WO2021074683A1

WO2021074683A1 - Bispecific anti-pd-l1 and anti-fcrn polypeptides

Info

Publication number: WO2021074683A1
Application number: PCT/IB2020/000848
Authority: WO
Inventors: Amrik Basran; Emma STANLEY; Emma JENKINS; Matthew P. Vincent; Estelle ADAM; Yeon Chul KIM
Original assignee: Avacta Life Sciences Limited
Priority date: 2019-10-16
Filing date: 2020-10-15
Publication date: 2021-04-22

Abstract

Provided herein, in some embodiments, are bispecific proteins comprising an AFFIMER® polypeptides that binds to PD-L1 and an AFFIMER® polypeptides that binds to FcRn. Also provided herein, in some embodiments, are compositions containing the bispecific proteins, methods of using the bispecific proteins, and methods of producing the bispecific proteins.

Description

BISPECIFIC ANTI-PD-U1 AND ANTI-FCRN POUYPEPTIDES

REUATED APPUICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 62/915,805, filed October 16, 2019, which is incorporated by reference herein in its entirety.

BACKGROUND

Specific antibodies to programmed death-ligand 1 (PD-L1) have been developed as anti cancer agents (see, e.g., U.S. Pat. NOS: 9,212,224 and 8,008,449). There exists a need however, for additional PD-L1 inhibitory activities useful in the treatment of cancer, infectious disease, and neurodegenerative disease, e.g., Alzheimer's disease.

SUMMARY

Provided herein, in some aspects, are therapeutic bispecific proteins comprising an (at least one) AFFIMER® polypeptide that binds (e.g., competitively or non-competitively) to PD- L1 and an (at least one) AFFIMER® polypeptide that binds (e.g., competitively or non- competitively) to FcRn (such as human FcRn). These polypeptides have been shown in in vivo pharmacokinetic (PK) studies to have a serum half-life of at least 7 days and can be made, for example, in bacterial cells (e.g., Escherichia coli).

Some aspects of the present disclosure provide a bispecific protein comprising an FcRn- binding AFFIMER® polypeptide and a PD-L1 -binding AFFIMER® polypeptide, wherein the FcRn-binding AFFIMER® polypeptide binds to human FcRn with a K_d of 1X10⁶M or less at pH 6.0 and optionally a K_d for binding human FcRn at pH 7.4 that is at least half a log greater than the K_d for binding at pH 6.0, and wherein the PD-L1 -binding AFFIMER® polypeptide binds to PD-L1 with a K_d of 1X10⁶M or less.

Other aspects of the present disclosure provide a bispecific protein comprising an FcRn- binding AFFIMER® polypeptide that binds to human FcRn and a PD-L1 -binding AFFIMER® polypeptide that binds to PD-L1, wherein the protein has a circulating half-life in human subjects of at least 7 days.

Yet other aspects of the present disclosure provide a bispecific protein comprising an FcRn-binding AFFIMER® polypeptide that binds to human FcRn and a PD-L1 -binding AFFIMER® polypeptide that binds to PD-L1, wherein the FcRn-binding AFFIMER® polypeptide facilitates transport of the protein across an epithelial tissue barrier. Yet further aspects of the present disclosure provide a bispecific protein comprising an FcRn-binding AFFIMER® polypeptide that binds to human FcRn and a PD-L1 -binding AFFIMER® polypeptide that binds to PD-L1, wherein the FcRn-binding AFFIMER® polypeptide has an amino acid sequence that is at least 75% identical to an AFFIMER® polypeptide selected from SEQ ID NOS: 671-964, and wherein the PD-Llbinding AFFIMER® polypeptide has an amino acid sequence that is at least 75% identical to an AFFIMER® polypeptide selected from SEQ ID NOS: 662-670.

Other aspects of the present disclosure provide a bispecific protein comprising an FcRn- binding AFFIMER® polypeptide that binds to human FcRn and a PD-L1 -binding AFFIMER® polypeptide that binds to PD-L1, wherein the FcRn-binding AFFIMER® polypeptide has an amino acid sequence that can be encoded by a polynucleotide having a coding sequence that hybridizes to any one of SEQ ID NOS: 974-1267 under stringent conditions of 6X sodium chloride/sodium citrate (SSC) at 45°C followed by a wash in 0.2X SSC at 65°C, and wherein the PD-L1 -binding AFFIMER® polypeptide has an amino acid sequence that can be encoded by a polynucleotide having a coding sequence that hybridizes to any one of SEQ ID NOS: 965-973 under stringent conditions of 6X sodium chloride/sodium citrate (SSC) at 45°C followed by a wash in 0.2X SSC at 65°C.

In some embodiments, the FcRn-binding AFFIMER® polypeptide binds to FcRn and/or the PD-L1 -binding AFFIMER® polypeptide binds to PDL1 with a Kd of lxlO ⁷ M, a Kd of lxlO ⁸ M, or Kd of lxlO ⁹ M.

In some embodiments, the FcRn-binding AFFIMER® polypeptide binds to FcRn at pH 7.4 with a K_dthat is at least one log greater than the K_d for binding to FcRn at pH 6.0, at least 1.5 logs greater than the K_d for binding to FcRn at pH 6, at least 2 logs greater than the K_d for binding to FcRn at pH 6, or at least 2.5 log greater than the K_d for binding to FcRn at pH 6.

In some embodiments, the bispecific protein has a serum half-life in human patients of greater than 10 hours, greater than 24 hours, greater than 48 hours, greater than 72 hours, greater than 96 hours, greater than 120 hours, greater than 144 hours, greater than 168 hours, greater than 192 hours, greater than 216 hours, greater than 240 hours, greater than 264 hours, greater than 288 hours, greater than 312 hours, greater than 336 hours or, greater than 360 hours.

In some embodiments, wherein the bispecific protein has a serum half-life in human subjects of greater than 50%, greater than 60%, greater than 70%, or greater than 80% of the serum half-life of IgG. In some embodiments, the protein has a serum half-life in human subjects of greater than 50%, greater than 60%, greater than 70%, or greater than 80% of the serum half- life of serum albumin. In some embodiments, the bispecific protein does not inhibit binding of human serum albumin to human FcRn. In some embodiments, the protein does not inhibit binding of IgG to human FcRn.

In some embodiments, binding of the bispecific protein to human FcRn facilitates transport of the polypeptide from an apical side to a basal side of an epithelial cell layer.

In some embodiments, the FcRn-binding AFFIMER® polypeptide and/or the PD-L1 the FcRn- binding AFFIMER® comprises an amino acid sequence represented in general formula (I)

FRl-(Xaa)_n-FR2-(Xaa)_m-FR3 (I), wherein FR1 is an amino acid sequence having at least 70% identity to MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 1); FR2 is an amino acid sequence having at least 70% identity to GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2); FR3 is an amino acid sequence having at least 70% identity to EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3); and Xaa, individually for each occurrence, is an amino acid, n is an integer from 3 to 20, and m is an integer from 3 to 20.

In some embodiments, FR1 has at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% identity to SEQ ID NO: 1; FR2 has at least 80%, at least 84%, at least 88%, at least 92%, or at least 96% identity to SEQ ID NO: 2; and/or FR3 has at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO: 3.

In some embodiments, FR1 comprises the amino acid sequence of SEQ ID NO: 1; FR2 comprises the amino acid sequence of SEQ ID NO: 2; and/or FR3 comprises the amino acid sequence of SEQ ID NO: 3.

In certain embodiments, the FcRn-binding AFFIMER® polypeptide sequence has an amino acid sequence wherein (Xaa)_n is an amino acid sequence represented in the general formula

-Xaa-Xaa2-Xaa3 -Xaa4-Xaa5-Xaa6-Xaa7 -Xaa-Xaa- wherein Xaa, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6 and Xaa7, individually for each occurrence, is an amino acid residue, with the caveat that (i) at least two of Xaa2, Xaa3, Xaa4 or Xaa5 are selected from His, Lys or Arg, or (ii) at least two of Xaa4, Xaa5, Xaa6 or Xaa7 are selected from His, Lys or Arg. In certain preferred embodiments, at least three, and preferably four of Xaa2, Xaa3, Xaa4, Xaa5, Xaa6 or Xaa7 are selected from His, Lys or Arg.

In some embodiments, (Xaa)_n of a FcRn-binding AFFIMER® polypeptide is at least 75% identical to a Loop 2 sequence selected from SEQ ID NOS: 74-367. In some embodiments, (Xaa)_n of a PD-L1 -binding AFFIMER® polypeptide is at least 75% identical to a Loop 2 sequence selected from SEQ ID NOS: 4-38. In certain embodiments, the FcRn-binding AFFIMER® polypeptide sequence has an amino acid sequence wherein (Xaa)_m is an amino acid sequence represented in the general formula

-Xaa-Xaa8-Xaa9-Xaa 10-Xaa 11 -Xaa 12-Xaa 13 -Xaa 14-Xaa- wherein Xaa, Xaa8, Xaa9, XaalO, Xaall, Xaal2, Xaal3 and Xaal4, individually for each occurrence, is an amino acid residue, with the caveat that at least three of Xaa8, Xaa9, XaalO, Xaal 1, Xaal2, Xaal3 and Xaal4 are selected from His, Lys or Arg, and at least an additional two of Xaa8, Xaa9, XaalO, Xaall, Xaal2, Xaal3 and Xaal4 are are selected from His, Lys,

Arg, Phe, Tyr or Trp.

In some embodiments, (Xaa)_m of a FcRn-binding AFFIMER® polypeptide is at least 75% identical to a Loop 4 sequence selected from SEQ ID NOS: 368-661. In some embodiments, (Xaa)_m of a PD-Ll-binding AFFIMER® polypeptide is at least 75% identical to a Loop 4 sequence selected from SEQ ID NOS: 39-73.

In some embodiments, the bispecific protein includes at least one cysteine, which is optionally available for chemical conjugation, and which (optionally) is located at the C-terminal end or the N-terminal end of the polypeptide.

In some embodiments, the FcRn-binding recombinantly engineered variant of stefin polypeptide binds to human FcRn with a K_d of 1X10⁸M or less at pH 6.0 and optionally a K_d for binding human FcRn at pH 7.4 that is at least half a log greater than the K_d for binding at pH 6.0, and wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide binds to PD-L1 with a K_d of 1X10 ⁸M.

In some embodiments, the FcRn-binding recombinantly engineered variant of stefin polypeptide binds to human FcRn with a K_d of 1X10⁹M or less at pH 6.0 and optionally a K_d for binding human FcRn at pH 7.4 that is at least half a log greater than the K_d for binding at pH 6.0, and wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide binds to PD-L1 with a K_d of 1X10 ⁹M.

In some embodiments, the FcRn-binding recombinantly engineered variant of stefin polypeptide binds to human FcRn with a K_d of lxlO ¹⁰M or less at pH 6.0 and optionally a K_d for binding human FcRn at pH 7.4 that is at least half a log greater than the K_d for binding at pH 6.0, and wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide binds to PD-L1 with a K_d of lxlO ¹⁰M.

In some embodiments, the bispecific protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 1268. In some embodiments, the bispecific protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 1269.

In some embodiments, the bispecific protein is encoded by a nucleic acid sequence having at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 1271.

In some embodiments, the bispecific protein is encoded by a nucleic acid sequence having at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 1272.

In some embodiments, the FcRn-binding recombinantly engineered variant of stefin polypeptide comprises a loop 2 amino acid sequence of any one of SEQ ID NOs: 74-367.

In some embodiments, the FcRn-binding recombinantly engineered variant of stefin polypeptide comprises a loop 4 amino acid sequence of any one of SEQ ID NOs: 368-661.

In some embodiments, the FcRn-binding recombinantly engineered variant of stefin polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%identity to the sequence of any one of SEQ ID NOs: 671-964.

In some embodiments, the PD-Ll-binding recombinantly engineered variant of stefin polypeptide comprises a loop 2 amino acid sequence of any one of SEQ ID NOs: 4-38.

In some embodiments, the PD-Ll-binding recombinantly engineered variant of stefin polypeptide comprises a loop 4 amino acid sequence of any one of SEQ ID NOs: 39-73.

In some embodiments, the PD-Ll-binding recombinantly engineered variant of stefin polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of any one of SEQ ID NOs: 662-670.

Also provided herein, in some aspects, is a pharmaceutical composition suitable for therapeutic use in a human subject, comprising a bispecific protein of any of any one of the preceding claims, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for pulmonary delivery. For example, the pharmaceutical composition may be formulated as an intranasal formulation. In other embodiments, the pharmaceutical composition is formulated for topical ( e.g ., transepithelial) delivery.

Further provided herein, in some aspects, is a polynucleotide comprising a sequence encoding a polypeptide (e.g., protein) of any of any one of the preceding claims. In some embodiments, the sequence encoding the polypeptide is operably linked to a transcriptional regulatory sequence. In some embodiments, the transcriptional regulatory sequence is selected from the group consisting of promoters and enhancers. In some embodiments, the polynucleotide further comprises an origin of replication, a minichromosome maintenance element (MME), and/or a nuclear localization element. In some embodiments, the polynucleotide further comprises a polyadenylation signal sequence operably linked and transcribed with the sequence encoding the polypeptide. In some embodiments, the sequence encoding the polypeptide comprises at least one intronic sequence. In some embodiments, the polynucleotide further comprises at least one ribosome binding site transcribed with the sequence encoding the polypeptide.

In some embodiments, the polynucleotide is a deoxyribonucleic acid (DNA). In some embodiments, the polynucleotide is a ribonucleic acid (RNA).

Also provided herein, in some aspects, is a viral vector comprising the polynucleotide of the present disclosure, a plasmid or minicircle comprising the polynucleotide of the present disclosure, a cell comprising the polypeptide of the present disclosure, the polynucleotide of the present disclosure, a viral vector of the present disclosure, and a plasmid or minicircle of the present disclosure.

Other aspects of the present disclosure provide a bispecific protein as described herein for use in a method for treating an autoimmune disease and/or an inflammatory disease, for use in a method for treating cancer, or for use in a method for treating cardiovascular or metabolic disease or disorder.

Further aspects provide a method of producing a bispecific protein of the present disclosure, the method comprising expressing in a host cell a nucleic acid encoding the polypeptide, and optionally isolating the polypeptide from the host cell.

Also provided herein is a pharmaceutical composition suitable for therapeutic use in a human subject, comprising the bispecific protein of the present disclosure, and a pharmaceutically acceptable excipient. The pharmaceutical composition, in some embodiments, is formulated for pulmonary ( e.g ., intranasal) delivery. The pharmaceutical composition, in other embodiments, is formulated for topical (e.g., transepithelial) delivery.

It should be understood that any one of the AFFIMER® polypeptides described herein may include or exclude a signal sequence (e.g., ~ 15-30 amino acids present at the N-terminus of the polypeptide) or a tag sequence (e.g., C-terminal polyhistadine (e.g., HHHHHH) (SEQ ID NO: 1292)).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1: SDS-PAGE analysis of purified anti-FcRn AFFIMER® polypeptides.

FIG. 2: Human FcRn EFISA of clones FcRn-15 (SEQ ID NO: 685), FcRn-35 (SEQ ID NO: 705) and FcRn-38 (SEQ ID NO: 708) at both pH (7.4 and 6). FIG. 3: Schematics of formatted bispecific AFFIMER® ILF (in-line fusion) polypeptide with PD-L1 and FcRn binders (PD-L1-251-FX3 (SEQ ID NO: 1268); PD-L1-251-FX6 (SEQ ID NO: 1269); and PD-L1-251-BH (SEQ ID NO: 1270)).

FIG. 4: SDS-PAGE and SEC-HPLC analysis of AFFIMER® ILF polypeptide formats (PD-L1-251-FX3 (SEQ ID NO: 1268); PD-L1-251-FX6 (SEQ ID NO: 1269)).

FIG. 5: Binding capacity of bispecific AFFIMER® polypeptide to human FcRn and human PD-L1 evaluated by ELISA (PD-L1-251-FX3 (SEQ ID NO: 1268); PD-L1-251-FX6 (SEQ ID NO: 1269); FcRN-38 (SEQ ID NO: 708), and PD-L1-251-BH (SEQ ID NO: 1270)).

FIGs. 6A-6B: Analytical SEC-HPLC traces of purified FcRn AFFIMER® monomers (FcRn-35 (SEQ ID NO: 705), FcRN-38 (SEQ ID NO: 708), FcRn-120 (SEQ ID NO: 790),

FcRn- 125 (SEQ ID NO: 795)), and PD-L1-251-FX6 (SEQ ID NO: 1269) (also referred to herein as AVA04-251 FX6, an FcRn- and PD-Ll-binding AFFIMER® fusion).

FIG. 7: SDS-PAGE analysis of purified FcRn AFFIMER® monomers and AVA04-FcRn binding AFFIMER® fusion (FcRn-35 (SEQ ID NO: 705), FcRN-38 (SEQ ID NO: 708), FcRn- 120 (SEQ ID NO: 790), FcRn- 125 (SEQ ID NO: 795)), and PD-L1-251-FX6 (SEQ ID NO:

1269) (also referred to herein as AVA04-251 FX6, an FcRn- and PD-Ll-binding AFFIMER® fusion).

FIGs. 8A-8B: FcRn binding ELISA showing the binding activity of purified FcRn AFFIMER® monomers and AVA04-FcRn binding AFFIMER® fusion at pH 6 and 7 (FcRn-35 (SEQ ID NO: 705), FcRN-38 (SEQ ID NO: 708), FcRn-120 (SEQ ID NO: 790), FcRn-125 (SEQ ID NO: 795)), and PD-L1-251-FX6 (SEQ ID NO: 1269) (also referred to herein as AVA04-251 FX6, an FcRn- and PD-Ll-binding AFFIMER® fusion).

FIG. 9: FcRn competition ELISA showing the activity of FcRn AFFIMER® monomers and AVA04-FcRn binding AFFIMER® fusion (FcRn-35 (SEQ ID NO: 705), FcRN-38 (SEQ ID NO: 708), FcRn-120 (SEQ ID NO: 790), and FcRn-125 (SEQ ID NO: 795)).

FIG. 10: Demonstration of FcRn mediated recycling of the FcRn binding AFFIMER® polypeptides as determined using the human endothelial cell-based recycling assay (FcRn-35 (SEQ ID NO: 705), FcRN-38 (SEQ ID NO: 708), FcRn-120 (SEQ ID NO: 790), FcRn-125 (SEQ ID NO: 795)), and PD-L1-251-FX6 (SEQ ID NO: 1269) (also referred to herein as AVA04-251 FX6, an FcRn- and PD-Ll-binding AFFIMER® fusion).

DETAILED DESCRIPTION

The present disclosure is based on the generation of a bispecific protein that includes an AFFIMER® polypeptide that binds to PD-L1 and an AFFIMER® polypeptide that binds to human neonatal Fc receptor (FcRn). The FcRn binding AFFIMER® polypeptide extends, in a controlled manner, the serum half-life of the PD-L1 binding AFFIMER® polypeptide to which it is conjugated.

Based on naturally occurring proteins (cy statins) that have been engineered to stably display two loops that create a binding surface, the AFFIMER® polypeptides of the present disclosure provide a number of advantages over antibodies, antibody fragments, and other non antibody molecule-binding proteins. One is the small size of the AFFIMER® polypeptide itself. In its monomeric form it is about 14 kDa, or 1/lOth the size of an antibody. This small size gives greater potential for increased tissue penetration, particularly in poorly vascularized and/or fibrotic target tissues (like tumors). AFFIMER® polypeptides have a simple protein structure (versus multi-domain antibodies), and as the AFFIMER® polypeptides do not require disulfide bonds or other post-translational modifications for function, these polypeptides can be manufactured in prokaryotic and eukaryotic systems.

Using libraries of AFFIMER® polypeptides (such as the phage display techniques described in the appended examples) as well as site directed mutagenesis, AFFIMER® polypeptides can be generated with tunable binding kinetics with ideal ranges for therapeutic uses. For instance, an AFFIMER® polypeptides can have high affinity for human FcRn or PD- Ll, such as single digit nanomolar or lower K_d for monomeric AFFIMER® polypeptides, and picomolar K_d and avidity in multi-valent formats. An AFFIMER® polypeptides can be generated with tight binding kinetics for human FcRn or PD-L1, such as slow K_0ff rates in the 10⁴ to 10⁵ (s-1) range, which benefits target tissue localization.

The bispecific proteins of the present disclosure include AFFIMER® polypeptides with exquisite selectivity.

Moreover, the AFFIMER® polypeptides can be readily formatted, allowing formats such as Fc fusions, whole antibody fusions, and in-line multimers to be generated and manufactured with ease.

The lack of need for disulfide bonds and post-translational modifications also permit many embodiments of bispecific proteins including the AFFIMER® polypeptides to be delivered therapeutically by expression of gene delivery constructs that are introduced into the tissues of a patient, including formats where the protein is delivered systemically (such as expression from muscle tissue) or delivered locally (such as through intratumoral gene delivery).

An AFFIMER® polypeptide (also referred to simply as an AFFIMER®) is a small, highly stable polypeptide (e.g., protein) that is a recombinantly engineered variant of stefin polypeptides. Thus, the term “AFFIMER® polypeptide” may be used interchangeably herein with the term “recombinantly engineered variant of stefin polypeptide”. A stefin polypeptide is a subgroup of proteins in the cystatin superfamily - a family that encompasses proteins containing multiple cy statin-like sequences. The stefin subgroup of the cy statin family is relatively small (~ 100 amino acids) single domain proteins. They receive no known post-translational modification, and lack disulfide bonds, suggesting that they will be able to fold identically in a wide range of extracellular and intracellular environments. Stefin A is a monomeric, single chain, single domain protein of 98 amino acids. The structure of stefin A has been solved, facilitating the rational mutation of stefin A into the AFFIMER® polypeptide. The only known biological activity of cystatins is the inhibition of cathepsin activity, has enabled exhaustively testing for residual biological activity of the engineered proteins.

AFFIMER® polypeptides display two peptide loops and an N-terminal sequence that can all be randomized to bind to desired target proteins with high affinity and specificity, in a similar manner to monoclonal antibodies. Stabilization of the two peptides by the stefin A protein scaffold constrains the possible conformations that the peptides can take, increasing the binding affinity and specificity compared to libraries of free peptides. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. Variations to other parts of the stefin A polypeptide sequence can be carried out, with such variations improving the properties of these affinity reagents, such as increase stability, make them robust across a range of temperatures and pH, for example. In some embodiments, an AFFIMER® polypeptide includes a sequence derived from stefin A, sharing substantial identify with a stefin A wild type sequence, such as human stefin A. In some embodiments, an AFFIMER® polypeptide has an amino acid sequence that shares at least 25%, 35%, 45%, 55% or 60% identity to the sequences corresponding to human stefin A. For example, an AFFIMER® polypeptide may have an amino acid sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95% identity, e.g., where the sequence variations do not adversely affect the ability of the scaffold to bind to the desired target, and e.g., which do not restore or generate biological functions such as those that are possessed by wild type stefin A, but which are abolished in mutational changes described herein.

Bispecific AFFIMER® Proteins

One aspect of the disclosure provides bispecific proteins comprising an AFFIMER® polypeptide that binds programmed death-ligand 1 (PD-L1) and an AFFIMER® polypeptide that binds human neonatal Fc receptor (FcRn).

PD-L1 is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified, Programmed Death Ligand- 1 (PD-L1) and Programmed Death Ligand-2 (PD-L2), that are expressed on antigen-presenting cells as well as many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et ah, J. Exp. Med. 192(7): 1027-34 (2000); Latchm an et al., Nat Immunol 2:261-8 (2001)).

PD-1 primarily functions in peripheral tissues where activated T-cells may encounter the immunosuppressive PD-L1 (also called B7-H1 or CD274) and PD-L2 (B7-DC) ligands expressed by tumor and/or stromal cells (Flies et al., Yale J Biol Med 84:409-21 (2011);

Topalian et al., Curr Opin Immuno 24:1-6 (2012)).

Inhibition of the PD-1/PD-L1 interaction mediates potent antitumor activity in preclinical models (U.S. Pat. NOS: 8,008,449 and 7,943,743). It appears that upregulation of PD-L1 may allow cancers to evade the host immune system. An analysis of 196 tumor specimens from patients with renal cell carcinoma found that high tumor expression of PD-L1 was associated with increased tumor aggressiveness and a 4.5-fold increased risk of death (Thompson et al.,

Proc Natl Acad Sci USA 101 (49): 17174-9 (2004)). Ovarian cancer patients with higher expression of PD-L1 had a significantly poorer prognosis than those with lower expression. PD- L1 expression correlated inversely with intraepithelial CD8+ T-lymphocyte count, suggesting that PD-L1 on tumor cells may suppress antitumor CD8+ T cells (Hamanishi et al., Proc Natl Acad Sci USA 104 (9): 3360-3365 (2007)).

PD-L1 has also been implicated in infectious disease, in particular chronic infectious disease. Cytotoxic CD8 T lymphocytes (CTLs) play a pivotal role in the control of infection. Activated CTLs, however, often lose effector function during chronic infection. PD-1 receptor and its ligand PD-L1 of the B7/CD28 family function as a T cell co-inhibitory pathway and are emerging as major regulators converting effector CTLs into exhausted CTLs during chronic infection with human immunodeficiency vims, hepatitis B virus, hepatitis C virus, herpes vims, and other bacterial, protozoan, and viral pathogens capable of establishing chronic infections. Such bacterial and protozoal pathogens can include E. coli, Staphylococcus sp., Streptococcus sp., Mycobacterium tuberculosis, Giardia, Malaria, Leishmania, and Pseudomonas aeruginosa. Importantly, blockade of the PD-1/PD-L1 pathway is able to restore functional capabilities to exhausted CTLs. PD1/PD-L1 is thus a target for developing effective prophylactic and therapeutic vaccination against chronic bacterial and viral infections (see, e.g., Hofmeyer et al, Journal of Biomedicine and Biotechnology, vol. 2011, Article ID 451694, 9 pages, doi: 10.1155/2011/451694).

Recent studies have also shown that systemic immune suppression may curtail the ability to mount the protective, cell-mediated immune responses that are needed for brain repair in neurodegenerative diseases. By using mouse models of Alzheimer's disease, immune checkpoint blockade directed against the programmed death- 1 (PD-1) pathway was shown to evoke an interferon g-dependent systemic immune response, which was followed by the recruitment of monocyte-derived macrophages to the brain. When induced in mice with established pathology, this immunological response led to clearance of cerebral amyloid- b (Ab) plaques and improved cognitive performance. These findings suggest that immune checkpoints may be targeted therapeutically in neurodegenerative disease such as Alzheimer's disease using antibodies to PD- L1 (see, e.g., Baruch et ak, Nature Medicine, January 2016, doi:10.1038/nm.4022).

Human neonatal Fc receptor, also known as the Brambell receptor, is a protein encoded by the FCGRT gene. This Fc receptor is similar in structure to the MHC class I molecule and also associates with beta-2-microglobulin. FcRn includes a 40 kDa alpha heavy chain that non- covalently associates with the 12 kDa light chain b-2-microgobulin. The FcRn heavy chain comprises three extracellular domains (al, a2, and a3), a transmembrane domain, and a 44 amino acid cytoplasmic tail. In humans, FcRn has a role in monitoring IgG and serum albumin turnover (Kuo TT et al. mAbs 2011;3(5):422-430; and Roopenian DC et al. Nature Reviews 2007;7(9):715-725). Neonatal Fc receptor expression is up-regulated by the proinflammatory cytokine, TNF-a, and down-regulated by IFN-g. A representative human FcRn sequence is provided by UniProtKB Primary accession number X and may include other human isoforms thereof.

FcRn-mediated transcytosis of IgG across epithelial cells is possible because FcRn binds IgG at acidic pH (<6.5) but not at neutral or higher pH. Thus, FcRn can bind IgG from the slightly acidic intestinal lumen and ensure efficient, unidirectional transport to the basolateral side where the pH is neutral to slightly basic (Kuo TT et al. Journal of Clinical Immunology 2010;30(6):777-89).

FcRn extends the half-life of IgG and serum albumin by reducing lysosomal degradation in endothelial cells (Roopenian DC et al. 2007) and bone-marrow derived cells (Akilesh S. et al. Journal of Immunology 2007;179(7):4580-4588). IgG, serum albumin and other serum proteins are continuously internalized through pinocytosis. Generally, serum proteins are transported from the endosomes to the lysosome, where they are degraded. The two most abundant serum proteins, IgG and serum albumin are bound by FcRn at the slightly acidic pH (<6.5) and recycled to the cell surface where they are released at the neutral pH (>7.0) of blood. In this way IgG and serum albumin avoids lysosomal degradation. This mechanism provides an explanation for the greater serum circulation half-life of IgG and serum albumin (Goebl NA et al. Molecular Biology of the Cell 2008;19(12):5490-505; and Roopenian DC et al. 2007) AFFIMER® polypeptides comprise an AFFIMER® polypeptide in which at least one of the solvent accessible loops is from the wild-type stefin A protein having amino acid sequences to enable an AFFIMER® polypeptide to bind PD-L1 or human FcRn, selectively, and in some embodiments, with a K_d of 10⁶M or less.

In some embodiments, an AFFIMER® polypeptide bind to PD-L1 or human FcRn with a K_d of lxlO ⁹ M to lxlO ⁶ M at pH 7.4 to 7.6. In some embodiments, the polypeptides bind to human FcRn with a K_d of lxlO ⁶ M or less at pH 7.4 to 7.6. In some embodiments, an AFFIMER® polypeptide bind to PD-L1 or human FcRn with a K_d of lxlO ⁷ M or less at pH 7.4 to 7.6. In some embodiments, an AFFIMER® polypeptide bind to PD-L1 or human FcRn with a K_d of lxlO ⁸ M or less at pH 7.4 to 7.6. In some embodiments, an AFFIMER® polypeptide bind to PD-L1 or human FcRn with a K_d of lxlO ⁹ M or less at pH 7.4 to 7.6. In some embodiments, an AFFIMER® polypeptide bind to PD-L1 or human FcRn with a K_d of lxlO ⁹ M to lxlO ⁶ M at pH 7.4. In some embodiments, an AFFIMER® polypeptide bind to PD-L1 or human FcRn with a K_d of lxlO ⁶ M or less at pH 7.4. In some embodiments, an AFFIMER® polypeptide bind to PD-L1 or human FcRn with a K_d of lxlO ⁷ M or less at pH 7.4. In some embodiments, an AFFIMER® polypeptide bind to PD-L1 or human FcRn with a K_d of lxlO ⁸ M or less at pH 7.4. In some embodiments, an AFFIMER® polypeptide bind to PD-L1 or human FcRn with a K_d of lxlO ⁹ M or less at pH 7.4.

In some embodiments, an AFFIMER® polypeptide at pH 5.8 to 6.2 binds to PD-L1 or human FcRn with a K_d of half a log to 2.5 logs less than the K_d for binding to PD-L1 or human FcRn at pH 7.4 to 7.6, respectively. In some embodiments, an AFFIMER® polypeptide at pH 5.8 to 6.2 binds to PD-L1 or human FcRn with a K_d of half a log less than the K_d for binding to PD-L1 or human FcRn at pH 7.4 to 7.6, respectively. In some embodiments, an AFFIMER® polypeptide at pH 5.8 to 6.2 binds to PD-L1 or human FcRn with a K_dof at least one log less than the K_d for binding to PD-L1 or human FcRn at pH 7.4 to 7.6, respectively. In some embodiments, an AFFIMER® polypeptide at pH 5.8 to 6.2 binds to PD-L1 or human FcRn with a K_dof at least 1.5 log less than the K_d for binding to PD-L1 or human FcRn at pH 7.4 to 7.6, respectively. In some embodiments, an AFFIMER® polypeptide at pH 5.8 to 6.2 binds to PD-L1 or human FcRn with a K_d of at least 2 log less than the K_d for binding to PD-L1 or human FcRn at pH 7.4 to 7.6, respectively. In some embodiments, an AFFIMER® polypeptide at pH 5.8 to 6.2 binds to PD-L1 or human FcRn with a K_d of at least 2.5 log less than the K_d for binding to PD-L1 or human FcRn at pH 7.4 to 7.6, respectively.

In some embodiments, an AFFIMER® polypeptide at pH 6 binds to PD-L1 or human FcRn with a K_d of half a log to 2.5 logs less than the K_d for binding to PD-L1 or human FcRn at pH 7.4. In some embodiments, an AFFIMER® polypeptide at pH 6 binds to PD-L1 or human FcRn with a K_dof at least half a log less than the K_d for binding to PD-L1 or human FcRn at pH 7.4. In some embodiments, an AFFIMER® polypeptide at pH 6 binds to PD-L1 or human FcRn with a K_d of at least one log less than the K_d for binding to PD-L1 or human FcRn at pH 7.4. In some embodiments, an AFFIMER® polypeptide at pH 6 binds to PD-L1 or human FcRn with a K_dof at least 1.5 log less than the K_d for binding to PD-L1 or human FcRn at pH 7.4. In some embodiments, an AFFIMER® polypeptide at pH 6 binds to PD-L1 or human FcRn with a K_dof at least two logs less than the K_d for binding to PD-L1 or human FcRn at pH 7.4. In some embodiments, an AFFIMER® polypeptide at pH 6 binds to PD-L1 or human FcRn with a K_dof at least 2.5 logs less than the K_d for binding to PD-L1 or human FcRn at pH 7.4.

In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 10 hours. In some embodiments, the polypeptides have a serum half-life in human patients of greater than 24 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 48 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 72 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 96 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 120 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 144 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 168 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 192 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 216 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 240 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 264 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 288 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 312 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 336 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of greater than 360 hours. In some embodiments, the proteins comprising the AFFIMER® polypeptides have a serum half-life in human patients of 24 to 360 hours, 48 to 360 hours, 72 to 360 hours, 96 to 360 hours, or 120 to 360 hours.

In some embodiments, an AFFIMER® polypeptide comprises an amino acid sequence represented in general formula (I)

In some embodiments, a PD-L1 binding AFFIMER® polypeptide comprises a loop 2 amino acid sequence selected from any one of SEQ ID NOS: 4-38 (Table 1). In some embodiments, a PD-L1 binding AFFIMER® polypeptide comprises a loop 4 amino acid sequence selected from any one of SEQ ID NOS: 39-73 (Table 1).

In some embodiments, (Xaa)_n comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 4-38. In some embodiments, (Xaa)_n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 4-38. In some embodiments, (Xaa)_n comprises the amino acid sequence of any one of SEQ ID NOS: 4-38.

In some embodiments, (Xaa)_m comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 39-73. In some embodiments, (Xaa)_m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 39-73. In some embodiments, (Xaa)_m comprises the amino acid sequence of any one of SEQ ID NOS: 39-73.

In some embodiments, an FcRn binding AFFIMER® polypeptide comprises a loop 2 amino acid sequence selected from any one of SEQ ID NOS: 74-367 (Table 2). In some embodiments, an FcRn binding AFFIMER® polypeptide comprises a loop 4 amino acid sequence selected from any one of SEQ ID NOS: 368-661 (Table 2).

In some embodiments, (Xaa)_n comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 74-367. In some embodiments, (Xaa)_n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 74-3678. In some embodiments, (Xaa)_n comprises the amino acid sequence of any one of SEQ ID NOS: 74-367.

In some embodiments, (Xaa)_m comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 368-661. In some embodiments, (Xaa)_m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 368-661. In some embodiments, (Xaa)_m comprises the amino acid sequence of any one of SEQ ID NOS: 368-661.

Table 1. Examples of PD-L1 Binding AFFIMER® Loop Sequences

Table 2. Examples of FcRn Binding AFFIMER® Loop Sequences

In some embodiments, an PD-L1 binding AFFIMER® polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOS: 662-670 (Table 3).

In some embodiments, an PD-L1 binding AFFIMER® polypeptide comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 662-670. In some embodiments, an PD-L1 binding AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 662-670. In some embodiments, an PD-L1 binding AFFIMER® polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 662-670.

In some embodiments, an FcRn binding AFFIMER® polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOS: 671-964 (Table 4).

In some embodiments, an FcRn binding AFFIMER® polypeptide comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 671-964. In some embodiments, an FcRn binding AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 671-964. In some embodiments, an FcRn binding AFFIMER® polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 671-964.

Table 3. Examples of PD-L1 Binding AFFIMER® Polypeptide Sequences

Table 4. Examples of FcRn Binding AFFIMER® Polypeptide Sequences

In some embodiments, an PD-L1 binding AFFIMER® polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from any one of SEQ ID NOS: 965- 973 (Table 5).

In some embodiments, an PD-Llbinding AFFIMER® polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence having at least 80% or at least 90% identity to the nucleic acid sequence of any one of SEQ ID NOS: 965-973. In some embodiments, an PD- Llbinding AFFIMER® polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence having 80% to 90% identity to the nucleic acid sequence of any one of SEQ ID NOS: 965-973. In some embodiments, an PD-Llbinding AFFIMER® polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence of any one of SEQ ID NOS: 965-973.

In some embodiments, an FcRn binding AFFIMER® polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from any one of SEQ ID NOS: 974- 1267 (Table 6).

In some embodiments, an FcRn binding AFFIMER® polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence having at least 80% or at least 90% identity to the nucleic acid sequence of any one of SEQ ID NOS: 974-1267. In some embodiments, an FcRn binding AFFIMER® polypeptide is encoded by polynucleotide comprising a nucleic acid sequence having 80% to 90% identity to the nucleic acid sequence of any one of SEQ ID NOS: 974-1267. In some embodiments, an FcRn binding AFFIMER® polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence of any one of SEQ ID NOS: 974-1267.

Table 5. Examples of PD-L1 Binding AFFIMER® Polynucleotide Sequences

Table 6. Examples of FcRn Binding AFFIMER® Polynucleotide Sequences

The bispecific proteins here may include any one or more of the PD-L1 binding AFFIMER® polypeptides and/or any one or more of the FcRn binding AFFIMER® polypeptides. For example, a bispecific protein may comprise of one, two, three or more PD-L1 binding AFFIMER® polypeptide molecules and one, two, three or more PD-L1 binding AFFIMER® polypeptide molecules. In some embodiments, a bispecific protein comprises three (at least three) PD-L1 binding AFFIMER® polypeptide molecules and one (at least one) FcRn binding AFFIMER® polypeptide molecules.

The bispecific proteins provided herein include an FcRn binding AFFIMER® polypeptide linked to a PD-L1 binding AFFIMER® polypeptide and has an extended half-life due to the presence of the binding AFFIMER® polypeptide. The term half-life refers to the amount of time it takes for a substance (e.g., a protein comprising a PD-L1 binding AFFIMER® polypeptide) to lose half of its pharmacologic or physiologic activity or concentration. Biological half-life can be affected by elimination, excretion, degradation (e.g., enzymatic degradation) of the substance, or absorption and concentration in certain organs or tissues of the body. Biological half-life can be assessed, for example, by determining the time it takes for the blood plasma concentration of the substance to reach half its steady state level (“plasma half-life”).

In some embodiments, an FcRn binding AFFIMER® polypeptide extends the serum half- life of the PD-L1 binding AFFIMER® polypeptide in vivo. For example, an FcRn binding AFFIMER® polypeptide may extend the half-life of the PD-L1 binding AFFIMER® polypeptide by at least 1.2-fold, relative to the half-life of the PD-L1 binding AFFIMER® polypeptide not linked to an FcRn binding AFFIMER® polypeptide. In some embodiments, an FcRn binding AFFIMER® polypeptide extends the half-life of the PD-L1 binding AFFIMER® polypeptide by at least 1.5-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, or at least 30-fold, relative to the half-life of the PD-L1 binding AFFIMER® polypeptide not linked to an FcRn binding AFFIMER® polypeptide. In some embodiments, an FcRn binding AFFIMER® polypeptide extends the half-life of the PD-L1 binding AFFIMER® polypeptide by 1.2-fold to 5- fold, 1.2-fold to 10-fold, 1.5-fold to 5-fold, 1.5-fold to 10-fold, 2-fold to 5-fold, 2-fold to 10-fold, 3 -fold to 5-fold, 3 -fold to 10-fold, 15-fold to 5-fold, 4-fold to 10-fold, or 5-fold to 10-fold, relative to the half-life of the PD-L1 binding AFFIMER® polypeptide not linked to an FcRn binding AFFIMER® polypeptide. In some embodiments, an FcRn binding AFFIMER® polypeptide extends the half-life of the PD-L1 binding AFFIMER® polypeptide by at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, for example, at least 1 week after in vivo administration, relative to the half-life of the PD-L1 binding AFFIMER® polypeptide not linked to an FcRn binding AFFIMER® polypeptide.

Table 7. Examples of PD-L1- FcRn Bispecific In-line fusion Polynucleotide Sequences

Table 8. Examples of PD-L1- FcRn Bispecific In-line fusion Polynucleotide Sequences

Polypeptides

A polypeptide is a polymer of amino acids (naturally-occurring or non-naturally occurring, e.g., amino acid analogs) of any length. The terms “polypeptide” and “peptide” are used interchangeably herein unless noted otherwise. A protein is one example of a polypeptide. It should be understood that a polypeptide may be linear or branched, it may comprise naturally- occurring and/or non-naturally-occurring (e.g., modified) amino acids, and/or it may include non-amino acids (e.g., interspersed throughout the polymer). A polypeptide, as provided herein, may be modified (e.g., naturally or non-naturally), for example, via disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. Polypeptides, in some instances, may contain at least one analog of an amino acid (including, for example, unnatural amino acids) and/or other modifications.

An amino acid (also referred to as an amino acid residue) participates in peptide bonds of a polypeptide. In general, the abbreviations used herein for designating the amino acids are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). For instance, Met, lie, Leu, Ala and Gly represent “residues” of methionine, isoleucine, leucine, alanine and glycine, respectively. A residue is a radical derived from the corresponding a-amino acid by eliminating the OH portion of the carboxyl group and the H portion of the a-amino group. An amino acid side chain is that part of an amino acid exclusive of the — CH(NH2)COOH portion, as defined by K. D. Kopple, “Peptides and Amino Acids”, W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33.

Amino acids used herein, in some embodiments, are naturally-occurring amino acids found in proteins, for example, or the naturally-occurring anabolic or catabolic products of such amino acids that contain amino and carboxyl groups. Examples of amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan, and those amino acids and amino acid analogs that have been identified as constituents of peptidylglycan bacterial cell walls.

Amino acids having basic sidechains include Arg, Lys and His. Amino acids having acidic sidechains include Glu and Asp. Amino acids having neutral polar sidechains include Ser, Thr, Asn, Gin, Cys and Tyr. Amino acids having neutral non-polar sidechains include Gly, Ala, Val, lie, Leu, Met, Pro, Trp and Phe. Amino acids having non-polar aliphatic sidechains include Gly, Ala, Val, lie and Leu. Amino acids having hydrophobic sidechains include Ala, Val, He, Leu, Met, Phe, Tyr and Trp. Amino acids having small hydrophobic sidechains include Ala and Val. Amino acids having aromatic sidechains include Tyr, Trp and Phe.

The term amino acid includes analogs, derivatives and congeners of any specific amino acid referred to herein; for instance, the AFFIMER® polypeptides (particularly if generated by chemical synthesis) can include an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminiopimelic acid, ornithine, or diaminobutyric acid. Other naturally-occurring amino acid metabolites or precursors having side chains that are suitable herein will be recognized by those skilled in the art and are included in the scope of the present disclosure.

Also included herein are the (D) and (L) stereoisomers of such amino acids when the structure of the amino acid admits of stereoisomeric forms. The configuration of the amino acids and amino acids herein are designated by the appropriate symbols (D), (L) or (DL); furthermore, when the configuration is not designated the amino acid or residue can have the configuration (D), (L) or (DL). It will be noted that the structure of some of the compounds of the present disclosure includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry are included within the scope of the present disclosure. Such isomers can be obtained in substantially pure form by classical separation techniques and by sterically controlled synthesis. For the purposes of this disclosure, unless expressly noted to the contrary, a named amino acid shall be construed to include both the (D) or (L) stereoisomers.

Percent identity, in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same (identical/ 100% identity) or have a specified percentage (e.g., at least 70% identity) of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the present disclosure are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40- 60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80- 1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

A conservative amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains ( e.g ., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and/or antibodies of the present disclosure do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site. Methods of identifying amino acid conservative substitutions that do not eliminate binding are well-known in the art.

Herein, it should be understood that an isolated molecule (e.g., polypeptide (e.g., soluble protein, antibody, etc.), polynucleotide (e.g., vector), cell, or other composition) is in a form not found in nature. Isolated molecules, for example, have been purified to a degree that is not possible in nature.

In some embodiments, an isolated molecule ( e.g ., polypeptide (e.g., soluble protein, antibody, etc.), polynucleotide (e.g., vector), cell, or other composition) is substantially pure, which refer to an isolated molecule that is at least 50% pure (e.g., free from 50% of contaminants associated with the unpurified form of the molecule), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

Conjugates, Including Polypeptide Fusions

The verb conjugate (used interchangeably with the verb link) herein refers to the joining together of two or more molecules (e.g., polypeptides and/or chemical moieties) to form another molecule. Thus, one molecule (e.g., a PD-L1 binding AFFIMER® polypeptide) conjugated to another molecule (e.g., a PD-L1 AFFIMER® polypeptide, drug molecule, or other therapeutic protein or nucleic acid) forms a conjugate. The joining of two or more molecules can be, for example, through a non-covalent bond or a covalent bond. Non-limiting examples of conjugates include chemical conjugates (e.g., joined through “click” chemistry or another chemical reaction) and fusions (two molecules linked by contiguous peptide bonds). In some embodiments, a conjugate is a fusion polypeptide, for example, a fusion protein.

A fusion polypeptide (e.g., fusion protein) is a polypeptide comprising at least two domains (e.g., protein domains) encoded by a polynucleotide comprising nucleotide sequences of at least two separate molecules (e.g., two genes). In some embodiments, a bispecific protein comprises two AFFIMER® polypeptides covalently linked (to an amino acid of the polypeptide) through an amide bond to form a contiguous fusion polypeptide (e.g., fusion protein). In some embodiments, AFFIMER® polypeptides are conjugated to each other through contiguous peptide bonds at the C-terminus or N-terminus of the FcRn binding AFFIMER® polypeptide.

A linker is a molecule inserted between a first polypeptide (e.g., an AFFIMER® polypeptide) and a second polypeptide (e.g., another AFFIMER® polypeptide, an Fc domain, a ligand binding domain, etc.). A linker may be any molecule, for example, one or more nucleotides, amino acids, chemical functional groups. In some embodiments, the linker is a peptide linker (e.g., two or more amino acids). Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. In some embodiments, linkers are not antigenic and do not elicit an immune response. An immune response includes a response from the innate immune system and/or the adaptive immune system. Thus, an immune response may be a cell-mediate response and/or a humoral immune response. The immune response may be, for example, a T cell response, a B cell response, a natural killer (NK) cell response, a monocyte response, and/or a macrophage response. Other cell responses are contemplated herein.

In some embodiments, linkers are non-protein-coding.

Empirical linkers designed by researchers are generally classified into 3 categories according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers. Besides the basic role in linking the functional domains together (as in flexible and rigid linkers) or releasing free functional domain in vivo (as in in vivo cleavable linkers), linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles. Linkers should not adversely affect the expression, secretion, or bioactivity of the fusion protein. Linkers should not be antigenic and should not elicit an immune response.

Suitable linkers are known to those of skill in the art and often include mixtures of glycine and serine residues and often include amino acids that are sterically unhindered. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. Linkers can range in length, for example from 1-50 amino acids in length, 1-22 amino acids in length, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3 amino acids in length. In some embodiments, the linker may comprise a cleavage site. In some embodiments, the linker may comprise an enzyme cleavage site, so that the second polypeptide may be separated from the first polypeptide.

In some embodiments, the linker can be characterized as flexible. Llexible linkers are usually applied when the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. See, for example, Argos P. (1990) “An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion” J Mol Biol. 211:943- 958. The small size of these amino acids provides flexibility and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the linker and the protein moieties. The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). An example of the most widely used flexible linker has the sequence of (Gly-Gly-Gly-Gly-Ser)n. By adjusting the copy number “n”, the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions. Besides the GS linkers, many other flexible linkers have been designed for recombinant fusion proteins. As These flexible linkers are also rich in small or polar amino acids such as Gly and Ser but can contain additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility.

In some embodiments, the linker can be characterized as rigid. While flexible linkers have the advantage to connect the functional domains passively and permitting certain degree of movements, the lack of rigidity of these linkers can be a limitation in certain fusion protein embodiments, such as in expression yield or biological activity. The ineffectiveness of flexible linkers in these instances was attributed to an inefficient separation of the protein domains or insufficient reduction of their interference with each other. Under these situations, rigid linkers have been successfully applied to keep a fixed distance between the domains and to maintain their independent functions.

Many natural linkers exhibited a-helical structures. The a-helical structure was rigid and stable, with intra-segment hydrogen bonds and a closely packed backbone. Therefore, the stiff a- helical linkers can act as rigid spacers between protein domains. George et al. (2002) “An analysis of protein domain linkers: their classification and role in protein folding” Protein Eng. 15(11):871-9. In general, rigid linkers exhibit relatively stiff structures by adopting a-helical structures or by containing multiple Pro residues. Under many circumstances, they separate the functional domains more efficiently than the flexible linkers. The length of the linkers can be easily adjusted by changing the copy number to achieve an optimal distance between domains. As a result, rigid linkers are chosen when the spatial separation of the domains is critical to preserve the stability or bioactivity of the fusion proteins. In this regard, alpha helix-forming linkers with the sequence of (EAAAK)n (SEQ ID NO: 1274) have been applied to the construction of many recombinant fusion proteins. Another type of rigid linkers has a Pro-rich sequence, (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu.

Merely to illustrate, exemplary linkers include:

Other linkers that may be used in the subject fusion proteins include, but are not limited to, SerGly, GGSG (SEQ ID NO: 1293), GSGS (SEQ ID NO: 1294), GGGS (SEQ ID NO: 1295), S(GGS)n (SEQ ID NO: 1296) where n is 1-7, GRA, poly(Gly), poly(Ala), GGGSGGG (SEQ ID NO: 1285), ESGGGGVT (SEQ ID NO: 1286), LESGGGGVT (SEQ ID NO: 1287), GRAQVT (SEQ ID NO: 1288), WRAQVT (SEQ ID NO: 1289), and ARGRAQVT (SEQ ID NO: 1290). The hinge regions of the Fc fusions described below may also be considered linkers.

Any conjugation method may be used, or readily adapted, for joining a molecule to an AFFIMER® polypeptide of the present disclosure, including, for example, the methods described by Hunter, et al, (1962) Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407.

Therapeutics

In some embodiments, a bispecific protein may be used, for example, to prevent and/or treat a disease in a subject, such as a human subject or other animal subject.

In some embodiments, the bispecific protein is for the treatment of an autoimmune disease (a condition in which a subject’s immune system mistaken attacks his/her body). Non limiting examples of autoimmune diseases include myasthenia gravis, pemphigus vulgaris, neuromyelitis optica, Guillain-Barre syndrome, rheumatoid arthritis, systemic lupus erythematosus (lupus), idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, antiphospholipid syndrome (APS), autoimmune urticarial, chronic inflammatory demyelinating polyneuropathy (CIDP), psoriasis, Goodpasture's syndrome, Graves' disease, inflammatory bowel disease, Crohn’s disease, Sjorgren’s syndrome, hemolytic anemia, neutropenia, paraneoplastic cerebellar degeneration, paraproteinemic polyneuropathies, primary biliary cirrhosis, stiff person syndrome, vitiligo, warm idiopathic haemolytic anaemia, multiple sclerosis, type 1 diabetes mellitus, Hashimoto’s thyroiditis, Myasthenia gravis, autoimmune vasculitis, pemicus anemia, and celiac disease. Other autoimmune diseases are contemplated herein.

In some embodiments, the bispecific protein is for the treatment of a cancer. Non-limiting examples of cancers include skin cancer ( e.g ., melanoma or non-melanoma, such as basal cell or squamous cell), lung cancer, prostate cancer, breast cancer, colorectal cancer, kidney (renal) cancer, bladder cancer, non-Hodgkin’s lymphoma, thyroid cancer, endometrial cancer, exocrine cancer, and pancreatic cancer. Other cancers are contemplated herein.

The term treat, as known in the art, refers to the process of alleviating at least one symptom associated with a disease. A symptom may be a physical, mental, or pathological manifestation of a disease. Symptoms associated with various diseases are known. To treat or prevent a particular condition, a conjugate as provided herein ( e.g ., a bispecific protein comprising an AFFIMER® polypeptide linked to a therapeutic molecule) should be administered in an effective amount, which can be any amount used to treat or prevent the condition. Thus, in some embodiments, an effective amount is an amount used to alleviate a symptom associated with the particular disease being treated. Methods are known for determining effective amounts of various therapeutic molecules, for example.

A subject may be any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, and rodents. A “patient” refers to a human subject.

In some embodiments, an AFFIMER® polypeptide is considered “pharmaceutically acceptable,” and in some embodiments, is formulated with a pharmaceutically-acceptable excipient. A molecule or other substance/agent is considered “pharmaceutically acceptable” if it is approved or approvable by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. An excipient may be any inert (inactive), non-toxic agent, administered in combination with an AFFIMER® polypeptide. Non-limiting examples of excipients include buffers (e.g., sterile saline), salts, carriers, preservatives, fillers, coloring agents.

Methods of Use

The bispecific proteins of the disclosure are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as immunotherapy for cancer. In some embodiments, bispecific proteins described herein are useful for activating, promoting, increasing, and/or enhancing an immune response, inhibiting tumor growth, reducing tumor volume, inducing tumor regression, increasing tumor cell apoptosis, and/or reducing the tumorigenicity of a tumor. In some embodiments, the polypeptides or agents of the disclosure are also useful for immunotherapy against pathogens, such as viruses. In some embodiments, the bispecific proteins described herein are useful for inhibiting viral infection, reducing viral infection, increasing virally-infected cell apoptosis, and/or increasing killing of virus-infected cells. The methods of use may be in vitro, ex vivo, or in vivo methods.

The present disclosure provides methods for activating an immune response in a subject using a bispecific protein. In some embodiments, the disclosure provides methods for promoting an immune response in a subject using a bispecific protein described herein. In some embodiments, the disclosure provides methods for increasing an immune response in a subject using a bispecific protein. In some embodiments, the disclosure provides methods for enhancing an immune response in a subject using a bispecific protein. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing cell- mediated immunity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing Thl-type responses. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD4+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD8+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CU activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of Treg cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of MDSCs. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing the number of the percentage of memory T-cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term immune memory function. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term memory. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of substantial side effects and/or immune-based toxicities. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of cytokine release syndrome (CRS) or a cytokine storm. In some embodiments, the immune response is a result of antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor cell. In some embodiments, the antigenic stimulation is cancer. In some embodiments, the antigenic stimulation is a pathogen. In some embodiments, the antigenic stimulation is a virally-infected cell.

In vivo and in vitro assays for determining whether a bispecific protein activates, or inhibits an immune response are known in the art. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a bispecific protein described herein, wherein the bispecific protein binds human PD-L1. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a bispecific protein described herein, wherein the bispecific protein including an AFFIMER® polypeptide that specifically binds to PD-L1. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an polynucleotide encoding a bispecific protein, wherein the polynucleotide encoding a bispecific protein, when expressed in the patient, produces a recombinant bispecific protein including an PD-L1 binding AFFIMER® polypeptide.

In some embodiments of the methods described herein, a method of activating or enhancing a persistent or long-term immune response to a tumor comprises administering to a subject a therapeutically effective amount of a bispecific protein which binds human PD-L1. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a bispecific protein described. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of an polynucleotide encoding a bispecific protein, wherein the polynucleotide encoding a bispecific protein, when expressed in the patient, produces a recombinant bispecific protein including an PD-L1 binding AFFIMER® polypeptide.

In some embodiments of the methods described herein, a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a bispecific protein which binds human PD-L1. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a bispecific protein described herein. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an polynucleotide encoding a bispecific protein, wherein the polynucleotide encoding a bispecific protein, when expressed in the patient, produces a recombinant bispecific protein including an PD-L1 binding AFFIMER® polypeptide.

In some embodiments of the methods described herein, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a bispecific protein which binds human PD-L1. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a bispecific protein described herein. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an polynucleotide encoding a bispecific protein, wherein the polynucleotide encoding a bispecific protein, when expressed in the patient, produces a recombinant bispecific protein including an PD-L1 binding AFFIMER® polypeptide.

In some embodiments, the tumor expresses or overexpresses a tumor antigen that is targeted by an additional binding entity provided in the bispecific protein along with the PD-L1 binding AFFIMER® polypeptide.

In some embodiments, the method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of a bispecific protein described herein. In some embodiments, the subject is a human. In some embodiments, the subject has a tumor, or the subject had a tumor which was removed.

In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a tumor selected from the group consisting of: colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, neuroendocrine tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In some embodiments, the tumor is a colorectal tumor. In some embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a lung tumor. In some embodiments, the tumor is a pancreatic tumor. In some embodiments, the tumor is a melanoma tumor. In some embodiments, the tumor is a bladder tumor.

To further illustrate, the subject bispecific proteins can be used to treat patients suffering from cancer, such as osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell cancer, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breast cancer (including triple negative breast cancer), prostate cancer, bone cancer, lung cancer (e.g., small cell or non-small cell lung cancer), gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell cancer, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, a primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelfibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer or liver cancer, breast cancer or gastric cancer. In some embodiments of the disclosure, the cancer is metastatic cancer, e.g., of the varieties described above. In some embodiments, the cancer is a hematologic cancer. In some embodiment, the cancer is selected from the group consisting of: acute myelogenous leukemia (AML), Hodgkin lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML), non- Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and cutaneous T-cell lymphoma (CTCL).

Pharmaceutical Compositions/F ormulations

The present disclosure also provides pharmaceutical compositions comprising a bispecific protein described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions find use in immunotherapy. In some embodiments, the pharmaceutical compositions find use in immuno-oncology. In some embodiments, the compositions find use in inhibiting tumor growth. In some embodiments, the pharmaceutical compositions find use in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the compositions find use in treating cancer. In some embodiments, the pharmaceutical compositions find use in treating cancer in a subject (e.g., a human patient).

Formulations are prepared for storage and use by combining a purified bispecific protein of the present disclosure with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those of skill in the art generally consider pharmaceutically acceptable carriers, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.

In some embodiments, a bispecific protein described herein is lyophilized and/or stored in a lyophilized form. In some embodiments, a formulation comprising a bispecific protein described herein is lyophilized.

Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.).

The pharmaceutical compositions of the present disclosure can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).

In some embopdiments, a composition is formulated for topical delivery such that the when applied to the skin, for example, the bispecific protein penetrates the skin (crosses epithelial and mucosal barriers) to function systemically.

The therapeutic formulation can be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions, such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form a solid preformulation composition containing a homogeneous mixture of a compound of the present disclosure, or a non-toxic pharmaceutically acceptable salt thereof. The solid preformulation composition is then subdivided into unit dosage forms of a type described above. The tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner composition covered by an outer component. Furthermore, the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

The bispecific proteins described herein can also be entrapped in microcapsules. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London.

In some embodiments, pharmaceutical formulations include a bispecific protein of the present disclosure complexed with liposomes. Methods to produce liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter.

In some embodiments, sustained-release preparations comprising bispecific proteins described herein can be produced. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing a bispecific protein, where the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl- methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L- glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3- hydroxybutyric acid.

For the treatment of a disease, the appropriate dosage of a bispecific protein of the present disclosure depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the bispecific protein is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on, all at the discretion of the treating physician. The bispecific protein can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is affected or a diminution of the disease state is achieved (e.g., reduction in tumor size). Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual agent. The administering physician can determine optimum dosages, dosing methodologies, and repetition rates. In some embodiments, dosage is from 0.01 mg to 100 mg/kg of body weight, from 0.1 mg to 100 mg/kg of body weight, from 1 mg to 100 mg/kg of body weight, from 1 mg to 100 mg/kg of body weight, 1 mg to 80 mg/kg of body weight from 10 mg to 100 mg/kg of body weight, from 10 mg to 75 mg/kg of body weight, or from 10 mg to 50 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is from about 0.1 mg to about 20 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 0.1 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 0.25 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 0.5 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 1 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 1.5 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 2 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 2.5 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 5 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 7.5 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 10 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 12.5 mg/kg of body weight. In some embodiments, the dosage of the bispecific protein is about 15 mg/kg of body weight. In some embodiments, the dosage can be given once or more daily, weekly, monthly, or yearly. In some embodiments, the bispecific protein is given once every week, once every two weeks, once every three weeks, or once every four weeks.

In some embodiments, a bispecific protein may be administered at an initial higher "loading" dose, followed by one or more lower doses. In some embodiments, the frequency of administration may also change. In some embodiments, a dosing regimen may comprise administering an initial dose, followed by additional doses (or "maintenance" doses) once a week, once every two weeks, once every three weeks, or once every month. For example, a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose. Or a dosing regimen may comprise administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week. Or a dosing regimen may comprise administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.

As is known to those of skill in the art, administration of any therapeutic agent may lead to side effects and/or toxicities. In some cases, the side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose. In some cases, drug therapy must be discontinued, and other agents may be tried. However, many agents in the same therapeutic class often display similar side effects and/or toxicities, meaning that the patient either has to stop therapy, or if possible, suffer from the unpleasant side effects associated with the therapeutic agent.

In some embodiments, the dosing schedule may be limited to a specific number of administrations or "cycles". In some embodiments, the bispecific protein is administered for 3, 4, 5, 6, 7, 8, or more cycles. For example, the bispecific protein is administered every 2 weeks for 6 cycles, the bispecific protein is administered every 3 weeks for 6 cycles, the bispecific protein is administered every 2 weeks for 4 cycles, the bispecific protein is administered every 3 weeks for 4 cycles, etc. Dosing schedules can be decided upon and subsequently modified by those skilled in the art.

Thus, the present disclosure provides methods of administering to a subject the polypeptides or agents described herein comprising using an intermittent dosing strategy for administering one or more agents, which may reduce side effects and/or toxicities associated with administration of a bispecific protein, chemotherapeutic agent, etc. In some embodiments, a method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of a bispecific protein in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a bispecific protein to the subject and administering subsequent doses of the bispecific protein about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a bispecific protein to the subject and administering subsequent doses of the bispecific protein about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a bispecific protein to the subject and administering subsequent doses of the bispecific protein about once every 4 weeks. In some embodiments, the bispecific protein is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly.

In some embodiments, the disclosure also provides methods for treating subjects using a bispecific protein of the disclosure, wherein the subject suffers from a viral infection. In some embodiments, the viral infection is infection with a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (A, B, or C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein Barr vims), adenovirus, influenza virus, flaviviruses, echovims, rhinovirus, coxsackie vims, coronavims, respiratory syncytial vims, mumps vims, rotavirus, measles vims, rubella vims, parvovirus, vaccinia vims, HTLV vims, dengue vims, papillomavims, molluscum vims, poliovims, rabies vims, JC vims or arboviral encephalitis vims.

In some embodiments, the disclosure provides methods for treating subjects using a bispecific protein thereof of the disclosure, wherein the subject suffers from a bacterial infection. In some embodiments, the bacterial infection is infection with a bacterium selected from the group consisting of Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae, Clostridium tetan, Clostridium botulinum, Bacillus anthricis, Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella.

In some embodiments, the disclosure provides methods for treating subjects using a bispecific protein of the disclosure, wherein the subject suffers from a fungal infection. In some embodiments, the fungal infection is infection with a fungus selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

In some embodiments, the disclosure provides methods for treating subjects using a bispecific protein of the disclosure, wherein the subject suffers from a parasitic infection. In some embodiments, the parasitic infection is infection with a parasite selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba,

Giardia lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii and Nippostrongylus brasiliensis.

Polynucleotides

A polynucleotide (also referred to as a nucleic acid) is a polymer of nucleotides of any length, and may include deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, a polynucleotide herein encodes a polypeptide, such as a bispecific protein comprising a FcRn binding AFFIMER® polypeptide and a PD-L1 binding AFFIMER® polypeptide. As known in the art, the order of deoxyribonucleotides in a polynucleotide determines the order of amino acids along the encoded polypeptide ( e.g ., protein).

A polynucleotide sequence may be any sequence of deoxyribonucleotides and/or ribonucleotides, may be single- stranded, double-stranded, or partially double-stranded. The length of a polynucleotide may vary and is not limited. Thus, a polynucleotide may comprise, for example, 2 to 1,000,000 nucleotides. In some embodiments, a polynucleotide has a length of 100 to 100,000, a length of 100 to 10,000, a length of 100 to 1,000, a length of 100 to 500, a length of 200 to 100,000, a length of 200 to 10,000, a length of 200 to 1,000, or a length of 200 to 500 nucleotides.

A vector herein refers to a vehicle for delivering a molecule to a cell. In some embodiments, a vector is an expression vector comprising a promoter (e.g. , inducible or constitutive) operably linked to a polynucleotide sequence encoding a polypeptide. Non-limiting examples of vectors include viral vectors (e.g., adenoviral vectors, adeno-associated vims vectors, and retroviral vectors), naked DNA or RNA expression vectors, plasmids, cosmids, phage vectors, DNA and/or RNA expression vectors associated with cationic condensing agents, and DNA and/or RNA expression vectors encapsulated in liposomes. Vectors may be transfected into a cell, for example, using any transfection method, including, for example, calcium phosphate-DNA co-precipitation, DEAE- dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, or biolistics technology (biolistics).

Gene Delivery

An alternative approach to the delivery of therapeutic bispecific protein would be to leave the production of the therapeutic polypeptide to the body itself. A multitude of clinical studies have illustrated the utility of in vivo gene transfer into cells using a variety of different delivery systems. In vivo gene transfer seeks to administer to patients the bispecific protein nucleotide sequence, rather than the bispecific protein itself. This allows the patient’s body to produce the bispecific protein of interest for a prolonged period of time, and secrete it either systemically or locally, depending on the production site. Gene-based nucleotides encoding bispecific proteins can present a labor- and cost-effective alternative to the conventional production, purification and administration of the polypeptide version of the bispecific protein. A number of antibody expression platforms have been pursued in vivo to which delivery of polynucleotides encoding bispecific proteins can be adapted: these include viral vectors, naked DNA and RNA. The use of gene transfer with polynucleotides encoding bispecific proteins cannot only enable cost-savings by reducing the cost of goods and of production but may also be able to reduce the frequency of drug administration. Overall, a prolonged in vivo production of the therapeutic bispecific protein by expression of the polynucleotides encoding bispecific proteins can contribute to (i) a broader therapeutic or prophylactic application of bispecific proteins in price- sensitive conditions, (ii) an improved accessibility to therapy in both developed and developing countries, and (iii) more effective and affordable treatment modalities. In addition to in vivo gene transfer, cells can be harvested from the host (or a donor), engineered with polynucleotides encoding bispecific proteins to produce bispecific proteins and re-administered to patients.

The tumor presents a site for the transfer of polynucleotides encoding bispecific proteins, targeted either via intravenous or direct injection/electroporation. Indeed, intratumoral expression of polynucletodies encoding bipecific proteins can allow for a local production of the therapeutic bispecific proteins, waiving the need for high systemic bispecific protein levels that might otherwise be required to penetrate and impact solid tumors. See, for example, Beckman et al. (2015) “Antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors” Cancer 109(2): 170-9 and Dronca et al. (2015) “Immunomodulatory antibody therapy of cancer: the closer, the better” Clin Cancer Res. 21(5):944-6.

The success of gene therapy has largely been driven by improvements in nonviral and viral gene transfer vectors. An array of physical and chemical nonviral methods have been used to transfer DNA and mRNA to mammalian cells and a substantial number of these have been developed as clinical stage technologies for gene therapy, both ex vivo and in vivo , and are readily adapted for delivery of the polynucleotides encoding bispecific proteins of the present disclosure. To illustrate, cationic liposome technology can be employed, which is based on the ability of amphipathic lipids, possessing a positively charged head group and a hydrophobic lipid tail, to bind to negatively charged DNA or RNA and form particles that generally enter cells by endocytosis. Some cationic liposomes also contain a neutral co-lipid, thought to enhance liposome uptake by mammalian cells. See, for example, Feigner et al. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. MNAS 84:7413-7417; San et al. (1983) “Safety and short-term toxicity of a novel cationic lipid formulation for human gene therapy” Hum. Gene Ther. 4:781-788; Xu et al. (1996) “Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection” Biochemistry 35,:5616-5623; and Legendre et al. (1992) “Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: comparison with cationic liposomes” Pharm. Res. 9, 1235-1242.

Similarly, other polycations, such as poly-l-lysine and polyethylene-imine, can be used to deliver polynucleotides encoding bispecific proteins. These polycations complex with nucleic acids via charge interaction and aid in the condensation of DNA or RNA into nanoparticles, which are then substrates for endosome-mediated uptake. Several of these cationic nucleic acid complex technologies have been developed as potential clinical products, including complexes with plasmid DNA, oligodeoxynucleotides, and various forms of synthetic RNA. Modified (and unmodified or “naked”) DNA and RNA have also been shown to mediate successful gene transfer in a number of circumstances and can also be used as systems for delivery of polynucleotides encoding bispecific proteins. These include the use of plasmid DNA by direct intramuscular injection, the use of intratumoral injection of plasmid DNA. See, for example, Rodrigo et al. (2012) “De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells” PNAS 109:15271-15276; Oishi et al. (2005) “Smart polyion complex micelles for targeted intracellular delivery of PEGylated antisense oligonucleotides containing acid-labile linkages” Chembiochem. 6:718-725; Bhatt et al. (2015) “Microbeads mediated oral plasmid DNA delivery using polymethacrylate vectors: an effectual groundwork for colorectal cancer” Drug Deliv. 22:849-861; Ulmer et al. (1994) Protective immunity by intramuscular injection of low doses of influenza virus DNA vaccines” Vaccine 12: 1541-1544; and Heinzerling et al. (2005) “Intratumoral injection of DNA encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy” Hum. Gene Ther. 16:35-48.

Viral vectors are currently used as a delivery vehicle in the vast majority of pre-clinical and clinical gene therapy trials and in the first to be approved directed gene therapy. See Gene Therapy Clinical Trials Worldwide 2017 (abedia.com/wiley/). The main driver thereto is their exceptional gene delivery efficiency, which reflects a natural evolutionary development; viral vector systems are attractive for gene delivery, because viruses have evolved the ability to cross through cellular membranes by infection, thereby delivering nucleic acids such as polynucleotides encoding bispecific proteins to target cells. Pioneered by adenoviral systems, the field of viral vector-mediated antibody gene transfer made significant strides in the past decades. The myriad of successfully evaluated administration routes, pre-clinical models and disease indications puts the capabilities of antibody gene transfer at full display through which the skilled artisan would readily be able to identify and adapt antibody gene transfer systems and techniques for in vivo delivery of polynucleotides constructs encoding bispecific proteinsln the context of vectored intratumoral polynucleotides encoding bispecific proteinsgene transfer, oncolytic viruses have a distinct advantage, as they can specifically target tumor cells, boost bispecific protein expression, and amplify therapeutic responses - such as to a PD-L1 AFFIMER® bispecifc proteins.

In vivo gene transfer of polynucleotides encoding bispecific proteins can also be accomplished by use of nonviral vectors, such as expression plasmids. Nonviral vectors are easily produced and do not seem to induce specific immune responses. Muscle tissue is most often used as target tissue for transfection, because muscle tissue is well vascularized and easily accessible, and myocytes are long-lived cells. Intramuscular injection of naked plasmid DNA results in transfection of a certain percentage of myocytes. Using this approach, plasmid DNA encoding cytokines and cytokine/IgGl chimeric proteins has been introduced in vivo and has positively influenced (autoimmune) disease outcome.

In some instances, in order to increase transfection efficiency via so-called intravascular delivery in which increased gene delivery and expression levels are achieved by inducing a short-lived transient high pressure in the veins. Special blood-pressure cuffs that may facilitate localized uptake by temporarily increasing vascular pressure and can be adapted for use in human patients for this type of gene delivery. See, for example, Zhang et al. (2001) “Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates” Hum. Gene Ther., 12:427-438

Increased efficiency can also be gained through other techniques, such as in which delivery of the nucleic acid is improved by use of chemical carriers — cationic polymers or lipids — or via a physical approach — gene gun delivery or electroporation. See Tranchant et al. (2004) “Physicochemical optimisation of plasmid delivery by cationic lipids” J. Gene Med., 6 (Suppl. 1): S24-S35; and Niidome et al. (2002) “Gene therapy progress and prospects: nonviral vectors” Gene Ther., 9:1647-1652. Electroporation is especially regarded as an interesting technique for nonviral gene delivery. Somiari, et al. (2000) “Theory and in vivo application of electroporative gene delivery” Mol. Ther. 2:178-187; and Jaroszeski et al. (1999) “In vivo gene delivery by electroporation” Adv. Drug Delivery Rev., 35:131-137. With electroporation, pulsed electrical currents are applied to a local tissue area to enhance cell permeability, resulting in gene transfer across the membrane. Research has shown that in vivo gene delivery can be at least 10- 100 times more efficient with electroporation than without. See, for example, Aihara et al. (1998) “Gene transfer into muscle by electroporation in vivo” Nat. Biotechnol. 16:867-870; Mir, et al. (1999) “High-efficiency gene transfer into skeletal muscle mediated by electric pulses” PNAS 96:4262-4267; Rizzuto, et al. (1999) “Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation” PNAS 96: 6417-6422; and Mathiesen (1999) “Electropermeabilization of skeletal muscle enhances gene transfer in vivo” Gene Ther., 6:508- 514.

Encoded bispecific proteins can be delivered by a wide range of gene delivery system commonly used for gene therapy including viral, non- viral, or physical. See, for example, Rosenberg et al., Science, 242:1575-1578, 1988, and Wolff et al., Proc. Natl. Acad. Sci. USA 86:9011-9014 (1989). Discussion of methods and compositions for use in gene therapy include Eck et al., in Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., eds., McGraw-Hill, New York, (1996), Chapter 5, pp. 77-101; Wilson, Clin.

Exp. Immunol. 107 (Suppl. 1):31-32, 1997; Wivel et al., Hematology/Oncology Clinics of North America, Gene Therapy, S. L. Eck, ed., 12(3):483-501, 1998; Romano et al., Stem Cells, 18:19- 39, 2000, and the references cited therein. U.S. Pat. No. 6,080,728 also provides a discussion of a wide variety of gene delivery methods and compositions. The routes of delivery include, for example, systemic administration and administration in situ.

An effective gene transfer approach should be directed to the specific tissues/cells where it is needed, and the resulting transgene expression should be at a level that is appropriate to the specific application. Promoters are a major cis-acting element within the vector genome design that can dictate the overall strength of expression as well as cell- specificity. In some embodiments, a viral vector is used to deliver a nucleic acid encoding a bispecific protein of the present disclosure. Non-limiting examples of viral vectors include adenoviral vectors, adeno-associated viral (AAV) vectors, and retroviral vectors. In other embodiments, a non-viral vector is used to deliver a nucleic acid encoding a bispecific protein of the present disclosure. Non-limiting examples of non-viral vectors include plasmid vectors (e.g., plasmid DNA (pDNA) delivered via, e.g., hydrodynamic-based transfection or electroporation), minicircle DNA, and RNA-mediate gene transfer (e.g., delivery of messenger RNA (mRNA) encoding a bispecific protein of the present disclosure).

Exemplary nucleic acids or polynucleotides for the encoded bispecific proteins of the present disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a b- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2 '-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof. mRNA presents an emerging platform for antibody gene transfer that can be adapted by those skilled in the art for delivery of polynucleotide constructs encoding bispecific proteinsof the present disclosure. Although current results differ considerably, in certain instances the mRNA constructs appear to be able to rival viral vectors in terms of generated serum mAb titers. Levels were in therapeutically relevant ranges within hours after mRNA administration, a marked shift in speed compared to DNA. The use of lipid nanoparticles (LNP) for mRNA transfection, rather than the physical methods typically required for DNA, can provide significant advantages in some embodiments towards application range.

Nucleic acids encoding bispecific proteins may be delivered g, for example, intravenously, intramuscularly, or intratumorally (e.g., by injection, electroporation or other means).

Nucleic acids encoding bispecific proteins may be formulated, for example, in lipid nanoparticles or liposomes (e.g., cationic lipid nanoparticles or liposomes), biodegradable microsphere, or other nano- or microparticle. Other lipid-based (e.g., PEG lipid) and polymeric- based formulations and delivery vehicles are contemplated herein. EXAMPLES

Example 1. AFFIMER® Phage Selections Process Overview Phage Selections

• Biopanning on captured Human FcRn

• Solution selection on biotinylated human FcRn

• Two (2) rounds of selection on human FcRn

• Enrichment monitored by output size and polyclonal Phage ELISA

Primary Screening

• Monoclonal Crude extract ELISA against captured human FcRn at pH6

Secondary Screening

• ELISA on human FcRn at pH 6.0 and 7.4

General Methods

Selection of human FcRn binding phage from the AFFIMER® library was carried out as described below using approximately 1 x 10¹² phage added from a library of size approximately 6 x 10¹⁰ diversity.

A peptide of the present disclosure, for example, a human FcRn binding component, may be identified by selection from a library of AFFIMER® polypeptides with two random loops, for example, generally but not exclusively of the same length of 9 amino acids.

As indicated above, the human FcRn binding peptides of the disclosure were identified by selection from a phage display library comprising random loop sequences nine amino acids in length displayed in a constant AFFIMER® framework backbone based upon the sequence for SQT. Such selection procedures are generally known. According to such procedures, suspensions of phage are incubated with target antigen (either biotinylated antigen captured on streptavidin beads or unbiotinylated antigen captured on a plate). Unbound phage are then washed away and, subsequently, bound phage are eluted either by incubating the antigen with low pH, high pH or trypsin. E. coli are then infected with released, pH neutralized phage or trypsin-inactivated phage and a preparation of first round phage is obtained. The cycle is performed repeatedly, for example, two or three times and, in order to enrich for targeting phage, the stringency conditions may be increased in the later rounds of selection, for example by increasing the number of wash steps, reducing the antigen concentration, and preselecting with blocked streptavidin beads or wells coated with blocking reagent. Antigens used herein were human FcRn (BPS # 71285), and biotinylated human FcRn (BPS # 71283). Following selection by successive rounds of phage amplification, human FcRn binding clones were identified by a crude extract ELISA as described below.

Following phage selections, individual bacterial clones containing the phagemid vector were picked from titration plates into 96 well cell culture format. Soluble AFFIMER® polypeptide in crude cell extract was prepared from lysis of bacterial cells overexpressing the AFFIMER® polypeptides with a C-terminal myc tag and used in a primary screening ELISA. These AFFIMER® polypeptides in extract were screened for binding to antigen at pH 6 and later also at pH 7.4, detecting AFFIMER® polypeptide bound to antigen immobilized on a plate with an HRP labelled anti-myc tag antibody (Abeam # abl261), developing the ELISA using 1-step Ultra TMB-ELISA substrate (Thermo Scientific). The screening was also carried out against non-target or related target molecules captured on the plate ( e.g ., blocking molecule, neutravidin or b-2microglobulin (Sigma #M4890) The non-target and target binding data were compared to identify library members that specifically bind to the target.

Example 2. Production and purification of FcRn binding AFFIMER® Polypeptides

AFFIMER® proteins were formatted produced from E. coli by transformation of expression plasmid pD861-CMH (C terminal Myc His; Atum) into BL21 E. coli cells (Millipore) using the manufacturers protocol. Total transformed cell mixture was plated onto LB agar plates containing 50pg/ml kanamycin (AppliChem) and incubated at 37°C overnight. The following day the lawn of transformed E. coli was transferred to a sterile flask of lx terrific broth media (Melford) & 50 pg/ml Kanamycin and incubated at 37°C shaking at 250 rpm. Expression was induced with 10 mM Rhamnose (Alfa Aesar) once the cells have reached an optical density OD600 of 0.8 and the culture incubated overnight at 30°C. Harvested cells by centrifuging and lysing cell pellet using lOx bugbuster (Millipore), lysozyme (Applichem), Benzonase nuclease (Millipore) and protease inhibitors (Roche) for 1 hour at room temperature and centrifuged to clarify solution at 20,000 rpm for lh. AFFIMER® polypeptide purification was performed using batch bind affinity purification using the C-terminal 6xHis tagged protein using 96 well His MultiTrap HP plates (GE Healthcare) using the manufacturers protocol using NPI-20 binding buffer; 50mM Sodium phosphate, 0.5M NaCl, 20mM Imidazole and NPI-400 elution buffer; 50mM Sodium phosphate, 0.5M NaCl, 0.4M Imidazole. Final eluted protein was buffer exchanged into PBS lx (Melford) using 96 well PD MultiTrap G25 (GE Healthcare) as described by the manufacturer or purified with a preparative size exclusion 26/600 column (GE) run in PBS lx at 3.6ml/min. Purity of AFFIMER® polypeptides expressed were analysed using SEC-HPLC running Acclaim SEC-300 column (Thermo) in PBS lx mobile phase on an Ultimate-3000 UPLC system (Thermo). The protein yield was estimated using Nanodrop (Thermo) A280 readings and the final product run on an SDS-PAGE Bolt Bis Tris plus 4-12% gel (Thermo) in Novex™ 20X Bolt™ MES SDS running buffer (Thermo) at 200 volts for 20 minutes, with samples heated at 95 °C in reducing buffer. AFFIMER® protein bands on gel were stained with Quick Commas sie (Generon). PageRuler prestained protein molecular weight marker (Thermo) was run on the gel to estimate the molecular weight of the final proteins at 15-16 kDa (FIG. 1).

Example 3. huFcRn Binding ELISA Assay at pH 6.0 and 7.4

To evaluate the binding capacity of AFFIMER® polypeptide to human FcRn at pH 6 and 7.4, enzyme linked immunosorbent assay (ELISA) in 384 well plate format was performed.

Briefly huFcRn (BPS Bioscience) was coated at 5pg/ml on the plate in 40 mM MES, pH 6. Plates were washed 3 times with 100 mΐ of washing buffer (PBS, Tween 200.05%, pH 6) with a plate washer and saturated with Casein 5% (Sigma) in MES pH6 for 60 minutes at room temperature (25 ±l°c). Plates were washed as described previously. AFFIMER® polypeptide and negative controls (mAb anti-huFcRn (clone ADM31), negative controls) were then diluted in duplicate, and loaded on the plate for 90 minutes at room temperature (25 ±l°c). Plates were washed 3 times as described previously. Biotinylated polyclonal antibody anti Cystatin (R&D Systems) was then diluted in dilution Buffer (1% casein, 0.05% Tween 20, and 8 mM MES. It is in pH6) and incubated 60 minutes at room temperature (25 ±l°c). Plates were washed 3 times as described previously and Streptavidin HRP (N200, thermo-Fisher) was incubated for 30 minutes at room temperature (25 ±l°c). Plates were washed and the substrate (TMB, Pierce Thermo- Scientific) was added in the plate for 8+1 minute. The reaction was stopped using an acidic solution and plates were read at 450 -630 nm. (FIG. 2).

Similarly, binding to huFcRn at pH 7.4 was assessed. huFcRn (BPS Bioscience) was coated at 5pg/ml on the plate in PBS, pH 7.4. Plates were washed 3 times with 100 mΐ of washing buffer (PBS, Tween 200.05%, pH 7.4) with a plate washer and saturated with Casein 5%

(Sigma) in MES pH 7.4 for 60 minutes at room temperature (25 ±l°c). Plates were washed as described previously. AFFIMER® polypeptide and controls (mAb anti-huFcRn (ADM31), blank) were then diluted in duplicate, and loaded on the plate for 90 minutes at room temperature (25 ±l°c). Plates were washed 3 times as described previously. Biotinylated polyclonal antibody anti Cystatin (R&D Systems) was then diluted in dilution Buffer (1% casein, 0.01% Tween 20, and 8 mM MES. It is in pH7.4) and incubated 60 minutes at room temperature (25 ±l°c). Plates were washed 3 times as described previously and Streptavidin HRP (N200, thermo-Fisher) was incubated for 30 minutes at room temperature (25 ±l°c). Plates were washed and the substrate (TMB, Pierce Thermo-Scientific) was added in the plate for 8+1 minute. The reaction was stopped using an acidic solution and plates were read at 450 -630 nm. The EC 50 value was then calculated using the interpolated non-linear four-parameters standard curve and pH was plotted on the same figure overlaying binding profile for each clone at both pH showing a lower binding capacity at pH 7.4 compared to pH 6. (FIG. 2, Table 9).

Table 9. Differential binding of LGC01 clones at pH 6 and 7.4 in a direct huFcRn ELISA.

Example 4. Formatted bispecific AFFIMER® in-line fusion (ILF) with hPD-Ll and FcFn binders

Two bispecific ILF AFFIMER® proteins PD-L1-251 FX3 (SEQ ID NO: 1268) and PD- Ll-251 FX6 (SEQ ID NO: 1269) (PD-L1-251 dimer (SEQ ID NO: 1270) genetically fused to FcRn-38 (SEQ ID NO: 708)) were expressed from E.coli as described in Example 1. Schematic representations of PD-L1 binding AFFIMER® polypeptide genetically fused to an FcRn-binding affimer with rigid linkers A(EAAAK)_ό (SEQ ID NO: 1291) compared to parent AFFIMER® PD-L1 251 BH (SEQ ID NO: 1270) dimer (FIG. 3). Two stage purified AFFIMER® ILF proteins were run on SEC-HPLC and SDS-PAGE to confirm >98% purity (FIG. 4).

Table 10. Nomenclature of PD-L1 binding AFFIMER® in-line fusion with FcRn binding AFFIMER®

To evaluate if the addition of FcRn-38 (SEQ ID NO: 708) at various positions in an AFFIMER® in-line fusion format impacted the binding to human PD-L1 of PD-L1-251 dimer, a PD-L1 binding ELISA was performed with the two (2) FcRn ILF formatted AFFIMER® polypeptides (FIG. 5). Briefly, human PD-Ll-Fc (R&D Systems) chimeric protein was coated in 96 well plates at 0.5 mg/ml in carbonate buffer. After saturation with 5% casein/PBS, plates were washed and a dilution of AFFIMER® polypeptides or controls were incubated for 90 minutes. Plates were then washed and a biotinylated polyclonal antibody anti-cystatin A (R&D Systems) was added for 1 hour. Plates were washed and AFFIMER® polypeptides were detected using Strepativin-HRP. After a last washing step, TMB was added for the development of the experiment and plates were read at 450 nm. The two (2) constructs tested exhibit similar EC50 (ranging from 0.01 to 0.04 nM) and are identical to the anti-PD-Ll parental ILF dimer molecule (PD-L1 -251 BH (SEQ ID NO: 1270).

Similarly, the binding to hu FcRn has been assessed at pH 6 (as described in example 2). The two (2) constructs tested exhibit similar EC50 (ranging from 0.03 to 0.49 nM) and decrease binding to target protein less than 20-fold compared to the parental molecule (FcRn-38; SEQ ID NO: 708) (FIG. 2).

Example 5. AFFIMER® expression and purification

All AFFIMER® constructs expressed in E. coli have been cloned with a C-terminal hexa-HIS tag (HHHHHH) (SEQ ID NO: 1292) to simplify protein purification with immobilized metal affinity chromatography resin (IMAC resin). When required, additional peptide sequences can be added between the AFFIMER® polypeptide and the HIS tag such as MYC (EQKLISEEDL) (SEQ ID NO: 1297) for detection or a TEV protease cleavage site (ENLYFQ(G/S)) (SEQ ID NO: 1298) to allow for the removal of tags. AFFIMER® proteins were expressed from E. coli and purified using IMAC, a second stage purification to remove endotoxin, CHT (Ceramic hydroxyapatite, BioRad) type I resin or cation ion exchange (HiTrap, Cytiva) with a triton 114x wash step (Sigma), and size exclusion chromatography (SEC; Cytiva). AFFIMER® monomer purification from E. coli was performed by transforming the expression plasmid pD861 (Atum) into BL21 E. coli cells (Millipore) using the manufacturer’s protocol.

The total transformed cell mixture was plated onto LB agar plates containing 50pg/ml kanamycin (AppliChem) and incubated at 37°C overnight. The following day, the lawn of transformed E. coli was transferred to a sterile flask of lx terrific broth media (Melford) and 50 pg/ml kanamycin and incubated at 30°C shaking at 250 rpm. Expression was induced with 10 mM rhamnose (Alfa Aesar) once the cells reached an optical density ODeoo of approximate 0.8- 1.0. The culture was then incubated for a further 5 hours at 37°C. Cells were harvested by centrifuging and lysing the resulting cell pellet. AFFIMER® purification was performed using batch bind affinity purification of His-tagged protein. Specifically, nickel agarose affinity resin (Super-NiNTA500; Generon) was used. The resin was washed with NPI20 buffer (50mM sodium phosphate, 0.5M NaCl, 20mM imidazole) and the bound protein was eluted with 5 column volumes (CV) of NPI400 buffer. Eluted protein was buffer exchanged for a second stage purification using CHT type I resin in running buffer lOmM sodium phosphate pH6.4-6.5 buffer, eluting with the addition of 2M NaCl over a linear gradient (SEQ ID NO: 705, 708, 790 and 965). Alternatively, a second stage purification using cation exchange was used with a SP HP ion exchange column (Cytiva) in running buffer 50mM MES pH 6.2 for clone FcRn-125 included a 0.1% triton 114x (Sigma) wash step and the protein was eluted with a 1M NaCl linear gradient (SEQ ID NO: 795). A third stage polishing purification was performed on a preparative SEC performed using the HiLoad 26/600 Superdex 75pg (Cytiva) run in PBS lx buffer. Expression and purity of clones was analysed using SEC-HPLC (FIGs. 6A-6B) with an Acclaim SEC-300 column (Thermo) using a PBS lx mobile phase. The protein yield was estimated using Nanodrop (Thermo) A280 readings and the final product was run on an SDS-PAGE Bolt Bis Tris plus 4-12% gel (Thermo) in Novex™ 20X Bolt™ MES SDS running buffer (Thermo) at 200 volts, with samples heated in reducing buffer at 95 °C for 5 minutes. Protein bands on the gel were stained with Quick Commassie (Generon). PageRuler prestained protein molecular weight marker (Thermo) was run on the gel to estimate the molecular weight of the fusion proteins (FIG. 7) following the three-stage purification. Endotoxin levels of final protein batches were measured using a LAL test on an Endosafe® Nexgen MCS system (Charles River) and were between 1-0.1 EU/mg for all protein batches.

Example 6. huFcRn Binding ELISA Assay at pH 6 for AFFIMER® Polypeptide Characterization

The binding of AFFIMER® polypeptide to hu-FcRn was evaluated by enzyme linked immunosorbent assay (ELISA) in 384 well plate format. Hu FcRn (BPS Bioscience) was coated at 5pg/ml on the plate in 40 mM MES, pH 6. Plates were washed 3 times with 100 mΐ of washing buffer (PBS, Tween 200.05%, pH 6) with a plate washer and saturated with Casein 5% (Sigma) in MES pH6 for 60 minutes at room temperature (25 ±1°C). Plates were washed as described previously. AFFIMER® polypeptide and negative controls (mAb anti hFcRn (clone ADM31), negative controls) were then diluted in duplicate, and loaded on the plate for 90 minutes at room temperature (25 ±l°c). Plates were washed 3 times as described previously. Biotinylated polyclonal antibody anti Cystatin (R&D Systems) was then diluted in dilution buffer (1% casein, 0.05% Tween 20, and 8 mM MES; pH 6) and incubated 60 minutes at room temperature (25 ±1°C). Plates were washed 3 times as described previously and Streptavidin HRP (N200, Thermo-Fisher) was incubated for 30 minutes at room temperature (25 ±1°C). Plates were washed and the substrate (TMB, Pierce Thermo-Scientific) was added in the plate for 8+1 minute. The reaction was stopped using an acidic solution and plates were read at 450-630 nm. The EC50 was then calculated using the interpolated non-linear four-parameters standard curve (FIGs. 8A-8B and Table 11).

Example 7. huFcRn Binding ELISA Assay at pH 7.4 for AFFIMER® Polypeptide Characterization

The binding of AFFIMER® polypeptide to hu-FcRn was evaluated by enzyme linked immunosorbent assay (ELISA) in 384 well plate format. Hu FcRn (BPS Bioscience) was coated at 5pg/ml on the plate in PBS, pH 7.4. Plates were washed 3 times with 100 mΐ of washing buffer (PBS, Tween 200.05%, pH 7.4) with a plate washer and saturated with Casein 5% (Sigma) in MES pH 7.4 for 60 minutes at room temperature (25 ±1°C). Plates were washed as described previously. AFFIMER® polypeptide and controls (mAb anti hFcRn (ADM31), blank) were then diluted in duplicate, and loaded on the plate for 90 minutes at room temperature (25 ±1°C).

Plates were washed 3 times as described previously. Biotinylated polyclonal antibody anti Cystatin (R&D Systems) was then diluted in dilution Buffer (1% casein, 0.01% Tween 20, and 8 mM MES. It is in pH7.4) and incubated 60 minutes at room temperature (25 ±l°c). Plates were washed 3 times as described previously and Streptavidin HRP (N200, thermo-Fisher) was incubated for 30 minutes at room temperature (25 ±l°c). Plates were washed and the substrate (TMB, Pierce Thermo-Scientific) was added in the plate for 8+1 minute. The reaction was stopped using an acidic solution and plates were read at 450 -630 nm. The EC 50 was then calculated using the interpolated non -linear four-parameters standard curve (FIGs. 8A-8B, Table 11).

Table 11. ECso at pH 6 and pH 7.4

Example 8. FcRn competition ELISA

To evaluate if the AFFIMER® polypeptide was competiting with IgGl, a competitive ELISA (huIgGl/huFcRn) was performed. Briefly, huIgGl isotype control (BioXcell) was coated overnight on the plate at 5 pg/ml in 40 mM MES, pH 6. Then plates were saturated using 40 mM MES + 5% casein, pH 6. In the meantime, huFcRn (His-tagged molecule, BPS) was pre incubated with a dilution of FcRn Binding AFFIMER® polypeptide and its control (human IgGl and HuSA. After saturation, plates were washed in PBS, 0.05% Tween at pH6, the mix was added to the plates and incubated for minimum an hour. Plates were then washed as previously and the detection monoclonal antibody, anti-B2M HRP (Biolegend), was added and incubated for minimum 1 hour. After a final wash, development of the reaction was performed using TMB (Pierce) and the plates were read using a plate reader at 450 nm and absorbance was plotted against log of AFFIMER® polypeptide and control concentration using a four-parameter fit.

FIG. 9 shows FcRn binding AFFIMER® polypeptides do not compete with huIgGl.

Example 9. Screening of lead FcRn binding AFFIMER® polypeptides for receptor mediated recycling in a human endothelial cell-based recycling assay

7.5 x 10⁵ endothelial cell line (HMEC1) stably expressing HA-hFcRn-EGFP were seeded into 24-well plates per well (Costar) and cultured for 2 days in growth medium. The cells were washed twice and starved for 1 hour in Hank’s balanced salt solution (HBSS) (ThermoFisher). Then, 800 nM of either hlgGl or AFFIMER® polypeptide were diluted in 125 pi HBSS (pH 7.4) and added to the cells followed by 4 h incubation. The media was removed and the cells were washed four times with ice cold HBSS (pH 7.4), before fresh warm HBSS (pH 7.4) or growth medium without FCS and supplemented with MEM non-essential amino acids (ThermoFisher) was added. The cells were incubated for 4 hours before sample were collected. The wells with uptake samples and residual amounts were then lysed prior to collection. Total protein lysates were obtained using RIPA lysis buffer (ThermoFisher) supplied with complete protease inhibitor tablets (Roche). The mixture was incubated (220 ul) with the cells on ice and a shaker for 10 min followed by centrifugation for 15 min at 10,000 x g to remove cellular debris. Rescued AFFIMER® polypeptides and controls were quantified by quantitative ELISA anti-cystatin (see Example 8) or anti-human IgG (FIG. 10).

Example 10. AFFIMER® polypeptide quantification by ELISA following HERA assay

96-well plates (Coming Costar, 3590) were coated with 50ul of lug/ml of Anti-His MAB050 diluted in coating buffer (Carbonate/bicarbonate) for 16 hours (+/- 2h) at 4°C. The plates were further washed 2x with 150ul wash buffer (lx PBS + 0.05% Tween) and blocked with lOOul lx PBS + 5% casein blocking buffer for 90 min (+/- 15 min) at room temperature (RT). Next, the HERA samples were added to the plates, diluted 1:1 in 6 steps in dilution buffer (PBS + 1% casein + 0.01% Tween) and matching AFFIMER® polypeptides were used as a standard for each variant (3.5nM - 0.0017nM). The HERA samples were incubated for 90 min (+/- 15 min) at RT. Plates were washed 3x with wash buffer. Binding was detected by using 0.05mg/ml BAF1470 1:1000 and lmg/ml poly streptavidin-HRP 1:5000. The two antibodies were pre incubated in a small volume for 20 min, before diluted in dilution buffer and added to the plates in 50ul volume and incubated for 90 min (+/- 15 min) at RT. Plates were washed 3x and binding was visualized by adding 50ul of RT TMB to each well. The reaction was stopped by adding 50ul 1M HC1 (after 20-30 min). Absorbance was read at 450nm and 620nm. Control IgGl was quantified using similar protocol using a goat polyclonal anti human Fc for capture and an alkaline phosphatase conjugated polyclonal antibody anti huIgGFc for detection.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

Where a range of values is provided, each value between the upper and lower ends of the range are specifically contemplated and described herein.

Claims

What is claimed is: CLAIMS

1. A bispecific protein comprising an FcRn-binding recombinantly engineered variant of stefin polypeptide and a PD-L1 -binding recombinantly engineered variant of stefin polypeptide, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide binds to human FcRn with a K_d of 1X10⁶M or less at pH 6.0 and optionally a K_d for binding human FcRn at pH 7.4 that is at least half a log greater than the K_d for binding at pH 6.0, and wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide binds to PD-L1 with a K_d of 1X10⁶M.

2. A bispecific protein comprising an FcRn-binding recombinantly engineered variant of stefin polypeptide that binds to human FcRn and a PD-Ll-binding rombinantly engineered variant of stefin polypeptide that binds to PD-L1, wherein the protein has a circulating half-life in human subjects of at least 7 days.

3. A bispecific protein comprising an FcRn-binding recombinantly engineered variant of stefin polypeptide that binds to human FcRn and a PD-Ll-binding recombinantly engineered variant of stefin polypeptide that binds to PD-L1, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide facilitates transport of the protein across an epithelial tissue barrier.

4. A bispecific protein comprising an FcRn-binding recombinantly engineered variant of stefin polypeptide that binds to human FcRn and a PD-L1 -binding recombinantly engineered variant of stefin polypeptide that binds to PD-L1, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide has an amino acid sequence that is at least 75% identical to an recombinantly engineered variant of stefin polypeptide selected from SEQ ID NOS: 671- 964, and wherein the PD-L1 -binding recombinantly engineered variant of stefin polypeptide has an amino acid sequence that is at least 75% identical to an recombinantly engineered variant of stefin polypeptide selected from SEQ ID NOS: 662-670.

5. A bispecific protein comprising an FcRn-binding recombinantly engineered variant of stefin polypeptide that binds to human FcRn and a PD-L1 -binding recombinantly engineered variant of stefin polypeptide that binds to PD-L1, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide has an amino acid sequence that can be encoded by a polynucleotide having a coding sequence that hybridizes to any one of SEQ ID NOS: 974-1267 under stringent conditions of 6X sodium chloride/sodium citrate (SSC) at 45°C followed by a wash in 0.2X SSC at 65°C, and wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide has an amino acid sequence that can be encoded by a polynucleotide having a coding sequence that hybridizes to any one of SEQ ID NOS: 965-973 under stringent conditions of 6X sodium chloride/sodium citrate (SSC) at 45°C followed by a wash in 0.2X SSC at 65°C.

6. The bispecific protein of any of the preceding claims, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide binds to FcRn with a K_d of lxlO ⁷ M or less at pH 6.0, a K_d of lxlO^-8 M or less at pH 6.0, or K_d of lxlO ⁹ M or less at pH 6.0.

7. The bispecific protein of any of the preceding claims, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide binds to FcRn at pH 7.4 with a K_dthat is at least one log greater than the K_d for binding to FcRn at pH 6.0, at least 1.5 logs greater than the K_d for binding to FcRn at pH 6, at least 2 logs greater than the K_d for binding to FcRn at pH 6, or at least 2.5 log greater than the K_d for binding to FcRn at pH 6.

8. The bispecific protein of any one of the preceding claims, wherein the protein has a serum half-life in human patients of greater than 10 hours, greater than 24 hours, greater than 48 hours, greater than 72 hours, greater than 96 hours, greater than 120 hours, greater than 144 hours, greater than 168 hours, greater than 192 hours, greater than 216 hours, greater than 240 hours, greater than 264 hours, greater than 288 hours, greater than 312 hours, greater than 336 hours or, greater than 360 hours.

9. The bispecific protein of any one of the preceding claims, wherein the bispecific protein has a serum half-life in human subjects of greater than 50%, greater than 60%, greater than 70%, or greater than 80% of the serum half-life of IgG.

10. The bispecific protein of any one of the preceding claims, wherein the bispecific protein has a serum half-life in human subjects of greater than 50%, greater than 60%, greater than 70%, or greater than 80% of the serum half-life of serum albumin.

11. The bispecific protein of any one of the preceding claims, wherein the bispecific protein does not inhibit binding of human serum albumin to human FcRn.

12. The bispecific protein of any one of the preceding claims, wherein the bispecific protein does not inhibit binding of IgG to human FcRn.

13. The bispecific protein of any one of the preceding claims, wherein binding of the FcRn- binding recombinantly engineered variant of stefin polypeptide to human FcRn facilitates transport of the polypeptide from an apical side to a basal side of an epithelial cell layer.

14. The bispecific protein of any one of the preceding claims, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide comprise an amino acid sequence represented in general formula (I)

FRl-(Xaa)_n-FR2-(Xaa)_m-FR3 (I), wherein

FR1 is an amino acid sequence having at least 70% identity to MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 1);

FR2 is an amino acid sequence having at least 70% identity to GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2);

FR3 is an amino acid sequence having at least 70% identity to EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3); and

Xaa, individually for each occurrence, is an amino acid, n is an integer from 3 to 20, and m is an integer from 3 to 20.

15. The bispecific protein of claim 14, wherein:

FR1 has at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% identity to SEQ ID NO: 1;

FR2 has at least 80%, at least 84%, at least 88%, at least 92%, or at least 96% identity to

SEQ ID NO: 2; and/or.

FR3 has at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:

3.

16. The bispecific protein of claim 14, wherein:

FR1 comprises the amino acid sequence of SEQ ID NO: 1;

FR2 comprises the amino acid sequence of SEQ ID NO: 2; and/or FR3 comprises the amino acid sequence of SEQ ID NO: 3.

17. The bispecific protein of any of claims 14-16, wherein (Xaa)_n is at least 75% identical to a Loop 2 sequence selected from SEQ ID NOS: 74-367.

18. The bispecific protein of any of claims 14-17, wherein (Xaa)_m is at least 75% identical to a Loop 4 sequence selected from SEQ ID NOS: 368-661.

19. The bispecific protein of any one of the preceding claims, wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide comprise an amino acid sequence represented in general formula (I)

FRl-(Xaa)_n-FR2-(Xaa)_m-FR3 (I), wherein

20. The bispecific protein of claim 19, wherein:

SEQ ID NO: 2; and/or.

3.

21. The bispecific protein of claim 20, wherein:

FR1 comprises the amino acid sequence of SEQ ID NO: 1;

22. The bispecific protein of any of claims 19-21, wherein (Xaa)_n is at least 75% identical to a Loop 2 sequence selected from SEQ ID NOS: 4-38.

23. The bispecific protein of any of claims 19-22, wherein (Xaa)_m is at least 75% identical to a Loop 4 sequence selected from SEQ ID NOS: 39-73.

24. The bispecific protein of any one of the preceding claims, wherein the bispecific protein includes at least one cysteine, which is optionally available for chemical conjugation, and which (optionally) is located at the C-terminal end or the N-terminal end of the bispecific protein.

25. A pharmaceutical composition suitable for therapeutic use in a human subject, comprising a bispecific protein of any of any one of the preceding claims, and a pharmaceutically acceptable excipient.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is formulated for pulmonary delivery or topical application.

27. The pharmaceutical composition of claim 26, wherein the pulmonary delivery is intranasal delivery.

28. A polynucleotide comprising a sequence encoding the bispecific protein of any of any one of the preceding claims.

29. The polynucleotide of claim 28, wherein the sequence encoding the bispecific protein is operably linked to a transcriptional regulatory sequence.

30. The polynucleotide of claim 29, wherein the transcriptional regulatory sequence is selected from the group consisting of promoters and enhancers.

31. The polynucleotide of any of claims 28-30, further comprising an origin of replication, a minichromosome maintenance element (MME), and/or a nuclear localization element.

32. The polynucleotide of any of claims 28-31, further comprising a polyadenylation signal sequence operably linked and transcribed with the sequence encoding the polypeptide.

33. The polynucleotide of any of claims 28-32, wherein the sequence encoding the polypeptide comprises at least one intronic sequence.

34. The polynucleotide of any of claims 28-33, further comprising at least one ribosome binding site transcribed with the sequence encoding the polypeptide.

35. The polynucleotide of any of claims 28-34, wherein the polynucleotide is a deoxyribonucleic acid (DNA).

36. The polynucleotide of any of claims 28-35, wherein the polynucleotide is a ribonucleic acid (RNA).

37. A viral vector comprising the polynucleotide of any of claims 28-36.

38. A plasmid or minicircle comprising the polynucleotide any of claims 28-37.

39. A cell comprising the polypeptide of any one of the preceding claims, the polynucleotide of any one of the preceding claims, the viral vector of claim 37, or the plasmid or minicircle of claim 38.

40. The bispecific protein of any one of the preceding claims for use in a method for treating an autoimmune disease and/or an inflammatory disease.

41. The bispecific protein of any one of the preceding claims for use in a method for treating cancer.

42. The bispecific protein of any one of the preceding claims for use in a method for treating cardiovascular or metabolic disease or disorder.

43. A method of producing the bispecific protein of any one of the preceding claims, the method comprising expressing in a host cell a nucleic acid encoding the polypeptide, and optionally isolating the polypeptide from the host cell.

44. The bispecific protein of any one of the preceding claims, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide binds to human FcRn with a K_d of 1X10⁸M or less at pH 6.0 and optionally a K_d for binding human FcRn at pH 7.4 that is at least half a log greater than the K_d for binding at pH 6.0, and wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide binds to PD-L1 with a K_d of 1X10^~8M.

45. The bispecific protein of any one of the preceding claims, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide binds to human FcRn with a K_d of 1X10⁹M or less at pH 6.0 and optionally a K_d for binding human FcRn at pH 7.4 that is at least half a log greater than the K_d for binding at pH 6.0, and wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide binds to PD-L1 with a K_d of 1X10 ⁹M.

46. The bispecific protein of any one of the preceding claims, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide binds to human FcRn with a K_d of lxlO ¹⁰M or less at pH 6.0 and optionally a K_d for binding human FcRn at pH 7.4 that is at least half a log greater than the K_d for binding at pH 6.0, and wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide binds to PD-L1 with a K_d of lxlO ¹⁰M.

47. The bispecific protein of any one of the preceding claims comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 1268.

48. The bispecific protein of any one of the preceding claims comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 1269.

49. The bispecific protein of claim 45 encoded by a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 1271.

50. The bispecific protein of claim 46 encoded by a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 1272.

51. The bispecific protein of any one of the preceding claims, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide comprises a loop 2 amino acid sequence of any one of SEQ ID NOs: 74-367.

52. The bispecific protein of any one of the preceding claims, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide comprises a loop 4 amino acid sequence of any one of SEQ ID NOs: 368-661.

53. The bispecific protein of any one of the preceding claims, wherein the FcRn-binding recombinantly engineered variant of stefin polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%identity to the sequence of any one of SEQ ID NOs: 671-964.

54. The bispecific protein of any one of the preceding claims, wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide comprises a loop 2 amino acid sequence of any one of SEQ ID NOs: 4-38.

55. The bispecific protein of any one of the preceding claims, wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide comprises a loop 4 amino acid sequence of any one of SEQ ID NOs: 39-73.

56. The bispecific protein of any one of the preceding claims, wherein the PD-Ll-binding recombinantly engineered variant of stefin polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of any one of SEQ ID NOs: 662-670.