of a first VHH that binds to a first tumor-associated antigen, the C-terminal of the first VHH linked directly or via a second linker to the N-terminal of a second VHH that binds to the first tumor-associated antigen, optionally wherein the first VHH and the second VHH have the same amino acid sequence;5.(ii) the second mRNA molecule comprises a second ORF encoding a second polypeptide comprising from the N-terminal to C-terminal a second VH and a second CHI linked via the C-terminal of the second CHI directly or via a third linker to the N-terminal of a third VHH that binds to a second tumor-associated antigen, the C-terminal of the third VHH linked directly or via a fourth linker to the N-terminal of a fourth VHH that binds to the second tumor-associated antigen, optionally wherein the third VHH and the fourth VHH have the same amino acid sequence,6.(iii) the third mRNA molecule comprises a third ORF encoding a third polypeptide comprising from the N-terminal to C-terminal a first VL and a first CL linked via the C-terminal of the CL directly or via a fifth linker to the N-terminal of a fifth VHH that binds to human serum albumin;7.wherein the first VH and first CHI of the first polypeptide associates with the first VL and first CL of the third polypeptide to form a Fab and wherein the Fab specifically bind to human CD3, optionally wherein the human CD3 is human CD3E, wherein the second VH and second CH I of the second polypeptide associates with the first VL and first CL of the third polypeptide to form a Fab and wherein the Fab specifically bind to human CD3, optionally wherein the human CD3 is human CD3E;8.optionally wherein the first VH and first CHI of the first polypeptide and the second VH and second CHI of the second polypeptide have the same amino acid sequence; and Attorney Docket No.: 45817-0187WO19.(iv) the fourth mRNA molecule comprises a fourth ORF encoding a fourth polypeptide comprising from the N-terminal to C-terminal a sixth VHH that binds to a third tumor-associated antigen linked directly or via a sixth linker to the N-terminal of an immunologically silent human Ig Fc domain comprising from N terminal to C-terminal a hinge, a CH2 domain, and a CH3 domain of an immunologically silent human Ig Fc domain, the C-terminal of the immunologically silent human Ig Fc domain linked directly or via a seventh linker to the N-terminal of a seventh VHH that binds to a costimulatory molecule on the surface of an activated T cell,10.wherein the fourth polypeptide associates with itself to form a homodimer; and11.wherein the first, second, third, and fourth mRNA molecules are formulated in one or more LNPs,12.optionally wherein the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain from a human IgG4PAA polypeptide, and13.optionally wherein all the uracils in the first, second, third, and fourth mRNAs are N 1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

2. The composition of claim 1, wherein:15.(i) the composition comprises a first lipid nanoparticle (LNP) and a second LNP, wherein the first LNP is formulated with the first, second, and third, mRNA molecules and the second LNP is formulated with the fourth mRNA molecule;16.(ii) the composition comprises a first lipid nanoparticle (LNP) and a second LNP, wherein the first LNP is formulated with the first, second, and third, mRNA molecules and the second LNP is formulated with the fourth mRNA molecule and the first LNP and second LNP are combined together to generate a drug product; or (iii) the first, second, third, and fourth mRNA molecules are formulated together in the same LNP.

3. The composition of any one of claims 1 and 2, wherein the first, second, and third tumor-associated antigens are solid tumor-associated antigens; optionally wherein:Attorney Docket No.: 45817-0187WO118.the first tumor-associated antigen is MUC16,19.the second tumor-associated antigen is CLDN6,20.the third tumor-associated antigen is TROP2 or MUC16; and21.the costimulatory molecule is 4-1BB.

4. The composition of any one of claims 1 to 3. wherein:23.the C-terminal of the first CHI is linked via the first linker to the N-terminal of the first VHH, optionally wherein the first linker is G4S (SEQ ID NO:4);24.the C-terminal of the first VHH is linked via the second linker to the N-terminal of the second VHH. optionally wherein the second linker is (G4S)3 (SEQ ID NO:5);25.the C-terminal of the second CHI is linked via the third linker to the N-terminal of the third VHH, optionally wherein the third linker is G4S (SEQ ID NO:4);26.the C-terminal of the third VHH is linked via the fourth linker to the N-terminal of the fourth VHH, optionally wherein the fourth linker is (G4S)3 (SEQ ID NO:4);27.the C-terminal of the first CL is linked via the fifth linker to the N-terminal of the fifth VHH, optionally wherein the fifth linker is G4S (SEQ ID NO:4); and the C-terminal of the immunologically silent human Ig Fc domain is linked via the seventh linker to the N-terminal of the seventh VHH, optionally wherein the seventh linker is G4S (SEQ ID NO:4).

5. The composition of any one of claims 1 to 4, wherein:29.the first CHI and the second CHI have the identical amino acid sequence, optionally wherein the first CHI and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:28; and30.the first CL and the second CL have the identical amino acid sequence, optionally wherein the first CL and the second CL comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least Attorney Docket No.: 45817-0187WO131.93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:30.

6. The composition of any one of claims 1 to 5, wherein the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain of a human IgG4PAA polypeptide that comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 33.

7. The composition of any one of claims 1 to 6, wherein:34.(a) the first VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40, optionally wherein the first VHH binds to the 5-SEA domain of human MUC16;35.the second VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40, optionally wherein the second VHH binds to the 5-SEA domain of human MUC16;36.the third VHH comprises a VHH-CDR1. a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43, optionally wherein the third VHH binds specifically to human CLDN6 relative to CLDN3, CLDN4, and CLDN9;37.the fourth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43, optionally wherein the fourth VHH binds specifically to human CLDN6 relative to CLDN3, CLDN4, and CLDN9;38.the fifth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:208;39.the sixth VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:

206. optionally wherein the sixth VHH binds specifically to human TROP2 relative to EpCAM; and40.the seventh VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:37; or Attorney Docket No.: 45817-0187WO141.(b) the first VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40;42.the second VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40;43.the third VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43;44.the fourth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43;45.the fifth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:208;46.the sixth VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:206; and47.the seventh VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 37.

8. The composition of any one of claims 1 to 7, wherein:Attorney Docket No.: 45817-0187WO149.(a) the first VH and second VH each comprise a CDR1, a CDR2, and a CDR3 of the VH set forth in SEQ ID NO:26, and50.the first VL and second VL each comprise a CDR1, a CDR2, and a CDR3 of the VL set forth in SEQ ID NO: 27; or51.(b) the first VH and second VH each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:26, and52.the first VL and second VL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:27.

9. The composition of any one of claims 1 to 8, wherein:54.the first VH and the first CHI and the second VH and the second CHI comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:31, and55.the first VL and first CL and second VL and second CL each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:32.

10. The composition of any one of claims 1 to 9, wherein:57.the first polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:55,58.the second polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at Attorney Docket No.: 45817-0187WO159.least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:56,60.the third polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 57, and61.the fourth polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:58.

11. The composition of any one of claims 1 to 10, wherein:63.the first ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:60 or 230,64.the second ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:61 or 231,65.the third ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:62 or 232, and66.the fourth ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:63 or 233.

12. A composition comprising:Attorney Docket No.: 45817-0187WO168.(i) a first mRNA molecule comprises a first ORF encoding a first polypeptide comprising from the N-terminal to C-terminal a first VH and a first CHI linked via the C-terminal of the first CHI directly or via a first linker to the N-terminal of a first VHH that binds to a first tumor-associated antigen, the C-terminal of the first VHH linked directly or via a second linker to the N-terminal of a second VHH that binds to the first tumor-associated antigen, optionally wherein the first VHH and the second VHH have the same amino acid sequence;69.(ii) a second mRNA molecule comprises a third ORF encoding a third polypeptide comprising from the N-terminal to C-terminal a first VL and a first CL linked via the C-terminal of the CL directly or via a third linker to the N-terminal of a third VHH that binds to human serum albumin;70.wherein the first VH and first CHI of the first polypeptide associates with the first VL and first CL of the second polypeptide to form a Fab and wherein the Fab specifically bind to human CD3, optionally wherein the human CD3 is human CD3E, wherein the first and second mRNA molecules are formulated in one or two LNPs,71.optionally wherein all the uracils in the first and second mRNAs are N1-methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines.

13. The composition of claim 12, wherein the first tumor-associated antigen is MUC16, and optionally wherein:73.(i) the first and the second VHH each comprise a VHH-CDR1, a VHH- CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:40, optionally wherein the first VHH binds to the 5-SEA domain of human MUC16;74.(ii) the first and the second VHH each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:40; Attorney Docket No.: 45817-0187WO175.(iii) the third VHH comprises a VHH-CDR1, a VHH-CDR2. and a VHH- CDR3 of the VHH set forth in SEQ ID NO:208;76.(iv) the third VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:208;77.(v) the first VH comprises a CDR1, a CDR2, and a CDR3 of the VH set forth in SEQ ID NO: 26, and the first VL comprises a CDR1, a CDR2, and a CDR3 of the VL set forth in SEQ ID NO:27;78.(vi) the first VH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:26, and the first VL comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:27; and / or79.(vii) the first CHI comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:28; and the first CL comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:30.

14. The composition of claim 12, wherein the first tumor-associated antigen is CLDN6, and optionally wherein:Attorney Docket No.: 45817-0187WO181.(i) the first and the second VHH each comprise a VHH-CDR1, a VHH- CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:43; optionally wherein the third VHH binds specifically to human CLDN6 relative to CLDN3, CLDN4, and CLDN9;82.(ii) the first and the second VHH each comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:43; (iii) the third VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH- CDR3 of the VHH set forth in SEQ ID NO:208;83.(iv) the third VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:208;84.(v) the first VH comprises a CDR1, a CDR2, and a CDR3 of the VH set forth in SEQ ID NO:26, and the first VL comprises a CDR1, a CDR2, and a CDR3 of the VL set forth in SEQ ID NO:27:85.(vi) the first VH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:26, and the first VL comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:27; and / or86.(vii) the first CHI comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:28; and the first Attorney Docket No.: 45817-0187WO187.CL comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 30.

15. A composition comprising:89.an mRNA molecule comprising an ORF encoding a polypeptide comprising from the N-terminal to C-terminal a first VHH that binds to a first tumor-associated antigen linked directly or via a first linker to the N-terminal of an immunologically silent human Ig Fc domain comprising from N terminal to C-terminal a hinge, a CH2 domain, and a CH3 domain of an immunologically silent human Ig Fc domain, optionally wherein the immunologically silent human Ig Fc domain is the hinge-CH2-CH3 domain from a human IgG4PAA polypeptide, the C-terminal of the immunologically silent human Ig Fc domain linked directly or via a second linker to the N-terminal of a second VHH that binds to a costimulatory molecule on the surface of an activated T cell,90.wherein the polypeptide associates with itself to form a homodimer; and optionally wherein all the uracils in the mRNA molecule are N1 -methylpseudouracils, or the uridines of the mRNA are N1 -methylpseudouridines and wherein the mRNA molecule is formulated in a LNP,91.optionally wherein:92.(a) the first VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:206, optionally wherein the first VHH binds specifically to human TROP2 relative to EpCAM; and93.the second VHH comprises a VHH-CDR1, a VHH-CDR2, and a VHH- CDR3 of the VHH set forth in SEQ ID NO:37; or94.(b) the first VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:206; and Attorney Docket No.: 45817-0187WO195.the second VHH comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:37; or96.(c) the polypeptide comprises an amino acid sequence that is 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 the sequence set forth in SEQ ID NO: 58; or97.(d) the ORF comprises a nucleic acid sequence that is 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 the sequence set forth in SEQ ID NO:63 or 233.

16. The composition of any one of claims 1 to 15, wherein the mRNA molecules further comprise:99.(i) a 5’ terminal cap, optionally comprising m7GpppGm;100.(ii) a 5’UTR, optionally comprising the sequence of SEQ ID NO:64;101.(iii) if a signal sequence is not included as part of the ORF, a signal sequence, optionally comprising the sequence of any one of SEQ ID NOs:225, 226. 68, or 227;102.(iv) a 3’UTR, optionally wherein the 3’UTR comprises one or more miR binding sites and / or an IDR, and further optionally comprising the sequence of any one of SEQ ID NOs:234, 235, 130, or 236; and103.(v) a poly A tail, optionally wherein the poly A tail is about 100 nucleotides in length, optionally comprising the sequence of SEQ ID NO: 132,104.optionally wherein all the uracils are N1 -methylpseudouracils, or the uridines of the mRNA are Nl-methylpseudouridines.

17. The composition of any one of claims 1 to 16, wherein the lipid nanoparticle(s) comprises an ionizable amino lipid, a structural lipid, a phospholipid, and a polyethylene glycol (PEG)-modified lipid, optionally wherein the ionizable amino lipid is present at about 40 to 50 mole ratio %, the structural lipid is present at 35 toAttorney Docket No.: 45817-0187WO1106.45 mole ratio %, the phospholipid is present at 5 to 15 mole ratio %, and the PEG-modified lipid is present at 1.5 to 5 mole ratio %, and further optionally wherein the ionizable amino lipid is present at about 47.5 mole ratio %, the structural lipid is present at about 39 mole ratio %, the phospholipid is present at about 10.5 mole ratio %, and the PEG-modified lipid is present at about 3 mole ratio %, and also optionally wherein the ionizable amino lipid is heptadecan-9-yl 8-((2 -hydroxy ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate and the PEG-modified lipid is 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate;107.optionally wherein the ionizable amino lipid is Compound 2 and is present at about 47.5 mole ratio %, the structural lipid is cholesterol and is present at about 39 mole ratio %, the phospholipid is DSPC and is present at about 10.5 mole ratio %, and the PEG-modified lipid is Compound I and is present at about 3 mole ratio %.

18. A pharmaceutical composition comprising the composition of any one of claims 1 to 17, and a pharmaceutically acceptable carrier.

19. A method of treating a solid tumor expressing one or more of MUC16, CLDN6, or TROP2 in a human subject in need thereof, optionally wherein the solid tumor is an epithelial cancer such as ovarian cancer, the method comprising administering to the human subject a therapeutically effective amount of the composition of any one of claims 1 to 17, or the pharmaceutical composition of claim 18, optionally wherein the ovarian cancer is epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer, further optionally wherein the treatment is a second, third, or later line treatment, and also optionally wherein if the composition comprises a first LNP and a second LNP. then the first LNP and the second LNP are administered at substantially the same time or sequentially.

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