PD-Ll BINDING FIBRONECTIN TYPE III DOMAINS

FIELD OF THE INVENTION

The present invention relates to fibronectin type III domains that specifically bind to PD-Ll and methods of making and using the molecules.

BACKGROUND OF THE INVENTION

The immune system is tightly controlled by a network of costimulatory and co- inhibitory ligands and receptors. These molecules provide secondary signals for T cell activation and provide a balanced network of positive and negative signals to maximize immune responses against infection and tumors, while limiting immunity to self (Wang et al., (Epub Mar. 7, 2011) J Exp Med 208(3): 577-92; Lepenies et al, (2008) EndocrMetab Immune Disord Drug Targets 8:279-288).

Programmed Death- 1 (PD-1) is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. The ligand for PD-1, PD-Ll, is expressed by antigen-presenting cells and many cancers such as lung, ovarian and colon carcinoma and various myelomas. Binding of PD-Ll to PD-1 on T cells downregulates T cell proliferation and activation and drives T cell anergy and exhaustion in the tumor microenvironment, facilitating tumor cell escape from T-cell mediated immune surveillance.

Therapeutic efficacy of PD-1 and PD-Ll antagonists has been validated in clinical trials. However, response rates remain low. For example, Opdivo® (Nivolumab) treatment achieved a 26% objective response rate (ORR) across the 27 clinical trials analyzed (Tie et al, Int J Cancer 2016 Nov 4 doi: 10.1002/ijc.30501. [Epub ahead of print])

Measuring the expression of PD-Ll protein in the tumor tissue may aid in the early detection of cancer pathologies and may help assess the efficacy and durability of PD-Ll and PD-1 antagonists. For example, PD-Ll expression in at least 50% of tumor cells correlated with improved efficacy of Keytruda® (pembrolizumab) (Garon et al., N Engl J Med 2015; 372:2018-2028), and PD-Ll expression has been correlated with poor prognosis (see for example Wang et al., Eur J Surg oncol 2015 Apr; 41(4):450-6).

However, the use of PD-Ll protein expression as an accurate predictor for cancer and/or the efficacy of anti-PD-1 and anti-PD-Ll directed therapies remain challenging partially due to observed variability in results depending on the detection reagent used. For example, the evaluation of PD-Ll expression in non-small cell lung cancer samples using commercially available assays such as PD-Ll (E1L3N®) XP® Rabbit mAb (Cell Signaling) and Ventana PD-Ll (SP142) Assay yielded discordant results (McLaughlin et al, JAMA Oncol 2016 Jan;2(l):46-54)

Therefore, there is a need for reagents to accurately detect PD-Ll in tumor tissues and other samples and new therapeutics that modulate the interaction between PD-Ll and PD-1.

SUMMARY OF THE INVENTION

The invention provides an isolated FN3 domain that specifically binds to PD-Ll.

The invention also provides an isolated FN3 domain that specifically binds to PD- Ll comprising the sequence of SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 or 124.

The invention also provides an isolated polynucleotide encoding the FN3 domain that specifically binds to PD-Ll of the invention.

The invention also provides a vector comprising the polynucleotide of the invention.

The invention also provides a host cell comprising the vector of the invention.

The invention also provides a method of producing the FN3 domain that specifically binds to PD-Ll of the invention, comprising culturing the isolated host cell of the invention under conditions that the FN3 domain that specifically binds to PD-Ll is expressed, and purifying the FN3 domain that specifically binds PD-Ll.

The invention also provides a pharmaceutical composition comprising the FN3 domain that specifically binds PD-Ll of the invention and a pharmaceutically acceptable carrier.

The invention also provides an anti-idiotypic antibody that specifically binds the FN3 domain that specifically binds to PD-Ll of the invention.

The invention also provides a kit comprising the FN3 domain of the invention.

The invention also provides a method of detecting PD-Ll -expressing cancer cells in a tumor tissue, comprising

obtaining a sample of the tumor tissue from a subject; and detecting whether PD-L1 is expressed in the tumor tissue by contacting the sample of the tumor tissue with the FN3 domain that specifically binds to PD-L1 comprising the sequence of any one of SEQ ID NOs: 34-124 and detecting the binding between PD- Ll and the FN3 domain.

The invention also provides a method of isolating or detecting PD-L1 expressing cells, comprising obtaining a sample from a subject;

contacting the sample with the FN3 domain that specifically binds to PD-L1 comprising the sequence of any one of SEQ ID NOs: 34-124, and

isolating or detecting the cells bound to the FN3 domains.

The invention also provides a method of detecting PD-L1 -expressing cancer cells in a tumor tissue, comprising

conjugating the FN3 domain that specifically binds to PD-L1 comprising the sequence of any one of SEQ ID NOs: 34-124 to a detectable label to form a conjugate;

administering the conjugate to a subject; and

visualizing the PD-L1 expressing cancer cells to which the conjugate is bound.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

"Fibronectin type III (FN3) domain" (FN3 domain) refers to a domain occurring frequently in proteins including fibronectins, tenascin, intracellular cytoskeletal proteins, cytokine receptors and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacterid 175: 1910-1918, 1993; Watanabe et ai, J Biol Chem 265: 15659-15665, 1990). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fibronectin (FN), and non-natural synthetic FN3 domains as described for example in U.S. Pat. No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, e.g., the 3 ^rd FN3 domain of tenascin (TN3), or the 10 ^th FN3 domain of fibronectin (FNIO).

"Centyrin" refers to a FN3 domain that is based on the consensus sequence of the 15 different FN3 domains present in human tenascin C.

The term "capture agent" refers to substances that bind to a particular type of cells and enable the isolation of that cell from other cells. Exemplary capture agents are magnetic beads, ferrofluids, encapsulating reagents, molecules that bind the particular cell type and the like.

"Sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are tissue biopsies, fine needle aspirations, surgically resected tissue, organ cultures, cell cultures and biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites fluids, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium and lavage fluids and the like.

"Substituting" or "substituted" or 'mutating" or "mutated" refers to altering, deleting of inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence.

"Variant" refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions or deletions.

"Specifically binds" or "specific binding" refers to the ability of the FN3 domain of the invention to bind PD-Ll with a dissociation constant (K _D ) of about lxlO ^"6 M or less, for example about lxlO ^"7 M or less, about lxlO ^"8 M or less, about lxlO ^"9 M or less, about lxlO ^"10 M or less, about lxlO ^"11 M or less, about lxlO ^"12 M or less, or about lxlO ^"13 M or less. Alternatively, "specific binding" refers to the ability of the FN3 domain of the invention to bind PD-Ll at least 5-fold above the negative control in standard ELISA assay. The isolated FN3 domain of the invention that specifically binds PD-Ll may, however, have cross-reactivity to other related antigens, for example to the same predetermined antigen from other species (homologs), such as Macaca Fascicularis (cynomolgous monkey, cyno) or Pan troglodytes (chimpanzee).

"Library" refers to a collection of variants. The library may be composed of polypeptide or polynucleotide variants.

"Stability" refers to the ability of a molecule to maintain a folded state under physiological conditions such that it retains at least one of its normal functional activities, for example, binding to a predetermined antigen such as PD-Ll . "PD-Ll" refers to human PD-Ll protein having the amino acid sequence of SEQ ID NO: 32. The extracellular domain of PD-Ll spans residues 1-220, the transmembrane domain spans residues 221-241 and the cytoplasmic domain spans residues 242-272.

"PD-1" refers to human PD-1 protein having the amino acid sequence of SEQ ID NO: 33. The extracellular domain of PD-1 spans residues 1-150, the transmembrane domain spans residues 151-171 and the cytoplasmic domain spans residues 172-268 of SEQ ID NO: 33

"Tencon" refers to the synthetic fibronectin type III (FN3) domain having the sequence shown in SEQ ID NO: 1 and described in U.S. Pat. Publ. No. 2010/0216708.

A "cancer cell" or a "tumor cell" refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, and in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is exemplified by, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, proliferation, malignancy, tumor specific markers levels, invasiveness, tumor growth or suppression in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).

"Vector" refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these.

"Expression vector" refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

"Polynucleotide" refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide. "Polypeptide" or "protein" refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than about 50 amino acids may be referred to as "peptides".

"Valent" refers to the presence of a specified number of binding sites specific for an antigen in a molecule. As such, the terms "monovalent", "bivalent", "tetravalent", and "hexavalent" refer to the presence of one, two, four and six binding sites, respectively, specific for an antigen in a molecule.

"Subject" includes any human or nonhuman animal. "Nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows chickens, amphibians, reptiles, etc. Except when noted, the terms "patient" or "subject" are used interchangeably.

"Isolated" refers to a homogenous population of molecules (such as synthetic polynucleotides or a polypeptide such as FN3 domains) which have been substantially separated and/or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. "Isolated FN3 domain" refers to an FN3 domain that is substantially free of other cellular material and/or chemicals and encompasses FN3 domains that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

Compositions of matter

The present invention provides fibronectin type III (FN3) domains that specifically bind PD-L1. These molecules can be used in therapeutic and diagnostic applications and in imaging. The present invention provides polynucleotides encoding the FN3 domains of the invention or complementary nucleic acids thereof, vectors, host cells, and methods of making and using them.

The invention provides an isolated FN3 domain that specifically binds PD-L1.

The FN3 domain of the invention may bind PD-L1 with a dissociation constant (K _D ) of less than about lxlO ^"7 M, for example less than about lxlO ^"8 M, less than about lxlO ^"9 M, less than about lxlO ^"10 M, less than about lxlO ^"11 M, less than about lxlO ^"12 M, or less than about lxlO ^"13 M as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art. The measured affinity of a particular FN3 domain-antigen interaction can vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., K _D , Kon, Koff) are made with standardized solutions of protein scaffold and antigen, and a standardized buffer, such as the buffer described herein.

The FN3 domain of the invention may bind PD-Ll at least 5 -fold above the signal obtained for a negative control in standard ELISA assay.

In some embodiments, the FN3 domain that specifically binds PD-Llcomprises an initiator methionine (Met) linked to the N-terminus of the molecule.

In some embodiments, the FN3 domain that specifically binds PD-Llcomprises a cysteine (Cys) linked to a C-terminus of the FN3 domain.

The addition of the N-terminal Met and/or the C-terminal Cys may facilitate expression and/or conjugation of half-life extending molecules.

In some embodiments, the FN3 domain that specifically binds PD-Ll is internalized into a cell.

Internalization of the FN3 domain may facilitate delivery of a cytotoxic agent into tumor cells.

In some embodiments, the FN3 domain that specifically binds PD-Ll inhibits binding of PD-Ll to PD-1.

Inhibition of binding of PD-Ll to PD-1 by the FN3 domains of the invention may be assessed using competition ELISA. In an exemplary assay, 1 μg/ml recombinant human PD-Ll extracellular domain is bound on wells of microtiter plates, the wells are washed and blocked, and 10 μg/ml of the test FN3 domain is added. Without washing, 7.5 μg/ml PD-1 extracellular domain is added into the wells and incubated for 30 min, after which 0.5 μg/ml anti-PD-1 antibody is added and incubated for 30 min. The plates are washed and 0.5 μg/mL neutravidin-HRP conjugate polyclonal antibody is added and incubated for 30 minutes. The plates are washed and POD Chemiluminescence substrate added immediately prior to reading the luminescence signal. The FN3 domains of the invention inhibit binding of PD-Ll to PD-1 when the binding of PD-1 is reduced by at least about 80%, 85%, 90%, 95% or 100%.

In some embodiments, the FN3 domain that specifically binds PD-Ll is a PD-Ll antagonist.

In some embodiments, the FN3 domain that specifically binds PD-Ll is a PD-Ll agonist.

"Antagonist" refers to a FN3 domain that specifically binds PD-Ll that suppresses at least one reaction or activity that is induced by PD-Ll binding PD-1. A molecule is an antagonist when the at least one reaction or activity is suppressed by at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than the at least one reaction or activity suppressed in the absence of the antagonist (e.g., negative control), or when the suppression is statistically significant when compared to the suppression in the absence of the antagonist. A typical reaction or activity that is induced by PD-L1 binding PD-1 is reduced antigen-specific CD4 ⁺ or CD8 ⁺ cell proliferation or reduced interferon-γ (IFN-γ) production by T cells.

The antagonistic FN3 domains that specifically bind PD-L1 may be used in the treatment of cancer or viral infections and in general in treatment of diseases in which activation of immune responses is desirable.

"Agonist" refers to a FN3 domain that specifically binds PD-L1 that induces at least one reaction or activity that is induced by PD-L1 binding PD-1. The FN3 domain is an agonist when the at least one reaction or activity is induced by at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% greater than the at least one reaction or activity induced in the absence of the agonist (e.g., negative control), or when the induction is statistically significant when compared to the induction in the absence of the agonist. A typical reaction or activity that is induced by PD-L1 binding PD-1 is reduced antigen-specific CD4 ⁺ or CD8 ⁺ cell proliferation or reduced interferon-γ (IFN-γ) production by T cells.

The agonistic FN3 domains that specifically bind PD-L1 may be used in the treatment of autoimmune or inflammatory diseases and in general diseases in which suppression of immune responses is desirable.

In some embodiments, the FN3 domain that specifically binds PD-L1 does not inhibit binding of PD-L1 to PD-1.

In some embodiments, the FN3 domain that specifically binds PD-L1 does not activate signaling downstream of PD-1.

In some embodiments, the FN3 domain that specifically binds PD-L1 is based on Tencon sequence of SEQ ID NO: 1 or Tencon 27 sequence of SEQ ID NO: 4, optionally having substitutions at residues positions 11, 14, 17, 37, 46, 73, and/or 86 (residue numbering corresponding to SEQ ID NO: 4).

The invention also provides an isolated FN3 domain that specifically binds PD-L1 comprising the amino acid sequence of SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 and/or 124. The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 34.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 35.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 36.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 37.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 38.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 39.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 40.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 41.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 42.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 43.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 44.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 45.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 46.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 47.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 48.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 49.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 50. The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 51.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 52.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 53.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 54.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 55.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 56.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 57.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 58.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 59.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 60.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 61.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 62.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 63.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 64.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 65.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 66.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 67. The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 68.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 69.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 70.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 71.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 72.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 73.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 74.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 75.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 76.

The invention also provides an isolated FN3 domain that specifically binds PD-L 1 comprising the amino acid sequence of SEQ ID NO: 77.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 78.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 79.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 80.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 81.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 82.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 83.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 84. The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 85.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 86.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 87.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 88.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 89.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 90.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 91.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 92.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 93.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 94.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 95.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 96.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 97.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 98.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 99.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 100.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 101. The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 102.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 103.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 104.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 105.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 106.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 107.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 108.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 109.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 110.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 111.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 112.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 113.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 114.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 115.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 116.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 117.

The invention also provides an isolated FN3 domain that specifically binds PD-Ll comprising the amino acid sequence of SEQ ID NO: 118. The invention also provides an isolated FN3 domain that specifically binds PD-L1 comprising the amino acid sequence of SEQ ID NO: 119.

The invention also provides an isolated FN3 domain that specifically binds PD-L1 comprising the amino acid sequence of SEQ ID NO: 120.

The invention also provides an isolated FN3 domain that specifically binds PD-L1 comprising the amino acid sequence of SEQ ID NO: 121.

The invention also provides an isolated FN3 domain that specifically binds PD-L1 comprising the amino acid sequence of SEQ ID NO: 122.

The invention also provides an isolated FN3 domain that specifically binds PD-L1 comprising the amino acid sequence of SEQ ID NO: 123.

The invention also provides an isolated FN3 domain that specifically binds PD-L1 comprising the amino acid sequence of SEQ ID NO: 124.

In some embodiments, the isolated FN3 domain that specifically binds PD-L1 comprises an initiator methionine (Met) linked to the N-terminus of the molecule.

in some embodiments, the isolated FN3 domain that specifically binds PD-L1 comprises an amino acid sequence that is 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 74.

In some embodiments, the isolated FN3 domain that specifically binds PD-L1 comprises an amino acid sequence that is 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 34-124.

Conjugates of the FN3 domains that specifically bind PD-L1 of the invention

The invention also provides an isolated FN3 domain that specifically binds PD-L1 conjugated to a heterologous molecule(s).

In some embodiments, the heterologous molecule is a detectable label or a cytotoxic agent.

The invention also provides an FN3 domain that specifically binds PD-L1 conjugated to a detectable label.

The invention also provides an FN3 domain that specifically binds PD-L1 conjugated to a cytotoxic agent.

In some embodiments, the detectable label is also a cytotoxic agent. The FN3 domains that specifically bind PD-L1 of the invention conjugated to a detectable label can be used to evaluate expression of PD-L1 on samples such as tumor tissue in vivo or in vitro.

Detectable label includes compositions that when conjugated to the FN3 domains that specifically bind PD-L1 of the invention renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

Exemplary detectable labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescent quenching agents, colored molecules, radioactive isotopes, cintillants, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni ²⁺ , Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors, acridinium esters, and colorimetric substrates.

A detectable label may emit a signal spontaneously, such as when the detectable label is a radioactive isotope. In other cases the detectable label emits a signal as a result of being stimulated by an external field.

Exemplary radioactive isotopes may be γ-emitting, Auger-emitting, β-emitting, an alpha-emitting or positron-emitting radioactive isotope. Exemplary radioactive isotopes include ³ H, ⁿ C, ¹³ C, ¹⁵ N, ¹⁸ F, ¹⁹ F, ⁵⁵ Co, ⁵⁷ Co, ⁶⁰ Co, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Ga, ⁷² As, ⁷⁵ Br, ⁸⁶ Y, ⁸⁹ Zr, ⁹⁰ Sr, ^94m Tc, ^99m Tc, ¹¹⁵ In, ¹²³ 1, ¹²⁴ 1, ¹²⁵ 1, ¹³¹ 1, ²¹¹ At, ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²²⁶ Ra, ²²⁵ Ac and ²²⁷ Ac.

Exemplary metal atoms are metals with an atomic number greater than 20, such as calcium atoms, scandium atoms, titanium atoms, vanadium atoms, chromium atoms, manganese atoms, iron atoms, cobalt atoms, nickel atoms, copper atoms, zinc atoms, gallium atoms, germanium atoms, arsenic atoms, selenium atoms, bromine atoms, krypton atoms, rubidium atoms, strontium atoms, yttrium atoms, zirconium atoms, niobium atoms, molybdenum atoms, technetium atoms, ruthenium atoms, rhodium atoms, palladium atoms, silver atoms, cadmium atoms, indium atoms, tin atoms, antimony atoms, tellurium atoms, iodine atoms, xenon atoms, cesium atoms, barium atoms, lanthanum atoms, hafnium atoms, tantalum atoms, tungsten atoms, rhenium atoms, osmium atoms, iridium atoms, platinum atoms, gold atoms, mercury atoms, thallium atoms, lead atoms, bismuth atoms, francium atoms, radium atoms, actinium atoms, cerium atoms, praseodymium atoms, neodymium atoms, promethium atoms, samarium atoms, europium atoms, gadolinium atoms, terbium atoms, dysprosium atoms, holmium atoms, erbium atoms, thulium atoms, ytterbium atoms, lutetium atoms, thorium atoms, protactinium atoms, uranium atoms, neptunium atoms, plutonium atoms, americium atoms, curium atoms, berkelium atoms, californium atoms, einsteinium atoms, fermium atoms, mendelevium atoms, nobelium atoms, or lawrencium atoms.

In some embodiments, the metal atoms may be alkaline earth metals with an atomic number greater than twenty.

In some embodiments, the metal atoms may be lanthanides.

In some embodiments, the metal atoms may be actinides.

In some embodiments, the metal atoms may be transition metals,

In some embodiments, the metal atoms may be poor metals.

In some embodiments, the metal atoms may be gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.

In some embodiments, the metal atoms may be metals with an atomic number of 53 (i.e. iodine) to 83 (i.e. bismuth).

In some embodiments, the metal atoms may be atoms suitable for magnetic resonance imaging.

The metal atoms may be metal ions in the form of +1 , +2, or +3 oxidation states, such as Ba ²⁺ , Bi ³⁺ , Cs ⁺ , Ca ²⁺ , Cr ²⁺ , Cr ³⁺ , Cr ⁶⁺ , Co ²⁺ , Co ³⁺ , Cu ⁺ , Cu ²⁺ , Cu ³⁺ , Ga ³⁺ , Gd ³⁺ , Au ⁺ , Au ³⁺ , Fe ²⁺ , Fe ³⁺ , F ³⁺ , Pb ²⁺ , Mn ²⁺ , Mn ³⁺ , Mn ⁴⁺ , Mn ⁷⁺ , Hg ²⁺ , Ni ²⁺ , Ni ³⁺ , Ag ⁺ , Sr ²⁺ , Sn ²⁺ , Sn ⁴⁺ , and Zn ²⁺ . The metal atoms may comprise a metal oxide, such as iron oxide, manganese oxide, or gadolinium oxide.

Suitable dyes include any commercially available dyes such as, for example, 5(6)- carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW, ruthenium polypyridyl dyes, and the like.

Suitable fluorophores are fluorescein isothiocyante (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIK) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.

The FN3 domains that specifically bind PD-L1 conjugated to a detectable label may be used as an imaging agent to evaluate tumor distribution, diagnosis for the presence of tumor cells and/or, recurrence of tumor.

In some embodiments, the FN3 domains that specifically bind PD-L1 of the invention are conjugated to a cytotoxic agent.

In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a

radioconjugate).

The FN3 domains that specifically bind PD-L1 conjugated to a cytotoxic agent of the invention may be used in the targeted delivery of the cytotoxic agent to PD-L1 expressing tumor cell, and intracellular accumulation therein, wherein systemic administration of these unconjugated cytotoxic agents may result in unacceptable levels of toxicity to normal cells.

In some embodiments, the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, bacterial toxins such as diphtheria toxin, ricin, geldanamycin, maytansinoids or calicheamicin. The cytotoxic agent may elict their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

In some embodiments, the cytotoxic agent is an enzymatically active toxins such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In some embodiments, the cytotoxic agent is a radionuclide, such as ²¹² Bi, ¹³¹ I, ^m In, ⁹⁰ Y, and ¹⁸⁶ Re.

In some embodiments, the cytotoxic agent is dolastatins or dolostatin peptidic analogs and derivatives, auristatin or monomethyl auristatin phenylalanine. Exemplary molecules are disclosed in U.S. Pat No. 5,635,483 and 5,780,588. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001) Antimicrob Agents and Chemother. 45(12):3580-3584) and have anticancerand antifungal activity. The dolastatin or auristatin drug moiety may be attached to the FN3 domain of the invention through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172), or via any cysteine engineered into the FN3 domain.

The FN3 domains that specifically bind PD-L1 of the invention may be conjugated to a detectable label using known methods.

In some embodiments, the detectable label is complexed with a chelating agent.

In some embodiments, the detectable label is conjugated to the FN3 domain that specifically binds PD-L1 of the invention via a linker. The detectable label or the cytotoxic moiety may be linked directly, or indirectly, to the FN3 domain that specifically binds PD-L1 of the invention using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, non- phenolic linkers (derivatives of N-succimidyl-benzoates; dodecaborate), chelating moieties of both macrocyclics and acyclic chelators, such as derivatives of 1,4,7,10- tetraazacyclododecane-l,4,7,10,tetraacetic acid (DOTA), derivatives of

diethylenetriaminepentaacetic avid (DTP A), derivatives of S-2-(4-Isothiocyanatobenzyl)- l,4,7-triazacyclononane-l,4,7-triacetic acid (NOT A) and derivatives of 1,4,8,11- tetraazacyclodocedan-l,4,8,l l-tetraacetic acid (TETA), N-succinimidyl-3-(2- pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p- azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene) and other chelating moieties. Suitable peptide linkers are well known.

In some embodiment, the FN3 domain that specifically binds PD-L1 is removed from the blood via renal clearance.

Isolation of PD-L1 binding FN3 domains from a library based on Tencon sequence

Tencon (SEQ ID NO: 1) is a non-naturally occurring fibronectin type III (FN3) domain designed from a consensus sequence of fifteen FN3 domains from human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25: 107-117, 2012; U.S. Pat. Publ. No. 2010/0216708). The crystal structure of Tencon shows six surface- exposed loops that connect seven beta-strands as is characteristic to the FN3 domains, the beta-strands referred to as A, B, C, D, E, F, and G, and the loops referred to as AB, BC, CD, DE, EF, and FG loops (Bork and Doolittle, Proc Natl Acad Sci USA 89:8990-8992, 1992; U.S. Pat. No. 6,673,901). These loops, or selected residues within each loop, may be randomized in order to construct libraries of fibronectin type III (FN3) domains that may be used to select novel molecules that bind Pd-Ll. Table 1 shows positions and sequences of each loop and beta-strand in Tencon (SEQ ID NO: 1).

Library designed based on Tencon sequence may thus have randomized FG loop, or randomized BC and FG loops, such as libraries TCL1 or TCL2 as described below. The Tencon BC loop is 7 amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may be randomized in the library diversified at the BC loop and designed based on Tencon sequence. The Tencon FG loop is 7 amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may be randomized in the library diversified at the FG loop and designed based on Tencon sequence. Further diversity at loops in the Tencon libraries may be achieved by insertion and/or deletions of residues at loops. For example, the FG and/or BC loops may be extended by 1-22 amino acids, or decreased by 1-3 amino acids. The FG loop in Tencon is 7 amino acids long, whereas the corresponding loop in antibody heavy chains ranges from 4-28 residues. To provide maximum diversity, the FG loop may be diversified in sequence as well as in length to correspond to the antibody CDR3 length range of 4-28 residues. For example, the FG loop can further be diversified in length by extending the loop by additional 1, 2, 3, 4 or 5 amino acids.

Library designed based on Tencon sequence may also have randomized alternative surfaces that form on a side of the FN3 domain and comprise two or more beta strands, and at least one loop. One such alternative surface is formed by amino acids in the C and the F beta-strands and the CD and the FG loops (a C-CD-F-FG surface). A library design based on Tencon alternative C-CD-F-FG surface is described in U.S. Pat. Publ. No. US2013/0226834. Library designed based on Tencon sequence also includes libraries designed based on Tencon variants, such as Tencon variants having substitutions at residues positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO: 1), and which variants display improve thermal stability. Exemplary Tencon variants are described in US Pat. Publ. No. 2011/0274623, and include Tencon27 (SEQ ID NO: 4) having substitutions El 1R, L17A, N46V and E86I when compared to Tencon of SEQ ID NO: 1.

Table 1.

DE loop 51-54

E strand 55-59

EF loop 60-64

F strand 65-74

FG loop 75-81

G strand 82-89

Tencon and other FN3 sequence based libraries may be randomized at chosen residue positions using a random or defined set of amino acids. For example, variants in the library having random substitutions may be generated using NNK codons, which encode all 20 naturally occurring amino acids. In other diversification schemes, DVK codons may be used to encode amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys. Alternatively, NNS codons may be used to give rise to all 20 amino acid residues and simultaneously reducing the frequency of stop codons. Libraries of FN3 domains with biased amino acid distribution at positions to be diversified may be synthesized for example using Slonomics® technology (http:_//www_sloning_com). This technology uses a library of pre-made double stranded triplets that act as universal building blocks sufficient for thousands of gene synthesis processes. The triplet library represents all possible sequence combinations necessary to build any desired DNA molecule. The codon designations are according to the well-known IUB code.

The FN3 domains that specifically bind PD-L1 of the invention may be isolated by producing the FN3 library such as the Tencon library using cis display to ligate DNA fragments encoding the scaffold proteins to a DNA fragment encoding RepA to generate a pool of protein-DNA complexes formed after in vitro translation wherein each protein is stably associated with the DNA that encodes it (U.S. Pat. No. 7,842,476; Odegrip et al., Proc Natl Acad Sci U S A 101, 2806-2810, 2004), and assaying the library for specific binding to PSMA by any method known in the art and described in the Example.

Exemplary well known methods which can be used are ELISA, sandwich immunoassays, and competitive and non-competitive assays (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). The identified FN3 domains that specifically bind PD-L1 are further characterized for their binding to PD-L1, modulation of PD-L1 activity, internalization, stability, and other desired characteristics. The FN3 domains that specifically bind PD-L1 of the invention may be generated using any FN3 domain as a template to generate a library and screening the library for molecules specifically binding PD-L1 using methods provided within. Exemplar FN3 domains that may be used are the 3rd FN3 domain of tenascin C (TN3) (SEQ ID NO: 125), Fibcon (SEQ ID NO: 126), and the 10 ^th FN3 domain of fibronectin (FN10) (SEQ ID NO: 127). Standard cloning and expression techniques are used to clone the libraries into a vector or synthesize double stranded cDNA cassettes of the library, to express, or to translate the libraries in vitro. For example ribosome display (Hanes and Pluckthun, Proc Natl Acad Sci USA, 94, 4937-4942, 1997), mRNA display (Roberts and Szostak, Proc Natl Acad Sci USA, 94, 12297-12302, 1997), or other cell-free systems (U.S. Pat. No. 5,643,768) can be used. The libraries of the FN3 domain variants may be expressed as fusion proteins displayed on the surface for example of any suitable bacteriophage. Methods for displaying fusion polypeptides on the surface of a bacteriophage are well known (U.S. Pat. Publ. No.

2011/0118144; Int. Pat. Publ. No. WO2009/085462; U.S. Pat. No. 6,969,108; U.S. Pat. No. 6,172,197; U.S. Pat. No. 5,223,409; U.S. Pat. No. 6,582,915; U.S. Pat. No. 6,472,147).

In some embodiments, the FN3 domain that specifically binds PD-L1 is based on Tencon sequence of SEQ ID NO: 1 or Tencon27 sequence of SEQ ID NO: 4, the SEQ ID NO: 1 or the SEQ ID NO: 4, optionally having substitutions at residues positions 11, 14, 17, 37, 46, 73, and/or 86.

The FN3 domains that specifically bind PD-L1 of the invention may be modified to improve their properties such as improve thermal stability and reversibility of thermal folding and unfolding. Several methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparison to highly similar thermostable sequences, design of stabilizing disulfide bridges, mutations to increase alpha-helix propensity, engineering of salt bridges, alteration of the surface charge of the protein, directed evolution, and composition of consensus sequences (Lehmann and Wyss, Curr Opin Biotechnol, 12, 371-375, 2001). High thermal stability may increase the yield of the expressed protein, improve solubility or activity, decrease immunogenicity, and minimize the need of a cold chain in manufacturing. Residues that may be substituted to improve thermal stability of Tencon (SEQ ID NO: 1) are residue positions 11, 14, 17, 37, 46, 73, or 86, and are described in US Pat. Publ. No.

2011/0274623. Substitutions corresponding to these residues may be incorporated to the FN3 domain containing molecules of the invention.

Measurement of protein stability and protein lability can be viewed as the same or different aspects of protein integrity. Proteins are sensitive or "labile" to denaturation caused by heat, by ultraviolet or ionizing radiation, changes in the ambient osmolarity and pH if in liquid solution, mechanical shear force imposed by small pore-size filtration, ultraviolet radiation, ionizing radiation, such as by gamma irradiation, chemical or heat dehydration, or any other action or force that may cause protein structure disruption. The stability of the molecule can be determined using standard methods. For example, the stability of a molecule can be determined by measuring the thermal melting ("T _m ") temperature, the temperature in ° Celsius (°C) at which half of the molecules become unfolded, using standard methods. Typically, the higher the T _m , the more stable the molecule. In addition to heat, the chemical environment also changes the ability of the protein to maintain a particular three dimensional structure.

In one embodiment, the FN3 domain that specifically binds PD-L1 of the invention may exhibit increased stability by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more compared to the same domain prior to engineering measured by the increase in the T _m .

Chemical denaturation can likewise be measured by a variety of methods.

Chemical denaturants include guanidinium hydrochloride, guanidinium thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride); reducing agents (e.g. dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene, and hydrides, such as sodium borohydride), non-ionic and ionic detergents, acids (e.g. hydrochloric acid (HQ), acetic acid (CH ₃ COOH), halogenated acetic acids), hydrophobic molecules (e.g.

phosopholipids), and targeted denaturants. Quantitation of the extent of denaturation can rely on loss of a functional property, such as ability to bind a target molecule, or by physiochemical properties, such as tendency to aggregation, exposure of formerly solvent inaccessible residues, or disruption or formation of disulfide bonds.

The FN3 domain that specifically binds PD-L1 of the invention may be generated as monomers, dimers, or multimers, for example, as a means to increase the valency and thus the avidity of target molecule binding, or to generate bi- or multispecific scaffolds simultaneously binding two or more different target molecules. The dimers and multimers may be generated by linking monospecific, bi- or multispecific protein scaffolds, for example, by the inclusion of an amino acid linker, for example a linker containing poly- glycine, glycine and serine, or alanine and proline. Exemplary linker include (GS)2, (SEQ ID NO: 128), (GGGS) ₂ (SEQ ID NO: 129), (GGGGS) ₅ (SEQ ID NO: 130), (AP) ₂ (SEQ ID NO: 131), (AP) ₅ (SEQ ID NO: 132), (AP)io (SEQ ID NO: 133), (AP) ₂₀ (SEQ ID NO: 134) and A(E AAAK) ₅ AA A (SEQ ID NO: 135). The dimers and multimers may be linked to each other in a N-to C-direction. The use of naturally occurring as well as artificial peptide linkers to connect polypeptides into novel linked fusion polypeptides is well known in the literature (Hallewell et al, J Biol Chem 264, 5260-5268, 1989; Alfthan et al, Protein Eng. 8, 725-731, 1995; Robinson & Sauer, Biochemistry 35, 109-116, 1996; U.S. Pat. No. 5,856,456).

Half-life extending moieties

The FN3 domains that specifically bind PD-L1 of the invention may incorporate other subunits for example via covalent interaction. In one aspect of the invention, the FN3 domains that specifically bind PD-L1 of the invention further comprise a half -life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin-binding proteins and/or domains, transferrin and fragments and analogues thereof, and Fc regions. An exemplary albumin variant is shown in SEQ ID NO: 136. Amino acid sequences of the human Fc regions are well known, and include IgGl, IgG2, IgG3, IgG4, IgM, IgA and IgE Fc regions.

All or a portion of an antibody constant region may be attached to the FN3 domain that specifically binds PD-L1 of the invention to impart antibody-like properties, especially those properties associated with the Fc region, such as Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody -dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (e.g., B cell receptor; BCR), and may be further modified by modifying residues in the Fc responsible for these activities (for review; see Strohl, Curr Opin Biotechnol. 20, 685-691, 2009).

Additional moieties may be incorporated into the FN3 domains that specifically bind PD-L1 of the invention such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties. These moieties may be direct fusions with the protein scaffold coding sequences and may be generated by standard cloning and expression techniques.

Alternatively, well known chemical coupling methods may be used to attach the moieties to recombinantly produced molecules of the invention.

A pegyl moiety may for example be added to the FN3 domain that specifically binds PD-L1 of the invention by incorporating a cysteine residue to the C-terminus of the molecule, or engineering cysteines into residue positions that face away from the PD-L1 binding face of the molecule, and attaching a pegyl group to the cysteine using well known methods.

FN3 domains that specifically bind PD-L1 of the invention incorporating additional moieties may be compared for functionality by several well-known assays. For example, altered properties due to incorporation of Fc domains and/or Fc domain variants may be assayed in Fc receptor binding assays using soluble forms of the receptors, such as the FcyRI, FcyRII, FcyRIII or FcRn receptors, or using well known cell-based assays measuring for example ADCC or CDC, or evaluating pharmacokinetic properties of the molecules of the invention in in vivo models.

Polynucleotides, vectors, host cells

The invention also provides nucleic acids encoding the FN3 domains specifically binding PD-L1 of the invention as isolated polynucleotides or as portions of expression vectors or as portions of linear DNA sequences, including linear DNA sequences used for in vitro transcription/translation, vectors compatible with prokaryotic, eukaryotic or filamentous phage expression, secretion and/or display of the compositions or directed mutagens thereof. Certain exemplary polynucleotides are disclosed herein, however, other polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the FN3 domains of the invention are also within the scope of the invention.

The invention also provides an isolated polynucleotide encoding the FN3 domain specifically binding PD-L1 comprising the amino acid sequence of SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 or 124.

The polynucleotides of the invention may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer and assembled into complete single or double stranded molecules. Alternatively, the polynucleotides of the invention may be produced by other techniques such as PCR followed by routine cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art. The polynucleotides of the invention may comprise at least one non-coding sequence, such as a promoter or enhancer sequence, intron, polyadenylation signal, a cis sequence facilitating RepA binding, and the like. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids that encode for example a marker or a tag sequence such as a histidine tag or an HA tag to facilitate purification or detection of the protein, a signal sequence, a fusion protein partner such as RepA, Fc or bacteriophage coat protein such as pIX or pill.

The invention also provides a vector comprising at least one polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the polynucleotides of the invention into a given organism or genetic background by any means. Such vectors may be expression vectors comprising nucleic acid sequence elements that can control, regulate, cause or permit expression of a polypeptide encoded by such a vector. Such elements may comprise transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of encoded polypeptides in a given expression system. Such expression systems may be cell-based, or cell-free systems well known in the art.

The invention also provides a host cell comprising the vector of the invention. The FN3 domain that specifically bind PD-L1 of the invention may be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001);

Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 ^nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al, Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

The host cell chosen for expression may be of mammalian origin or may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2/0, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell may be selected from a species or organism incapable of glycosylating polypeptides, e.g. a prokaryotic cell or organism, such as BL21, BL21(DE3), BL21-GOLD(DE3), XLl-Blue, JM109, HMS174,

HMS174(DE3), and any of the natural or engineered E. coli spp, Klebsiella spp., or Pseudomonas spp strains. The invention also provides a method of producing the isolated FN3 domain that specifically binds PD-Ll of the invention, comprising culturing the isolated host cell of the invention under conditions such that the isolated FN3 domain that specifically binds PD- Ll is expressed, and purifying the FN3 domain.

The FN3 domains that specifically bind PD-Ll may be purified from recombinant cell cultures by well-known methods, for example by protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography, or high performance liquid chromatography (HPLC).

Anti-idiotypic antibodies

The present invention also provides an anti-idiotypic antibody binding to the FN3 domain of the invention.

The invention also provides an anti-idiotypic antibody that specifically binds the FN3 domain comprising any one of SEQ ID NOs: 34-124.

Kits

The invention also provides a kit comprising the FN3 domain that specifically binds PD-Ll of the invention.

The kit may be used for therapeutic uses and as a diagnostic kit.

In some embodiments, the kit comprises the FN3 domain that specifically binds PD-Ll of the invention and reagents for detecting the FN3 domain. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, an agent useful for chelating, or otherwise coupling, a radioprotective composition; devices or other materials for preparing the FN3 domain that specifically binds PD-Ll of the invention for administration for imaging, diagnostic or therapeutic purpose;

pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

In some embodiments, the kit comprises the FN3 domain that specifically binds PD-Ll comprising any one of SEQ ID NOs: 34-124.

Uses of PD-Ll binding FN3 domains of the invention

The FN3 domains that specifically bind PD-Ll of the invention may be used to diagnose, monitor, modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of human disease or specific pathologies in cells, tissues, organs, fluid, or, generally, a host. The FN3 domains that specifically bind PD-Ll of the invention may also be used in imaging PD-Ll positive tumor tissue in a subject. The methods of the invention may be used with an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals.

The invention provides a method of diagnosing a subject having, or who is likely to develop cancer of a tissue based on the expression of PD-Ll by cells of the cancer tissue, methods of predicting success of immunotherapy, methods of prognosis, and methods of treatment.

The invention also provides a method of detecting PD-Ll -expressing cancer cells in a tumor tissue, comprising

obtaining a sample of the tumor tissue from a subject;

detecting whether PD-Ll is expressed in the tumor tissue by contacting toe sample of the tumor tissues with the FN3 domain that specifically binds PD-Ll comprising the sequence of any one of SEQ ID NOs: 34-124 and detecting the binding between PD-Ll and the FN3 domain.

The tissue can be tissue of any organ or anatomical system, for example lung, epithelial, connective, vascular, muscle, neural, skeletal, lymphatic, prostate, cervical, breast, spleen, gastric, intestinal, oral, esophageal, uterine, ovarian, renal or testicular tissue.

PD-Ll expression may be evaluated using known methods such as

immunohistochemistry or ELISA.

The invention also provides a method of isolating PD-Ll expressing cells, comprising

obtaining a sample from a subject;

contacting the sample with the FN3 domain that specifically binds PD-Ll comprising the sequence of any one of SEQ ID NOs: 34-124, and

isolating the cells bound to the FN3 domains.

The invention also provides a method of detecting PD-Ll -expressing cancer cells in a tumor tissue, comprising

conjugating the FN3 domain that specifically binds PD-Ll comprising the sequence of any one of SEQ ID NOs: 34-124 to a detectable label to form a conjugate;

administering the conjugate to a subject; and

visualizing the PD-Ll expressing cancer cells to which the conjugate is bound. The invention also provides a method of treating a subject having cancer, comprising administering to the subject a FN3 domain that specifically binds PD-Ll of the invention.

In some embodiments, the subject has a solid tumor.

n some embodiments the subject has a hematological malignancy,

n some embodiments the solid tumor is a melanoma,

n some embodiments the solid tumor is a lung cancer.

n some embodiments the solid tumor is a non-small cell lung cancer (NSCLC). n some embodiments. the solid tumor is a squamous non-small cell lung cancer

(NSCLC)

n some embodiments the so id tumor is a non-squamous NSCLC.

n some embodiments, the so id tumor is a lung adenocarcinoma.

n some embodiments the so id tumor is a renal cell carcinoma (RCC).

n some embodiments the so id tumor is a mesothelioma.

n some embodiments the so id tumor is a nasopharyngeal carcinoma (NPC). n some embodiments the so id tumor is a colorectal cancer.

n some embodiments the so id tumor is a prostate cancer.

n some embodiments the so id tumor is castration-resistant prostate cancer. n some embodiments the so id tumor is a stomach cancer.

n some embodiments the so id tumor is an ovarian cancer.

n some embodiments the so id tumor is a gastric cancer.

n some embodiments the so id tumor is a liver cancer.

n some embodiments the so id tumor is pancreatic cancer.

n some embodiments the so id tumor is a thyroid cancer.

n some embodiments the so id tumor is a squamous cell carcinoma of the head and neck

n some embodiments the so id tumor is a carcinomas of the esophagus or gastroin estinal tract,

n some embodiments the sol lid tumor is a breast cancer.

n some embodiments the sol lid tumor is a fallopian tube cancer.

n some embodiments the sol lid tumor is a brain cancer.

n some embodiments the sol lid tumor is an urethral cancer.

n some embodiments the sol lid tumor is a genitourinary cancer.

n some embodiments the sol lid tumor is an endometriosis.

n some embodiments the sol lid tumor is a cervical cancer. In some embodiments, the solid tumor is a metastatic lesion of the cancer.

In some embodiments, the hematological malignancy is a lymphoma, a myeloma or a leukemia.

In some embodiments, the hematological malignancy is a B cell lymphoma. In some embodiments, the hematological malignancy is Burkitt's lymphoma. In some embodiments, the hematological malignancy is Hodgkin's lymphoma. In some embodiments, the hematological malignancy is a non-Hodgkin's lymphoma.

In some embodiments, the hematological malignancy is a myelodysplastic syndrome.

In some embodiments, the hematological malignancy is an acute myeloid leukemia (AML).

In some embodiments, the hematological malignancy is a chronic myeloid leukemia (CML).

In some embodiments, the hematological malignancy is a chronic myelomoncytic leukemia (CMML).

In some embodiments, the hematological malignancy is a multiple myeloma

(MM).

In some embodiments, the hematological malignancy is a plasmacytoma.

In some embodiments, the cancer is kidney cancer.

"Treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the FN3 domains that specifically bind PD-L1 of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual. Exemplary indicators of an effective FN3 domain that specifically binds PD-Ll is improved well-being of the patient, decrease or shrinkage of the size of a tumor, arrested or slowed growth of a tumor, and/or absence of metastasis of cancer cells to other locations in the body.

Administration/ Pharmaceutical Compositions

The invention provides for pharmaceutical compositions of the FN3 domains that specifically bind PD-Ll, optionally conjugated to a detectable label or a cytotoxic drug of the invention and a pharmaceutically acceptable carrier. For therapeutic use, the FN3 domains that specifically bind PD-Llof the invention may be prepared as pharmaceutical compositions containing an effective amount of the domain or molecule as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the molecules of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21 ^st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the FN3 domains of the invention may be any suitable route that delivers the agent to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal, rectal), using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art. Site specific administration may be achieved by for example intrarticular, intrabronchial, intraabdominal, intracapsular,

intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intrapro static, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

Pharmaceutical compositions can be supplied as a kit comprising a container that comprises the pharmaceutical composition as described herein. A pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.

Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a pharmaceutical composition. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.

While having described the invention in general terms, the embodiments of the invention will be further disclosed in the following examples that should not be construed as limiting the scope of the claims.

EXAMPLE 1. Construction of Tencon libraries with randomized loops

Tencon (SEQ ID NO: 1) is an immunoglobulin-like scaffold, fibronectin type III (FN3) domain, designed from a consensus sequence of fifteen FN3 domains from human tenascin-C (Jacobs et ah, Protein Engineering, Design, and Selection, 25 : 107-117, 2012; U.S. Pat. No. 8,278,419). The crystal structure of Tencon shows six surface-exposed loops that connect seven beta-strands. These loops, or selected residues within each loop, can be randomized in order to construct libraries of fibronectin type III (FN3) domains that can be used to select novel molecules that bind to specific targets.

Tencon:

LP APKNL VVSE VTED SLRL S WT APD AAFD SFLIQYQE SEKVGE AINLT VPGSERS Y DLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO 1):

Various libraries were generated using the tencon scaffold and various design strategies. In general, libraries TCL1 and TCL2 produced good binders. Generation of TCL1 and TCL2 libraries are described in detail in Int. Pat. Publ. No. WO/2014081944A2. Construction of TCL1 library

A library designed to randomize only the FG loop of Tencon (SEQ ID NO: 1), TCL1, was constructed for use with the c/s-display system (Jacobs et al, Protein Engineering, Design, and Selection, 25: 107-117, 2012). In this system, a single-strand DNA incorporating sequences for a Tac promoter, Tencon library coding sequence, RepA coding sequence, cis-element, and ori element is produced. Upon expression in an in vitro transcription/translation system, a complex is produced of the Tencon-RepA fusion protein bound in cis to the DNA from which it is encoded. Complexes that bind to a target molecule are then isolated and amplified by polymerase chain reaction (PCR), as described below.

Construction of the TCL1 library for use with czs-display was achieved by successive rounds of PCR to produce the final linear, double-stranded DNA molecules in two halves; the 5' fragment contains the promoter and Tencon sequences, while the 3' fragment contains the repA gene and the cis- and ori elements. These two halves are combined by restriction digest in order to produce the entire construct. The TCL1 library was designed to incorporate random amino acids only in the FG loop of Tencon, KGGHRSN (SEQ ID NO: 55). NNS codons were used in the construction of this library, resulting in the possible incorporation of all 20 amino acids and one stop codon into the FG loop. The TCL1 library contains six separate sub -libraries, each having a different randomized FG loop length, from 7 to 12 residues, in order to further increase diversity.

TCL1 library (SEQ ID NO: 2)

LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY DLTGLKPGTEYTVSIYGVX1X2X3X4X5X6X7X8X9X10X11X12 PLSAEFTT; wherein Xi, X2, X3, X4, X5, Χδ, X7 is any amino acid; and

Xg, X9, X10, Xn and X12 are any amino acid or deleted

Construction of TCL2 Library

TCL2 library was constructed in which both the BC and the FG loops of Tencon were randomized and the distribution of amino acids at each position was strictly controlled. Table 3 shows the amino acid distribution at desired loop positions in the TCL2 library. The designed amino acid distribution had two aims. First, the library was biased toward residues that were predicted to be structurally important for Tencon folding and stability based on analysis of the Tencon crystal structure and/or from homology modeling. For example, position 29 was fixed to be only a subset of hydrophobic amino acids, as this residue was buried in the hydrophobic core of the Tencon fold. A second layer of design included biasing the amino acid distribution toward that of residues preferentially found in the heavy chain HCDR3 of antibodies, to efficiently produce high- affinity binders (Birtalan et al., J Mol Biol 377: 1518-28, 2008; Olson et al., Protein Sci 16:476-84, 2007). Towards this goal, the "designed distribution" in Table 2 refers to the distribution as follows: 6% alanine, 6% arginine, 3.9% asparagine, 7.5% aspartic acid, 2.5% glutamic acid, 1.5% glutamine, 15% glycine, 2.3% histidine, 2.5% isoleucine, 5% leucine, 1.5% lysine, 2.5% phenylalanine, 4% proline, 10% serine, 4.5% threonine, 4% tryptophan, 17.3% tyrosine, and 4% valine. This distribution is devoid of methionine, cysteine, and STOP codons.

TCL2 library (SEQ ID NO: 3)

LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVGEAINLTVPGS ERSYDLTGLKPGTEYTVSIYGVX ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ SX ₁₄ X ₁₅ LSAEFTT; wherein

Xi is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X2 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X3 Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X4 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X5 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

Xe is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X7 is Phe, He, Leu, Val or Tyr;

Xg is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; Xio is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

Xii is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

Xi2 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

Xi3 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

Xi4 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and

Xi5 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

Table 2.

WT residues Distribution in the TCL2 library

22 T designed distribution

23 A designed distribution

24 P 50% P + designed distribution

25 D designed distribution

26 A 20% A + 20% G + designed distribution

27 A designed distribution

28 F 20% F, 20% I, 20% L, 20% V, 20% Y

29 D 33% D, 33% E, 33% T

75 K designed distribution

76 G designed distribution

77 G designed distribution

78 H designed distribution

79 R designed distribution

80 S 100% S 81 N designed distribution

82 P 50% P + designed distribution

*residue numbering is based on Tencon sequence of SEQ ID NO: 1

Subsequently, these libraries were improved by various ways, including building of the libraries on a stabilized Tencon framework (U.S. Pat. No. 8,569,227) that incorporates substitutions El 1R/L17A/N46V/E86I (Tencon27; SEQ ID NO: 4) when compared to the wild type tencon as well as altering of the positions randomized in the BC and FG loops. Tencon27 is described in Int. Pat. Appl. No. WO2013049275. From this, new libraries designed to randomize only the FG loop of Tencon (library TCL9), or a combination of the BC and FG loops (library TCL7) were generated. These libraries were constructed for use with the cis-display system (Odegrip et al., Proc Natl Acad Sci U S A 101: 2806-2810, 2004). The details of this design are shown below:

Stabilized Tencon (Tencon27) (SEQ ID NO: 4)

LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

TCL7 (randomized FG and BC loops) (SEQ ID NO: 5)

LPAPKNLVVSRVTEDSARLSWX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ FDSFLIQYQESEKVGEAIVLT VPGSERSYDLTGLKPGTEYTVSIYGVX ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ SNPLSAIFTT; wherein

Xi, X2, X3, X4, X5, Χδ, Xio, X11, X12, Xi3, Xi4, Xi5 and Xi6 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and

Xv, Xs, X9, Xi7, Xi8 and X19, is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.

TCL9 (randomized FG loop) (SEQ ID NO: 6) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGV X1X2X3X4X5X6X7X8X9 X10X11X12SNPLSAIFTT;

Xi, X ₂ , X ₃ , X4, Xs, X ₆ and X ₇ , is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and

X ₈ , X ₉ , X10, X11 and X12 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.

For library construction, DNA fragments encoding randomized BC loops (lengths 6-9 positions) or FG loops (lengths 7-12 positions) were synthesized using Slonomics technology (Sloning Biotechnology GmbH) so as to control the amino acid distribution of the library and to eliminate stop codons. Two different sets of DNA molecules randomizing either the BC loop or the FG loops were synthesized independently and later combined using PCR to produce the full library product.

Construction of FG loop libraries (TCL9)

A set of synthetic DNA molecules consisting of a 5 ' Tac promoter followed by the complete gene sequence of Tencon with the exception of randomized codons in the FG loop was produced (SEQ ID NOs: 26-31). For FG loop randomization, all amino acids except cysteine and methionine were encoded at equal percentages. The lengths of the diversified portion are such that they encode for 7, 8, 9, 10, 11, or 12 amino acids in the FG loop. Sub-libraries of each length variation were synthesized individually at a scale of 2ug and then amplified by PCR using oligos Sloning-FOR (SEQ ID NO: 9) and Sloning- Rev (SEQ ID NO: 10)

The 3 ' fragment of the library is a constant DNA sequence containing elements for display, including a PspOMI restriction site, the coding region of the repA gene, and the cis- and ori elements. PCR reactions were performed to amplify this fragment using a plasmid (pCR4Blunt) (Invitrogen) as a template with Ml 3 Forward and Ml 3 Reverse primers. The resulting PCR products were digested by PspOMI overnight and gel- purified. To ligate the 5' portion of library DNA to the 3' DNA containing rep A gene, 2 pmol (~540ng to 560ng) of 5' DNA was ligated to an equal molar (-1.25 μg) of 3' repA DNA in the presence of Notl and PspOMI enzyme and T4 ligase at 37°C overnight. The ligated library product was amplified by using 12 cycles of PCR with oligos POP2250 (SEQ ID NO: 11) and DigLigRev (SEQ ID NO: 12). For each sub-library, the resulting DNA from 12 PCR reactions were combined and purified by Qiagen spin column. The yield for each sub-library of TCL9 ranged from 32-34 μg. Construction of FG/BC Loop libraries (TCL7)

The TCL7 library provides for a library with randomized Tencon BC and FG loops. In this library, BC loops of lengths 6-9 amino acids were mixed combinatorially with randomized FG loops of 7-12 amino acids in length. Synthetic Tencon fragments BC6, BC7, BC8, and BC9 (SEQ ID NOs: 13-16, respectively) were produced to include the Tencon gene encoding for the N-terminal portion of the protein up to and including residue VX such that the BC loop is replaced with either 6, 7, 8, or 9 randomized amino acids. These fragments were synthesized prior to the discovery of L17A, N46V and E83I mutations (CEN5243) but these mutations were introduced in the molecular biology steps described below. In order to combine this fragment with fragments encoding for randomized FG loops, the following steps were taken.

First, a DNA fragment encoding the Tac promoter and the 5' sequence of Tencon up to the nucleotide encoding for amino acid A17 (130mer-L17A, SEQ ID NO: 17 ) was produced by PCR using oligos POP2222ext (SEQ ID NO: 18 ) and LSI 114 (SEQ ID NO: 19). This was done to include the L17A mutation in the library (CEN5243). Next, DNA fragments encoding for Tencon residues R18-V75 including randomized BC loops were amplified by PCR using BC6, BC7, BC8, or BC9 as a templates and oligos LSI 115 (SEQ ID NO: 20) and LSI 117 (SEQ ID NO: 21). This PCR step introduced a Bsal site at the 3' end. These DNA fragments were subsequently joined by overlapping PCR using oligos POP2222ext and LSI 117 as primers. The resulting PCR product of 240bp was pooled and purified by Qiagen PCR purification kit. The purified DNA was digested with Bsal-HF and gel purified.

Fragments encoding the FG loop were amplified by PCR using FG7, FG8, FG9, FG10, FGl l, and FG12 as templates with oligonucleotides SDG10 (SEQ ID NO: 22) and SDG24 (SEQ ID NO: 23) to incorporate a Bsal restriction site and N46V and E86I variations (CEN5243).

The digested BC fragments and FG fragments were ligated together in a single step using a 3-way ligation. Four ligation reactions in the 16 possible combinations were set up, with each ligation reaction combining two BC loop lengths with 2 FG loop lengths. Each ligation contained -300 ng of total BC fragment and 300 ng of the FG fragment. These 4 ligation pools were then amplified by PCR using oligos POP2222 (SEQ ID NO: 24) and SDG28 SEQ ID N: 25). 7.5 μg of each reaction product were then digested with Notl and cleaned up with a Qiagen PCR purification column. 5.2 μg of this DNA, was ligated to an equal molar amount of RepA DNA fragment (~14 μg) digested with PspOMI and the product amplified by PCR using oligos POP2222.

EXAMPLE 2: Generation of Tencon libraries having alternative binding surfaces

The choice of residues to be randomized in a particular library design governs the overall shape of the interaction surface created. X-ray crystallographic analysis of an FN3 domain containing scaffold protein selected to bind maltose binding protein (MBP) from a library in which the BC, DE, and FG loops were randomized was shown to have a largely curved interface that fits into the active site of MBP (Koide et al., Proc Natl Acad Sci U S A 104: 6632-6637, 2007). In contrast, an ankyrin repeat scaffold protein that was selected to bind to MBP was found to have a much more planar interaction surface and to bind to the outer surface of MBP distant from the active (Binz et al., Nat Biotechnol 22: 575-582, 2004). These results suggest that the shape of the binding surface of a scaffold molecule (curved vs. flat) may dictate what target proteins or specific epitopes on those target proteins are able to be bound effectively by the scaffold. Published efforts around engineering protein scaffolds containing FN3 domains for protein binding has relied on engineering adjacent loops for target binding, thus producing curved binding surfaces. This approach may limit the number of targets and epitopes accessible by such scaffolds.

Tencon and other FN3 domains contain two sets of CDR-like loops lying on the opposite faces of the molecule, the first set formed by the BC, DE, and FG loops, and the second set formed by the AB, CD, and EF loops. The two sets of loops are separated by the beta-strands that form the center of the FN3 structure. If the image of the Tencon is rotated by 90 degrees, an alternative surface can be visualized. This slightly concave surface is formed by the CD and FG loops and two antiparallel beta- strands, the C and the F beta-strands, and is herein called the C-CD-F-FG surface. The C-CD-F-FG surface can be used as a template to design libraries of protein scaffold interaction surfaces by randomizing a subset of residues that form the surface. Beta-strands have a repeating structure with the side chain of every other residue exposed to the surface of the protein. Thus, a library can be made by randomizing some or all surface exposed residues in the beta strands. By choosing the appropriate residues in the beta-strands, the inherent stability of the Tencon scaffold should be minimally compromised while providing a unique scaffold surface for interaction with other proteins.

Library TCL14 (SEQ ID NO: 7), was designed into Tencon27 scaffold (SEQ ID NO: 4). A full description of the methods used to construct this library is described in US. Pat. Publ. No. US2013/0226834.

TCL14 library (SEQ ID NO: 7):

LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIVLTVPGS ERSYDLTGLKPGTEYX ₈ VX ₉ IX ₁₀ GVKGGX ₁₁ X ₁₂ SX ₁₃ PLSAIFTT;

wherein

Xi, X ₂ , X ₃ , X ₄ , Xs, ¾, Xv, Xs, X _9> Xio, X11, X12 and X _J3 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, C or M.

The two beta strands forming the C-CD-F-FG surface in Tencon27 have a total of 9 surface exposed residues that could be randomized; C-strand: S30, L32, Q34, Q36; F- strand: E66, T68, S70, Y72, and V74, while the CD loop has 6 potential residues: S38, E39, K40, V41, G42, and E43 and the FG loop has 7 potential residues: K75, G76, G77, H78, R79, S80, and N81. Select residues were chosen for inclusion in the TCL14 design due to the larger theoretical size of the library if all 22 residues were randomized.

Thirteen positions in Tencon were chosen for randomizing: L32, Q34 and Q36 in C-strand, S38, E39, K40 and V41 in CD-loop, T68, S70 and Y72 in F-strand, H78, R79, and N81 in FG-loop. In the C and F strands S30 and E66 were not randomized as they lie just beyond the CD and FG loops and do not appear to be as apparently a part of the C- CD-F-FG surface. For the CD loop, G42 and E43 were not randomized as glycine, providing flexibility, can be valuable in loop regions, and E43 lies at the junction of the surface. The FG loop had K75, G76, G77, and S80 excluded. The glycines were excluded for the reasons above while careful inspection of the crystal structures revealed S80 making key contacts with the core to help form the stable FG loop. K75 faces away from the surface of the C-CD-F-FG surface and was a less appealing candidate for randomization. Although the above mentioned residues were not randomized in the original TCL14 design, they could be included in subsequent library designs to provide additional diversity for de novo selection or for example for an affinity maturation library on a select TCL14 target specific hit.

Subsequent to the production of TCL14, 3 additional Tencon libraries of similar design were produced. These two libraries, TCL19, TCL21 and TCL23, are randomized at the same positions as TCL14 (see above) however the distribution of amino acids occurring at these positions is altered (Table 3). TCL19 and TCL21 were designed to include an equal distribution of 18 natural amino acids at every position (5.55% of each), excluding only cysteine and methionine. TCL23 was designed such that each randomized position approximates the amino acid distribution found in the HCDR3 loops of functional antibodies (Birtalan et al., J Mol Biol 377: 1518-1528, 2008) as described in Table 3. As with the TCL21 library, cysteine and methionine were excluded.

A third additional library was built to expand potential target binding surface of the other libraries library. In this library, TCL24, 4 additional Tencon positions were randomized as compared to libraries TCL14, TCL19, TCL21, and TCL23. These positions include N46 and T48 from the D strand and S84 and 186 from the G strand. Positions 46, 48, 84, and 86 were chosen in particular as the side chains of these residues are surface exposed from beta-strands D and G and lie structurally adjacent to the randomized portions of the C and F strand, thus increasing the surface area accessible for binding to target proteins. The amino acid distribution used at each position for TCL24 is identical to that described for TCL19 and TCL21 in Table 3.

TCL24 Library (SEQ ID NO: 8)

LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9VPG SERSYDLTGLKPGTEYX10VX11IX12GVKGGX13X14SX15PLX16AX17FTT;

wherein

Xi, X2, X3, X4, X5, Χδ, Xio, Xn, X12, Xi3, Xi4, Xi5, X½and X17 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, Y or W.

Table 3. Amino acid frequency (%) at each randomized position for TCL21, TCL23, and TCL24.

Asn 5.6 5.6 3.9 5.6

Asp 5.6 5.6 7.5 5.6

Cys 0.0 0.0 0.0 0.0

Gin 5.6 5.6 1.5 5.6

Glu 5.6 5.6 2.5 5.6

Gly 5.6 5.6 15.0 5.6

His 5.6 5.6 2.3 5.6

lie 5.6 5.6 2.5 5.6

Leu 5.6 5.6 5.0 5.6

Lys 5.6 5.6 1.5 5.6

Met 0.0 0.0 0.0 0.0

Phe 5.6 5.6 2.5 5.6

Pro 5.6 5.6 4.0 5.6

Ser 5.6 5.6 10.0 5.6

Thr 5.6 5.6 4.5 5.6

Trp 5.6 5.6 4.0 5.6

Tyr 5.6 5.6 17.3 5.6

Val 5.6 5.6 4.0 5.6

Generation of TCL21, TCL23, and TCL24 libraries

The TCL21 library was generated using Colibra library technology (Isogenica) in order to control amino acid distributions. TCL19, TCL23, and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control amino acid distributions. PCR was used to amplify each library following initial synthesis followed by ligation to the gene for RepA in order to be used in selections using the CIS-display system (Odegrip et al., Proc Natl Acad Sci U S A 101: 2806-2810, 2004) as described above for the loop libraries. EXAMPLE 3: Selection of fibronectin type III (FN3) domains that bind PD-L1 Panning

FN3 domains specific for human PD-L1 were selected via CIS-Display (Odegrip et al 2004) using recombinant biotinylated PD-L1 (rhPD-Ll/Fc Chimera, R&D Systems 156-B7). For in vitro transcription and translation (ITT), 3 μg of DNA from libraries TCL18, TCL19, TCL21, TCL23, and TCL24 were incubated at 30°C with 0.1 mM complete amino acids, IX S30 premix components, and 15 iL of S30 extract (Isogenica) in a total volume of 50 After 1 hour, 375 iL of blocking solution (2% BSA in PBS, Invitrogen) was added and reactions were incubated on a cold block for 15 minutes.

Unbound library members were removed by successive washes with TBST and TBS. After washing, DNA was eluted from the target protein by heating to 75°C for 10 minutes and amplified by PCR using KOD polymerase for further rounds of panning. High affinity binders were isolated by successively lowering the concentration of target PD-L1 during each round from 400 nM to 100 nM and increasing the washing stringency.

Outputs from the fifth round panning were subjected to four additional rounds of off-rate selection. Library transcription and translation was performed as described above after which the ITT reactions were incubated with biotinylated recombinant PD-L1 proteins and captured on neutravidin or streptavidin coated magnetic beads, before being washed in TBST extensively then subsequently washed in 5 μΜ cold recombinant PD-L1 protein for 1 hour. The biotinylated target antigen concentration was reduced from 25 nM in rounds 6 and 7 to 2.5 nM in rounds 8 and 9.

Following panning, genes encoding the selected FN3 domains were amplified by PCR, subcloned into a pET vector modified to include a ligase independent cloning site, and transformed into BL21 (DE3) (Stratagene) cells for soluble expression in E. coli using standard molecular biology techniques. A gene sequence encoding a C-terminal poly- histidine tag was added to each FN3 domain to enable purification and detection. Cultures were grown to an optical density of 0.6-0.8 in TB medium supplemented with 100 μg/mL carbenicillin in 1 mL 96-well blocks at 37°C before the addition of IPTG to 1 mM, at which point the temperature was reduced to 30°C. Cells were harvested approximately 16 hours later by centrifugation and frozen at -20°C. Cell lysis was achieved by incubating each pellet in 0.6 mL of BugBuster® HT lysis buffer (Novagen EMD Biosciences) supplemented with 0.2 mg/mL lysozyme with shaking at room temperature for 30 minutes. Biochemical Screening for FN3 domains that bind Recombinant PD-L1

Streptavidin-coated Maxisorp plates (Nunc catalog 436110) were blocked for lh in Starting Block T20 (Pierce) and then coated with biotinylated PD-L1 (using same antigen as in panning) or negative controls (an unrelated Fc-fused recombinant protein and human serum albumin) for lh. Plates were rinsed with TBST and diluted lysate was applied to plates for lh. Following additional rinses, wells were treated with HRP- conjugated anti-FN3 domain antibody (PAB25) for lh and then assayed with POD (Roche catalog 11582950001). The DNA from FN3 domain lysates with ELISA binding signals to PD-L1 at least 5-fold above both Fc and HSA controls were sequenced resulting in 57 (Table 4) and 37 (Table 5) unique, readable FN3 domain sequences isolated from Round 5 and Round 9 screening respectively.

High-throughput Expression of anti-PD-Ll FN3 domains

40 isolated clones from unique hits identified by biochemical binding ELISA from Round 9 were combined for growth into 96-well block plate; clones grew in 1 mL cultures (LB media supplemented with kanamycin for selection) at 37°C overnight with shaking. For protein expression in 96-block plates, 1 mL TB media supplemented with kanamycin was inoculated with 50 μΐ. of the overnight culture and grown at 37°C with continual shaking at 300rpm until OD ₆ oo = 0.6-1. Once the target OD was reached, protein expression was induced with addition of IPTG to 1 mM; plates were transferred to 30°C (300 rpm) for overnight growth. Overnight cultures were centrifuged to harvest the cells; bacterial pellets were stored at -80°C until ready for use. Pellets were lysed with BugBuster® HT lysis buffer (Novagen EMD Biosciences) and His-tagged Centyrins purified from the clarified lysates with His MultiTrap™ HP plates (GE Healthcare) and eluted in buffer containing 20 mM sodium phosphate, 500 mM sodium chloride, and 250 mM imidazole at pH 7.4. Purified samples were exchanged into PBS pH 7.4 for analysis using PD MultiTrap™ G-25 plates (GE Healthcare).

Size Exclusion Chromatography Analysis

Size exclusion chromatography was used to determine the aggregation state of anti-PD-Ll FN3 domains. Aliquots (10 μΚ) of each purified FN3 domain were injected onto a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min in a mobile phase of PBS pH 7.4. Elution from the column was monitored by absorbance at 280 nm. Tencon protein was included in each run as a control. Agilent ChemStation software was used to analyse the elution profiles. 20 anti-PD-Ll FN3 domains demonstrated a retention time between 5.2 and 6.4 minutes and only a single SEC peak indicative of monomeric protein (Table 6).

Table 4.

ISOP121BR5P1D1 186240 1040 960 60

ISOP121BR5P1C9 226400 1120 1040 61

ISOP121GR5P1G11 239600 960 1040 62

ISOP121BR5P1A7 388640 800 1120 63

ISOP121BR5P1C3 177040 640 480 64

ISOP121AR5P1D11 392800 640 1040 65

ISOP121ER5P1E7 251120 480 560 66

ISOP121GR5P1G7 367760 800 800 67

ISOP121AR5P1A8 515920 560 1040 68

ISOP121BR5P1E7 411760 800 640 69

ISOP121FR5P1H8 430640 560 640 70

ISOP121GR5P1D2 513280 720 640 71

ISOP121AR5P1H2 926720 880 1120 72

ISOP121GR5P1F10 577120 640 640 73

ISOP121BR5P1A2 742800 720 800 74

ISOP121GR5P1F7 697200 640 720 75

ISOP121AR5P1B8 591600 640 560 76

ISOP121GR5P1D7 791920 720 720 77

ISOP121BR5P1G3 770800 560 640 78

ISOP121AR5P1C5 732480 640 560 79

ISOP121FR5P1H9 1195520 720 880 80

ISOP121AR5P1A10 788560 1120 560 81

ISOP121HR5P1F2 906960 480 640 82

ISOP121AR5P1H1 1475280 880 880 83

ISOP121BR5P1D10 1538800 480 880 84

ISOP121BR5P1F10 1422880 560 720 85

ISOP121BR5P1D11 2442960 800 1120 86

ISOP121AR5P1E11 1842000 720 720 87

ISOP121BR5P1D6 2435760 560 880 88

ISOP121BR5P1B5 1483520 720 400 89

Table 5. (RSU)

ISOP194ER9P1G3 4288320 560 720 90

ISOP194AR9P1F2 16271040 1920 7520 91

ISOP194AR9P1H 10 5212800 4400 2400 92

ISOP194BR9P1H4 4064960 3040 3840 93

ISOP194AR9P1D8 923200 12000 6560 94

ISOP194BR9P1D1 2152080 1360 1280 95

ISOP194AR9P1E8 3404480 6960 67680 96

ISOP194AR9P1E9 19719920 5520 1600 97

ISOP194AR9P1H9 2592720 21280 11360 98

ISOP194BR9P1A9 19046640 2320 3200 99

ISOP194BR9P1A5 3182000 800 1280 100

ISOP194BR9P1F7 15151120 1920 1760 101

ISOP194AR9P1G7 15914000 1280 560 102

ISOP194AR9P1E3 4566880 1120 800 103

ISOP194AR9P1C5 4371120 3440 1040 104

ISOP194AR9P1H3 17746800 9200 4880 105

ISOP194GR9P1E9 2821920 720 1200 106

ISOP194H R9P1B10 385360 560 1840 107

ISOP194ER9P1A11 4352240 800 880 108

ISOP194ER9P1A3 2360160 560 800 109

ISOP194ER9P1H9 3042800 720 880 110

ISOP194H R9P1B2 5656400 400 1840 111

ISOP194H R9P1D11 6620160 480 1680 112

ISOP194GR9P1F6 319200 400 1200 113

ISOP194GR9P1F9 105280 320 800 114

ISOP194GR9P1C11 164320 1040 1440 115

ISOP194ER9P1E6 8982160 240 720 116

ISOP194BR9P1G9 14376560 640 960 117

ISOP194BR9P1E4 9791680 640 1440 118

ISOP194AR9P1H 1 21445040 15680 6800 119

ISOP194BR9P1D10 1666880 720 1120 120

ISOP194BR9P1C8 6110640 640 1280 121

ISOP194AR9P1C10 13863040 38240 14960 122

ISOP194AR9P1D11 1043280 28160 12720 123

ISOP194AR9P1C3 3548240 56400 5920 124 Table 6.

ISOP194HR9P1B2 6.274 2.33 FALSE 111

ISOP194HR9P1D11 6.002 433.98 FALSE 112

ISOP194GR9P1F6 6.12 29.42 TRUE 113

ISOP194GR9P1F9 No peak FALSE 114

ISOP194GR9P1C11 12.458 2.90 FALSE 115

ISOP194ER9P1E6 6.125 149.28 TRUE 116

ISOP194BR9P1G9 6.622 84.28 FALSE 117

ISOP194BR9P1E4 5.714 456.33 TRUE 118

ISOP194AR9P1H1 6.247 12.76 FALSE 119

ISOP194BR9P1D10 6.059 10.60 FALSE 120

ISOP194BR9P1C8 No peak FALSE 121

ISOP194AR9P1C10 5.715 98.64 TRUE 122

ISOP194AR9P1D11 No peak FALSE 123

ISOP194AR9P1C3 5.588 700.26 TRUE 124

Sequences

SEQ ID No. 1= Original Tencon Sequence

LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY DLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT

SEQ ID No. 2= TCL1 library

LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY

DLTGLKPGTEYTVSIYGVX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ PLSAEFTT;

wherein

Xi, X2, X3, X4, X5, Χδ, X7 is any amino acid; and

Xg, X9, X10, X11 and X12 are any amino acid or deleted

SEQ ID No. 3=TCL2 library

LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVGEAINLTVPGS

ERSYDLTGLKPGTEYTVSIYGVX ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ SX ₁₄ X ₁₅ LSAEFTT;

wherein

Xi is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X2 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X3 Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X4 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X5 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

Xe is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

Val or Tyr;

Xg is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

Xio is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X11 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X12 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

Xi3 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X14 IS Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and

Xi5 is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

SEQ ID No. 4= Stabilized Tencon

LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

SEQ ID No. 5= TCL7 (FG and BC loops)

LPAPKNLVVSRVTEDSARLSWX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ FDSFLIQYQESEKVGEAIVLT VPGSERSYDLTGLKPGTEYTVSIYGVX ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ SNPLSAIFTT; wherein

Xi, X2, X3, X4, X5, Χδ, Xio, X11, X12, Xi3, Xi4, Xi5 and Xi6 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and

X ₇ , Xs, X9, Xi7, Xi8 and Xi9, are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted

SEQ ID No. 6= TCL9 (FG loop)

LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSY

DLTGLKPGTEYTVSIYGVX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ SNPLSAIFTT;

wherein

Xi, X ₂ , X ₃ , X4, Xs, X ₆ and X ₇ , is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and X ₈ , X ₉ , Xio, X11 and X12 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.

TCL14 library (SEQ ID NO: 7):

LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFXiIX2YX3EX ₄ X ₅ X6X7GEAIVLTVPGS

ERSYDLTGLKPGTEYX ₈ VX ₉ IX ₁₀ GVKGGX ₁₁ X ₁₂ SX ₁₃ PLSAIFTT;

wherein Xi, X _¾ X ₃ , X ₄ , Xs, ¾, Xv, Xs, X _9> Xio, Xii, Xi2 and X _J3 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, C or M.

TCL24 Library (SEQ ID NO: 8)

LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9VPG

SERSYDLTGLKPGTEYX10VX11IX12GVKGGX13X14SX15PLX16AX17FTT;

wherein

Xi, X2, X3, X4, X5, Χδ, Xio, X11, X12, Xi3, Xi4, Xi5, Xi6and Xn are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, Y or W.

SEQ ID No. 9 = Sloning-FOR

GTGACACGGCGGTTAGAAC

SEQ ID No. 10 = Sloning-REV

GCCTTTGGGAAGCTTCTAAG

SEQ ID No. 11 = POP2250

CGGCGGTTAGAACGCGGCTACAATTAATAC

SEQ ID No. 12 = DigLigRev

CATGATTACGCCAAGCTCAGAA

SEQ ID No. 13 = BC9

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA

TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG

TTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNN

NNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGT

TGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGA

CCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGA

AGCTTCCCAAAGGC

SEQ ID No. 14 = BC8

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA

TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG

TTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNN

NNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGG

TGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCG

GTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGC

TTCCCAAAGGC

SEQ ID No. 15 = BC7

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA

TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG

TTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNN

NNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGA

AGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTC

TGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCC

CAAAGGC SEQ ID No. 16 = BC6

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA

TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG

TTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNN^

NNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC

GATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA

AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCA

AAGGC

SEQ ID No. 17 = 130mer-L17A

CGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCAC ACAGGAAACAGGATCTACCATGCTG

SEQ ID No. 18 = POP222ext

CGG CGG TTA GAA CGC GGC TAC AAT TAA TAC SEQ ID No. 19 = LSI 114

CCA AGA CAG ACG GGC AGA GTC TTC GGT AAC GCG AGA AAC AAC CAG GTT TTT CGG CGC CGG CAG CAT GGT AGA TCC TGT TTC

SEQ ID No. 20 = LSI 115

CCG AAG ACT CTG CCC GTC TGT CTT GG

SEQ ID No. 21 = LSI 117

CAG TGG TCT CAC GGA TTC CTG GTA CTG GAT CAG GAA AGA GTC GAA SEQ ID No. 22 = SDG10

CATGCGGTCTCTTCCGAAAAAGTTGGTGAAGCGATCGTCCTGACCGTTCCGGG T

SEQ ID No. 23 = SDG24

GGTGGTGAAGATCGCAGACAGCGGGTTAG

SEQ ID No. 24 = POP2222

CGGCGGTTAGAACGCGGCTAC

SEQ ID No. 25 = SDG28

AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCACCGCCGGTGGTGAAG ATCGCAGAC

SEQ ID No. 26 = FG12

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA

TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG

TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG

GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC

GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA

AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACC GGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAAC TGATCTTGGC

SEQ ID No. 27 = FG11

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA

TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG

TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG

GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC

GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA

AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGC

GGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGA

TCTTGGC

SEQ ID No. 28 = FG10

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA

TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG

TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG

GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC

GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA

AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTC

ACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTT

GGC

SEQ ID No. 29 = FG9

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA

TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG

TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG

GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC

GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA

AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNN

NNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACC

ATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

SEQ ID No. 30 = FG8

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA

TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG

TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG

GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC

GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA

AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNN

NNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATC

ACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

SEQ ID No. 31 = FG7

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG

TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG

GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC

GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA

AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTN TCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACC

ATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

SEQ ID NO: 32 = human mature PD-L1

FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEED LKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRIT VKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTT NSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER

SEQ ID NO: 33 = human mature PD-1

PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT DKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPK AQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVW VLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCV PEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPE DGHCSWPL

ISOP121AR5P1G6 40 LPAPKNLVVSRVTEDSARLSWHATFGDPFDSFLIQYQESEKVG EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLS AIFTT

ISOP121BR5P1B7 41 LPAPKNLVVSRITEDSARLSWKWEEGFFDSFLIQYQESEKVGEA

IVLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRHASAFVSNPLSA IFTT

ISOP121FR5P1G1 42 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIHYTEAPVHGE

AIVLTVPGSERSYDLTGLKPGTEYTVVIWGVKGGTWSSPLSAIF

TT

ISOP121GR5P1B4 43 LPAPKNLIVSRVTEDSARLSWTAPDAAFDSFPINYGERATKGEA

INLYVPGSERSYDLTGLKPGTEYWVLIGGVKGGLKSSPLWAWF TT

ISOP121BR5P1G2 44 LPAPKNLVVSRVTEDSARLSWADELHHANHFDSFLIQYQESEK

VGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYDRHYEIHFYS NPLSAIFTT

ISOP121HR5P1H2 45 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYLEYDYSGEA

IVLTVPGSERSYDLTGLKPGTEYDVLIIGVKGGSLSTPLSAIFTT

ISOP121FR5P1G11 46 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIWYLEIVAHGE

AIVLTVPGSERSYDLTGLKPGTEYEVIIHGVKGCGPSGPLSAIFTT

ISOP121AR5P1E7 47 LPAPKNLVVSRVTEDSARLSWHVYHEIDYFDSFLIQYQESEKVG

EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRKVEFYSNPLS AIFTT

ISOP121GR5P1F6 48 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIRYHEYTWPGE

AIVLLVPGSERSYDLTGLKPGTEYGVYINGVKGGFRSKPLFAWF TTGG

ISOP121BR5P1E9 49 LPAPKNLVVSRVTEDSARLSWDSYRDYFDSFLIQYQESEKVGEA

IVLTVPGSERSYDLTGLKPGTEYTVSIYGVYSRKHVVFVQSNPLS AIFTT

ISOP121AR5P1F2 50 LPAPKNLVISRVTEDSARLSWGWSELIATHFDSFLIQYQESEKV

GEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYNRKVNFYSNPL

SAIFTT

ISOP121AR5P1F7 51 LPAPKNLVVSRVTEDSARLSWQEHWDTSSNFDSFLIQYQESEK

VGEAIVLTVPGSERSYDLTGLKPGTEYTISIYGVYNRKVLFYSNPL SAIFTT

ISOP121BR5P1H6 52 LPAPKNLVVSRVTEDSARLSWGYIDVSYFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYSRPKAEFTSNPLSA

IFTT

ISOP121GR5P1A2 53 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIQYIERYIPGEAI

QLNVPGSERSYDLTGLKPGTEYSVIIPGVKGGRNSFPLWAWFT T

ISOP121BR5P1D3 54 LPAPKNLVVSRVTEDSARLSWYEDNTERFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYIRVQVLWFSNPLS AIFTT

ISOP121AR5P1F9 55 LPAPKNLVVSRVTEDSARLSWGWSELIATHFDSFLIQYQESEKV

GEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYNRKVNFYSNPL SAIFTT ISOP121AR5P1H5 56 LPAPKNLVVSRVTEDSARLSWEDAVKHIWFDSFLIQYQESEKV GEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVWIASVWRSNPL SAIFTT

ISOP121AR5P1G10 57 LPAPKNLVVSRVTEDSARLSWEWLEHFDSFLIQYQESEKVGEAI

VLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRKVEFHSNPLSAIF TTT

ISOP121AR5P1F3 58 LPAPKNLVVSRVTEDSARLSWPFNNYSEHFDSFLIQYQESEKVG

EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYERKTAFYSNPLSA IFTT

ISOP121BR5P1E2 59 LPAPKNLVVSRVTEDSARLSWWFPLEWFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYTREHKSVWASNP LSAIFTT

ISOP121BR5P1D1 60 LPAPKNLVVSRVTEDSARLSWKWGGEFFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRNWHHWYSNP LSAIFTT

ISOP121BR5P1C9 61 LPAPKNLVVSRVTEDSARLSWIWPDKHEFFDSFLIQYQESEKVG

EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYDRKYANWSSNP

LSAIFTT

ISOP121GR5P1G11 62 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQINYHEYGQNG

EAIQLIVPGSERSYDLTGLKPGTEYGVWIWGVKGGIRSKPLWA FFTT

ISOP121BR5P1A7 63 LPAPKNLVVSRVTEDSARLSWTTAFHNEYFDSFLIQYQESEKVG

EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYSRPKAEFTSNPLS AIFTT

ISOP121BR5P1C3 64 LPAPKNLVVSRVTEDSARLSWASARDYFDSFLIQYQESEKVGEA

IVLTVPGSERSYDLTGLKPGTEYTVSIYGVLAIAQITHWFSNPLS

AIFTT

ISOP121AR5P1D11 65 LPAPKNLVVSRVTEDSARLSWEWLEHFDSFLIQYQESEKVGEAI

VLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRKVEFHSNPLSAIF TT

ISOP121ER5P1E7 66 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIGYTETPPRGEA

IVLTVPGSERSYDLTGLKPGTKYYVSILGVKGGLGSWPLSAIFTT

ISOP121GR5P1G7 67 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIRYHEYDKNGE

AIQLYVPGSERSYDLTGLKPGTEYGVYIHGVKGGGRSKPLWAH FTT

ISOP121AR5P1A8 68 LPAPKNLVVSRVTEDSARLSWGLEWAYQFFDSFLIQYQESEKV

GEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYLRAIEFYSNPLS AIFTT

ISOP121BR5P1E7 69 LPAPKNLVVSRVTEDSARLSWRKQEQYFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKKWPSI 11 ISNPLS AIFTT

ISOP121FR5P1H8 70 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIYYSEQHFYGE

AIVLTVPGSERSYDLTGLKPGTEYVVKIYGVKGGETSKPLSAIFTT

ISOP121GR5P1D2 71 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHILYQERAQSGE

AIGLVVPGSERSYDLTGLKPATEYSVQIFGVKGGKLSNPLWAW FTT ISOP121AR5P1H2 72 LPAPKNLVVSRVTEDSARLSWVIDEFIPLFDSFLIQYQESEKVGE AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVLAKNIGISNPLSAIFT T

ISOP121GR5P1F10 73 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIDYVERATVGE

AIALNVPGSKRSYALTGLKPGTEYFVKIRGVKGGLKSKPLWAW FTT

ISOP121BR5P1A2 74 LP AP KN LVVS RVTE DSARLSW RFSQEW F DS F LI QYQES E KVG E

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYARGIHKWLSNPLS AIFTT

ISOP121GR5P1F7 75 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGINYVERASEGE

AIDLGVPGSERSYDLTGLKPGTEYFVKIFGVKGGIPSVPLWAWF

TT

ISOP121AR5P1B8 76 LPAPKNLVISRVTEDSARLSWDKRTQFAFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVPTWSGRTQSNPLS AIFTT

ISOP121GR5P1D7 77 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIWYQERSIVGE

AIFLLVPGSERSYDLTGLKPGTEYIVQIFGVKGGPYSNPLWAPFT

T

ISOP121BR5P1G3 78 LP AP KN LVVS RVTE DSARLSW KQRTS F H F DS F LI QYQES E KVG E

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVPFWQQWQPESNP LS AIFTT

ISOP121AR5P1C5 79 LPAPKNLVVSRVTEDSARLSWKRSDDEWFDSFLIQYQESEKVG

E A 11 LTVP GS E RS Y D LTG LK P GTE YTVS 1 YG VYQRAA LW FS N P LS AIFTT

ISOP121FR5P1H9 80 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSILYGETAPIGEAI

VLTVPGSERSYDLTGLKPGTEYVVYIQGVKGGNYSQPLSAIFTT

ISOP121AR5P1A10 81 LPAPKNLVVSRVTEDSARLSWPDWSNSEYFDSFLIQYQESEKV

GEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYARHRLFVSNPL SAIFTT

ISOP121HR5P1F2 82 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTILYGETYSGGEA

IVLTVPGSERSYDLTGLKPGTEYVVYIFGVKGGKWSRPLSAIFTT

ISOP121AR5P1H1 83 LPAPKNLVVSRVTEDSARLSWKQATKFVFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVPDWFVLESNPLSAI

FTT

ISOP121BR5P1D10 84 LPAPKNLVVSRVTEDSARLSWGKKSHFFDSFLIQYQESEKVGEA

IVLTVPGSERSYDLTGLKPGTEYTVSIYGVYTRGQCEWESNQLS

AIFFTT

ISOP121BR5P1F10 85 LPAPKNLVVSRVTEDSARLSWPLNLEYFDSFLIQYQESEKVGEAI

VLTVPGSERSYDLTGLKPGTEYTVSIYGVYGRYGGPFVSNPLSAI FTT

ISOP121BR5P1D11 86 LPAPKNLVVSRVTEDSARLSWFNADEEYFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYVRAVRFVSSNPLS

AIFTT

ISOP121AR5P1E11 87 LPAPKNLVVSRVTEDSARLSWSVQTSFVFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVPLWHGFDSNPLSAI FTT ISOP121BR5P1D6 88 LP AP KN LVVS RVTE DSARLSW KQGTS F H F DS FLIQYQES E KVG E AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVQLLANDIISSNPLSAI FTT

ISOP121BR5P1B5 89 LPAPKNLVVSRVTEDSARLSWRKQEQYFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRGYHNWFSNPL

SAIFTT

ISOP194ER9P1G3 90 LPAPKNLIVSRVTEDSARLSWTAPDAAFDSFRIAYYETMVSGEA

1 VLTVPGSERSYD LTG LKPGTEYAVI 1 KG VKGG KPSWP LSAI FTT

ISOP194AR9P1F2 91 LPAPKNLVISRVTEDSARLSWEWLEHFDSFLIQYQESEKVGEAI

VLTVPGSERSYDLTGLKPGTEYTVSIYGVYNRKVNFYSNPLSAIF TT

ISOP194AR9P1H10 92 LPAPKNLVISRVTEDSARLSWPAHYHSAFFDSFLIQYQESEKVG

EAI VLTVPGSERSYD LTG LKPGTEYTVSIYGVYQRKVEFHSNPLS AIFTT

ISOP194BR9P1H4 93 LPAPKNLVVSRVTEDSACLSWTTAFHNEYFDSFLIQYQESEKVG

EAI VLTVPGSERSYD LTG LKPGTEYTVSIYGVYSRPKAEFTSNPLS AIFTT

ISOP194AR9P1D8 94 LPAPKNLVVSRVTEDSARLSWDTWNDFFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRKVIWLSNPLSA IFTT

ISOP194BR9P1D1 95 LPAPKNLVVSRVTEDSARLSWEHSLLNDQWFDSFLIQYQESEK

VG EAI VLTVPGSERSYD LTG LKPGTEYTVSIYGVYQRGRALWYS NPLSAIFTT

ISOP194AR9P1E8 96 LPAPKNLVVSRVTEDSARLSWEWLEHFDSFLIQYQESEKVGEAI

VLTIPGSERSYDLTGLKPGTEYTVSIYGVYQRKVEFHSNPLSAIFT T

ISOP194AR9P1E9 97 LPAPKNLVVSRVTEDSARLSWEWLEHFDSFLIQYQESEKVGEAI

VLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRKVNFYSNPLSAIF TT

ISOP194AR9P1H9 98 LPAPKNLVVSRVTEDSARLSWEWLEHFDSFQIQYQESEKVGEA

IVLTVPGSERSYD LTG LKPGTEYTVSIYGVYQRKVEFHSNP LSAI F TT

ISOP194BR9P1A9 99 LPAPKNLVVSRVTEDSARLSWFNADEEYFDSFLIQYQESEKVGE

Al VLTVPGSERSYD LTG LKPGTEYTVSIYGVYDRKVKFVQSNPLS AIFTT

ISOP194BR9P1A5 100 LPAPKNLVVSRVTEDSARLSWFNADEEYFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRGYHNWFSNPL SAIFTT

ISOP194BR9P1F7 101 LPAPKNLVVSRVTEDSARLSWFNADEEYFDSFLIQYQESEKVGE

AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYTRGRYEWRESNPL SAIFTT

ISOP194AR9P1G7 102 LPAPKNLVVSRVTEDSARLSWGDDFNSEYFDSFLIQYQESEKV

GEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYTRAVVFTSNPL

SAIFTT

ISOP194AR9P1E3 103 LPAPKNLVVSRVTEDSARLSWKRSDDEWFDSFLIQYQESEKVG

EAI VLTVPGSERSYD LTG LKPGTEYTVSIYGVYQRAALWFSNPLS AIFTT ISOP194AR9P1C5 104 LPAPKNLVVSRVTEDSARLSWLRDFNGRAFFDSFLIQYQESEKV GEAIVLTVPGSERSYDPTGLKPGTEYTVSIYGVFITWIHVRSNPL SAIFTT

ISOP194AR9P1H3 105 LPAPKNLVVSRVTEDSARLSWNASWISHNFFDSFLIQYQESEK

VGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYERKTAFYSNP LSAIFTT

ISOP194GR9P1E9 106 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIRYHEYDKNGE

AIQLYVPGSERSYDLTGLKPGTEYGVFIWGVKGGLKSKPLWAW FTT

ISOP194HR9P1B10 107 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIRYYERANGEAI

VLTVPGSERSYDLTGLKPGTEYIVWIYGVKGGGRSGPLSAIFTT

ISOP194ER9P1A11 108 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIAYYETMVSGE

AIVLTVPGSERSYDLTGLKPGTEYAVIIKGVKGGKPSWPLSAIFT T

ISOP194ER9P1A3 109 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIAYYETMVSGE

AIVLTVPGSERSYDLTGLKPGTEYAVIIKGVKGGMVSWPLSAIFT

T

ISOP194ER9P1H9 110 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIAYYETMVSGE

AIVLTVPGSERSYDLTGPKPGTEYAVIIKGVKGGKPSWPLSAIFT T

ISOP194HR9P1B2 111 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSILYGELIGDGEAI

VLTVPGSERSYDLTGLKPGSEYTVYIFGVKGGRYSRP LSAIFTT

ISOP194HR9P1D11 112 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSILYGELIGDGEAI

VLTVPGSERSYDLTGLKPGTEYTVYIFGVKGGRYSRPLSAIFTT

ISOP194GR9P1F6 113 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIDYWERLSEGE

AIALRVPGSERSYDLTGLKPGTEYYVWIVGVKGGKFSQPLRAW FTT

ISOP194GR9P1F9 114 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIFYNERWQNG

EAIRLIVPGSERSYDLTGLKPGTEYSVIIPGVKGGRNSFPLWAWF TT

ISOP194GR9P1C11 115 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIFYNERWQNG

EAIRLTVPGSERSYDLTGLKPGTEYWVLIGGVKGGLKSSPLWA

WFTT

ISOP194ER9P1E6 116 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIKYYEKRNPGE

AIVLTVPGSERSYDLTGLKPGTEYLVIISGVKGGSRSVPLSAIFTT

ISOP194BR9P1G9 117 LPAPKNLVVSRVTEDSARLSWTTAFHNEYFDSFLIQYQESEKVG

EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYIRVQVLWFSNPL SAIFTT

ISOP194BR9P1E4 118 LPAPKNLVVSRVTEDSARLSWTTAFHNEYFDSFLIQYQESEKVG

EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRGYHNWFSNP LSAIFTT

ISOP194AR9P1H1 119 LPAPKNLVVSRVTEDSARLSWWRVLGHSHFFDSFLIQYQESEK

VGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYNRKVNFYSNP

LSAIFTT ISOP194BR9P1D10 120 LPAPKN LVVSRVTEDSARLSWYEDNTERFDSFLIQYQESEKVVE AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYI RVQVLWFSN PLS AI FTT

ISOP194BR9P1C8 121 LPAPKN LVVSRVTEDSARLSWYFAGELWFDSFLIQYQESEKVG

EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRGYH NWFSNP LS AI FTT

ISOP194AR9P1C10 122 LPAPKN LVVSRVTEDSARPSWEWLEH FDSFLIQYQESEKVGEAI

VLTVPGSERSYDLTGLKPGTEYTVSIYGVYN RKVN FYSN PLSAI F TT

ISOP194AR9P1D11 123 LPAPKN LVVSRVTEDSG RLSWQH H ISFFDSFLIQYQESEKVGEA

IVLTVPGSERSYDLTGLKPGTEYTVSIYGVYN RKVN FYSN PLSAI F TT

ISOP194AR9P1C3 124 LPAPKN LVVSRVTQNSARLSWEWLEH FDSFLI HYQESEKVGEA

IVLTVPGSERSYDLTGLKPGTEYTVSIYGVYQRKVEFHSNPLSAI F TT

3rd FN3 domain of 125 DAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVP tenascin C (TN3 GDRTTIDLTEDENQYSIGNLKPDTEYEVSLISRR

GDMS SNPAKETFTT

Fibcon 126 LDAPTDLQVTNVTDTSITVSWTPPSATITGYRITYTPSNG

PGEPKELTVPPSSTSVTITGLTPGVEYWSLYAL KDNQESPPLVGTQTT

10 ^th FN3 domain of 127 VSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGET fibronectin GGNSPVQEFTVPGSKSTATISGLKPGVDYTITVY

AVTGRGD SPASSKPISINYRT

Linker 128 GSGS

Linker 129 GGGSGGGS

Linker 130 GGGGSGGGGSGGGGSGGGGSGGGGS

Linker 131 APAP

Linker 132 APAPAPAPAP

Linker 133 APAP APAP APAP APAP APAP

Linker 134 APAP APAP APAP APAP APAP APAP APAP APAP APAP APAP

Linker 135 E AAA K E AAA K E AAA K E AAA K EAAA K AAA Albumin variant DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFED HVKLV EVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFL QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL KKYLYEIARRHPYFYAPELLFFAKRYKAAFTEC CQAADKAACLLPKLDELRDEGKAS SAKQRLKC ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSK LVTDLTKVHTECCHGDLLECADDRADLAKYICE NQD SIS SKLKECCEKPLLEK SH CI AE VENDEMP A DLPSLAADFVESKDVCKNYAEAKDVFLGMFLY EYARRHPDYSVVLLLRLAKTYETTLEKCCAAAD PHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLG EYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLH EKTPVSDRVT CCTESLV RRPCFSALEVDETY VPKEFN AETFTFH ADICTL SEKERQIKKQTAL VE LVKHKPKATKEQLKAVMDDFAAFVEKCCKAD DKETCFAEEGKKLVAASQAALGL