Multivalent antibody constructs for targeting prostate cancer cells

Abstract

Disclosed are multispecific antibody constructs, and polynucleotides encoding for same, the proteins and antibody constructs being useful for the treatment of prostate cancer. The disclosed proteins and antibody constructs comprise at least one antigen binding domain that specifically binds to at least one prostate cancer cell specific antigen. The disclosed proteins and antibody constructs may further comprise at least one binding domain that specifically binds to a T-cell specific antigen, which may be used to form a multispecific T-cell engager molecule useful for the treatment of prostate cancer. Further disclosed are methods of making and using the disclosed compositions.

Description

MULTIVALENT ANTIBODY CONSTRUCTS FOR TARGETING PROSTATE CANCER CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present patent application claims priority from U.S. Provisional Application No. 63 / 631.088 filed April 8, 2024, and U.S. Provisional Application No. 63 / 607,994 filed December 8, 2023, which are incorporated herein by reference in their entirety.REFERENCE TO SEQUENCE LISTING

[0002] A Sequence Listing submitted as an XML text file via Patent Center is hereby incorporated by reference. The name of the XML file for the Sequence Listing is 0795697. xml, the date of the creation of the XML file is December 2. 2024, and the size of the XML file is 109,475 bytes.BACKGROUND

[0003] The subject matter discussed in this section should not be assumed to be prior art merely as a result of its mention in this section. Similarly, a problem mentioned in this section or associated with the subject matter provided as background should not be assumed to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which in and of themselves may also correspond to implementations of the claimed technology’.

[0004] Prostate cancer is the most common cancer in American men and the second leading cause of cancer death in men in western countries. Most metastatic patients develop castration resistant prostate cancer, which despite advances in immunotherapy, is limited in treatment options and represents a critical unmet need. Bi-specific T-cell engagers (TCEs) targeting prostate cancer antigens represent a modality that has demonstrated preliminary clinical activity.

[0005] Clinically validated antigens that are highly expressed in prostate cancers include Prostate-Specific Membrane Antigen (PSMA) and Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1). TCEs targeting either PSMA or STEAP1 individually have shown promising clinical activity, but challenges with durability and toxicity have been reported, potentially attributable to antigen escape, target-independent T-cell activation (bystander activation) and specificity7issues.

[0006] Contrary to its name, PSMA expression is not exclusive to prostate cells and can be found in several other tissues and / or conditions, including normal nonprostatic epithelial cells, inflammation / infection, nonprostatic neoplastic cells and nonprostatic tumor-associated neovasculature. The widespread expression of PSMA may limit the therapeutic window of PSMA-specific TCEs and drive immunotoxicity by increased on-target, off-tumor binding.

[0007] Bystander or target independent activation results when immune cells are engaged by the multivalent antibody in the absence of target cells. This, in turn, can result in the release of inflammatory’ cytokines and chemokines and an overall inflammatory environment that can compromise the therapeutic effect of the multivalent antibody therapy. Thus, while multivalent antibody therapies hold promise for treating certain cancers, prevention or reduction of unwanted bystander activation is needed to improve the use of multivalent antibodies. The present disclosure seeks to address one or more of the aforementioned needs in the art.BRIEF SUMMARY

[0008] In one aspect, the present invention includes novel multispecific TCE polypeptides that target both PSMA and STEAP1. The multispecific polypeptides may be administered as recombinant proteins, or as mRNA polynucleotides encoding the polypeptides. The polypeptides may be designed with attenuated PSMA and CD3 binding to mitigate on-target, off-tumor binding, and target-independent T-cell activation.

[0009] The polypeptides are preferably capable of engaging CD3 T-cells but display minimal T-cell activation in the absence of STEAP1 or PSMA. e g., as assayed with STEAP1 or PSMA expressing target cells. In some embodiments, the polypeptides have attenuated PSMA and / or and CD3 binding but maintain robust target cell killing. In some embodiments, the polypeptides are tri-specific TCE polypeptides with minimal bystander activity7that target both STEAP1 and PSMA on tumor cells, but only engage with supraphysiological levels of PSMA, as is found on prostate cancer cells. In some embodiments, the polypeptides display superior anti-tumor activity7over single tumor antigen targeting TCEs. Exemplary polypeptides described herein, encoded by polynucleotides, such as polynucleotides described herein, target both STEAP1 and PSMA on tumor cells with minimal bystander activity, and are capable of engaging CD3 T-cells but with minimal activity’ in the absence of STEAP1 or PSMA expressing target cells. Such constructs preferably retain robust target cell killing activity7.

[0010] One aspect of the present disclosure is a polynucleotide sequence encoding a multispecific antibody construct comprising a heavy chain portion comprising (a) an anti-prostate cancer antigen domain; (b) an anti-hCD3 domain; and (c) a human IgG Fc domain. In one aspect, the Fc domain lacks the effector function.

[0011] In one aspect, the anti-prostate cancer antigen domain may be selected from an antiProstate Specific Membrane Antigen (anti-PSMA) domain, an anti-Six-Transmembrane Epithelial Antigen of the Prostate 1 (anti-STEAPl) domain, an anti-Kallikrein-related peptidase 2 (KLK-2) domain, and combinations thereof.

[0012] In one aspect, the anti-prostate cancer antigen domain comprises at least two domains selected from an anti-Prostate Specific Membrane Antigen (anti-PSMA) domain, an anti-Six- Transmembrane Epithelial Antigen of the Prostate 1 (anti-STEAPl) domain, and an anti-KLK- 2 domain.

[0013] In one aspect, the anti-PSMA domain encodes for one or more amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 63, SEQ ID NO: 71, and SEQ ID NO: 72 .

[0014] In one aspect, the anti-STEAPl domain encodes for one or more amino acid sequence having at least 90% sequence identity to SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68. SEQ ID NO: 75, and SEQ ID NO: 76.

[0015] In one aspect, the anti-KLK-2 domain encodes for one or more amino acid sequence having at least 90% sequence identity to SEQ ID NO: 62, SEQ ID NO: 69, and SEQ ID NO: 70.

[0016] In one aspect, the anti-hCD3 domain encodes for one or more amino acid sequence having at least 90% sequence identity to SEQ ID NO: 64, SEQ ID NO: 73, and SEQ ID NO: 74.

[0017] In one aspect, the human IgG Fc domain comprises a native IgG sequence or a variant IgG sequence. In one aspect, the variant sequence lacks the effector function.

[0018] In one aspect, the polynucleotide sequence encoding for an amino acid sequence has at least 90% sequence identity to a sequence selected from SEQ ID NO: 71, wherein the prostate cancer targeting domain specifically binds to PSMA.

[0019] In one aspect, the polynucleotide sequence encoding for an amino acid sequence has at least 90% sequence identity to a sequence selected from SEQ ID NO: 67, wherein the prostate cancer targeting domain specifically binds to STEAP1.

[0020] In one aspect, the polynucleotide sequence encodes for an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 69, wherein the prostate cancer targeting domain specifically binds to KLK-2.

[0021] In one aspect, the anti-hCD3 domain is located at the N-terminus of the heavy chain.

[0022] In one aspect, the anti-hCD3 domain is located at the C-terminus of the heavy chain.

[0023] In one aspect, the prostate cancer targeting domain is located at the N-terminus of the heavy chain.

[0024] In one aspect, the prostate cancer targeting domain is located at the C-terminus of the heavy chain.

[0025] In one aspect, the anti-hCD3 domain is located at the N-terminus of the heavy’ chain.

[0026] In one aspect, the anti-hCD3 domain is located at the C-terminus of the heavy chain.

[0027] In one aspect, the prostate cancer targeting domain is located at the N-terminus of the heavy chain.

[0028] In one aspect, the prostate cancer targeting domain is located at the C-terminus of the heavy chain.

[0029] In one aspect, the IgG Fc domain comprises a knob or hole mutation.

[0030] In one aspect, the IgG Fc domain comprises a mutation that introduces a cysteine residue.

[0031] In one aspect, the polynucleotide is DNA.

[0032] In one aspect, the polynucleotide is RNA.

[0033] In one aspect, the polynucleotide is mRNA.

[0034] One aspect of the present disclosure is a polynucleotide sequence encoding an antibody construct comprising a light chain portion, the light chain portion comprising adomain selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, an anti-CD3 domain, and combinations thereof.

[0035] In one aspect, the polynucleotide sequence comprises a single domain.

[0036] In one aspect, the polynucleotide sequence comprises at least two domains.

[0037] In one aspect, the at least two domains are the same.

[0038] In one aspect, the at least two domains are different.

[0039] In one aspect, the polynucleotide sequence encodes for at least one amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID NO: 74.

[0040] In one aspect, the polynucleotide is DNA.

[0041] In one aspect, the polynucleotide is RNA.

[0042] In one aspect, the polynucleotide is mRNA.

[0043] One aspect of the disclosure is a multispecific antibody construct comprising at least one heavy chain portion and at least one light chain portion, wherein the at least one heavy chain portion comprises a prostate cancer targeting domain.

[0044] In one aspect, the prostate cancer targeting domain of the multispecific antibody construct is selected from an anti-Prostate Specific Membrane Antigen (anti-PSMA) domain, an anti-Six-Transmembrane Epithelial Antigen of the Prostate 1 (anti-STEAPl) domain, an anti-Kallikrein-related peptidase 2 (KLK-2) domain, and combinations thereof.

[0045] In one aspect, the heavy chain portion of the multispecific antibody construct comprises at least one prostate cancer targeting domain.

[0046] In one aspect, the heavy chain portion of the multispecific antibody construct comprises at least two prostate cancer targeting domains.

[0047] In one aspect, the heavy chain portion of the multispecific antibody construct comprises at least three prostate cancer targeting domains.

[0048] In one aspect, the heavy chain portion of the multispecific antibody construct comprises an anti-hCD3 domain at one or both of the N-terminus and the C-terminus of the heavy chain portion.

[0049] In one aspect, the heavy chain portion of the multispecific antibody construct comprises an anti-hCD3 domain at the N-terminus of the heavy chain portion.

[0050] In one aspect, the heavy chain portion of the multispecific antibody construct comprises an anti-hCD3 domain at the C-terminus of the heavy chain portion.

[0051] In one aspect, the heavy chain portion of the multispecific antibody construct comprises a human IgG Fc domain. In one aspect, the Fc domain lacks the effector function.

[0052] In one aspect, the heavy chain portion of the multispecific antibody construct comprises a human IgG Fc domain comprising a knob or a hole mutation.

[0053] In one aspect, the heavy chain portion of the multispecific antibody construct comprises a human IgG Fc domain comprising a mutation that introduces a cysteine residue.

[0054] One aspect of the disclosure is a light chain protein comprising a domain selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, an anti-CD3 domain, and combinations thereof.

[0055] In one aspect, the light chain region comprises a single domain.

[0056] In one aspect, the single domain of the light chain protein is an anti-PSMA domain.

[0057] In one aspect, the single domain of the light chain protein is an anti-STEAPl domain.

[0058] In one aspect, the single domain of the light chain protein is an anti-KLK-2 domain.

[0059] In one aspect, the single domain of the light chain is an anti-CD3 domain.

[0060] In one aspect, the light chain region comprises two domains.

[0061] In one aspect, the at least two domains of the light chain protein are selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, and an anti-CD3 domain, and the at least two domains are the same.

[0062] In one aspect, the at least two domains of the light chain protein are selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, and an anti-CD3 domain, and the at least two domains are different.

[0063] In one aspect, the light chain protein has at least 90% sequence identity to a sequence selected from SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, and SEQ ID NO: 76.

[0064] One aspect of the disclosure is a heavy chain protein comprising a domain selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, an anti-CD3 domain, and combinations thereof.

[0065] In one aspect, the heavy chain region comprises a single domain.

[0066] In one aspect, the single domain of the heavy chain protein is an anti-PSMA domain.

[0067] In one aspect, the single domain of the heavy chain protein is an anti-STEAPl domain.

[0068] In one aspect, the single domain of the heavy chain protein is an anti-KLK-2 domain.

[0069] In one aspect, the single domain of the heavy chain is an anti-CD3 domain.

[0070] In one aspect, the heavy chain region comprises two domains.

[0071] In one aspect, the heavy' chain region comprises three domains.

[0072] In one aspect, the at least two domains of the heavy chain protein are selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, and an anti-CD3 domain, and the at least two domains are the same.

[0073] In one aspect, the at least two domains of the heavy chain protein are selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, and an anti-CD3 domain, and the at least two domains are different.

[0074] In one aspect, the antibody construct comprises three different antigen-binding domains selected from an anti-PSMA domain, an anti-STEAPl domain, and an anti-CD3 domain.

[0075] In one aspect, the antibody construct with three different antigen-binding domains comprises an anti-PSMA domain and an anti-STEAPl domain at the N-terminus, and an anti- CD3 domain at the C-terminus.

[0076] One aspect of the disclosure is a multispecific antibody construct comprising at least two heavy chains and one light chain.

[0077] In one aspect, the at least two heavy chains of the multispecific antibody construct are any two heavy chains as described above, and at least two light chains.

[0078] In one aspect, the multi-specific antibody construct has a structure selected from a homodimeric antibody, and a heterodimeric antibody.

[0079] In one aspect, the heavy chain region of the multispecific antibody construct is encoded by a polynucleotide of any one of paragraphs

[0008] -

[0036] ,

[0080] In one aspect, the light chain region of the multispecific antibody construct is encoded by a polynucleotide of any of paragraphs

[0008] -

[0036] ,

[0081] In one aspect, a first heavy chain of the multispecific antibody construct comprises a knob mutation, and a second heavy chain of the multispecific antibody construct comprises a hole mutation.

[0082] In one aspect, the at least two heavy chains of the multispecific antibody construct comprise a mutation that introduces a cysteine residue.

[0083] In one aspect, the antibody construct is selected from the group consisting of an IgGl , IgG2, IgG3, or IgG4 isotype.

[0084] In one aspect, the IgGl, IgG2, IgG3, or IgG4 isotype is a variant moditying Fc functions.

[0085] In one aspect, the IgG variant does not bind to the Fc receptor (i. e.. the variant lacks the effector function).

[0086] One aspect of the disclosure is a tandem scFv antibody construct comprising a domain selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, an anti- CD3 domain, and combinations thereof.

[0087] In one aspect, the anti-PSMA domain of the tandem scFv antibody comprises an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% sequence identity to SEQ ID NO: 63.

[0088] In one aspect, the anti-STEAP domain of the tandem scFv antibody has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76.

[0089] In one aspect, the anti-KLK-2 domain of the tandem scFv antibody has an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% sequence identity to SEQ ID NO: 62.

[0090] In one aspect, the anti-hCD3 domain of the tandem scFv antibody has an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% sequence identity to SEQ ID NO: 64.

[0091] In one aspect, the tandem scFv antibody construct comprises at least two domains, at least one domain being an anti-CD3 domain.

[0092] In one aspect, the tandem scFv antibody construct comprises at least three domains, at least one domain being an anti-CD3 domain.

[0093] In one aspect, the tandem scFv antibody construct comprises at least four domains, at least one domain being an anti-CD3 domain.

[0094] In one aspect, the anti-CD3 domain of the tandem scFv antibody construct is located at the N-terminus.

[0095] In one aspect, the at least one or two anti-CD3 domain(s) of the tandem scFv antibodyconstruct are located at the C-terminus.

[0096] In one aspect of the disclosure is a therapeutic composition comprising at least two polynucleotides of paragraphs

[0008] -

[0036] and a delivery vehicle molecule comprising an amino-lipi dated peptoid.

[0097] In one aspect, the delivery vehicle of the therapeutic composition is selected from an amphipathic molecule, an amino-lipidated peptide, a tertiary amino lipidated cationic peptide, a cationic component, a peptoid, a lipoid, a liposome, a lipoplex, a lipid nanoparticle, a cationic lipid nanoparticle, a polymeric compound, a conjugate, and combinations thereof.

[0098] In one aspect, the delivery vehicle of the therapeutic composition has a particle size less than or equal to about 200 nm.

[0099] In one aspect of the disclosure is a lipid nanoparticle comprising an mRNA encoding a multispecific antibody construct of any one of claims 51 through 57; and a delivery vehicle encapsulating the mRNA.

[0100] In one aspect, the polynucleotides of therapeutic composition or lipid nanoparticle comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity to a sequence selected from at least one of SEQ ID NOS: 1-61; and a second mRNA encoding the amino acid having at least 90% sequence identity to a sequence selected from at least one of SEQ ID NOS: l-61;wherein said first mRNA and said second mRNA are the same or are different.

[0101] In one aspect, the polynucleotides of therapeutic composition or lipid nanoparticle comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity to a sequence selected from at least one of SEQ ID NOS: 1-61; a second mRNA encoding the amino acid having at least 90% sequence identity to a sequence selected from at least one of SEQ ID NOS: 1-61; and a third mRNA encoding the amino acid having at least 90% sequence identity to a sequence selected from at least one of SEQ ID NOS:1-61 ; wherein said first, second, and third mRNA are the same or are different.

[0102] In one aspect, a therapeutic composition or lipid nanoparticle is disclosed, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 10; and a second mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 28.

[0103] In one aspect, a therapeutic composition or lipid nanoparticle is disclosed, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 10; and a second mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 33.

[0104] In one aspect, a therapeutic composition or lipid nanoparticle is disclosed, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 10; and a second mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 34.

[0105] In one aspect, a therapeutic composition or lipid nanoparticle is disclosed, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 11; and a second mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 28.

[0106] In one aspect, a therapeutic composition or lipid nanoparticle is disclosed, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 19;a second mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 40; and a third mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 33.

[0107] In one aspect, the antibody construct is Ml 096, as shown in FIG. 6 A.

[0108] In one aspect, the antibody construct is Ml 096, as shown in FIG. 6A.

[0109] In one aspect, the antibody construct is M1097, as shown in FIG. 6B.

[0110] In one aspect, the antibody construct is Ml 098, as shown in FIG. 6C.

[0111] In one aspect, the antibody construct is Ml 102, as shown in FIG. 7A.

[0112] In one aspect, the antibody construct is Ml 103, as shown in FIG. 7B.

[0113] In one aspect, the antibody construct is Ml 104, as shown in FIG. 7C.

[0114] In one aspect, the antibody construct is Ml 107, as shown in FIG. 8A.

[0115] In one aspect, the antibody construct is Ml 108, as shown in FIG. 8B.

[0116] In one aspect, the antibody construct is Ml 109, as shown in FIG. 8C.

[0117] In one aspect, the antibody construct is Ml 110, as shown in FIG. 8D.

[0118] In one aspect, the antibody construct is M1093, as shown in FIG. 9A.

[0119] In one aspect, the antibody construct is M1094, as shown in FIG. 9B.

[0120] In one aspect, the antibody construct is M1095, as shown in FIG. 9C.

[0121] In one aspect is disclosed a method of treating an individual having a disorder or condition, comprising administering to the individual an effective amount of an antibody construct or multispecific antibody construct, a lipid nanoparticle, or a therapeutic composition of any preceding paragraph. In one aspect, the disorder is prostate cancer. In one aspect, the prostate cancer is metastatic and / or castration-resistant prostate cancer (mCRPC).

[0122] In one aspect, the administration of the method is via a route selected from a topical route (such as epicutaneous, inhalational, nasal, ophthalmic, auricular / aural, vaginal, mucosal); an enteral route (such as oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and a parenteral route (such as intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, epidural, intrathecal, subcutaneous, intraperitoneal, extra-amniotic, intraarticular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intrasynovial, intraluminal). In one aspect, the administration of the method is intravenous (IV) administration.BRIEF DESCRIPTION OF THE DRAWINGS

[0123] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0124] A better understanding of the features and advantages of the methods and apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative aspects, and the accompanying drawings of which:

[0125] FIG. 1A depicts a monoclonal antibody specific for PSMA, identified as M1063.

[0126] FIG. IB depicts a monoclonal antibody specific for KLK2, identified as Ml 064.

[0127] FIG. 1C depicts a monoclonal antibody specific for STEAP1, identified as M1065. FIG. 1C also illustrates different regions of a typical antibody. The light chain comprises a variable region (VL), and a constant region (CL). The heavy chain comprises a variable region (VH), a hinge region with disulfide bonds, and three constant regions (CHI, CH2, CH3). CH2 and CH3 comprise the Fc portion of the antibody, while the VH and CHI regions, together with VL and CL, comprise the Fab portions of the antibody.

[0128] FIG. ID depicts a heterodimer antibody construct identified as 1066, where one chain comprises an anti-CD3 scFv (formed by linking the VH and VL portions of the anti-CD3 antibody together with a linker) connected to a hinge region and then CH23 and CH3, and the other chain comprises a hinge region, CH2, and CH3. Heterodimer formation is facilitated by incorporating "knob-into-hole" mutations (shown as “H K”) in the CH3 domains of the two antibody chains.

[0129] FIG. IE depicts a homodimer antibody construct identified as 1408, where the chains each comprise a hinge region, CH2, and CH3, with an anti-CD3 scFv at the C-terminal.

[0130] FIGS. 2A-2F depict various arrangements of scFvs.

[0131] FIG. 2A depicts a tandem scFv identified as 1428, comprising an anti-CD3 scFv, an anti-PSMA scFv, and an anti-KLK2 scFv.

[0132] FIG. 2B depicts a tandem scFv identified as 1429, comprising an anti-CD3 scFv, an anti-PSMA scFv, and an anti-STEAPl scFv.

[0133] FIG. 2C depicts a tandem scFv identified as 1430, comprising an anti-CD3 scFv and an anti-PSMA scFv.

[0134] FIG. 2D depicts atandem scFv identified as 1431, comprising an anti-CD3 scFv, and an anti-KLK2 scFv.

[0135] FIG. 2E depicts a tandem scFv identified as 1432, comprising an anti-CD3 scFv and an anti-STEAPl scFv.

[0136] FIG. 2F depicts an anti-CD3 scFv identified as 1433.

[0137] FIG. 3 A depicts a homodimeric monovalent antibody construct, identified as M 1087, having anti-CD3 VH domains (located at the N-terminus) and common light chains.

[0138] FIG. 3B depicts a homodimeric monovalent antibody construct, identified as M1088, having anti-PSMA VH domains and common light chains.

[0139] FIG. 3C depicts a common light chain heterodimeric multivalent antibody construct, identified as Ml 089, with one heavy chain (HC) comprising an anti-CD3 VH domain and the other HC comprising an anti-PSMA VH domain; and common light chains (VL). Heterodimer formation is facilitated by incorporating "knob-mto-hole" mutations (shown as “H K’') in the CH3 domains, and is further facilitated by incorporating cysteines to enable formation of a disulfide bond between the two chains.

[0140] FIG. 3D depicts a heterodimeric multivalent antibody construct, identified as Ml 084, having an anti-CD3 VH domain at the N-terminus of one heavy chain, an anti-PSMA VH domain at the N-terminus of the other heavy chain, and common light chains (VL).Heterodimer formation is facilitated by incorporating ‘'knob-into-hole” mutations (shown as “H K”) in the CH3 domains.

[0141] FIG. 4A depicts a bivalent antibody construct, identified as Ml 105, having an anti- CD3 scFv at the N-terminus of one heavy chain and an anti-PSMA domain (comprising an aPSMA VH and an aPSMA VL) at the N-terminus of the other heavy chain. Heterodimer formation is facilitated by incorporating “knob-into-hole” mutations (shown as “H K”) in the two CH3 domains, and is further facilitated by incorporating cysteines to enable formation of a disulfide bond between the two chains.

[0142] FIG. 4B depicts a bivalent antibody construct, identified as Ml 106, having an anti- CD3 scFv at the N-terminus of one heavy chain and an anti-STEAPl domain (comprising an aSTEAP 1 VH and an aSTEAP 1 VL) at the N-terminus of the other heavy chain. Heterodimer formation is facilitated by incorporating “knob-into-hole” mutations (shown as “H K”) in the two CH3 domains and is further facilitated by incorporating cysteines to enable formation of a disulfide bond between the two chains.

[0143] FIG. 4C depicts a bivalent antibody construct, identified as M1099, having an anti- CD3 scFv at the N-terminus of one heavy chain and an anti-PSMA domain (comprising an aPSMA VH and an aPSMA VL) at the N-terminus of the other heavy chain. Heterodimer formation is facilitated by incorporating “knob-into-hole” mutations (shown as “H K”) in the two CH3 domains.

[0144] FIG. 4D depicts a bivalent antibody construct, identified as Ml 100, having an anti- CD3 scFv at the N-terminus of one heavy chain and an anti-KLK2 domain (comprising an aKLK2 VH and an aKLK2 VL) at the N-terminus of the other heavy chain. Heterodimer formation is facilitated by incorporating “knob-into-hole” mutations (shown as “H K”) in the two CH3 domains.

[0145] FIG. 4E depicts a bivalent antibody construct, identified as Ml 101, having an anti- CD3 scFv at the N-terminus of one heavy chain and an anti-STEAPl domain (comprising an aSTEAP 1 VH and an aSTEAP 1 VL) at the N-terminus of the other heavy chain. Heterodimer formation is facilitated by incorporating “knob-into-hole” mutations (shown as “H K”) in the two CH3 domains.

[0146] FIG 5A depicts a trivalent antibody construct, identified as M1085, having an anti- CD3 VH domain at the N-terminus of one heavy chain, an anti-PSMA VH domain at the N-terminus of the other heavy chain, common light chains (VL), and an anti-KLK2 scFv at the C-terminus of each heavy chain. Heterodimer formation is facilitated by incorporating “knob- into-hole” mutations (shown as “H K’') in the CH3 domains.

[0147] FIG 5B depicts a trivalent antibody construct, identified as M1086, having an anti- CD3 VH domain at the N-terminus of one heavy chain, an anti-PSMA VH domain at the N- terminus of the other heavy chain, common light chains (VL), and an anti-STEAPl scFv at the C-terminus of each heavy chain. Heterodimer formation is facilitated by incorporating “knob- into-hole” mutations (shown as ’H KT) in the CH3 domains.

[0148] FIG 5C depicts a trivalent antibody construct, identified as Ml 115, having an anti- CD3 VH domain at the N-terminus of one heavy chain, an anti-PSMA VH domain at the N- terminus of the other heavy chain, common light chains (VL), and an anti-STEAPl domain at the C-terminus of the molecule, with a aSTEAPl VH on one heavy chain, and a aSTEAPl VL on the other heavy chain. Heterodimer formation is facilitated by incorporating "knob-into- hole” mutations (shown as “H K”) in the CH3 domains.

[0149] FIG. 6A depicts a trivalent antibody construct, identified as M1096, comprising an anti-KLK2 scFv at the N-terminal of one chain; and an anti-PSMA Fab (VH+VL) at the N- terminal and an anti-CD3 scFv at the C-terminal of the other chain. Heterodimer formation is facilitated by incorporating “knob-into-hole” mutations (shown as “H KT) in the CH3 domains.

[0150] FIG. 6B depicts a trivalent antibody construct, identified as M1097, comprising an anti-KLK2 scFv at the N-terminal of one chain; and an anti-STEAPl Fab (VH+VL) at the N- terminal and an anti-CD3 scFv at the C-terminal of the other chain. Heterodimer formation is facilitated by incorporating "knob-into-hole” mutations (shown as “H KT) in the CH3 domains.

[0151] FIG. 6C depicts a trivalent antibody construct, identified as M1098, comprising an anti-PSMA scFv at the N-terminal of one chain; and an anti-STEAPl Fab (VH+VL) at the N- terminal and an anti-CD3 scFv at the C-terminal of the other chain. Heterodimer formation is facilitated by incorporating “knob-into-hole” mutations (shown as “H KT) in the CH3 domains.

[0152] FIG. 7A depicts a trivalent antibody construct, identified as Ml 102. comprising an anti-KLK2 scFv at the N-terminal of one chain; and an anti-PSMA Fab (VH+VL) at the N- terminal and an anti-CD3 scFv at the C-terminal of the other chain. Heterodimer formation is facilitated by incorporating “knob-into-hole” mutations (shown as “H K”) in the CH3 domains,and is further facilitated by incorporating cysteines to enable formation of a disulfide bond between the two chains.

[0153] FIG. 7B depicts a trivalent antibody construct, identified as Ml 103, comprising an anti-KLK2 scFv at the N-terminal of one chain; and an anti-STEAPl Fab (VH+VL) at the N- terminal and an anti-CD3 scFv at the C-terminal of the other chain. Heterodimer formation is facilitated by incorporating “knob-into-hole” mutations (shown as “H K”) in the CH3 domains, and is further facilitated by incorporating cysteines to enable formation of a disulfide bond between the two chains.

[0154] FIG. 7C depicts a trivalent antibody construct, identified as Ml 104. comprising an anti-PSMA scFv at the N-terminal of one chain; and an anti-STEAPl Fab (VH+VL) at the N- terminal and an anti-CD3 scFv at the C-terminal of the other chain. Heterodimer formation is facilitated by incorporating “knob-into-hole"’ mutations (shown as “H K”) in the CH3 domains, and is further facilitated by incorporating cysteines to enable formation of a disulfide bond between the two chains.

[0155] FIG. 8A depicts a homodimeric bivalent antibody construct, identified as Ml 107, having anti-PSMA VH at the N-terminus of each heavy chain, and an anti-CD3 scFv at the C- terminus of each heavy chain. anti-PSMA light chains combine with the anti-PSMA VH to form anti-PSMA domains at the N terminal of the molecule.

[0156] FIG. 8B depicts a homodimeric trivalent antibody construct, identified as Ml 108. Each heavy chain comprises an anti-PSMA VH domain at the N-terminus and an anti-CD3 scFv at the C-terminus. Each light chain comprises an anti-PSMA VL at the N-terminus, and an anti-STEAPl scFv at the C terminus.

[0157] FIG. 8C depicts a homodimeric trivalent antibody construct, identified as Ml 109. Each heavy chain comprises an anti-PSMA VH domain at the N-terminus and an anti-CD3 scFv at the C-terminus. Each light chain comprises an anti-STEAPl scFv at the N-terminus, and an anti-PSMA VL at the C terminus.

[0158] FIG. 8D depicts a homodimeric trivalent antibody construct, identified as Ml 110. Each heavy chain comprises an anti-STEAPl scFv at the N-terminus, followed by an anti- PSMA VH domain and then an anti-CD3 scFv at the C-terminus. Each light chain comprises an anti-PSMA VL.

[0159] FIG. 9A depicts a trivalent antibody construct, identified as M1093, comprising anti- PSMA VH and VL domains, with an anti-STEAPl scFv (VH+VL) at the C-terminal of the anti-PSMA light chain, and an anti-CD3 domain at the C-terminal of the molecule; the anti- CD3 domain consists of a VH at the C-terminal of one heavy chain, and a VL at the C-terminal of the other heavy chain. Heterodimer formation of the heavy chains is facilitated by incorporating “knob-into-hole” mutations (shown as “KH”) in the CH3 domains.

[0160] FIG. 9B depicts a trivalent antibody construct, identified as Ml 094, comprising anti- PSMA VH and VL domains, with an anti-STEAPl scFv (VH+VL) at the C-terminal of the anti-PSMA light chain, and an anti-CD3 domain at the C-terminal of the molecule; the anti- CD3 domain consists of a VH at the C-terminal of one heavy chain, and a VL at the C-terminal of the other heavy chain. A CD3 CHI domain is connected to the VH, and a CD3 CK domain is connected to the VL as shown. Heterodimer formation of the heavy chains is facilitated by incorporating “knob-into-hole” mutations (shown as “KH ”) in the CH3 domains.

[0161] FIG. 9C depicts a trivalent antibody construct, identified as Ml 095, comprising anti- PSMA VH and VL domains, with an anti-STEAPl scFv (VH+VL) at the C-terminal of the anti-PSMA light chain, and an anti-CD3 domain at the C-terminal of the molecule; the anti- CD3 domain consists of a VH at the C-terminal of one heavy chain, and a VL at the C-terminal of the other heavy chain. A CD3 CHI domain is connected to the VL, and a CD3 CK domain is connected to the VH as shown. Heterodimer formation of the heavy chains is facilitated by incorporating “knob-into-hole'’ mutations (shown as “KH”) in the CH3 domains.

[0162] FIG. 9D is a plot showing target receptor (PSMA and / or STEAP-1) density on LNCaP cells. 22Rvl cells, and PC3-STEAP1 cells.

[0163] FIG. 10A is a plot showing binding of the indicated molecules (S1408, Ml 107, Ml 108, Ml 109 & Ml 110) at the indicated concentrations in a PSMA ELSIA assay.

[0164] FIG. 10B is a plot showing binding of the indicated molecules (S1408, Ml 107, Ml 108, Ml 109 & Ml 110) to cells engineered to express STEAP1.

[0165] FIG. 10A is a plot showing binding of the indicated molecules (S1408, Ml 107. Ml 108, Ml 109, & Ml 110) at the indicated concentrations to prostate cancer cell line 22Rvl expressing PSMA and STEAP1.

[0166] FIG. 10B is a plot showing binding of S 1408, Ml 107, Ml 108, Ml 109, & Ml 110 to prostate cancer cell line LNCaP expressing PSMA and STEAP1.

[0167] FIG 10C is a plot showing binding of the indicated molecules (S1408, Ml 107, Ml 108, Ml 109 & Ml 110) to prostate cancer cell line LNCaP22Rvl expressing PSMA and STEAP1.

[0168] FIG. 10D is a plot showing results from an ex vivo functional assessment of sera from mice injected with mRNAs encoding the indicated constructs (S1408, Ml 107, and Ml 110) on LNCaP binding at the indicated dilutions.FIG. 11A is a plot showing results from an ex vivo functional assessment of sera from mice injected with mRNAs encoding the indicated constructs (S1408, Ml 107, and Ml 110) on LNCaP binding at the indicated dilutions.

[0169] FIG. 1 IB is a plot showing binding assay data from a T-cell dependent cytotoxicity assay with human T-cells derived from healthy donor PBMCs performed using sera from mice injected with mRNAs encoding the indicated constructs (S1408, Ml 107, and Ml 110)T-cell.

[0170] FIG. 11C is a plot showing T-cell Dependent Cellular Cytotoxicity (TDCC; shown as % killing of the engineered LNCaP target cells) with the indicated dilutions of sera from mice injected with mRNAs encoding Ml 110, Ml 107 or S 1408 in a co-culture ofT-cells with LNCaP engineered to express PSMA and STEAP1.

[0171] FIG. 1 I D is a plot showing T-cell Activation (shown as %CD69+CD137+CD8 T- cells) by the indicated dilutions of sera from mice injected with mRNAs encoding Ml 110, M1107 or S1408.

[0172] FIG. 12A shows % killing of LNCaP target cells (engineered to express PSMA and STEAP1) following 48-hour culture with T-cells and one of the following multivalent constructs: S1408, Ml 107, Ml 108, Ml 109 and Ml 110 at the indicated concentrations.

[0173] Fig. 12B shows % killing of 22Rvl target cells following 72-hour culture with T- cells and one of the following multivalent constructs: S1408. Ml 107, Ml 108, Ml 109 and Ml 110 at the indicated concentrations.

[0174] FIG. 13 is a plot showing T-cell bystander activation (shown as %CD69+ CD137+ CD8 T-cells) following an overnight culture with one of the following molecules: Ml 110, M1086, M1098, S 1429 and M1093.

[0175] FIG. 14 shows serum expression of the indicated constructs (quantified by ProteinSimple’s Jess automated Western blot system) 24 hours following injection with mRNAs encoding the constructs.

[0176] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.DETAILED DESCRIPTION

[0177] DEFINITIONS

[0178] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The methods may comprise, consist of, or consist essentially of the elements of the compositions and / or methods as described herein, as well as any additional or optional element described herein or otherwise useful in the administration of multivalent antibody constructs for treatment of a disease state such as a cancer, particularly a prostate cancer.

[0179] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art.

[0180] “Amelioration” as used herein refers to any improvement of the disease state of a patient, by the administration of an antibody construct according to the invention to a subject in need thereof. Such an improvement may also be seen as a slowing or stopping of the progression patient's disease.

[0181] “Antibody construct” refers to a molecule in which the structure and / or function is / are based on the structure and / or function of an antibody, e.g. of a full-length or wholeimmunoglobulin molecule capable of binding one or more specific target(s) or antigen(s). In one aspect, the antibody construct comprises the minimum structural requirements of an antibody which allow for the target binding, e.g. be defined by the presence of at least the three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or the three heavy chain CDRs (i.e. CDR1 , CDR2 and CDR3 of the VH region). In one aspect, an '‘antibody construct” includes one or more Antibody fragments(s), as defined below.

[0182] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds an antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab1, Fab'-SH, F(ab')2; diabodies; bispecific diabodies (e.g., bsDb); single chain diabodies (e.g., scDb); linear antibodies; single-chain antibody molecules (e.g. scFv); tandem single-chain antibody molecules (e.g., TaFv); dual affinity retargeting molecules (e.g., DART); bispecific T-cell engagers; variable domain of heavy chain-only antibody molecules (e.g., VHH); and multivalent antibodies formed from antibody fragments. “Antibody constructs” include monovalent, bivalent and polyvalent / multivalent constructs and monospecific constructs, specifically binding to only one antigenic structure, as well as bispecific and polyspecific / multivalent constructs, which specifically bind more than one antigenic structure, e.g. two, three or more, through distinct binding domains. The term “antibody construct” includes molecules consisting of only one polypeptide chain as well as molecules having more than one polypeptide chain, which chains can be either identical (homodimers, homotrimers or homo oligomers) or different (heterodimer, heterotrimer or heterooligomer). In one aspect, the antibody constructs may be an “in vitro generated antibody constructs.”

[0183] The term “antigen binding domain” or “antigen-binding site” refers to the part of an antigen binding molecule (e.g., an antigenic fragment or an antibody construct) that specifically binds to an antigenic determinant. More particularly, the term “antigen-binding domain” refers the part of an antibody, antibody construct, or antibody fragment that comprises the area which specifically binds to part or all of an antigen. An antigen binding domain may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions), and may, in one aspect, comprise a light chain variable region (VL) and a heavy chain variable region (VH). In another aspect, an antigen binding domain may comprise an scFv.

[0184] The term “Fab fragment” refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CHI) of a heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.

[0185] The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of a “classic” antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Particularly, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The amino acid sequences of the heavy chains are always presented with the C- terminal lysine, however variants without the C-terminal lysine are included in the inv ention. The Fc region may be modified to lack an effector function. A number of examples of Fc modifications lacking the effector function are known in the art. Exemplary Fc regions lacking the effector function are disclosed herein in the disclosed sequences that include the Fc region.

[0186] An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain. The “CH2 domain” of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one aspect, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain. The “CH3 domain” comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e., from an amino acid residue at about position 341 to an amino acid residue at about position 447 of an IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with an introduced “protuberance” (“knob”) in one chain thereof and a corresponding introduced “cavity” (“hole”) in the other chain thereof; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as herein described. Unless otherw ise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda. Md., 1991.

[0187] The term “binding domain” means a domain which (specifically) binds to / interacts with / recognizes a given target epitope. “Specific binding” means that the binding is selectivefor the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding molecule to bind to a specific antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one aspect, a molecule that binds to the antigen has a dissociation constant (Kd) of <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 107M or less, e.g. from 107M to 10’13M, e.g. from 109M to 10’13M). The terms “(specifically) binds to”, (specifically) recognizes”, “is (specifically) directed to”, and “(specifically) reacts with” mean that a binding domain interacts or specifically interacts with one or more, or at least two, or at least three, or at least four amino acids of an epitope located on the target protein or antigen. In one aspect, the disclosed antibodies or antibody constructs have high affinity for a target. As used herein, the term “high affinity” of an antibody refers to an antibody having a Kd of ICT9M or less and even more particularly 1010M or less for a target antigen. The term “low affinity” of an antibody refers to an antibody having a Kd of 1 (T8or higher.

[0188] “Disease” refers to any condition that would benefit from treatment with the antibody construct or the pharmaceutic composition described herein. This includes chronic and acute disorders or diseases including those pathological conditions that predispose the mammal to the disease in question.

[0189] “Effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0190] “Epitope” refers to a site on an antigen to which a binding domain, such as an antibody or immunoglobulin or derivative or fragment of an antibody or of an immunoglobulin, specifically binds. An “epitope” is antigenic and thus the term epitope is sometimes also referred to herein as “antigenic structure” or “antigenic determinant."

[0191] The term “Fab fragment” refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CHI) of a heavy chain.

[0192] The terms “individual,” “host,” “subject.” and “patient” may be used interchangeably to refer to an animal that is the object of treatment, observation and / or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals.

[0193] As used herein, the term “isolated” refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). A polynucleotide, vector, polypeptide, cell, or any composition disclosed herein which is “isolated” is a polynucleotide, vector, polypeptide, cell, or composition which is in a form not found in nature. Isolated polynucleotides, vectors, polypeptides, or compositions include those that have been purified to the degree that they are no longer in a form in which they are found in nature. In some aspects, a polynucleotide, vector, polypeptide, or composition that is isolated is substantially pure.

[0194] The term “multispecific” as used with respect to a fusion protein, antibody, antibody construct, or the like, means that the protein comprises multiple binding specificities, allowing it to interact with two or more distinct antigens or epitopes.

[0195] The terms “peptide”, “polypeptide” and “protein” refer to natural or naturally modified peptides / polypeptides / proteins wherein the modification is effected e.g. by post- translational modifications like glycosylation, acetylation, phosphorylation and the like. A “peptide”, “polypeptide” or “protein” when referred to herein may also be chemically modified such as pegylated.

[0196] “Polynucleotide” refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g. an amide bond, such as found in peptide nucleic acids (PNA). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, e g., DNA or RNA fragments, present in a polynucleotide.

[0197] "Percent (%) amino acid sequence identity " with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary7, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) 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, a nucleic acid sequence may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference nucleic acid sequence. The length of comparison sequences will generally be at least 5 contiguous nucleotides, or at least 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23. 24, or 25 contiguous nucleotides.

[0198] '‘Treatment” (and grammatical variations thereof such as “treat” or “treating”), as used herein refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0199] The term “cluster of differentiation 3” or “CD3.” as used herein, refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated, including, for example, CD3s, CD3y, CD3a, and CD3[1 chains, and encompasses full-length, "unprocessed" CD3 (e.g., unprocessed or unmodified CD3s or CD3y), as well as any form of CD3 that results from processing in the cell, such as a "processed" CD3s polypeptide without all or a portion of its signal peptide, including, in particular, a CD3e polypeptide without the first 21 or 22 amino acids of the sequence ofNCBI Ref SeqNo. NP 000724 (human CD3sprotein). The term also encompasses naturally other occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, both human CD3s protein (NCBI RefSeq No.NP 000724), which is 207 amino acids in length, and human CD3y protein (NCBI RefSeq No. NP_000064), which is 182 amino acids in length, in unprocessed or processed form.

[0200] The term “variable region’" or “variable domain"’ refers to the domain of an antibody heavy or light chain that is involved in binding the antigen binding molecule to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).

[0201] Multi-specific Antibody Constructs and Polynucleotides Encoding Same

[0202] Disclosed herein are multi-specific antibody constructs, also referred to as “multivalent antibody constructs”, “multivalent T-cell engager molecules”, or “multispecific T-cell engager molecules”, which may be used for engaging at least one prostate cancer cell and a T- cell, for the selective killing of prostate cancer cells. In aspects, the multispecific antibody constructs comprise at least one domain having binding specificity for at least one prostate cancer cell antigen and at least one domain having binding specificity for a T-cell antigen. The T-cell antigen may be CD3.

[0203] The multi-specific antibody constructs may comprise at least one prostate cancer antigen binding domain specific for a first prostate cancer antigen, or at least two prostate cancer antigen binding domains specific for a first and a second prostate cancer antigen, wherein the first and second prostate cancer antigen are different, or at least three cancer antigen binding domains specific for a first, second, and third prostate cancer antigen, wherein the first, second, and third prostate cancer antigens are different. Exemplary' prostate cancer antigens are described herein.

[0204] In one aspect, the multi-specific antibody constructs may comprise a heavy chain region comprising at least one, or at least tw o, or at least three distinct prostate cancer binding domains, at least one anti-hCD3 domain, and a human IgG FC domain.

[0205] In one aspect, the multi-specific antibody constructs may comprise a light chain region comprising a domain selected from at least one or at least two distinct prostate cancer binding domains and, optionally, an anti-hCD3 domain.

[0206] In a further aspect, disclosed is a polynucleotide, such as an RNA, more particularly an mRNA, encoding for an above-described multi-specific antibody construct.

[0207] In a further aspect, disclosed are multivalent antibody constructs, such as bispecific or tri-specific antibody constructs, which can be in a homodimeric or heterodimeric format, formed from the described multi-specific antibody constructs. Further disclosed are polynucleotides encoding the disclosed multi-specific antibody constructs.

[0208] In other aspects, the multivalent antibody constructs may be in the form of a multispecific scFv antibody construct comprising at least one prostate cancer targeting domain. The disclosed multi-specific antibody constructs may be co-expressed or co-delivered to form multispecific antibody constructs useful for treatment of a prostate cancer in an individual in need thereof.

[0209] In one aspect, the disclosed multi-specific antibody constructs may comprise at least two binding domains, each binding domain having specificity for a unique antigen (i.e., a “bi- specific” antibody construct), or at least three unique binding domains (i.e., a “tri-specific” antibody construct) or at least four unique binding domains, or more than four unique binding domain. Exemplary antigens useful for the disclosed multi-specific antibody constructs and multispecific antibody constructs are described below.

[0210] In one aspect, the disclosed multi-specific antibody constructs may be combined to create a multi-specific antibody construct that is capable of simultaneously engaging a cancer cell, via a cell specific antigen, and a T-cell antigen, bringing the target cancer cell and T-cell in close proximity, which in turn results in cell-specific toxicity. The disclosed antibody constructs may be combined such that at least two, or even three, distinct prostate cancer antigens may be targeted, such that in theory, the vast majority of prostate cancer cells will be targeted and killed via proximity to the bound T-cells. The prostate cancer specific antigens may be any prostate cancer specific antigen. In some aspects, however, the antigen may be one or more of PSMA, STEAP1, and KLK-2, as described below.

[0211] Prostate Specific Membrane Antigen (PSMA). In one aspect, the targeted prostate cancer antigen may be Prostate Specific Membrane Antigen (PSMA), a Type II integral membrane protein expressed on the surface of prostate epithelial cells, which is upregulated in most prostate tumors. Greater than 85% of prostate cancer cells are PSMA-positive, and increased expression of PSMA correlates with higher grade malignancies, metastatic disease, and CRPC.

[0212] Thus, in one aspect, the disclosed multi-specific antibody constructs may comprise a region that specifically binds human PSMA (an anti-PSMA binding domain). The sequence of an exemplaiy anti-PSMA scFv is disclosed herein as SEQ ID NO: 58. Exemplary anti-PSMA antibodies are disclosed herein as SEQ ID NO:47 (VH) and SEQ ID NO:48 (VL). Additional anti-PSMA antibodies have been described, for example, in US9695248, US1 1155633, US20210023094, US20230250189, US11623957, US20220168386, US20220306764, US8461308, US20220323619, US10844134, US10100126, US11472886, US11464877, US9587036, US20230272110, US20220119525, US20230242668, US20190070322, US8114965, US20090297438, US11555078, US1 1773182, US10751346, US11414497, US 10781264, US11612646, US20200024353, US9687572, US11746157, US20230322961, US20230140397, US20150152187, US20200399372, US11548947, US9242012,US20080279868, US20180016346, US 11401342. US20220411530, US20230131727, US20230201366, US7666414, US6150508. US7045605, US20110189093, US7514078. US20060062793, US20170306045, US8703918, US7201900, US20180022819, US7381407, US11400121, US10800856, US9238694, US8629247, US7476513, US11649286, US11434302, and US11697684. The VH and VL sequences from antibodies described in the foregoing patent publications, or from other patent and non-patent publications and sequence databases describing such antibodies, may be used to generate PSMA binding domains for use as detailed herein.

[0213] The anti-PSMA binding domain may be formed by the VH and VL of the scaffold antibody, or may be connected, e.g., as an scFv, to the N-terminus or the C-terminus of a heavy chain, or on the N-terminus or a C-terminus of alight chain, as described herein and illustrated in the Figures. The anti-PSMA binding domain may be immediately adjacent to another described domain, or may be connected via a linker as described herein.

[0214] Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1). In one aspect, the targeted prostate cancer antigen may be Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1), a transmembrane protein predominantly expressed in prostate epithelium. STEAP1 has low or absent expression in normal tissues but is overexpressed in > 80% of prostate cancers, including bone and lymph node metastases.

[0215] Thus, in one aspect, the disclosed multi-specific antibody constructs may comprise a region that specifically binds human STEAP1 (an anti-STEAPl binding domain). The sequences of exemplary anti-STEAPl scFvs are disclosed herein as SEQ ID NO:60 and SEQID N0:61. Exemplary anti-STEAPl antibodies are disclosed herein as SEQ ID NO:51 (VH) and SEQ ID NO:52 (VL); and as SEQ ID NO:53 (VH) and SEQ ID NO:54 (VL). Additional anti-STEAPl antibodies have been described, for example, in US20220348686, US11530274, US20210277148, WO2023154890. US20210315986. US20170043034. US20230270857, W0201 1721 0, US9632091 , US 11401347, US 1 1685790, and US902951 .

[0216] The anti-STEAPl binding domain may be formed by the VH and VL of the scaffold antibody, or may be connected, e.g., as an scFv, to the N-terminus or the C-terminus of a heavychain, or on the N-terminus or a C-terminus of alight chain, as described herein and illustrated in the Figures. The anti-STEAPl binding domain may be immediately adjacent to another described domain, or may be connected via a linker as described herein.

[0217] Kallikrein-related peptidase 2 (KLK-2). KLK-2 is a serine protease enzyme that is primarily produced in the prostate gland. KLK-2 expression is elevated in prostate cancer. In one aspect, the disclosed multi-specific antibody constructs may comprise a region that specifically binds human KLK-2 (an anti-KLK-2 binding domain). The sequence of an exemplary anti-KLK-2 scFv is disclosed herein as SEQ ID NO:57. An exemplary anti-KLK-2 antibody is disclosed herein as SEQ ID NO:49 (VH) and SEQ ID NO:50 (VL). Additional anti- KLK-2 antibodies have been described, for example, in US20110177059, US20230114808, and WO2022 / 098972.

[0218] The anti-KLK-2 binding domain may be formed by the VH and VL of the scaffold antibody, or may be connected, e.g., as an scFv, to the N-terminus or the C-terminus of a heavy chain, or on the N-terminus or a C-terminus of alight chain, as described herein and illustrated in the Figures. The anti-KLK-2 binding domain may be immediately adjacent to another described domain, or may be connected via a linker as described herein.

[0219] Anti-hCD3 Domain. In addition to the anti -prostate cancer antigen domains described above, the disclosed multispecific antibody constructs and antibody constructs comprise an anti-hCD3 domain which is capable of targeting a T-cell. In one aspect, the disclosed multispecific antibody constructs and antibody constructs may comprise a T-cell binding domain that specifically binds to a CD3 antigen. The CD3 antigen, or “Cluster of Differentiation 3” is a protein complex associated with T-cell receptor (TCR) on both CD4+ and CD8+ T-cells. CD3 facilitates signal transduction following TCR antigen recognition and is required for T-cell activation. Agonistic binding of CD3 can induce T-cell signaling and activation.

[0220] Thus, in one aspect, the disclosed multi-specific antibody constructs comprise a region that specifically binds human CD3 (an anti-CD3 binding domain). The sequence of an exemplary anti-CD3 scFv is SEQ ID NO:59. An exemplary anti-CD3 antibody is SEQ ID NO: 55 (VH) and SEQ ID NO: 56 (VL). Additional anti-CD3 antibodies have been described, for example, in US1 1732054, US11 155633, US1 1 155621, US 1 1802158, US 1 1633501 , US20220242953, US20210253701, US11542330, WO2023125611, WO2016204966, US11485793, US11434300, US11440962, WO202314809, W02016179003. US11590223, US20230212289, US10662244, US20200339686, US11007267, US20180057593, and US 10941202.

[0221] The anti-CD3 binding domain may be formed by the VH and VL of the scaffold antibody, or may be connected, e.g., as an scFv, to the N-terminus or the C-terminus of a heavy chain, or on the N-terminus or a C-terminus of alight chain, as described herein and illustrated in the Figures. The anti-CD3 binding domain may be immediately adjacent to another described domain, or may be connected via a linker as described herein.

[0222] Experiments detailed herein demonstrate that a preferred arrangement of the CD3 binding domain is at the C-terminus of the antibody or core antibody scaffold. This location permits activation of T-cells to kill antigen-presenting cells, but minimizes bystander T-cell activation (activation in the absence of the tumor antigen(s)).

[0223] Human IgG Fc Domain. The disclosed multispecific antibody constructs and antibody constructs may further comprise a heavy chain region comprising a human IgG Fc domain. In some aspects, the IgG Fc region may be a heterogenous or homogenous combination of immunoglobulins (Ig). For example, an Fc region may include 2 CH2. 2 CH3 (IgA, IgD, IgG). In further aspects, the IgG Fc domain may be modified to comprise a knob and / or hole feature, or set of mutations, to assist in heterodimerization. “Knob and Holes” or “Knob-into-Holes” terms are used interchangeably herein. A CH3 domains of an Fc-region of can be altered by the “knob-into-holes” technology which is described in detail with several examples in e.g., US 8679785, WO 96 / 027011, Ridgway, J. B., et al.. Protein Eng. 9 (1996) 617-621; and Merchant, A. M., et al., Nat. Biotechnol. 16 (1998) 677-681. In this method the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of both heavy chains containing these two CH3 domains. Each of the two CHy domains (of the two heavy chains) can be the “knob”, while the other is the “hole”. The introduction of a disulfide bridge may further stabilize the heterodimers (Merchant, A. M., et al., Nature Biotech.16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield. For example, the mutation T366W in the CH3 domain of an antibody heavy chain is denoted as ‘‘knob mutation” and the mutations T366S, L368A, Y407V in the CH3 domain of an antibody heavy chain are denoted as “mutations hole” (numbering according to Kabat EU index). An additional interchain disulfide bridge between the CH3 domains can also be used (Merchant, A. M., et aL, Nature Biotech. 16 (1998) 677-681) e.g. by introducing a Sy54C mutation into the CHy domain of the heavy chain with the “knob mutation” (denotes as “knob- cys mutations” or “mutations knob-cys”) and by introducing a Yy49C mutation into the CH3 domain of the heavy chain with the “hole mutations” (denotes as “hole-cys mutations” or “mutations hole-cys”) (numbering according to Kabat EU index) or vice versa. The disclosed constructs may comprise a “knob-into-hole” modification, wherein a “knob” modification in is present in one of the two subunits of the Fc domain and a “hole” modification is present in the other one of the two subunits of the Fc domain. Knob-into-hole technology is described e.g.. in U.S. Pat. Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory' cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). Other knobs-in-holes technologies as described by EP 1 870 459, can be used alternatively or additionally. In one aspect the multivalent antibody construct may comprise the mutations R409D and K370E in the CH3 domain of the “knobs chain” and the mutations D399K and E357K in the CH3 domain of the “hole-chain” (numbering according to Kabat EU index). In one aspect, the bispecific antibody comprises a T366W mutation in the CH3 domain of the “knobs chain” and the mutations T366S, L368A and Y407V in the CH3 domain of the “hole chain” and additionally the mutations R409D and K370E in the CH3 domain of the “knobs chain” and the mutations D399K and E357K in the CH3 domain of the “hole chain” (numbering according to the Kabat EU index). The bispecific antibody may comprise the mutations Y349C and T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains, or the multivalent antibodycomprises the mutations Y349C and T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains and additionally the mutations R409D and K370E in the CH3 domain of the “knobs chain” and the mutations D399K and E357K in the CEB domain of the “hole chain” (numbering according to the Kabat EU index). Other techniques for modifying the CEB domains of the heavy chains of a multivalent antibody to enforce heterodimerization may include those described in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954 and WO 2013 / 096291 are contemplated herein as alternatives to the “knob-into- hole technology” in combination with a bispecific antibody.

[0224] In further aspects, the IgG Fc domain may further comprise a modification in which one or more cysteine residues are introduced to facilitate di-sulfide binding of complementary heavy chain regions. That is. the heavy chains of the multivalent antibody construct may modified to contain one or more cysteine residues, in which one or more residues of the molecule are substituted with cysteine residues. In one aspect, the substituted residues may be positioned such that reactive thiol groups are located at accessible sites of the multivalent antibody construct to form disulfide bonds with a desired heavy chain. For example, cysteine residues may be added to the heavy chain portions of the multivalent antibody construct to stabilize the structure. In one aspect, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy’ chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antigen binding molecules may be generated as described, e.g.. in U.S. Pat. No. 7,521,541. Techniques to introduce unnatural disulfide bridges for stabilization are described e.g., in WO 94 / 029350.

[0225] scFv Antibody Constructs. In one aspect, the disclosed multi-specific antibody constructs may take the form of an scFv antibody construct. A “single-chain variable fragment (scFv)” refers to a fusion of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected via a linker peptide. In one aspect, the linker is rich in glycine for flexibility’, and may also include serine or threonine for solubility'. Examples of linkers are described herein. The linker may either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. The scFv protein retains the specificity for a target epitope or antigen, despite removal of the constant regions and the introduction of the linker.

[0226] scFv antibody constructs may be expressed as a single chain polypeptide. An scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. The scFv can comprise VH and VL sequences from any suitable species, such as murine, human or humanized VH and VL sequences. To create an scFv-encoding nucleic acid, the VH and VL-encoding DNA fragments are operably linked to another fragment encoding a linker, such that the VH and VL sequences can be expressed as a contiguous singlechain protein, with the VL and VH regions joined by the flexible linker.

[0227] Table 1. Exemplary Multi-specific Antibody construct Sequences.

[0228] Table 2. Exemplary multivalent T-cell engager molecules

[0229] Exemplary tandem scFv antibody constructs are shown in FIGS. 5A-5C. Exemplary amino acid sequences that encode for scFv antibody constructs include, e.g.. those in the following Table 3.

[0230] Table 3. Exemplary Sequences

[0231] Properties of the disclosed Multi-specific Antibody Constructs

[0232] Reduced Bystander Activation. In one aspect, the disclosed multispecific antibody constructs formed from the disclosed multi-specific antibody constructs may be characterized by having reduced bystander activation upon binding to a T-cell surface. Bystander activation refers to a phenomenon in immunology in which T-cells, for example cytotoxic T-cells, indirectly impact neighboring cells to which they do not specifically bind. Bystander T-cell activation can lead to off-target cytotoxicity and cytokine release syndrome, including excessive inflammation and tissue damage, leading to tissue injury and autoimmune responses, thereby reducing the overall benefit of a therapy using the multivalent antibody construct. Without wishing to be limited by theory, it is believed that the C-terminal location of the cell antigen binding domain that specifically binds to an hCD3 domain serves to reduce this deleterious bystander effect.

[0233] Binding Affmi ty

[0234] The multivalent antibody constructs contemplated herein may comprise a first heavy chain, a second heavy chain, a first light chain, and a second light chain.

[0235] In general, the multivalent antibody constructs may comprise at least one prostate cancer cell binding domain, which binds specifically to a prostate cancer cell antigen, for example one or more, or two or more, or at least all three of PSMA, STEAP1. and KLK-2, and a binding domain that binds to hCD3.

[0236] In one aspect, the antigen binding domain that binds to PSMA has a KD value of the binding affinity of <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g.103M or less, e.g. from 103M to 1013M, e.g., from 19M to 10'13M).

[0237] In one aspect, the antigen binding domain that binds to STEAP1 has a KD value of the binding affinity of <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g. 1sM or less, e.g. from 13M to Itr13M, e.g., from HE9M to 10'13M).

[0238] In one aspect, the antigen binding domain that binds to KLK-2 has a KD value of the binding affinity of <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g. 1 O'3M or less. e.g. from 103M to 101 3M. e.g., from 109M to 10'13M).

[0239] Likewise, the antigen binding domains that binds to an hCD3 domain has a KD value of the binding affinity of <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g. 13M or less, e.g. from 103M to 1013M, e.g., from 109M to 10‘13M).

[0240] Tumor Cell Killing Effect

[0241] In some aspects, disclosed herein is a method of inducing a tumor cell killing effect in a target cell population. In some aspects, the method may comprise contacting the target cell population comprising at least one tumor cell with a multivalent antibody construct described above or a pharmaceutical composition described herein for a time sufficient to induce a cell killing effect, thereby killing the at least one tumor cell in the target cell population.

[0242] In some aspects, the time sufficient to induce a cell killing effect is about 5 minutes. 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, or more.

[0243] In some aspects, the tumor cell is a cell from a solid tumor, particularly a prostate cancer.

[0244] In some aspects, the multivalent antibody construct or the pharmaceutical composition comprising the multivalent antibody construct decreases tumor cells in the target cell population by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.

[0245] In some aspects, the multivalent antibody or the pharmaceutical composition comprising the multivalent antibody decreases tumor cells in the target cell population by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, or more.

[0246] In some aspects, the multivalent antibody construct or the pharmaceutical composition comprising the multivalent antibody construct decreases tumor cell proliferation in the target cell population by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.

[0247] In some aspects, the multivalent antibody construct or the pharmaceutical composition comprising the multivalent antibody construct decreases tumor cell proliferation in the target cell population by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more.

[0248] Monoclonal Antibodies

[0249] In one aspect, the multivalent antibody construct may be a monoclonal antibody. The term “monoclonal antibody’7as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci,such methods and other exemplar}- methods for making monoclonal antibodies being described herein.

[0250] Linkers

[0251] In one aspect, the one or more binding domains and / or the human IgG Fc domain may be connected via a peptide linker comprising one or more amino acids, ty pically about 2 to 20 amino acids. Exemplary linker peptides include, for example, (G4S)n, (So4)nor Gn(SG4)nor Gnpeptide linkers, wherein “n” is generally a number between 1 and 10, ty pically between 2 and 4, in particular 2, i.e. the peptides selected from the group consisting of GGGGS (SEQ ID NO:77) GGGGSGGGGS (SEQ ID NO:78), SGGGGSGGGG (SEQ ID NO:79) and GGGGSGGGGSGGGG (SEQ ID NO:80), but also include the sequences GSPGSSSSGS (SEQ ID NO: 81), (G4S)3(SEQ ID NO:82), (G4S)4(SEQ ID NO: 83), GSGSGSGS (SEQ ID NO: 84), GSGSGNGS (SEQ ID NO: 85), GGSGSGSG (SEQ ID NO: 86), GGSGSG (SEQ ID NO: 87), GGSG (SEQ ID NO: 88), GGSGNGSG (SEQ ID NO: 89), GGNGSGSG (SEQ ID NO: 90) and GGNGSG (SEQ ID NO: 91). In one aspect, peptide linkers may be between 5 and 24 ammo acids in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids in length), or between 5-15 amino acids in length (e.g., 5.6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length). In one aspect, the LM is highly flexible and may be rich in glycine (G) and / or serine (S) residues, which may be present in the form of GS repeats.

[0252] In one aspect, the disclosed polynucleotide encoding for the multispecific antibody construct is RNA.RNA is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP) and cytidine monophosphate (CMP) monomers or analogues thereof, which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e., ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e., the order of the bases linked to the sugar / phosphate-backbone, is called the RNA sequence. Usually, RNA may be obtainable by transcription of aDNA sequence, e.g., inside acell. In eukai otic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo, transcription of DN A usually results in the so-called premature RNA (also called pre-mRNA, precursor mRNA or heterogeneous nuclear RNA) which has to be processed into so-called messenger RNA, usually abbreviatedas mRNA. Processing of the premature RNA, e.g., in eukaryotic organisms, comprises a variety of different posttranscriptional modifications such as splicing, 5'-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA. The mature messenger RNA usually provides the nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein. Typically, a mature mRNA comprises a 5'-cap, optionally a 5' untranslated region ("5'UTR"), an open reading frame, optionally a 3' untranslated region ("3'UTR") and a poly(A) tail.

[0253] In addition to messenger RNA (mRNA). several non-coding types of RNA exist which may be involved in regulation of transcription and / or translation, and immunostimulation. Within the present disclosure the term "RNA" further encompasses any ty pe of single stranded (ssRNA) or double stranded RNA (dsRNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA). ribozymes, aptamers, riboswitches, immunostimulating / immunostimulatory RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and PlWI-interacting RNA (piRNA).

[0254] 5'-CAP-Structure: A 5'-CAP is typically a modified nucleotide (CAP analogue), particularly a guanine nucleotide, added to the 5' end of an mRNA molecule. In certain aspects, the 5 -CAP may be added using a 5'-5'-triphosphate linkage (also named m7GpppN). Further examples of 5'-CAP structures include glycery l, inverted deoxy abasic residue (moiety ), 4', 5' methylene nucleotide, l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide. 1,5-anhydrohexitol nucleotide. L-nucleotides. alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4- dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3 '-3 '-inverted abasic moiety, 3'-2'-inverted nucleotide moiety', 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3 -phosphate, 3'phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety. These modified 5'-CAP structures may be used in the context of the present disclosure to modify the RNA sequence of the present disclosure. Further modified 5 - CAP structures which may be used in the context of the present disclosure are CAP1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN). CAP2(additional methylation of the ribose of the 2nd nucleotide downstream of the nfZGpppN), CAP3 (additional methylation of the ribose of the 3rdnucleotide downstream of the m7GpppN), CAP4 (additional methylation of the ribose of the 4thnucleotide downstream of the m7GpppN), ARCA (anti-reverse CAP analogue), modified ARCA (e.g. phosphothioate modified ARCA), inosine, Nl -methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2- aminoguanosine, LNA-guanosine, and 2-azido-guanosine.

[0255] In the context of the present disclosure, a 5' cap structure may also be formed in chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping) using cap analogues, or a cap structure may be formed in vitro using capping enzymes (e.g., commercially available capping kits). A cap analogue refers to a non-polymerizable di-nucleotide that has cap functionality7in that it facilitates translation or localization, and / or prevents degradation of the RNA molecule when incorporated at the 5' end of the RNA molecule. Non-polymerizable means that the cap analogue will be incorporated only at the 5'terminus because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a templatedependent RNA polymerase.

[0256] Cap analogues include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG. m7GpppA, m7GpppC; unmethylated cap analogues (e.g., GpppG); dimethylated cap analogue (e.g., m2,7GpppG), trimethylated cap analogue (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g., m7Gpppm7G), or anti reverse cap analogues (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives). The synthesis of N7-(4- chlorophenoxy ethyl) substituted dinucleotide cap analogues is known.

[0257] A poly(A) tail also called "3'-poly(A) tail" or "Poly(A) sequence" is typically a long homopolymeric sequence of adenosine nucleotides of up to about 400 adenosine nucleotides, e.g. from about 25 to about 400, from about 50 to about 400, from about 50 to about 300, from about 50 to about 250, or from about 60 to about 250 adenosine nucleotides, added to the 3' end of an mRNA. In certain aspects, the poly(A) tail of an mRNA or srRNA is derived from a DNA template by RNA in vitro transcription. Alternatively, the poly(A) sequence may' also be obtained in vitro by common methods of chemical synthesis without being necessarily transcribed from a DNA-progenitor. Moreover, poly(A) sequences, or poly(A) tails may be generated by enzymatic polyadenylation of the RNA.

[0258] A stabilized nucleic acid, typically, exhibits a modification increasing resistance to in vivo degradation (e.g., degradation by an exo- or endo-nuclease) and / or ex vivo degradation (e.g., by the manufacturing process prior to composition administration, e.g., in the course of the preparation of the composition to be administered). Stabilization of RNA can, e.g.. be achieved by providing a 5'-CAP-Structure, a poly(A) tail, or any other UTR-modification. Stabilization can also be achieved by backbone-modification (e.g., use of synthetic backbones such as phosphorothioate) or modification of the G / C-content or the C-content of the nucleic acid. Various other methods are known in the art and conceivable in the context of the disclosure, to stabilize or otherwise improve the function of the nucleic acid. Provided herein, therefore, are polynucleotides which have been designed to improve one or more of the stability and / or clearance in tissues, receptor uptake and / or kinetics, cellular access, engagement with translational machinery. RNA half-life, translation efficiency, immune evasion, immune induction (for vaccines), protein production capacity, secretion efficiency (when applicable), accessibility to circulation, protein half-life and / or modulation of a cell's status, function and / or activity.

[0259] A 5'-UTR is typically understood to be a particular section of RNA. It is located 5' of the open reading frame of the mRNA. In the case of srRNA, the open reading frame encodes the viral non-structural proteins while the sequence of interest is encoded in the subgenomic fragment of the viral RNA. Thus, the 5’UTR is upstream of nsPl open reading frame. In addition, the subgenomic RNA of the srRNA has a 5'UTR. Thus, the subgenomic RNA containing a sequence of interest encoding a protein of interest contains a 5’UTR. Typically, the 5'-UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame. The 5'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory' elements may be, for example, ribosomal binding sites or a 5'-Terminal Oligopyrimidine Tract. The 5'-UTR may be post transcriptionally modified, for example by addition of a 5'-CAP. In the context of the present disclosure, a 5'UTR corresponds to the sequence of a mature mRNA or srRNA which is located between the 5'-CAP and the start codon. In one implementation, the 5'-UTR corresponds to the sequence which extends from a nucleotide located 3' to the 5'-CAP, and in certain aspects from the nucleotide located immediately 3' to the 5'-CAP, to a nucleotide located 5' to the start codon of the protein coding region and in some aspects to the nucleotide located immediately 5' to the start codon of the protein coding region. The nucleotide located immediately 3' to the 5'-CAP of a mature mRNA or srRNA typically corresponds to the transcriptional start site. The term"corresponds to" means that the 5'-UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 5'-UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In the context of the present disclosure, the term "a 5'- UTR of a gene’?is the sequence which corresponds to the 5'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the premature mRNA. The term "5'-UTR of a gene" encompasses the DNA sequence and the RNA sequence of the 5 -UTR. Generally, the term "3'-UTR" refers to a part of the nucleic acid molecule which is located 3' (i.e., "downstream") of an open reading frame and which is not translated into protein. Typically, a 3'-UTR is the part of an RNA which is located between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly (A) sequence of the mRNA.

[0260] In the context of the present disclosure, the term 3'-UTR may also comprise elements, which are not encoded in the template, from which an RNA is transcribed, but which are added after transcription during maturation, e.g., a poly(A) sequence. A 3'-UTR of the RNA is not translated into an amino acid sequence.

[0261] With respect to srRNA, the 3'-UTR sequence is generally encoded by the viral genomic RNA, which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into pre-mature mRNA. The pre-mature mRNA is then further processed into mature mRNA in a maturation process. This maturation process comprises 5'capping. In the context of the present disclosure, a 3'- UTR corresponds to the sequence of a mature mRNA or srRNA (and the srRNA subgenomic RNA), which is located between the stop codon of the protein coding region, preferably immediately 3' to the stop codon of the protein coding region for the sequence of interest, and the poly(A) sequence of the mRNA.

[0262] The term "corresponds to" means that the 3 -UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 3'-UTR sequence, or a DNA sequence, which corresponds to such RNA sequence. In the context of the present disclosure, the term "a 3 -UTR of a gene" is the sequence which corresponds to the 3 '-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term "3'-UTR of a gene" encompasses the DNA sequence and the RNA sequence (both sense and antisense strand and both mature and immature) of the 3'-UTR.

[0263] Polynucleotide Synthesis

[0264] Methods of making polynucleotides of a predetermined sequence are well-known. Solid-phase synthesis methods are known for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods of synthesizing DNA are also useful for synthesizing RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can be incorporated into the polynucleotide, as well.

[0265] Any method known in the art for making RNA is contemplated herein for making the RNAs. Illustrative methods for making RNA include but are not limited to, chemical synthesis and in vitro transcription.

[0266] In certain aspects, the RNA for use in the methods herein is chemically synthesized. Chemical synthesis of relatively short fragments of oligonucleotides with defined chemical structure provides rapid and inexpensive access to custom-made oligonucleotides of any desired sequence. Whereas enzymes synthesize DNA and RNA only in the 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it is most often carried out in the opposite, i.e., the 3' to 5' direction. In certain aspects, the process is implemented as solid-phase synthesis using the phosphorami dite method and phosphoramidite building blocks derived from protected nucleosides (A, C, G. and U), or chemically modified nucleosides.

[0267] In some implementations, modifications are included in the modified nucleic acid or in one or more individual nucleoside or nucleotide. For example, modifications to a nucleoside may include one or more modifications to the nucleobase, the sugar, and / or the intemucleoside linkage. In some implementations having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of: pseudouridine-alpha-thio-MP, 1 -methyl-pseudouridine-alpha-thio-MP, 1 -ethyl- pseudouridine-MP, 1-propyl-pseudouridine-MP, l-(2,2,2-trifluoroethyl)-pseudouridine-MP, 2-amino-adenine-MP, xanthosine-MP. 5-bromo-cytidine-MP. 5-aminoallyl-cytidine-MP. or 2- aminopurineriboside-MP.

[0268] In other implementations having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of: pseudouridine-alpha-thio-MP, 1-methyl-pseudouridine-alpha-thio-MP. or 5-bromo-cytidine- MP.

[0269] Nucleoside and nucleotide modifications contemplated for use in the present disclosure are known in the art. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain on a solid phase in the order required by the sequence of the product in a fully automated process. Upon the completion of the chain assembly, the product is released from the solid phase to the solution, deprotected, and collected. The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues), because the number of errors increases with the length of the oligonucleotide being synthesized. Products are often isolated by HPLC to obtain the desired oligonucleotides in high purity.

[0270] In certain aspects, RNA is made using in vitro transcription. The terms "RNA in vitro transcription" or "in vitro transcription" relate to a process wherein RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as template for the generation of RNA transcripts. RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which in certain aspects is a linearized plasmid DNA template. The promoter for controlling in vitro transcription can be any promoter for any DNA- dependent RNA polymerase. Particular examples of DNA-dependent RNA polymerases are the T7. T3. and SP6 RNA polymerases. A DNA template for in vitro RNA transcription may be obtained by cloning of a nucleic acid, in particular cDNA corresponding to the respective RNA to be in vitro transcribed, and introducing it into an appropriate vector for in vitro transcription, for example into plasmid DNA. In one implementation of the present disclosure, the DNA template is linearized with a suitable restriction enzyme, before it is transcribed in vitro. The cDNA may be obtained by reverse transcription of mRNA or chemical synthesis. Moreover, the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis.

[0271] Methods for in vitro transcription are known in the art. Reagents used in the methods ty pically include: 1) a linearized DNA template with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases; 2) ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); 3) in some aspects, a cap analogue as defined above (e.g. m7G(5')ppp(5')G (m7G)); 4) a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the linearized DNA template (e.g. T7, T3 or SP6 RNA polymerase); 5) optionally a ribonuclease (RNase) inhibitor to inactivate any contaminating RNase; 6) optionally apyrophosphatase to degrade pyrophosphate, which may inhibit transcription; 7) MgC12, which supplies Mg2+ ions as a co-factor for the polymerase; 8) a buffer to maintain a suitable pH value, which can also contain antioxidants (e.g. DTT), and / or polyamines such as spermidine at optimal concentrations.

[0272] Methods of making polynucleotides of a predetermined sequence are well-known. Solid-phase synthesis methods are known for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods of synthesizing DNA are also useful for synthesizing RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can be incorporated into the polynucleotide, as well. Any method known in the art for making RNA is contemplated herein for making the RNAs. Illustrative methods for making RNA include but are not limited to, chemical synthesis and in vitro transcription.

[0273] In certain aspects, the RNA for use in the methods herein is chemically synthesized. Chemical synthesis of relatively short fragments of oligonucleotides with defined chemical structure provides rapid and inexpensive access to custom-made oligonucleotides of any desired sequence. Whereas enzy mes synthesize DNA and RNA only in the 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it is most often earned out in the opposite, i.e., the 3' to 5' direction. In certain aspects, the process is implemented as solid-phase synthesis using the phosphoramidite method and phosphorami di te building blocks derived from protected nucleosides (A, C, G, and U), or chemically modified nucleosides.

[0274] In some implementations, modifications are included in the modified nucleic acid or in one or more individual nucleoside or nucleotide. For example, modifications to a nucleoside may include one or more modifications to the nucleobase, the sugar, and / or the intemucleoside linkage. In some implementations having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of: pseudouridine-alpha-thio-MP, 1 -methyl-pseudouridine-alpha-thio-MP, 1 -ethy 1- pseudouridine-MP, 1-propyl-pseudouridine-MP, l-(2,2,2-trifluoroethyl)-pseudouridine-MP, 2-amino-adenine-MP, xanthosine-MP, 5-bromo-cytidine-MP, 5-aminoallyl-cytidine-MP, or 2- aminopurineriboside-MP.

[0275] In other implementations having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase. sugar, and intemucleoside linkage of: pseudouridine-alpha-thio-MP, 1-methyl-pseudouridine-alpha-thio-MP, or 5-bromo-cytidine-MP. Nucleoside and nucleotide modifications contemplated for use in the present disclosure are known in the art.

[0276] To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain on a solid phase in the order required by the sequence of the product in a fully automated process. Upon the completion of the chain assembly, the product is released from the solid phase to the solution, deprotected, and collected. The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues), because the number of errors increases with the length of the oligonucleotide being synthesized. Products are often isolated by HPLC to obtain the desired oligonucleotides in high purity.

[0277] In certain aspects, RNA is made using in vitro transcription. The terms “RNA in vitro transcription"’ or “in vitro transcription"’ relate to a process wherein RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as template for the generation of RNA transcripts. RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which in certain aspects is a linearized plasmid DNA template. The promoter for controlling in vitro transcription can be any promoter for any DNA- dependent RNA polymerase. Particular examples of DNA-dependent RNA polymerases are the T7, T3, and SP6 RNA polymerases. A DNA template for in vitro RNA transcription may be obtained by cloning of a nucleic acid, in particular cDNA corresponding to the respective RNA to be in vitro transcribed and introducing it into an appropriate vector for in vitro transcription, for example into plasmid DNA. In one implementation of the present disclosure, the DNA template is linearized with a suitable restriction enzyme, before it is transcribed in vitro. The cDNA may be obtained by reverse transcription of mRNA or chemical synthesis. Moreover, the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis.

[0278] Methods for in vitro transcription are known in the art. Reagents used in the methods A pically include: 1) a linearized DNA template with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases; 2) ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); 3) in some aspects, a cap analogue as defined above (e.g. m7G(5')ppp(5')G (m7G)); 4) a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the linearized DNA template (e.g. T7, T3 or SP6 RNA polymerase); 5) optionally aribonuclease (RNase) inhibitor to inactivate any contaminating RNase; 6) optionally a pyrophosphatase to degrade pyrophosphate, which may inhibit transcription; 7) MgCh, which supplies Mg2+ ions as a co-factor for the polymerase; 8) a buffer to maintain a suitable pH value, which can also contain antioxidants (e.g. DTT), and / or polyamines such as spermidine at optimal concentrations.

[0279] mRNA-Based Therapeutics

[0280] In one aspect, an mRNA encoding for a polynucleotide, which may further encode for an amino acid sequence as described above, is disclosed. The mRNA may encode a heavy chain region of a multivalent antibody construct, the heavy chain region comprising (a) an antiprostate cancer antigen domain; (b) an anti hCD3 domain, and (c) a human IgG Fc domain, as disclosed herein.

[0281] In further aspect, an mRNA-based therapeutic is disclosed. In this aspect, a synthetic mRNA-based therapeutic (e.g., mRNA vaccines or mRNA encoding an antibody constructs) provide a template for the synthesis of proteins, protein fragments or peptides with the potential for significant benefit in a broad range of therapeutic applications.

[0282] Methods of Making and Multivalent Antibody Constructs

[0283] In one aspect, disclosed is a method for producing a multivalent antibody construct as disclosed herein, the method comprising a) transforming a host cell with vectors comprising polynucleotides encoding the bispecific antibody construct, b) culturing the host cell according under conditions suitable for the expression of the bispecific antibody construct and c) recovering the bispecific antibody construct from the culture.

[0284] Expression Vectors

[0285] In one aspect, disclosed is a vector, such as an expression vector, comprising one or more polynucleotides as disclosed herein. The term “expression cassette” refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In one aspect, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof. The term “vector” or “expression vector” may be used synonymously with “expression construct” and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a target cell. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. The expression vector may comprise an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and / or translation machinery. In one aspect, the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.

[0286] As used herein, an expression cassette includes a promoter sequence, an open reading frame, and a termination sequence. Expression cassettes may be configured for administration directly or to be encoded in one or more polynucleotides for expression in a cell and may be encoded in DNA, RNA, or mRNA for administration. In some aspects, the expression cassette may comprise a first mRNA encoding for a first binding region, a second mRNA encoding for a second binding region, and / or a third mRNA encoding a third region, and / or a fourth mRNA encoding for a fourth binding region.

[0287] For example, in one aspect, the cassette may include a first isolated mRNA having at least about 80%, or at least about 85%, or at least about 90% or at least about 95%, or at least 100% to sequence identity to SEQ ID NO: 10 and a second isolated mRNA having at least about 80% , or at least about 85%, or at least about 90% or at least about 95%, or at least 100% to sequence identity to SEQ ID NO: 28. In this aspect, a representative antibody construct encoded by the cassette is Ml 107 (see FIG 8A).

[0288] For example, in one aspect, the cassette may include a first isolated mRNA having at least about 80%, or at least about 85%, or at least about 90% or at least about 95%, or at least 100% to sequence identity' to SEQ ID NO: 10 and a second isolated mRNA having at least about 80% , or at least about 85%, or at least about 90% or at least about 95%, or at least 100% to sequence identity to SEQ ID NO: 33. In this aspect, a representative antibody construct encoded by the cassette is Ml 108 (see FIG 8B).

[0289] For example, in one aspect, the cassette may include a first isolated mRNA having at least about 80%, or at least about 85%, or at least about 90% or at least about 95%, or at least 100% to sequence identity to SEQ ID NO: 10 and a second isolated mRNA having at least about 80% , or at least about 85%, or at least about 90% or at least about 95%, or at least 100% to sequence identity to SEQ ID NO: 34. In this aspect, a representative antibody construct encoded by the cassette is Ml 109 (see FIG 8C).

[0290] For example, in one aspect, the cassette may include a first isolated mRNA having at least about 80%, or at least about 85%, or at least about 90% or at least about 95%, or at least 100% to sequence identity to SEQ ID NO: 1 1 and a second isolated mRNA having at least about 80% , or at least about 85%, or at least about 90% or at least about 95%, or at least 100% to sequence identity to SEQ ID NO: 28. In this aspect, a representative antibody construct encoded by the cassette is Ml 110 (see FIG 8D).

[0291] For example, in one aspect, the cassette may include a first isolated mRNA having at least about 80%, or at least about 85%, or at least about 90% or at least about 95%, or at least 100% to sequence identity to SEQ ID NO: 19; a second isolated mRNA having at least about 80% , or at least about 85%, or at least about 90% or at least about 95%, or at least 100% to sequence identity to SEQ ID NO: 40; and a third isolated mRNA having at least about 80%, or at least about 85%, or at least about 90% or at least about 95%, or at least 100% sequence identity7to SEQ ID NO: 33. In this aspect, a representative antibody construct encoded by the cassette is Ml 093 (see FIG 9A).

[0292] In some aspects, a cassette including the one or more mRNA is at least partially encapsulated by a delivery vehicle. In another aspect, a multimodal mRNA therapeutic may include at least two or three of the disclosed cassettes.

[0293] Delivery Vehicles

[0294] The delivery vehicle complexes disclosed herein can be used to deliver the nucleic acid compositions, such as a nucleic acid (e.g., RNA) to a cell. In one aspect, the method may comprise contacting the cell with the delivery vehicle complex or pharmaceutical composition disclosed herein. In some aspects, the cell may be obtained from a subject for whom treatment is desired. In some aspects, the cell is a tumor cell.

[0295] In some aspects, the nucleic acid composition may be delivered for therapeutic uses. As used herein, "delivery vehicle” refers to any substance that facilitates, at least in part, the invivo, in vitro, or ex vivo delivery of a polynucleotide (e.g., therapeutic polynucleotide) to targeted cells or tissues (e.g., tumors, etc.). Referring to something as a delivery7vehicle need not exclude the possibility of the delivery vehicle also having therapeutic effects. Some versions of a delivery vehicle may provide additional therapeutic effects. In some versions, a delivery vehicle may be a peptoid molecule, such as an amino-lipi dated peptoid molecule, that may be used to at least partially encapsulate mRNA. The term "DV " may also be used herein as a shorthand for “delivery7vehicle.” In some aspects, the mRNA for use in the delivery vehicle complexes herein comprise an mRNA comprising at least one region encoding a peptide (e.g., a polypeptide), or protein, or functional fragment as described herein. As used herein, “functional fragment” refers to a fragment of a peptide, (e.g., a polypeptide), or protein that retains the ability to induce an immune response.

[0296] The disclosed delivery vehicle compositions useful for the delivery of one or more polynucleotides or amino acids disclosed herein may comprise hydroxyethyl-capped cationic peptoids, including, for example, hydroxyethyl-capped tertiary amino lipidated cationic peptoids. The delivery vehicle compositions of the disclosure can form an electrostatic interaction between the hydroxyethyl-capped tertiary amino lipidated cationic peptoids of the delivery vehicle composition and a polyanionic compound, such as a nucleic acid, to form a delivery vehicle complex, wherein the polyanionic compound functions as the cargo of the complex. The delivery7vehicle complex is useful for the delivery of polyanionic compounds, such as nucleic acids (e.g., mRNA), into cells. When the mRNA of the delivery7vehicle complex encodes, e.g., for a viral antigen, the delivery vehicle complexes can elicit humoral and cellular immune responses in vivo, thus functioning as a vaccine. The delivery vehicle complexes disclosed herein are further advantages in that they are stable, and demonstrate good tolerability7and low toxicity7.

[0297] In one aspect, a composition as described herein may be formulated with a vehicle, a delivery agent, a delivery vehicle molecule, or a delivery7vehicle composition to make delivery vehicle complexes or pharmaceutical formulations. Such polyanionic compounds, e.g., polynucleotides disclosed herein, may also be referred to as poly anionic cargo compounds or cargos of a delivery7vehicle complex (also referred to as a multicomponent delivery system), which complex or system also includes delivery vehicle compositions.

[0298] In some aspects, the deliver}' vehicle molecule or delivery vehicle composition may comprise a peptoid, a lipoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric compound, or a conj ugate.

[0299] In some aspects, the delivery vehicle molecule or delivery vehicle composition is suitable for use with systemic delivery, for example, where the delivery vehicle molecule or delivery vehicle composition is preferably taken up and / or expressed by the liver. Examples of deliver}' vehicles having such characteristics are known in the art.

[0300] Lipid Nanoparticles

[0301] In some aspects, the delivery vehicle molecule or delivery vehicle composition comprises lipid nanoparticles (LNPs), such as cationic lipid nanoparticles. Exemplary cationic lipid nanoparticles are described, for example, in W02020 / 219941 and W02020 / 097548, each of which is incorporated herein by reference.

[0302] Peptoid-based Delivery Vehicles

[0303] As used herein, "peptoid” refers to a peptidomimetic compound in which one or more of the nitrogen atoms of the peptide backbone are substituted with side chains. As used herein, “lipidated peptoid” refers to a peptoid in which one or more of the side chains on the nitrogen atom comprises a lipid. As used herein, “polyanionic’' refers to a compound having at least two negative charges, such as nucleic acids. Peptoids are a unique class of N-substituted alphaamino acids structurally related to peptides, but which incorporate their diverse side chain functionality on the amide nitrogen rather than alpha carbon allowing for modular synthesis. In a non-limiting embodiment, peptoids can be synthesized by the submonomer approach wherein repeating cycles of acylation using bromoacetic acid and nucleophilic addition of primary amines can be accomplished on a solid support, producing peptoids with high yield and fidelity7. Peptoids have been successfully used in many applications including anti-fouling and anti-bacterial agents, to drug delivery and anti-freeze additives in tissue storage, and even as complexing agents for nucleic acids, but they have never been explored as the ionizable component of a lipid nanoparticle for mRNA delivery.

[0304] Exemplary delivery vehicle compositions suitable for use with the instant invention comprise one or more hydroxyethyl-capped tertiary amino lipidated cationic peptoids. These positively charged peptoids can associate with a polyanionic compound, such as a nucleic acid, to form a delivery vehicle complex. In some aspects, the delivery vehicle compositions furthercomprise one or more of an anionic or zwitterionic component, such as a phospholipid; a neutral lipid, such as a sterol; and a shielding lipid, such as a PEGylated lipid. In various aspects, the delivery vehicle compositions further comprise an anionic or zwitterionic component (e.g.. a phospholipid), a neutral lipid (e.g., a sterol), and a shielding lipid (e.g., a PEGylated lipid). In some aspects, the delivery' vehicle compositions consist essentially of a hydroxyethyl-capped tertiary' amino lipidated cationic peptoid, an anionic or zwitterionic component (e.g., a phospholipid), a neutral lipid (e.g., a sterol), and a shielding lipid (e.g., a PEGylated lipid).

[0305] For instance, the delivery vehicle may be formed with, for example, an amino lipidated peptide that may include tertiary amino lipidated cationic peptides, such as any of those described in PCT application, PCT / US19 / 53661, titled “LIPID NANOPARTICLE FORMULATIONS COMPRISING LIPIDATED CATIONIC PEPTIDE COMPOUNDS FOR NUCLEIC ACID DELIVERY ”, filed on September 27. 2019, and in PCT / US 19 / 53655, titled “TERTIARY AMINO LIPIDATED CATIONIC PEPTIDES FOR NUCLEIC ACID DELIVERY” filed on September 27, 2019, the contents of each of which are incorporated herein by reference in their entirety. The nanoparticle delivery vehicle may comprise additional lipids / components. For example, the amino lipidated peptides can include one or more phospholipids, e.g., MSPC or DSPC. The lipid composition can also comprise a quaternary amine compound such as DOTAP. In some aspects, the delivery vehicle has a particle size less than or equal to about 200 nm.

[0306] When formulated with mRNA. DSPC, Cholesterol and DMG-PEG, delivery’ vehicles are generated and characterized for physical properties and biological activity. Through iterative synthesis and particle screening, the relationship between peptoid structure and delivery' vehicle activity' can be explored.

[0307] Delivery vehicle compositions useful for delivering mRNA encoding multivalent T- cell engagers of the disclosure may comprise hydroxyalkyl-capped cationic peptoids, such as 2-aminopropane-l,3-diol-capped cationic peptoids (“cationic component”, sometimes referred to as an "ionizable lipid"). In some implementations, the hydroxyalkyl-capped cationic peptoids comprise a compound of Formulawherein n is n is1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R1 is H or C2-5 alkyl optionally substituted with 1-3 OH; R2 is H or C2-5alk lene-OH substituted with 1-3 additional OH; and each R3 independently is C6- 24alkyl or C6-24alkenyl. As used herein, “alkyl’" refers to straight chained and branched saturated hydrocarbon groups containing one to thirty carbon atoms, for example, one to twenty four carbon atoms (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 carbon atoms). The term Cn means the alkyl group has “n” carbon atoms. For example, C3 alkyl refers to an alkyl group that has 3 carbon atoms. Cl-24alkyl refers to an alkyl group having a number of carbon atoms encompassing the entire range (i.e., 1 to 24 carbon atoms), as well as all subgroups. Nonlimiting examples of alkyl groups include, methyl, ethyl, npropyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), and / -butyl (1,1 -dimethylethyl). Unless otherwise indicated, an alky l group can be an unsubstituted alkyl group or a substituted alkyl group. As used herein, “alkenyl” refers to straight chained and branched hydrocarbon groups having a double bond and containing two to thirty carbon atoms, for example, two to twenty four carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, or 24 carbon atoms). The term Cn means the alkenyl group has “n” carbon atoms. For example. C3 alkenyl refers to an alkenyl group that has 3 carbon atoms. C2-C24 alkenyl refers to an alkenyl group having a number of carbon atoms encompassing the entire range (i.e., 2 to 24 carbon atoms), as well as all subgroups. Nonlimiting examples of alkenyl groups include ethenyl, propenyl, butenyl, geranyl, and oleyl. Unless otherwise indicated, an alkenyl group can be an unsubstituted alkenyl group or a substituted alkenyl group.

[0308] In some implementations, n is 2 to 5. In various implementations, n is 3 to 4. In some implementations, n is 1. In various implementations, n is 2. In some cases, n is 3. In various cases, n is 4. In some implementations n is 5. In various implementations, n is 6. In various cases, n is 7. In various cases, n is 8. In various cases, n is 9. In various cases, n is 10.

[0309] In some implementations, R1 is H. In various implementations, R1 is C2-5alkyl optionally substituted with 1-3 OH. In some cases, R1 is methyl or ethyl. In some implementations, R1 is ethyl. In various implementations, R1 is C2-5alkylene-OH substituted with 0-2 additional OH. In some cases, R1 is(hydroxyethyl). In various cases, R1 is ethyl or hydroxy ethyl. In some cases, C2-5alkyl is substituted with 1 OH. In some cases, C2-5alkyl is substituted with 2 OH. In some cases, C2-5alkyl is substituted with 3 OH.

[0310] R2 is C2-5alkylene-OH substituted with 1-3 additional OH. In some cases, R2 is C2alkylene-OH substituted with 1-3 additional OH. In some cases, R2 is C3alkylene-OH substituted with 1-3 additinoal OH. In some cases, R2 is C4alkylene-OH substituted with 1-3 additional OH. In some cases, R2 is C5alkylene-OH substituted with 1-3 additional OH. In some cases, C2-5alkylene-OH is substituted with 1 additional OH. In some cases, C2- 5alkylene-OH is substituted with 2 additional OH. In some cases, C2-5alkylene-OH is substituted with 3 additional OH. In some cases, R2 is propyl-l,3-diol. In some cases, R2 is

[0311] In some implementations, each R3 independently is C8-18alkyl or C8-18alkenyl. In various implementations, each R3 independently is C8-16alkyl or C10-18alkenyl. In some cases, each R3 independently is C6-18alkyl or C6-18alkenyl. In some cases, each R3 independently is C10-12alkyl or C10-18alkenyl. In some implementations, each R3 independently is: C8-18alkyl, or C8-16alkyl, or C8-14alkyl, or C8-12alkyl. In various implementations, each R3 independently is selected from the group consisting ofcases, each R3 independently is selected from the group consisting ofIn various cases, each R3 independently is selected from the group consisting ofsome implementations, each R3 independently isContemplated compounds of Formula (I) include, but are not limited to, the compounds listed in Table 4:

[0312] Table 4. Examples of hydroxyalkyl-capped cationic peptoids.

[0313] In some implementations the compound of Formula (I) is compound 1, 6, 21, or 30. In some cases, the compound of Formula (I) is compound 1. In some cases, the compound of Formula (I) is compound 6. In some cases, the compound of Formula (I) is compound 21. In some cases, the compound of Formula (I) is compound 30. In some cases, the compound of Formula (I) is compound 41.

[0314] In implementations, the delivery' vehicle composition may comprise between about 25 mol% to about 70 mol% of the hydroxyalkyl-capped cationic peptoids, such as 2- aminopropane-1.3-diol-capped cationic peptoids (e.g., a compound of Formula (I), such as compound 1 , 6, 21 , or 30), based on the total number of moles of components in the delivery vehicle composition. The unit “mol%” or “molar percentage” refers to the number of moles of a particular component of the delivery vehicle composition divided by the total number of moles of all components in the delivery vehicle composition, times 100%. The poly anionic cargo is not calculated as part of the total number of moles of the delivery vehicle composition. In some cases, the delivery vehicle composition comprises between about 30 mol% to about 60 mol%, or about 35 mol% to about 55 mol%, or about 30 mol% to about 45 mol%, or about 35 mol% to about 40 mol%, or about 45 mol% to about 60 mol%, or about 50 mol% to about 55 mol%, or about 38 mol% to about 52 mol%, or about 38 mol%, or about 52 mol% of the hydroxyalkylcapped cationic peptoids, such as 2-aminopropane-l,3-diol-capped cationic peptoids (e.g., a compound of Formula (I), such as compound 1, 6, 21, or 30), based on the total number of moles of components in the delivery' vehicle composition.

[0315] Delivery Vehicles

[0316] Delivery vehicles, or “DV’‘s, may comprise one or more polyanionic compounds, and lipid components, wherein the lipid components comprise one or more lipidated cationic peptide compounds, one or more phospholipids, one or more shielding lipids, and optionally one or more structural lipids. The physical properties of the DVs and complexes described herein may be influenced by the particular selection of lipid components for a given poly anionic compound as well as by the quantities of each component within the DVs and complexes. In some embodiments, the lipid components within the DVs and complexes thereof may be characterized by the mass percentages of the lipid components (alone or in combination) with respect to mass of the total lipid components present and / or mass ratios of individual lipid components with respect to one another.

[0317] In some embodiments, the DVs are characterized by the mass percentages of the lipid components present. As described herein, the total mass or weight of the lipid components is the sum of the individual masses of any lipidated cationic peptide compounds, any structural lipids, any phospholipids, and any shielding lipids present.

[0318] In some embodiments, the DVs or compositions as described herein comprise complexes comprising one or more lipidated cationic peptide compounds. In some variations of the foregoing, the compositions and complexes therein comprise 40-80% w / w one or more lipidated cationic peptide compounds of the total weight of the lipid components. In certain variations, compositions and complexes therein comprise 40-70% w / w one or more lipidated cationic peptide compounds of the total weight of the lipid components. In other variations of the foregoing, the compositions and complexes therein comprise 40-80% w / w one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of Formula (I) or salts thereof of the total weight of the lipid components. In certain variations, the compositions and complexes therein comprise 40-70% w / w one or more tertiary' amino lipidated and / or PEGylated cationic peptide compounds of Formula (I) or salts thereof of the total weight of the lipid components. As described herein, the DVs or compositions comprising complexes may optionally include one or more structural lipids. In some embodiments which may be combined with any of the foregoing embodiments, the compositions and complexes therein comprise 0- 25% w / w one or more structural lipids of the total weight of the lipid components. In certain variations, the compositions and complexes therein comprise 0-25% w / w cholesterol of the total weight of the lipid components.

[0319] The DVs and complexes as described herein also comprise one or more phospholipids. In still other embodiments, the DVs and complexes therein comprise 10-60% w / w one or more phospholipids of the total weight of the lipid components. In certain variations, the DVs and complexes therein comprise 20-40% w / w one or more phospholipids of the total weight of the lipid components. In some embodiments, the DVs and complexes therein comprise 10-60% w / w l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) of the total weight of the lipid components. In certain embodiments, the DVs and complexes therein comprise 20-40% w / w l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) of the total weight of the lipid components.

[0320] In further embodiments, the DVs and complexes therein comprise one or more shielding lipids. In some embodiments, the DVs and complexes therein comprise 1-5% one or more shielding lipids of the total weight of the lipid components. In certain embodiments, the DVs and complexes therein comprise 1-5% one or more PEG lipids of the total weight of the lipid components. In still other embodiments, the DVs and complexes therein comprise 1-5% l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) of the total weight of the lipid components.

[0321] In still other embodiments, the DVs and complexes therein of the present disclosure comprise 40-80% w / w one or more hpidated cationic peptide compounds; 0-25% w / w one or more structural lipids; 10-60% w / w phospholipids; and 1-5% one or more shielding lipids of the total w eight of the lipid components. In yet further embodiments, the DVs and complexes therein of the present disclosure comprise 40-70% w / w one or more lipidated cationic peptide compounds; 0-25% w / w one or more structural lipids; 20-40% w / w phospholipids; and 1-5% one or more shielding lipids of the total weight of the lipid components.

[0322] In other embodiments, the DVs and complexes therein of the present disclosure comprise 40-80% w / w one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of Formula (I) or salts thereof; 0-25% w / w cholesterol; 10-60% w / w DOPE; and 1 -5% DMG-PEG2000 of the total weight of the lipid components. In yet further embodiments, the DVs and complexes therein of the present disclosure comprise 40-70% w / w one or more tertiary amino hpidated and / or PEGylated cationic peptide compounds of Formula (I) or salts thereof; 0-25% w / w cholesterol: 20-40% w / w DOPE; and 1-5% DMG-PEG2000 of the total w eight of the lipid components.

[0323] In some embodiments wherein the complex comprises a lipidated cationic peptide compound, a nucleic acid, a phospholipid, a PEG lipid, and optionally also a structural lipid, the complex may be characterized by the mass ratio of the individual components to each other, or a mass ratio of one or more components to the one or more other components. For example, in some embodiments, the DVs and complexes of the present disclosure may be described by a first mass ratio of the lipidated cationic peptide compound to the phospholipid to the structural lipid (if present) to the shielding lipid and a second mass ratio of the lipidated cationic peptide compound to nucleic acid. Alternatively, in other embodiments, the DVs and complexes provided herein may be described by a mass ratio of the total lipid components to nucleic acid, wherein the total lipid components include any lipidated cationic peptide compounds, structural lipids, phospholipids, and shielding lipids present.

[0324] In another aspect, the complexes and DVs can be characterized by mass ratios of individual components to one another. For example, as described herein, the complexes of the present disclosure comprise one or more phospholipids and optionally one or more structural lipids. In some embodiments wherein the composition comprises complexes comprising one or more structural lipids and one or more phospholipids, the composition can be characterized by the mass ratio of the one or more structural lipids to the one or more phospholipids. In some embodiments, the mass ratio of the one or more structural lipids, when present, to the one or more phospholipids in between 0.5: 1 and 2: 1. In certain embodiments wherein the composition comprises cholesterol and DOPE, the mass ratio of cholesterol to DOPE is between 0.5:1 and 2: 1.

[0325] With regard to the poly anionic compounds present in the DVs and complexes of the present disclosure, the quantity of polyanionic compounds within the complexes, and therefore also the compositions, may be characterized in a number of ways. In some embodiments, the DVs and complexes described herein may be characterized by the ratio of the number cationic groups on the lipidated cationic peptide compounds to the number of anionic phosphate groups on the nucleic acid. In some embodiments, the complex comprises the lipidated cationic peptide compound and the nucleic acid at a cation: anion charge ratio of between 0.5: 1 and 20: 1, between 0.5: 1 and 10: 1, between 0.5: 1 and 5:1, between 1 :1 and 20: 1, between 1 : 1 and 10: 1, between 1 : 1 and 5: 1. between 2: 1 and 20: 1, between 2: 1 and 10: 1, or between 2:1 and 5: 1. In certain embodiments, the complex comprises the lipidated cationic peptide compound and the nucleic acid at a cation: anion charge ratio of between 2: 1 and 5: 1. In still yet otherembodiments, the complex comprises the lipidated cationic peptide compound and the nucleic acid at a cation: anion charge ratio of 3: 1.

[0326] Alternatively, the DVs and complexes comprising the complexes described herein may be characterized by the relative mass ratio of the lipidated cationic peptide compound(s) to the polyanionic compound(s) and / or other cargoes in the complex. Mass ratios of the components in the complex can be readily calculated based upon the know n concentrations and volumes of stock solutions of each component used in preparing the complex. Moreover, if non-anionic cargoes are present in the complex, mass ratios may provide a more accurate representation of the relative amounts of lipidated cationic peptide compound to the overall cargo than cation:anion charge ratios, which do not account for non-anionic material.

[0327] In some embodiments, the complex comprises the one or more lipidated cationic peptide compounds and one or more polyanionic compounds and / or non-anionic compounds at a mass ratio of between 0.5: 1 and 20: 1. between 0.5: 1 and 10: 1, between 0.5: 1 and 5: 1, between 1: 1 and 20: 1, between 1: 1 and 10: 1, between 1: 1 and 5: 1, between 2: 1 and 20: 1, between 2: 1 and 10:1, or between 2: 1 and 5: 1. In certain embodiments, the complex comprises the one or more lipidated cationic peptide compounds and the one or more polyanionic compounds and / or non-anionic compounds at a mass ratio of between 2: 1 and 5: 1. In still yet other embodiments, the complex comprises the one or more lipidated cationic peptide compounds and the one or more polyanionic compounds and / or non-anionic compounds at a mass ratio of 3: 1.

[0328] In certain embodiments wherein the complex comprises a nucleic acid, the complex comprises the lipidated cationic peptide compound and the nucleic acid at a mass ratio of between 0.5: 1 and 20: 1, between 0.5: 1 and 10: 1, between 0.5: 1 and 5: 1, between 1 : 1 and 20: 1, between 1 :1 and 10: 1, between 1:1 and 5: 1, between 2: 1 and 20: 1, between 2: 1 and 10: 1, or between 2: 1 and 5: 1. In certain embodiments, the complex comprises the lipidated cationic peptide compound and the nucleic acid at a mass ratio of between 2: 1 and 5: 1. In still yet other embodiments, the complex comprises the lipidated cationic peptide compound and the nucleic acid at a mass ratio of 3 : 1.

[0329] In still other embodiments, the amount of poly anionic compounds present in the DVs and complexes thereof may be characterized by a mass ratio of the lipid components (lipidated cationic peptide compound, phospholipid, shielding lipid, and structural lipid if present) to the one or more polyanionic compounds. In some embodiments, the mass ratio of the lipidcomponents to the one or more polyanionic compounds is between 0.5:1 and 20:1, between 0.5: 1 and 10:1, between 0.5:1 and 5: 1, between 1: 1 and 20: 1, between 1 : 1 and 10: 1, between 1 : 1 and 5: 1, between 2: 1 and 25: 1, between 2: 1 and 20: 1, between 2: 1 and 10: 1, or between 2: 1 and 5: 1. In certain embodiments, the mass ratio of the lipid components to the one or more polyanionic compounds is between 5: 1 and 10: 1 or between 6: 1 and 7: 1.

[0330] The complexes of the present disclosure will comprise at least one lipidated cationic peptide compound complexed with at least one polyanionic compound or other suitable cargo compound. In some embodiments, the complexes described herein may comprise one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of Formula (I). The use of mixtures and combinations of multiple lipidated cationic peptide compounds of the present disclosure in a single complex may enable the preparation of formulations tailored for specific pharmacokinetic and pharmacodynamics properties. Pharmacokinetic and pharmacodynamics properties of relevance may include but are not limited to biodistribution, immunogenicity, formulation stability, encapsulation percentage, transfection efficiency, plasma half-life, etc. Different combinations of these properties may be required for different applications.

[0331] For example, in certain embodiments, the complex may comprise a combination of one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds which are cation-rich with one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds which are highly lipidated. In theory, such a combination could confer improved delivery7of polyanionic compounds by providing greater charge stabilization (via the cationrich peptide compounds) along with greater lipophilic shielding (by virtue of the lipidated peptide compounds). It should further be recognized that the individual amounts of each of the individual lipidated cationic peptide compounds may be adjusted to achieve the desired properties.

[0332] Components of the DV complex can be prepared through a variety of physical and / or chemical methods to modulate their physical, chemical, and biological properties. These may involve rapid combination of the hydroxyethyl-capped tertiary amino lipidated cationic peptoids in water or a water-miscible organic solvent with the desired polyanionic cargo compound (e.g., oligonucleotides or nucleic acids) in water or an aqueous buffer solution. These methods can include simple mixing of the components by pipetting, or microfluidic mixing processes such as those involving T-mixers, vortex mixers, or other chaotic mixingstructures. In some aspects, the multicomponent delivery system is prepared on a microfluidic platform.

[0333] It is to be understood that the particular process conditions for preparing the delivery vehicle complexes described herein may be adjusted or selected accordingly to provide the desired physical properties of the complexes. For example, parameters for mixing the components of the delivery system complex that may influence the final compositions may include, but are not limited to, order of mixing, temperature of mixing, mixing speed / rate. flow rate, physical dimensions of the mixing structure, concentrations of starting solutions, molar ratio of components, and solvents used.

[0334] Formulation of the delivery vehicle complexes can be accomplished in many ways. In some aspects, all components can be pre-mixed prior to addition of the nucleic acid cargo, which can result in a uniform distribution of components throughout the delivery particle.

[0335] Exemplary mixing methods are detailed in, e.g., US Patent Numbers 11,278,895 and 1 1,325,122, incorporated herein by reference.

[0336] In other aspects, the components can be added sequentially to produce a core-shell type structure. For example, a cationic component could be added first to begin particle condensation, followed by a lipid component to allow the particle's surface to associate with target cells, followed by a shielding component to prevent particle aggregation. For example, the hydroxyethyl-capped tertiary amino lipidated cationic peptoid can be premixed with the nucleic acid cargo to form a core structure. Then, the lipid components (such as lipid components comprising phospholipids and cholesterol) can be added to influence cell / endosomal membrane association. Because the shielding component is primarily useful on the outside of the multicomponent delivery system, this component can be introduced last, so that it does not disrupt the internal structure of the system, but rather provides a coating of the system after it is formed.

[0337] Additional components in the complexes and composition, such as the additional components of polymers, surface-active agents, targeting moieties. and / or excipients, may be admixed and combined with the rest of the components before, during, or after the principal components of the nucleic acid cargo, the cationic component, the lipid component and the shielding component have been combined.

[0338] In some aspects, the deliver}’ vehicle molecule or delivery vehicle composition comprises peptoids, such as tertiary’ amino lipidated and / or PEGylated cationic peptoids. Exemplary cationic peptoids are described, for example, in WO 2020 / 069442, WO 2020 / 069445, WO 2021 / 030218, WO 2022 / 32058, and W02023 / 014931, each of which is incorporated herein by reference.

[0339] Some example delivery vehicle compositions of the disclosure comprise one or more hydroxyalkylcapped cationic peptoids. such as 2-aminopropane-l,3-diol-capped cationic peptoids. These positively charged peptoids can associate with a polyanionic compound, such as a nucleic acid, to form a delivery vehicle complex. In some implementations, the delivery vehicle compositions further comprise one or more of an anionic or zwitterionic component, such as a phospholipid; a neutral lipid, such as a sterol; and a shielding lipid, such as a PEGylated lipid. In various implementations, the delivery vehicle compositions further comprise an anionic or zwitterionic component (e.g.. a phospholipid), a neutral lipid (e.g., a sterol), and a shielding lipid (e g., a PEGylated lipid). In some cases, the delivery vehicle compositions consist essentially of hydroxyalkyl-capped cationic peptoids, such as 2- aminopropane-l,3-diol-capped cationic peptoids, an anionic or zwitterionic component (e.g., a phospholipid), a neutral lipid (e.g., a sterol), and a shielding lipid (e.g., a PEGylated lipid).

[0340] Pharmaceutical / Therapeutic Compositions

[0341] In one aspect, disclosed is a therapeutic composition comprising a therapeutic agent. The therapeutic agent may comprise a polynucleotide, such as a synthetic mRNA, a polypeptide, or combination s thereof. The therapeutic agent may be a multivalent antibody construct as disclosed herein. The therapeutic agents may be combined with at least one pharmaceutically acceptable excipient to form a therapeutic composition. The therapeutic composition may optionally include one or more therapeutically acceptable carriers, diluents, or excipients such as salts, buffering agents, preservatives, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film formers or coatings, flavors, fragrances, glidants, lubricants, sorbents, suspending or dispersing agents, sweeteners, waters of hydration, and / or other therapeutic agents. As used herein, the term ‘‘excipient” means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API) (and typically in addition to components of the delivery vehicle compositions), suitably selected with respect tothe intended form of administration, and consistent with conventional pharmaceutical practices.

[0342] Methods of Treatment

[0343] In some aspects, the disclosed composition is configured to be administered to a subject in need thereof. In one aspect, disclosed is a method comprising administering at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight or more polynucleotides as described above. In one aspect, disclosed is a method comprising administering a synthetic mRNA as described above. In one aspect, disclosed is a method compnsing administering a pharmaceutical composition compnsing a polynucleotide, such as a synthetic mRNA, and / or a recombinant protein as described above. Such administration is to an individual in need thereof, for use in treating a prostate cancer, for example, a prostate cancer characterized by increased expression of one or more of an Prostate Specific Membrane Antigen (PSMA), a Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1), KLK2, or combinations thereof.

[0344] In one aspect, disclosed is a method comprising administering a pharmaceutical composition comprising a polynucleotide as disclosed herein, to an individual in need thereof, for use in treating prostate cancer, wherein the composition is administered in combination with an additional therapeutic agent.

[0345] In one aspect, the additional therapeutic agent may be a chemotherapeutic agent, radiation and / or other agents for use in cancer immunotherapy. In other aspects, the additional therapeutic agent may be one or more of a chemotherapy, a Cytokine Release Syndrome (CRS) Management active (such as tocilizumab (Actemra) or siltuximab (Sylvant)), a corticosteroid or other immunosuppressive agent, an antibiotic, an antiviral, and combinations thereof.

[0346] In some aspects, the multivalent antibody construct and the additional therapeutic agent may be administered simultaneously. In other aspects, the multivalent antibody construct and the additional therapeutic agent may be administered sequentially. In some aspects, the multivalent antibody construct may be administered to the subject prior to administering the additional therapeutic agent. In some aspects, the additional therapeutic agent may be administered to the subject prior to administering the multivalent antibody construct. In some aspects, the multivalent antibody construct and the additional therapeutic agent may beadministered as a combination. In some aspects, the multivalent antibody construct and the additional therapeutic agent may be administered as separate dosage forms.

[0347] Routes of Administration

[0348] The compositions, including the polynucleotides, polypeptides, multivalent antigen constructs, delivery vehicle complexes, and the therapeutic compositions described herein can be administered to a subject or patient by any suitable route, “Administering”, as used herein, is meant a method of giving a dosage of a compound or a composition to an individual. The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously. intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularily, mucosally. intrapericardially, intraumbilically. intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

[0349] Injectable Formulations

[0350] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables. Exemplary buffers include citrate, succinate, acetate, malate, succinate, and histidine. In addition, stabilizers such as sucrose, may be included. For example, the therapeutic compositions may be suspended in a sucrose-containing citrate buffer at a pH between pH 5 and pH 6, e.g., at about pH 5.5.

[0351] Injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable media prior to use. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to the compositions.

[0352] To prolong the effect of active ingredients, it is often desirable to slow' the absorption of active ingredients from subcutaneous or intramuscular injections. This may be accomplished using liquid suspensions of crystalline or amorphous material with poor water solubility. The rate of absorption of active ingredients depends upon the rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. Microorganism contamination can be prevented by adding various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like.

[0353] Dosages

[0354] The compounds and / or compositions of the disclosure can be administered to a subject or patient at predetermined dosage level, in an amount effective to achieve the desired response, for example, a therapeutic response that including amelioration of a disease or condition such as prostate cancer, an improvement in the health of an individual having prostate cancer, a slowing or stopping of the progression of a patient's disease, and / or the avoidance of occurrence or re-occurrence of prostate cancer, as a result of the administration of an antibody construct as described herein.

[0355] Therapeutic Regimens

[0356] In some aspects, one or more pharmaceutical compositions described herein comprising the multivalent antibody constructs may be administered for therapeuticapplications. In some aspects, the pharmaceutical composition may be administered once per day, twice per day, three times per day, or more. The pharmaceutical composition may be administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or more. The pharmaceutical composition may be administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months. 2 years, 3 years, or more.

[0357] Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary . Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.

[0358] In some aspects, the amount of a given agent that correspond to such an amount varies depending upon factors such as the particular compound, the severity of the disease, the identity (e g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In some aspects, the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[0359] The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages may be altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0360] Kits and Articles of Manufacture

[0361] Disclosed herein are kits and articles of manufacture suitable for carrying out the methods disclosed herein. In some aspects, the kit may comprise two or more components required for performing a therapeutic method described herein. In some aspects, kit components include, but are not limited to. one or more multivalent antibody constructs, or composition described herein, appropriate reagents, and / or equipment. In some aspects, the kitmay be packaged in a vial, pouch, ampoule, and / or any container suitable for a therapeutic method. Additional examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and optionally intended mode of administration and treatment. In some aspects, kit components may be provided as concentrates (including lyophilized compositions), which may be further diluted prior to use or provided at the concentration of use. In some aspects, when the one or more multivalent antibody constructs is provided for use in vivo, a single dosage is provided in a sterilized container having the desired amount and concentration of the multivalent antibody construct.

[0362] In some aspects, a kit includes labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also ty pically be included.

[0363] In some aspects, a label is on or associated with the container. In one aspect, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one aspect, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label may also indicate directions for use of the contents, such as in the methods described herein.

[0364] The kit may also include additional therapeutic nucleic acids, drug, therapeutic agent, diagnostic agent, prophylactic agent, and / or any other agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect.EXAMPLES

[0365] The following non-limiting examples are provided to further illustrate aspects of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific aspects that are disclosed and still obtain a like or similar result without departing from the spirit and scope ofthe invention. By providing these specific examples, it is not intended limit the scope and spirit of the present technology.

[0366] Example 1. Generation of exemplary multivalent T-cell engager molecules

[0367] To generate multispecific molecules with scFv components, target specific tandem scFvs (see Example 2) were connected to either N- or C- terminal heavy chains (CH3) using linkers, different linker lengths. In some instances, target specific scFvs were connected to either -N or C terminal light chains. In addition, some molecular formats were generated byfusing scFvs in tandem. Further, some multispecifics may have either have common light chains or specific light chains. To generate heterodimeric Fc molecules, knob and hole mutations were used. In certain constructs, the heterodimeric Fc molecules were further stabilized using disulfide bridges. The constructs were cloned into pcDNA3.4 vector.

[0368] The heavy chain of an exemplary multivalent T-cell engager molecules can comprise the following format: N — VH-CHl-CH2-CH3-peptide-scFv-C. where N and C denote the N- terminal and C-terminal ends of the construct. The heavy chain is an IgGl, IgG2, IgG3, or IgG4 isotype or their variants modifying Fc functions, for example, IgGs that do not bind to Fc receptor (so as to lack the effector function) and can further be cloned into a mammalian expression vector such as pcDNA3.4. The 5'-end of the heavy chain is cloned into the 3'-end of the leader sequence of the vector to enable secretion from the mammalian expression cells. The light chain is constructed in a separate expression vector where VL is cloned either with Kappa or Lamda CL domain. Similarly, the 5 '-end of the DNA sequence of the light chain may be cloned into the 3 '-end of the leader sequence of the vector. The plasmids containing the heavy and light chains are transiently co-expressed in HEK293, CHO, or Expi293 cells. The cells are grown in flasks on an orbital shaker platform rotating at 140 rpm at 37° C, 5% CO2 and sub-cultured following the manufacturer's protocol. Co-transfection is performed with polyethyleneimine (PEI) as the transfection reagent. Briefly, HEK293 / CHO cells are subcultured to a cell density of 0.5-0.7* 106cells / ml for 24 hours before transfection. Immediately before transfection, cell density is adjusted to 1 * 106cells / ml. Five hundred micrograms of each purified plasmid (1 mg / ml) is added to 19 ml Optipro (Invitrogen). Two milliliters of 1 mg / ml PEI, pH 7.0 (molecular weight (MW) of 25 000) dissolved in water is added to 18 ml Optipro. Both the solutions are incubated at room temperature for 5 min. The DNA / Optipro solution is added to the PEI / Optipro solution and incubated for 10 min at room temperature and added drop wise to 1 L HEK293 / CHO culture. The supernatant is collected 6-8 days after transfection.Multivalent T-cell engager molecules are purified and checked for expression, purity and quality7, as described in the following examples.

[0369] Example 2. Generation of exemplary scFvs

[0370] Expi293 cells were transfected with pcDNA3.4 (CMV promoter) expression vectors encoding tandem scFvs with His-tags using standard protocols provided by Invitrogen (LifeTech). Cell culture supernatants were collected 5 days post transfection and clarified by centrifugation. At 3000xg for 20 min at room temperature. 2mL of Ni IMAC magnetic beads (Genscript; Cat. L00295) were added to lOOmL of clarified supernatant, and the tandem scFvs were purified following the protocol provided by Genscript.

[0371] Example 3. Purification of Recombinant Multispecific Antibody Constructs

[0372] Lipofectamine™ MessengerMAX Reagent (ThermoFisher) was used to transfect mRNA Expi293 (ThermoFisher) to express the desired antibody fusion constructs using the manufacturer's instructions. The cells were grown Expi293 Expression Medium (ThermoFisher), and protein constructs were purified with benchtop scale Protein A (HiTrap MabSelect Prisma SuRe, Cytiva) using the manufacturer’s instructions.

[0373] Example 4. ELISA assay for PSMA binding

[0374] An ELISA was developed to assess PSMA binding in the presence of molecules S1408. M1107. M1108. M1109 and M1110. Briefly, recombinant PSMA protein (ACRObiosy stems) was coated on MaxiSorp ELISA plates (Nunc) in sodium bicarbonate buffer overnight at 4°C. Plates were washed, blocked and multispecific T-cell engager molecules were titrated in four-fold dilution series and incubated shaking at room temperature. Plates were washed and an HRP-Goat Anti-Human IgG (Jackson ImmunoResearch) was used as a detection antibody, incubated at room temperature. Plates were washed and developed with 1-Step TMB ELISA Substrate (ThermoFisher) and optical density7450nm was measured. As expected, S1408 did not bind PSMA and Ml 107 bound the strongest. Ml 108 and Ml 109 bound PSMA with similar affinity to eachother and with slightly decreased affinity compared to Ml 107. Ml 110 demonstrated the least amount of PSMA binding. The data are shown in Fig 10 A.

[0375] Example 5. Binding of Multispecific Molecules on Prostate Cancer cells

[0376] Binding of multivalent molecules to PSMA and / or STEAP1 was assessed in the prostate cancer cell line LNCaP (a well-characterized human prostate cell line, available from American Type Culture Collection (ATCC)), the cell line 22Rvl (a human prostate carcinoma epithelial cell line (expressing both STEAP1 and PSMA), available from ATCC), and PC3- STEAP1 (a PC3 cell line lacking endogenous PSMA or STEAP1 expression that was engineered to stably express STEAP1). Binding was measured with an anti-IgG-Fc-PE stain followed by flow cytometry. Both LNCaP and 22Rvl express both PSMA and STEAP1 protein natively, while PC3-STEAP1 only expresses STEAP1.

[0377] The relative receptor density values were quantified using QuantumTM MESF (Molecules of Equivalent Soluble Fluorochrome) fluorescence quantitation on LNCaP, 22Rvl and PC3-STEAP1 cells. The results are shown in Fig. 9D.

[0378] Approximately 50,000 cells (PC3-STEAP1, LNCaP or 22RV1) were seeded in 100 pL of media per well in a 96-well round bottom plate. Cells were centrifuged at 500 Relative Centrifugal Force (RCF) for 5 min at room temperature (RT). Cells were washed once with PBS and incubated with serially diluted multivalent molecules for 30 min in PBS at RT. The starting concentration of the multivalent molecules is 200 nM with 3-fold dilution and 12 total dilutions. Cells are washed twice with cell staining buffer (BioLegend, catalog # 420201) and incubated with anti-human IgG Fc PE conjugated antibody (ThermoFisher, catalog # 12-4998- 82) for 30 min at RT in the dark. Cells are washed once with cell staining buffer and then with PBS, and then resuspended in PBS. Cells were processed via flow cytometry' (Cytek® flow7cytometer) and analyzed using FlowJo™ software. Graphs were generated using GraphPad Prism. Peripheral Blood Mononuclear Cells (PBMC, Donor 10) are thawed, washed once with PBS and resuspended in MEM Complete Medium (Sigma Aldrich). Cells were rested overnight at 37° C. The next day, T-cells are isolated by negative selection with an EasySep™ Human T Cell Isolation Kit (Stemcell™, Cat# 17951). Binding is assessed using the same protocol described above, except that CD3 binding was performed on primary7isolated human T-cells.

[0379] Applying the above methods, binding was determined for various multivalent T-cell engager molecules. FIG. 10B depicts binding to PC3-STEAP1 cells as evidenced by Mean Fluorescent Intensity (MFI) for the following constructs: S1408, M1107, M1108, M1109 and Ml 110. FIG. 10C and 10D show binding to 22Rvl cells (10C) or LNCaP cells (10D). The data show that negative control S1408, having predicted CD3 binding only, did not show- I l l - appreciable binding to either 22Rvl or LNCaP cells, while each of Ml 107, Ml 108, Ml 109 and Ml 110 showed specific, dose-dependent binding to both 22Rvl and LNCaP.

[0380] Example 6. Activity of serum from mice injected with mRNAs encoding multispecific molecules.

[0381] Balb / c mice (n=3) were dosed intravenously with mRNA encoding multispecific T- cell engagers formulated in a peptoid delivery vehicle using Compound 41 described herein. M1107 and M1110 were encoded in 2 mRNAs formulated at ratios of either 2: 1 or 1 : 1 while S1408 was encoded by a single mRNA. 24 hours post injection, terminal bleeds were performed and serum was harvested using serum separator microtainer tubes (BD Biosciences). Serum from groups receiving 1 : 1 mRNA chain ratio formulations were pooled and used for functional assessment ex vivo. Serum was diluted in cell culture medium (RPMI + 10% FBS + Pen / Strep) and used in LNCaP binding studies as previously described (HA), in binding assays with human T-cells derived from healthy donor PBMCs (1 IB), or in T-cell dependent cytoxicity (TDCC) studies in which both LNCaP cells and human T-cells are incubated and killing of LNCaP cells was measured (Fig. 11C) as well as T-cell activation (Fig, 1 ID).

[0382] Fig. 11A shows data using sera from mice that were injected with mRNAs encoding Ml 110, Ml 107, or S1408. A dose-dependent response and incubated with LNCaP cells. Multispecific T-cell engager binding was measured with an anti-lgG-Fc-PE stain and detected by flow cytometry. Dose-dependent binding signal can be seen with responses to Ml 110 and M1107. but not with S1408, as expected.

[0383] Fig. 11B shows results from experiments where sera from mice that were injected with mRNAs encoding Ml 110, Ml 107, or S1408, were incubated with human T-cells. Multispecific T-cell engager binding was measured with an anti-lgG-Fc-PE stain and detected by flow cytometry. Dose-dependent binding signal can be seen with Ml 110, Ml 107 and S1408.

[0384] Fig. 11C shows results from experiments where sera from mice that were injected with mRNAs encoding Ml 110, Ml 107. or S 1408 and incubated with LNCaP cells and human T-cells. T-cell-dependent LNCaP target cell killing was observed in a dose-dependent manner with Ml 110 and Ml 107 but not with S1408, as expected.

[0385] Fig 11 D shows results from experiments where sera from mice that were inj ected with mRNAs encoding Ml 110. Ml 107, or S1408 and incubated with LNCaP cells and human T-cells. T-cell-activation was observed in a dose-dependent manner with Ml 110 and Ml 107 but not with S1408, as expected.

[0386] Example 7. T-cell dependent cytotoxicity (TDCC) and T-cell phenotyping

[0387] A prostate cancer cell line, LNCaP, expressing PSMA and STEAP1, was co-cultured with human T-cells in the presence of dose-titrated prostate cancer multivalent T-cell engager molecules and target cell killing and T-cell activation were evaluated.

[0388] Target cell killing and T-cell activation were evaluated according to the following protocol. In brief, thirty thousand CellTrace™ Violet (BioLegend)-labeled LNCaP (ATCC) target cells per well were seeded in a round bottom 96 well plate in 50 pl volume of supplied buffer. Prostate cancer multivalent constructs described herein were thawed and normalized to 300 nM concentration in T-cell culture media (50% AIM-V, 50% RPMI, 5% FBS, pen-strep, 2-mercaptoethanol) and used to perform a five-fold dilution series consisting of seven dilution points. 50 pl per well was added and the plate was incubated at 37° C + 5% CO2 for approximately 1 hour. Human T-cells were isolated from donor PBMCs by negative selection with an EasySep™ Human T Cell Isolation Kit (Stemcell technologies) and 2.41e5 cells per well in 50ul volume was added to the plate containing target cells and multivalent T- cell engager molecules to a final volume of 150 pl resulting in an effector to target cell ratio of approximately 8. The plate was spun at 300g for 5 minutes and incubated at 37°C + 5% CO2 for approximately 48 hrs.

[0389] After 2 days incubation, the co-culture was evaluated for target cell killing using the Zombie NIR Fixable Viability Kit (BioLegend) and T-cells were phenotyped by CD4, CD8, CD69, CD137, CD95 and CD107a surface stain. Percent killing of LNCaP target cells was calculated by (%NIR+ cells - %NIR+target cells only) / [(100 - %NIR+target cells only)xl00].

[0390] FIG. 12A depicts % killing of LNCaP target cells following 48-hour culture with T- cells and one of the following multivalent constructs: S1408, Ml 107, Ml 108, Ml 109 and Ml 110. As shown in FIG. 12 A, Ml 110 showed high efficacy at concentrations as low as 10'2through 10'LS1408, which lacks aprostate antigen binding protein entirely showed no LNCaP killing effect. The experiment was repeated using 22Rvl cells, and the results are shown in Fig. 12B shows killing of 22Rvl target cells following 72-hour culture with T-cells and one of the following multivalent constructs: S1408, Ml 107, Ml 108, Ml 109 and Ml 110 was observed. As seen in FIG. 12B, Ml 110 showed high efficacy at concentrations as low as 10‘2through 10’1. S 1408, which lacks a prostate antigen binding protein entirely showed no 22Rvl killing. The multispecific Ml 110 outperformed the bispecific Ml 107 in both cell lines.

[0391] Example 8. Bystander T-cell activation.

[0392] CD69 is a sensitive marker that reflects early T-cell activation events. An increase in the population of CD69+ and CD137+ T-cells is indicative of T-cell activation and can be used to measure bystander activation. Here, human T-cells were incubated with dose-titrated recombinantly expressed constructs in the absence of target cells overnight and CD69 expression was assessed on CD8+ T-cells as a surrogate for activation.

[0393] Human T-cells were isolated from donor PBMCs by negative selection with an EasySep™ Human T Cell Isolation Kit (Stemcell Technologies), and 3e4 cells per well in 100 pl volume of EasySep™ Buffer (Catalog #20144; Stemcell Technologies) was added to the plate. Multivalent construct molecules were thawed and normalized to 300 nM concentration in supplemented RPMI 1640 cell culture medium and used to perform a 7.5-fold dilution series. 50 pl per well was added and the plate was incubated at 37°C+ 5% CO2 overnight.

[0394] After approximately 24 hours incubation, T-cells were phenotyped with CD4, CD8, CD69 and CD137. Activation was evaluated by CD69 upregulation and CD69+CD137+ population increase assessed by flow cylometiy. Supernatant was collected and secreted cytokines were detected by Luminex using an Inflammation 20-Plex Human ProcartaPlex™ Panel (ThermoFisher).

[0395] FIG. 13 shows T-cell bystander activation as measured by % CD69+ CD137+CD8 T-cells at increasing concentrations of the following multivalent tri-specific constructs: Ml 110, M1086, M1098, S1429 and M1093. Ml 110 showed the least amount of bystander activation, while SI 429 showed bystander effect activity at concentrations as low as 1 nm, an effect that dropped off at higher concentrations of 10 and 100 nm. Ml 086, common light chain displayed robust bystander activation and Ml 093, heterodimeric format displayed intermediate bystander activation. Ml 098 knob and hole format displayed moderate bystander activity, but slightly greater than Ml 110 homodimeric format.

[0396] All percentages and ratios are calculated by weight unless otherwise indicated.

[0397] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0398] Example 9. Activity of multispecific constructs from serum of mice injected with mRNAs encoding the constructs

[0399] Balb / c mice (n=3) were dosed intravenously with formulated mRNA encoding multispecific T-cell engager molecules. Ml 107 and Ml 110 were encoded in 2 mRNAs formulated at ratios of either 2: 1 or 1 : 1 while S 1408 was encoded by a single mRNA. 24 hours post injection, serum was harvested and expression quantified by ELISA. The results are shown in Fig. 14.

[0400] Serum from groups receiving 1: 1 mRNA chain ratio formulations were pooled and used for functional assessment ex vivo. LNCaP cells or human T-cells derived from healthy donor PBMCs were incubated with titrated serum dilutions and multispecific T-cell engager molecule binding was measured with an anti-IgG-Fc-PE stain followed by flow cytometry . The data are shown in Fig. 11A for the LNCaP cell experiments, and in Fig. 11B for the human T- cell experiments.

[0401] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every' minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numencal range given throughout this specification will include every' narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0402] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”

[0403] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety- unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of anydocument is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0404] While particular aspects of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

[0405] Underlined and / or italicized headings and subheadings are used for convenience only, do not limit the subject technology, and are not referred to in connection with the interpretation of the description of the subject technology. All structural and functional equivalents to the elements of the various implementations described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and intended to be encompassed by the subject technology’. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the above description.

[0406] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.

Claims

CLAIMSWhat is claimed is:1 . A polynucleotide sequence encoding a multi-specific antibody construct comprising a heavy chain portion comprising(a) an anti-prostate cancer antigen domain;(b) an anti-hCD3 domain; and(c) a human IgG Fc domain.

2. The polynucleotide sequence of claim 1, wherein the anti-prostate cancer antigen domain is selected from an anti-Prostate Specific Membrane Antigen (anti-PSMA) domain, an anti-Six-Transmembrane Epithelial Antigen of the Prostate 1 (anti-STEAPl) domain, an anti-Kallikrein-related peptidase 2 (KLK-2) domain, and combinations thereof.

3. The polynucleotide sequence of claim 1, wherein the anti-prostate cancer antigen domain comprises at least two domains selected from an anti-Prostate Specific Membrane Antigen (anti-PSMA) domain, an anti-Six-Transmembrane Epithelial Antigen of the Prostate 1 (anti-STEAPl) domain, and an anti -KLK-2 domain.

4. The polynucleotide sequence of claim 2 or 3, wherein the anti-PSMA domain encodes for one or more amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 63, SEQ ID NO: 71, and SEQ ID NO: 72 .

5. The polynucleotide sequence of claim 2 or 3, wherein the anti-STEAPl domain encodes for one or more amino acid sequence having at least 90% sequence identity to SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76.

6. The polynucleotide sequence of claim 2 or 3, wherein the anti-KLK-2 domain encodes for one or more amino acid sequence has at least 90% sequence identity to SEQ ID NO: 62, SEQ ID NO: 69, and SEQ ID NO: 70.

7. The polynucleotide sequence of any preceding claim, wherein the anti-hCD3 domain encodes for one or more amino acid sequence has at least 90% sequence identity to SEQ ID NO: 64, SEQ ID NO: 73, and SEQ ID NO: 74.

8. The polynucleotide of any preceding claim, wherein the human IgG Fc domain comprises a native IgG sequence or a variant IgG sequence.

9. The polynucleotide sequence of claim 1, the polynucleotide sequence encoding for an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 71, wherein the prostate cancer targeting domain specifically binds to PSMA.

10. The polynucleotide sequence of claim 1, the polynucleotide sequence encoding for an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO:

67. wherein the prostate cancer targeting domain specifically binds to STEAP1.

11. The polynucleotide sequence of claim 1. the polynucleotide sequence encoding for an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 69, wherein the prostate cancer targeting domain specifically binds to KLK-2.

12. The polynucleotide sequence of any preceding claim, wherein the anti-hCD3 domain is located at the C-terminus of the heavy chain.

13. The polynucleotide sequence of claim 1, wherein the prostate cancer targeting domain is located at the N-terminus of the heavy chain.

14. The polynucleotide sequence of claim 1, wherein the prostate cancer targeting domain is located at the C-terminus of the heavy chain.

15. The polynucleotide sequence of claim 1. wherein the IgG Fc domain comprises a knob or hole mutation.1 . The polynucleotide sequence of claim 1, wherein the IgG Fc domain comprises a mutation that introduces a cysteine residue.

17. The polynucleotide sequence of any preceding claim, wherein the polynucleotide is DNA.

18. The polynucleotide sequence of any preceding claim, wherein the polynucleotide is RNA.

19. The polynucleotide sequence of any preceding claim, wherein the polynucleotide is mRNA.

20. A polynucleotide sequence encoding an antibody construct comprising a light chain portion, the light chain portion comprising a domain selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, an anti-CD3 domain, and combinations thereof.

21. The polynucleotide sequence of claim 20, wherein the polynucleotide sequence comprises a single domain.

22. The polynucleotide sequence of claim 20 or 21, wherein the polynucleotide sequence comprises at least two domains.

23. The polynucleotide sequence of claim 22. wherein the at least two domains are the same.

24. The polynucleotide sequence of claim 22, wherein the at least two domains are different.

25. The polynucleotide sequence of claim 20. wherein the polynucleotide sequence encodes for at least one amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID NO: 74.

26. The polynucleotide sequence of any preceding claim, wherein the polynucleotide is DNA.

27. The polynucleotide sequence of any preceding claim, wherein the polynucleotide is RNA.

28. The polynucleotide sequence of any preceding claim, wherein the polynucleotide is mRNA.

29. A multispecific antibody construct comprising at least one heavy chain portion and at least one tight chain portion, wherein the at least one heavy chain portion comprises a prostate cancer targeting domain.

30. The multispecific antibody construct of claim 29, the prostate cancer targeting domain being selected from an anti-Prostate Specific Membrane Antigen (anti-PSMA) domain, an anti-Six-Transmembrane Epithelial Antigen of the Prostate 1 (anti-STEAPl) domain, an anti- Kallikrein-related peptidase 2 (KLK-2) domain, and combinations thereof.

31. The multispecific antibody construct of claim 29 or 30, the heavy chain portion comprising at least one prostate cancer targeting domain.

32. The multispecific antibody construct of any of claims 29 through 31, the heavy chain portion comprising at least two prostate cancer targeting domains.

33. The multispecific antibody construct of any of claims 29 through 32, the heavy chain portion comprising at least three prostate cancer targeting domains.

34. The multispecific antibody construct of any of claims 29 through 33, the heavy chain portion comprising an anti-hCD3 domain at one or both of the N-terminus and the C-terminus of the heavy chain portion.

35. The multispecific antibody construct of any of claims 29 through 34, the heavy chain portion comprising an anti-hCD3 domain at the N-terminus of the heavy chain portion.

36. The multispecific antibody construct of any of claims 29 through 35, the heavy chain portion comprising an anti-hCD3 domain at the C-terminus of the heavy chain portion.

37. The multispecific antibody construct of any of claims 29 through 36, the heavy chain portion comprising a human IgG Fc domain.

38. The multispecific antibody construct of any of claims 29 through 37, the heavy chain portion comprising a human IgG Fc domain comprising a knob or a hole mutation.

39. The multispecific antibody construct of any of claims 29 through 38, the heavy chain portion comprising a human IgG Fc domain comprising a mutation that introduces a cysteine residue.

40. A light chain protein comprising a domain selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, an anti-CD3 domain, and combinations thereof.

41. The light chain protein of claim 40, the light chain region comprising a single domain.

42. The light chain protein of claim 40 or 41, the single domain being an anti-PSMA domain.

43. The light chain protein of claim 40 or 41, the single domain being an anti-STEAPl domain.

44. The light chain protein of claim 40 or 41, the single domain being an anti-KLK-2 domain.

45. The light chain protein of claim 40 or 41, the single domain being an anti-CD3 domain.

46. The light chain protein of any of claims 40 through 45, the light chain region comprising two domains.

47. The light chain protein of any of claims 40 through 46, comprising at least two domains selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, and an anti-CD3 domain, and wherein the at least two domains are the same.

48. The light chain protein of any of claims 40 through 46, wherein the light chain protein comprises at least two domains selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, and an anti-CD3 domain, and wherein the at least two domains are different.

49. The light chain protein of any of claims 40 through 48, the light chain protein having at least 90% sequence identity to a sequence selected from SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, and SEQ ID NO: 76.

50. A multispecific antibody construct comprising at least two heavy chains and one light chain.

51. The multispecific antibody construct of claim 50, comprising at least two heavy chains according to any of claims and at least two light chains.

52. The multispecific antibody construct of claims 50 or 51, the multi-specific antibody construct having a structure selected from a monoclonal antibody, an antibody fragment, a homodimeric antibody, and a heterdimenc antibody.

53. The multispecific antibody construct of any of claims 50 through 52 , wherein the heavy chain region is encoded by a polynucleotide of any one of claims 1 through 28.

54. The multispecific antibody construct of any of claims 50 through 53, wherein the light chain region is encoded by a polynucleotide of any of claims 1 through 28.

55. The multispecific antibody construct of any of claims 50 through 54, wherein a first heavy chain comprises a knob mutation, and a second heavy chain comprises a hole mutation.

56. The multispecific antibody construct of any of claims 50 through 55, wherein the at least two heavy’ chains comprise a mutation that introduces a cysteine residue.

57. A tandem scFv antibody construct comprising a domain selected from an anti-PSMA domain, an anti-STEAPl domain, an anti-KLK-2 domain, an anti-CD3 domain, and combinations thereof.

58. The tandem scFv antibody construct of claim 57, said anti-PSMA domain comprising an ammo acid sequence having at least 80%, or at least 85%, or at least 90%. or at least 95%, or 100% sequence identity to SEQ ID NO: 63.

59. The tandem scFv antibody construct of claim 57, said anti-STEAP domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76.

60. The tandem scFv antibody construct of claim 57, said anti-KLK-2 domain comprising an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% sequence identity' to SEQ ID NO: 62.

61. The tandem scFv antibody construct of claim 57, said anti-CD3 domain comprising an amino acid sequence having at least 80%, or at least 85%, or at least 90%. or at least 95%, or 100% sequence identity to SEQ ID NO: 64.

62. The tandem scFv antibody construct of any of claims 57 through 61, said tandem scFv antibody construct comprising at least two domains, at least one domain being an anti-CD3 domain.

63. The tandem scFv antibody construct of any of claims 57 through 62, said tandem scFv antibody construct comprising at least three domains, at least one domain being an anti-CD3 domain.

64. The tandem scFv antibody construct of any of claims 57 through 63, said tandem scFv antibody construct comprising at least four domains, at least one domain being an anti-CD3 domain.

65. The tandem scFv antibody construct of any of claims 57 through 64, said anti-CD3 domain being at the N-terminus.

66. The tandem scFv antibody construct of any of claims 57 through 65, said anti-CD3 domain being at the C -terminus.

67. A therapeutic composition comprising at least two polynucleotides of claims 1 through 28 and a delivery vehicle molecule comprising an amino-lipidated peptoid.

68. The therapeutic composition of claim 67, wherein the delivery' vehicle is selected from an amphipathic molecule, an amino-lipidated peptide, a tertiary amino lipidated cationic peptide, a cationic component, a peptoid, a lipoid, a liposome, a lipoplex. a lipid nanoparticle, a cationic lipid nanoparticle, a polymeric compound, a conjugate, and combinations thereof.

69. The therapeutic composition of claim 67 or 68, wherein the delivery vehicle has a particle size less than or equal to about 200 nm.

70. The therapeutic composition of any of claims 67 through 69, said delivery vehicle comprising ahydroxyalkyl-capped cationic peptoid compound according to Table 4.

71. A lipid nanoparticle comprising an mRNA encoding a multispecific antibody construct of any one of claims 50 through 56; and a delivery vehicle encapsulating the mRNA.

72. The therapeutic composition or lipid nanoparticle of any one of claims 67 through 71, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity7to a sequence selected from at least one of SEQ ID NOS: 1-61; and a second mRNA encoding the amino acid having at least 90% sequence identity to a sequence selected from at least one of SEQ ID NOS: 1-61; wherein said first mRNA and said second mRNA are the same or are different.

73. The therapeutic composition or lipid nanoparticle of any of claims 67 through 71, wherein the polynucleotides comprisea first mRNA encoding an amino acid sequence having at least 90% sequence identity to a sequence selected from at least one of SEQ ID NOS: 1-61; a second mRNA encoding the amino acid having at least 90% sequence identity to a sequence selected from at least one of SEQ ID NOS: 1-61; and a third mRNA encoding the amino acid having at least 90% sequence identity to a sequence selected from at least one of SEQ ID NOS: 1-61; wherein said first, second, and third mRNA are the same or are different.

74. The therapeutic composition or lipid nanoparticle of any of claims 67 through 71, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity' to a sequence selected from SEQ ID NO: 10; and a second mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 28.

75. The therapeutic composition or lipid nanoparticle of any of claims 67 through 71, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 10; and a second mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 33.

76. The therapeutic composition or lipid nanoparticle of any of claims 67 through 71, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 10; anda second mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity7, or 100% sequence identity to a sequence selected from SEQ ID NO: 34.

77. The therapeutic composition or lipid nanoparticle of any one of claims 67 through 71, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 11; and a second mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 28.

78. The therapeutic composition or lipid nanoparticle of any one of claims 71 and 72, wherein the polynucleotides comprise a first mRNA encoding an amino acid sequence having at least 90% sequence identity7, or at least 95% sequence identity, or 100% sequence identity7to a sequence selected from SEQ ID NO: 19; a second mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 40; and a third mRNA encoding an amino acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NO: 33.

79. A method of treating an individual having a disorder or condition, comprising administering to the individual an effective amount of a multispecific antibody construct, a lipid nanoparticle, or a therapeutic composition of any preceding claim.

80. The method of claim 79 wherein the disorder is prostate cancer.

81. The method of claim 80, wherein the prostate cancer is metastatic and / or castrationresistant prostate cancer (mCRPC).

82. The method of any of claims 79 through 81, wherein the administration is via a route selected from a topical route (such as epi cutaneous, inhalational, nasal, ophthalmic, auricular / aural. vaginal, mucosal); an enteral route (such as oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and a parenteral route (such as intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, epidural, intrathecal, subcutaneous, intraperitoneal, extra-amniotic, intraarticular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intrasynovial, intraluminal).

83. The method of any of claims 79 through 82, the administration being intravenous (IV) administration.

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