EA043883B1 - POLYPEPTIDES CONTAINING DE NOVO BINDING DOMAIN AND THEIR APPLICATION - Google Patents

Description

Related applications

The present patent application claims priority from the U.S. provisional patent application.

62/143,772, filed April 6, 2015, which is incorporated herein by reference in its entirety. All references, patents and patent applications cited in the present invention are incorporated by reference in their entirety.

Link to sequence list

The present invention is filed with an accompanying Sequence Listing in electronic format. The sequence listing is provided in a file called ENC002WOSEQUENCELISTING.TXT, created on April 4, 2016, which is 144 kilobytes in size. The information in the Sequence Listing is incorporated by reference into this application in its entirety.

State of the art

Antibody-based reagents have accelerated the pace of biological scientific research. Antibody compositions represent one of the most important and effective classes of therapeutic and diagnostic agents used in the pharmaceutical industry. However, cost, time, and efficiency have motivated the development of alternative affinity reagents.

A variety of non-immunoglobulin binding formats have emerged for applications that have historically used antibodies. Although many successful results have been reported for unstructured linear peptides, more encouraging results have been obtained by imposing a structural constraint on the peptide sequence, typically through the introduction of a disulfide bond. This restriction allows for higher affinity and specificity with more favorable thermodynamics of complementarity of fixed spatial conformations and surface presentation of residues (e.g., hydrophobic amino acids) that might otherwise be buried and thus not facing the target (Ladner, Trends in Biotech. 13 (10):426-430, 1995). On the other hand, formats that contain disulfide bonds are typically susceptible to mispairing of cysteines within or between domains, which can result in decreased expression, product yield, and product quality.

The structure found in protein subdomains provided another source of structural constraint. Structures such as type III fibronectin repeats (adnectins), z-proteins (affitebodies), nottins, lipocalins (anticalins), and ankyrin repeats (darpins) have been engineered with antibody-like affinities for a variety of different targets (Hey et al., Trends in Biotech 23(10):514-422, 2005). Such domains typically contain two elements that are analogous to the framework and complementarity determining regions (CDRs) present in antibody variable domains: structural frameworks that confer high thermodynamic stability, and residues or loops that form the basis of display library variability.

The essence of the invention

Overall, there remains a significant unmet need for new target-binding agents and compositions, and in particular those that contain alternative binding scaffolds (eg, non-immunoglobulin scaffolds). In several embodiments, agents of particular interest may feature, for example, substantially reduced manufacturing costs and/or comparable or superior reactive, diagnostic and/or therapeutic properties compared to antibodies. The present description proposes similar required means in several embodiments. For example, in several embodiments, the present disclosure provides certain polypeptide agents that have high binding affinity for the target and a non-immunoglobulin structural framework. Alternatively or additionally, in several embodiments, target binding agents, such as the polypeptides disclosed herein, for example, produced using the production methods disclosed herein, have advantages including, for example, highly specific target binding. In some embodiments, this may be advantageously used to target therapeutics (eg, immunocytes) to specific cells (eg, diseased cells), reducing or eliminating off-target effects. In some embodiments, the agents of the present invention, such as target-specific polypeptides, can be used as protein therapeutics to bind cells or soluble factors involved in a disease. In some embodiments, the present agents may be used to purify targets (eg, proteins or other targets) with a high degree of specificity, which may, for example, result in higher purity and/or reduced need for downstream processing to purify the target.

Several embodiments of the inventions disclosed herein relate to agents that specifically bind targets of interest, such as the de novo binding domain (DBD) polypeptides (DBDpp) disclosed herein. Also provided are nucleic acids encoding DBDpp, and vectors and host cells containing nucleic acids, such as DBDpp libraries, and methods for preparing and screening such libraries, as well as DBDpp identified in such libraries and/or screens. Also provided are DBDpp, including DBDpp fusion proteins, as well as methods for making and using DBDpp. Non-limiting examples of such applications include, but are not limited to, affinity purification, target analysis, diagnostic and/or therapeutic applications.

In several embodiments, a binding agent is provided that binds with a high degree of specificity to a target of interest. In several embodiments, the binding agent is a non-immunoglobulin agent. In several embodiments, the binding agent is a polypeptide. In several embodiments, polypeptides are provided for binding to a target of interest that has a sequence that differs at least at one position from the sequence of SEQ ID NO: 1. In several such embodiments, the agent (e.g., polypeptide) exhibits specific binding to the target of interest. a target of interest, wherein such binding is greater than the binding of the polypeptide corresponding to SEQ ID NO: 1 to the target of interest. In several embodiments, a polypeptide for binding a target of interest is provided, comprising an amino acid sequence comprising MGSWXsEFXgXgRLXisAIXisXisRLXigALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEXssL RX58X59AAX ₆₂ IRX ₆ 5X ₆₆ LQAYRHN (seq id NO: 4), wherein the sequence differs in sequence from SEQ ID NO: 1 (for example, modifications in amino acid sequence SEQ ID NO: 1). In several embodiments, the polypeptide specifically binds a target of interest (such as a tumor marker or other distinctive marker associated with the target of interest), wherein the specific binding of the polypeptide to the target of interest is greater than the binding of the polypeptide corresponding to SEQ ID NO: 1 to the target of interest. interest target. In several embodiments, the polypeptide does not contain the sequence of SEQ ID NO: 50.

In several embodiments, the polypeptide has a sequence that differs from SEQ ID NO: 1 because certain selected amino acid positions have been changed. In some embodiments, modifications include substitutions. In several embodiments, the substitutions are conservative substitutions, while in some embodiments, the substitutions are non-conservative substitutions. In additional embodiments, combinations of conservative and non-conservative substitutions are used. In some embodiments, the substitutions do not include a cysteine substitution (eg, no cysteines are added to the sequence). In some embodiments, where the substitutions do not include a proline substitution (eg, no prolines are added to the sequence). In some embodiments, there are no cysteine or proline substitutions in the polypeptide sequence.

Various targets of interest can be coupled by the means disclosed in the present invention. For example, in several embodiments, the target of interest specifically bound by the polypeptide is a cancer antigen. In some embodiments, the cancer antigen specifically bound by the polypeptide is PD-L1. In several such embodiments, the target binding polypeptide comprises or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44. In some embodiments, the cancer antigen specifically bound polypeptide is CD137. In some such embodiment, the polypeptide comprises or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 , SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In some embodiments, the cancer antigen specifically bound polypeptide is CD123. In some such embodiments, the polypeptide includes or essentially consists of an amino acid sequence selected from SEQ ID NOs: 92-127. In some embodiments, the combination of cancer antigens is subjected to targeted interaction, eg, by binding or otherwise combining, with different target-binding polypeptides. In some embodiments, two, three, four, or more different cancer antigens are targeted. In some embodiments, multiple target-binding polypeptides are used to enhance the ability and/or ability to bind a single target (eg, dimers, trimers, etc.).

Additionally, several embodiments provide a method of converting a reference polypeptide into a polypeptide having specific binding to a target of interest, where the method includes modifying a plurality of amino acid residues from the reference polypeptide to produce a plurality of candidate binding polypeptides, packaging the plurality of candidate binding polypeptides into a plurality of vectors to produce a candidate library and screening the candidate library for candidate binding polypeptides that exhibit

- 2 043883 specific binding to a target of interest. In several embodiments, the reference polypeptide includes a variant of a non-naturally occurring polypeptide and includes three antiparallel alpha helices that are connected by linker peptides. In several embodiments, the amino acid residues being modified are solvent accessible or solvent inaccessible amino acids. In several embodiments, more solvent accessible amino acids are changed, while in some embodiments, more solvent accessible amino acids are changed. In some embodiments, the modification includes amino acid substitutions. As discussed above, substitutions may include conservative amino acid substitutions, non-conservative amino acid substitutions, and/or combinations thereof. Optionally, in several embodiments, the substitution does not include a cysteine substitution, does not include a proline substitution, and, in some cases, does not include a cysteine or proline substitution.

In several embodiments, the method further includes identifying potentially immunogenic amino acid residues in candidate binding polypeptides and modifying at least one of the potentially immunogenic amino acid residues (eg, to reduce the potential immunogenicity of the resulting polypeptides that bind a target of interest). In several embodiments, the modification to reduce immunogenicity includes an amino acid substitution (eg, conservative and/or non-conservative substitutions).

In several embodiments, a de novo binding domain polypeptide (DBDpp) is provided that includes or is substantially composed of three antiparallel alpha helices, wherein DBDpp is a synthetic polypeptide variant wherein DBDpp immunospecifically binds to a protein that is at least 95% identical to CD123. In several embodiments, DBDpp has a dissociation constant (KD) of about 10 ^-4 M to about 10 ^-12 M. In some embodiments, the target to which DBDpp immunospecifically binds includes amino acids 19-305 of CD123 (SEQ ID NO: 187) . Also provided in the present invention is DBDpp having the amino acid sequence NGSWX ₅ EFX ₈ X ₉ RLXi ₂ AIX ₁₅ Xi ₆ RLX ₁₉ ALG GSEAELAAFEKEIAAFESELQAYKGKGNPEVEX ₅₅ LRX ₅₈ X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO: 4), and where Xn is natural or unnatural amino acid. Also provided is DBDpp having an amino acid sequence that is at least 85% identical to the amino acid sequence of any of SEQ ID NO: 60 through SEQ ID NO: 136. In other embodiments, a fusion protein is provided that binds to CD123 (or other protein of interest target disclosed in the present invention) and further includes one or more additional DBDpps demonstrating binding specificity for the tumor target.

In several embodiments, the target-binding agent (eg, a polypeptide with specificity for a target of interest) is labeled. Depending on the embodiment, various labels may be used, including, but not limited to, an enzyme label, a fluorescent label, a luminescent label, and a bioluminescent label. In some embodiments, the label is a biotin molecule. In several embodiments, a streptavidin molecule may be used. In some embodiments, a His tag, FLAG tag, or other tag is used. In some embodiments, the label is luciferase, green fluorescent protein, red fluorescent protein, or the like.

In several embodiments, the target-binding agent (eg, polypeptide) is conjugated to a therapeutic or cytotoxic agent (eg, chemotherapeutic agent, radiotherapy agent, etc.). Depending on the embodiment, the target binding agent may optionally include a pharmaceutically acceptable carrier.

In several embodiments, kits are provided that include any of the target-binding agents disclosed herein (eg, a therapeutic kit, a diagnostic kit, a research kit, etc.).

In several embodiments, isolated nucleic acid molecules encoding any of the target-binding polypeptides disclosed in the present invention are also provided. In other additional embodiments, a vector (eg, a plasmid, a viral vector, or a non-viral vector) containing an isolated nucleic acid molecule is provided. Several such embodiments may also include standard components for expressing the protein encoded by the nucleic acid (eg, promoters, packaging components, etc.). For example, in several embodiments, the vector further includes an additional nucleotide sequence that directs expression of the polypeptide encoded by the nucleic acid molecule. In several embodiments, the additional nucleic acid sequence is an inducible promoter.

Also provided in several embodiments are host cells that include nucleic acid molecules encoding any of the target-binding polypeptides disclosed in

- 3 043883 of the present invention. In several such embodiments, a host cell (eg, a cell line) is genetically engineered to express the target-binding polypeptides disclosed herein. In some embodiments, expression of target-binding polypeptides by host cells allows for the production and release of target-binding polypeptides. In some embodiments, expression results in target-binding polypeptides expressed on the surface and/or membrane bound of cells.

Also provided by the present invention are de novo binding domain polypeptides (DBDpp) that compete with the polypeptides disclosed herein for binding to CD123 (or other targets of interest). In several embodiments, polypeptides are also provided that compete with the polypeptides disclosed herein for binding to other targets of interest, including CD123, PDL1, CD19, CD22, and the like. (or other targets disclosed in the present invention). Competitive agents that have been proposed include full or partial agonists, full or partial antagonists, and the like. Those agents that compete for binding to the target of interest (or to the same epitope, overlapping epitope, or non-overlapping epitope, resulting in steric or other hindrance in the binding of the agent to the target of interest) can be identified using competitive binding assays.

The present invention also provides polypeptides (either alone or expressed by a cell) that bind to a tumor. In several embodiments, the binding is based on a polypeptide prepared and identified as having specific binding to one or more markers expressed by the tumor. The tumor, depending on the embodiment, may be a suspension tumor or a solid tumor.

In several embodiments, a chimeric antigen receptor (CAR) is also provided, wherein the CAR includes a targeting domain, a transmembrane domain, and an intracellular signaling domain. In several embodiments, the targeting domain consists at least in part of a target binding polypeptide as disclosed herein. In several embodiments, the intracellular signaling domain is selected from the group consisting of a human CD3 zeta domain, a 41BB domain, a CD28 domain, and any combination thereof. Depending on the embodiment, the costimulatory signaling region comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, lymphocyte function-associated antigen 1 1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and any combination thereof. In several embodiments, the CAR includes a fusion protein that includes an additional target-binding polypeptide. Also provided are isolated nucleic acid sequences encoding CAR receptors that include target binding polypeptides as part (or all) of the targeting region.

The present invention further provides cells comprising a nucleic acid sequence encoding a CAR, wherein the CAR includes an antigen binding domain consisting at least in part of a polypeptide that binds a target of interest, a transmembrane domain, and a signaling domain. In several embodiments, the polypeptide specifically binds to a tumor antigen (and thus functions to deliver the CAR-expressing cell to the tumor). In several embodiments, the tumor antigen is associated with a hematologic malignancy. In additional embodiments, the tumor antigen is associated with a solid tumor. Both solid and hematologic tumors may be simultaneously targeted in some embodiments. In several embodiments, the tumor antigen is selected from the group consisting of CD137, PD-L1, CD123, CTLA4, CD47, KIR, DR5, TIM3, PD-1, EGFR, TCR, CD19, CD20, CD22, ROR 1, mesothelin, CD33 /IL3Ra, cMet, PSMA, glycolipid F77, EGFRvIII, GD2, NY-SO-1, MAGE A3 and combinations thereof. Depending on the embodiment, the cell expressing the CAR may be a T cell or a natural killer (NK) cell. In several embodiments, a cell (T cell, NK cell, or other cell type) exhibits antitumor immunity when the polypeptide binds to its corresponding tumor antigen.

In other additional embodiments, amino acids having the sequence SEQ ID 4 are provided, wherein X _n is not a cysteine or a proline.

Also provided in several embodiments are mammalian cells that produce membrane-bound virus-like particles (VLPs), wherein the mammalian cell is genetically engineered to express a fusion protein comprising a de novo binding domain polypeptide (DBDpp) fused to a chimeric antigen receptor (CAR) ), wherein the fusion protein is expressed on the resulting VLPs (eg, as transmembrane proteins). Depending on the embodiments, VLPs produced by mammalian cells are suitable for use as immunogens for the production of antibodies. In some such embodiments, the antibodies are directed against a de novo binding domain polypeptide (DBDpp) (eg, the antibodies bind to DBDpp and can be used to detect DBDpp, isolate DBDpp, etc.).

The target-binding polypeptides disclosed in the present invention may also be used in a therapeutic context, for example, for the treatment and/or diagnosis of a disease such as cancer (eg, solid tumor or hematologic malignancy). Thus, in several embodiments, methods are provided for treating a subject having cancer, comprising administering to the subject an immunocyte comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a target binding domain, wherein the target binding domain comprises a polypeptide having an amino acid sequence comprising:

MGSWX ₅ EFX ₈ X ₉ RLX12AIX1 ₅ X1 ₆ RLX1 ₉ ALGG seaelaafekeiaafeselqaykgkgnpevex ₅₅ lrx ₅₈ x ₅₉ aax ₆₂ irx ₆₅ x ₆₆ lqayrhn (SEQ ID NO: 4) transmembrane domain and intracellular domain (including the signaling domain). When administered to a subject having cancer, the target binding domain specifically binds to a target of interest expressed by the cancer cell, wherein binding of the target of interest induces the immune cell to generate cytotoxic signals that result in a cytotoxic effect on the cancer cell, resulting in treatment of the cancer . In several embodiments, the polypeptide has a sequence that differs from SEQ ID NO: 1 (eg, the polypeptide is obtained by modifying the amino acid sequence of SEQ ID NO: 1). As a result of the sequence change, the specific binding of the polypeptide to the target of interest is greater than the binding of the polypeptide corresponding to SEQ ID NO: 1 to the target of interest.

Depending on the embodiment, the immunocyte may be a T cell. In some embodiments, the immunocyte is an NK cell. Optionally, other immunocytes and/or combinations of different types of immunocytes may be used. In some embodiments, combinations of cell types (eg, NK cells and T cells) provide an advantage because they act synergistically in treating cancer. When combinations are used, different cell types can target the same or different (or overlapping) tumor antigens.

In several embodiments that employ T cells, binding of a target of interest stimulates the T cell to initiate intracellular signaling, cytokine production, and degranulation, resulting in a cytotoxic effect on the cancer cell. Additionally, in several embodiments, the T cell proliferates in response to binding of a target of interest. Preferably, however, the T cell activity does not result in T cells exhibiting a phenotype associated with T cell exhaustion. In several embodiments that employ T cells, the transmembrane domain of the CAR includes 41BB or CD28, and the cytoplasmic domain includes the alpha, beta, or zeta chain of the T cell receptor.

In several embodiments where NK cells are used, the transmembrane domain includes CD28 and the cytoplasmic domain includes the T cell receptor zeta chain.

In several embodiments, the CAR-containing immunocytes are engineered to bind to a target of interest expressed by a cancer cell, such as a tumor antigen selected from the group consisting of CD137, PD-L1, CD123, CTLA4, CD47, KIR, DR5, TIM3, PD -1, EGFR, TCR, CD19, CD20, CD22, ROR 1, mesothelin, CD33/lL3Ra, cMet, PSMA, glycolipid F77, EGFRvIII, GD2, NY-SO-1, MAGE A3 and combinations thereof.

In several embodiments, the CAR further includes a second polypeptide having the amino acid SEQ ID NO: 4, wherein the polypeptide is capable of specifically binding to a second target of interest expressed by the cancer cell, and wherein the second polypeptide's specific binding to the second target of interest is greater than the binding of the polypeptide corresponding to SEQ ID NO: 1, with a second target of interest. In several embodiments, the production of a polypeptide that constitutes at least a portion of a CAR targeting domain does not involve substitution of a cysteine or a proline in SEQ ID NO: 1.

In several embodiments, administration of the CAR immunocytes is intravenous, although other routes, such as intra-arterial, intramuscular, local or other suitable route, may be used for a given treatment regimen.

Also provided in several embodiments are methods of treating a subject having cancer, comprising administering to the subject an immunocyte comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a target binding domain, wherein the target binding domain comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, wherein no cysteine or proline residue is replaced in any of SEQ ID NO: 2, SEQ ID NO : 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, wherein the polypeptide specifically binds a target of interest expressed by a cancer cell, and wherein the specific binding of the polypeptide to the target of interest is greater than the binding of the polypeptide corresponding to SEQ ID NO: 1, with a target of interest, a transmembrane domain and an intracellular domain, wherein the intracellular domain includes a signaling domain, wherein when administered to a subject having cancer, the target binding domain specifically binds to the target of interest expressed by the cancer cell, and wherein binding of a target of interest induces the immunocyte to generate cytotoxic signals that result in a cytotoxic effect on the cancer cell, resulting in treatment of the cancer. As discussed above, depending on the embodiment, the immunocyte may be a T cell, an NK cell, or another type of immunocyte (or a combination of different types). In one embodiment, the transmembrane domain includes 41BB or CD28, where the cytoplasmic domain includes the alpha, beta or zeta chain of the T cell receptor, and where the immunocyte is a T cell. In some such embodiments, upon binding to a target of interest, the T cell is stimulated to initiate intracellular signaling, cytokine production, proliferation, and degranulation, resulting in a cytotoxic effect on the cancer cell without the T cells exhibiting a phenotype associated with T cell exhaustion.

In other embodiments, a method of treating a subject having cancer is provided, comprising intravenously administering to the subject an immunocyte comprising a chimeric antigen receptor (CAR) expressed on a T cell, wherein the CAR includes a target binding domain comprising a polypeptide having an amino acid sequence comprising a polypeptide, having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, and no cysteine or proline residue is replaced in any of the SEQ IDs NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, wherein the polypeptide is capable of specifically binding to a target of interest expressed by the cancer cell, wherein binding to the target of interest is greater than binding a polypeptide corresponding to SEQ ID NO: 1 to a target of interest, a transmembrane domain selected from 41BB and CD28, and an intracellular domain, wherein the intracellular domain includes a signaling domain selected from T cell receptor alpha, beta or zeta chain wherein, when administered to a subject having cancer, the target binding domain specifically binds to a target of interest expressed by the cancer cell, and wherein binding of the target of interest induces the T cell to generate cytotoxic signals that result in a cytotoxic effect on the cancer cell. In several embodiments, the cytotoxic effect occurs as a result of T cell degranulation. Preferably, in several embodiments, T cell activation and cytotoxic activity are not associated with T cells exhibiting a T cell exhaustion phenotype. In several embodiments, the CAR optionally also includes a second target binding domain including a second polypeptide having a target other than the target binding domain. In other embodiments, additional targeting domains may optionally be included to increase marker binding ability or impart binding specificity to other markers.

Also provided in several embodiments is the use of an immunocyte comprising a chimeric antigen receptor (CAR) for the treatment of cancer, wherein the CAR comprises a target binding domain comprising a polypeptide having an amino acid sequence comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, wherein no cysteine or proline residue is replaced in any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, wherein the polypeptide specifically binds a target of interest expressed by the cancer cell, and wherein the specific binding of the polypeptide to the target of interest is greater than the binding of the polypeptide corresponding to SEQ ID NO: 1, with a target of interest, a transmembrane domain selected from 41BB and CD28, and an intracellular domain, wherein the intracellular domain includes a signaling domain selected from T cell receptor alpha, beta or zeta chain, wherein when administered to a subject, having a cancer, the target-binding domain specifically binds to a target of interest expressed by the cancer cell, and wherein binding of the target of interest induces the immune cell to generate cytotoxic signals that result in a cytotoxic effect on the cancer cell. Depending on the embodiment, the immunocytes may be a T cell or a natural killer (NK) cell.

In addition to binding domain compositions, methods for their preparation, screening and use, methods for purifying targets of interest are also provided. Thus, the present invention, in several embodiments, provides a method for purifying a target of interest, comprising contacting a sample comprising the target of interest with a composition comprising a polypeptide agent attached to a solid support, wherein the polypeptide agent has an amino acid sequence comprising MGSWX5EFX ₈ X9RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX55L RX ₅₈ X59AAX ₆ 2lRX ₆₅ X66LQAYRHN (SEQ ID NO: 4), where the polypeptide has an amino acid sequence that differs from SEQ ID NO: 1, where the polypeptide specifically binds a target of interest, where the specific tying polypeptide with a target of interest greater than the binding of the polypeptide corresponding to SEQ ID NO: 1 to the target of interest, the contact being carried out under conditions that ensure binding of the composition to the target of interest and removal of the portion of the sample that is not bound with composition. In several embodiments, the method further includes dissociating the composition from the target of interest and isolating the target of interest. In several embodiments, the target of interest may be eluted from the composition, resulting in purification (in whole or in part) of the target of interest.

Depending on the embodiment, the solid support can be a sphere, glass slide, chip, gelatin or agarose. In some embodiments, combinations of substrates may be used. In several embodiments, the polypeptide agent is connected to the solid support through non-covalent association, while in other embodiments, the polypeptide agent is connected to the solid support through covalent association. Depending on the embodiment, substrates and target of interest, combinations of covalent and non-covalent association may also be used.

In several embodiments, the polypeptide agent of the composition further includes a peptide tag, where the peptide tag includes a hexahistidine group or a FLAG tag. In some embodiments, the polypeptide agent of the composition further includes a streptavidin molecule. Other types of labels may be used, such as enzymatic, colorimetric, bioluminescent and/or fluorescent labels, depending on the embodiment.

In some embodiments, the solid support includes a sphere and the composition is suitable for use in affinity chromatography to purify a target of interest.

In several embodiments, a nucleic acid molecule encoding a polypeptide is packaged in an expression vector that is used to transduce a cell line to cause the cell line to express the polypeptide. Such embodiments provide production of the polypeptide on a larger scale for use in protein purification.

Also provided in several embodiments is a method for purifying a target of interest, comprising contacting a sample comprising the target of interest with a composition comprising a virus-like particle coupled to a solid support, wherein the virus-like particle expresses a polypeptide as a membrane protein, wherein the polypeptide has an amino acid sequence, selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, wherein the polypeptide has an amino acid sequence that differs from SEQ ID NO: 1 wherein the polypeptide specifically binds a target of interest, wherein the specific binding of the polypeptide to the target of interest is greater than the binding of the polypeptide corresponding to SEQ ID NO: 1 to the target of interest; wherein the contact is carried out under conditions that ensure binding of the composition to the target of interest; and removing a portion of the sample that is not associated with the composition. In several embodiments, wherein no cysteine or proline residue is replaced in any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 upon receipt polypeptide.

In several embodiments, the solid support includes one or more of a sphere, glass slide, chip, gelatin, or agarose. In several embodiments, the polypeptide of the composition further includes a peptide tag, where the peptide tag includes a hexahistidine group or a FLAG tag. As discussed herein, other types of labels may be used in additional embodiments.

In some embodiments, the portion of the sample that has not bound to the composition is removed. In some embodiments, the portion of the sample that has not bound to the composition is contacted with the composition a second time to capture additional target of interest, thereby increasing the overall purification yield.

In several embodiments, the method further includes contacting a portion of the sample that has not bound the composition with an antibody directed against a polypeptide of the composition, wherein the antibody is derived from membrane-bound virus-like particles (VLPs) expressing a polypeptide released from a genetically engineered mammalian cell for expressing a fusion protein comprising a polypeptide fused to a chimeric antigen receptor (CAR), wherein the fusion protein is expressed on the resulting VLPs, wherein the antibodies are suitable for use in an assay to detect residual polypeptides separated from a solid support.

The present invention not only provides methods for purifying a target (e.g., removing the target from a larger volume sample), but in several embodiments provides a method for removing one or more impurities from a sample including a target of interest, wherein the method includes contacting a sample including the target of interest , with a composition comprising a virus-like particle coupled to a solid support, wherein the virus-like particle expresses a polypeptide as a membrane protein, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, wherein no cysteine or proline residue is replaced in any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 5 and SEQ ID NO: 6, where the polypeptide has an amino acid

- 7 043883 sequence, which is different from SEQ ID NO: 1, where the polypeptide specifically binds one or more impurities removed from a sample comprising a target of interest, where the specific binding of the polypeptide to one or more impurities is greater than the binding of the polypeptide corresponding to SEQ ID NO: 1, with one or more impurities; where the contact is carried out under conditions that ensure binding of the composition with one or more impurities; and collecting the portion of the sample that has not associated with the composition. As discussed above, in several embodiments, the polypeptide of the composition further includes a label, such as a peptide tag. In several embodiments, the peptide tag includes a hexahistidine group or a FLAG tag. Depending on embodiments, the solid support may include a sphere, glass slide, chip, gelatin, or agarose, wherein the virus-like particles are attached to the solid support through non-covalent association. In some embodiments, the portion of the sample that is collected is contacted with the composition a second time to remove additional impurities from the sample.

The present invention also provides compositions for use in protein purification. In several embodiments, an affinity resin is provided comprising a polypeptide agent having an amino acid sequence including a sequence selected from the group consisting of:

MGSWX ₅ X ₆ FKX ₉ XioLAXi3lKXi ₆ Xi7LEALGGSEAELAX3oFEX33X ₃₄ IAX37FEX ₄ oX ₄ iLQX ₄₄ YKGK

GNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2),

MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX32X33EIX36AFX ₃ 9X4oELX ₄₃ AYKGKGNPEVEAL

X ₅ 7X ₅₈ EAX ₆ iAIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO:3),

MGSWX ₅ EFX ₈ X ₉ RLX1 ₂ AIX1 ₅ X1 ₆ RLX1 ₉ ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX ₅₅ L

RX ₅₈ X ₅₉ AAX ₆₂ IRX65X66LQAYRHN (SEQ ID NO:4),

MGSWX ₅ X ₆ FKX ₉ XioLAXi3lKXi ₆ Xi7LEALGGSEAELAAFX3 ₂ X33EIX3 ₆ AFX3 ₉ X ₄ oELX ₄₃ AYKGKG

NPEVEX ₅₅ LRX ₅₈ X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO:5),

MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALGGSEAELAX3oFEX33X ₃₄ IAX37FEX ₄ oX ₄ iLQX ₄₄ YKGK

GNPEVEALX ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO:6),

MGSWX5X ₆ FKX ₉ X ₁ oLAX ₁ 3lKX ₁₆ Xi7LEALZ ₁ EAELAX ₂₈ FEX3 ₁ X3 ₂ IAX35FEX3 ₈ X3 ₉ LQX ₄₂ YZ ₂ NP

EVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7),

MGSWAEFKQRLAAIKTRLEALZ ₁ EAELAAFX ₃ oX3iEIX3 ₄ AFX37X ₃₈ ELX ₄₁ AYZ ₂ NPEVEALX ₅₂

X ₅₃ EAX ₅₆ AIX ₅₉ X ₆ oELX ₆₃ AYRHN (SEQ ID NO:8),

MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALZ ₁ EAELAAFEKEIAAFESELQAYZ ₂ NPEVEX ₅₀ LRX ₅ 3

X ₅₄ AAX ₅₇ IRX ₆ oX ₆ iLQAYRHN (SEQ ID NO:9),

MGSWX ₅ X ₆ FKX ₉ X ₁ oLAX ₁ 3lKX ₁₆ X ₁ 7LEALZ ₁ EAELAAFX3oX3iEIX3 ₄ AFX37X ₃₈ ELX ₄₁ AYZ ₂ NPEV

EX _5O LRX ₅₃ X ₅₄ AAX ₅ 7lRX ₆ oX6iLQAYRHN (SEQ ID NO: 10) and MGSWX ₅ EFX ₈

X ₉ RLXi ₂ AIXi5Xi ₆ RLXi ₉ ALZiEAELAX ₂₈ FEX3iX3 ₂ IAX35FEX3 ₈ X3 ₉ LQX ₄₂ YZ ₂ NPEVEALX5 ₂ X53

EAX ₅₆ AIX ₅₉ X ₆₀ ELX ₆₃ AYRHN (SEQ ID NO: 11), as well as combinations thereof, and where the amino acid sequence is not SEQ ID NO: 1.

In any of the above sequences, any of the X positions (eg, Xn) may be a natural or unnatural amino acid; where each Xn is the same or different natural or unnatural amino acids. Additionally, in several embodiments, Z1 and/or Z2 may include from about 2 to about 30 natural or unnatural amino acids.

In several embodiments, the polypeptide agent has an amino acid sequence that differs from SEQ ID NO: 1 by amino acid substitution at one or more residues. Depending on embodiments, an amino acid substitution at one or more residues may include a conservative substitution or a non-conservative substitution. Combinations of conservative and non-conservative substitutions may also be used in several embodiments. Additionally, in several embodiments, an amino acid substitution at one or more residues includes a substitution at a solvent accessible residue. In some embodiments, an amino acid substitution at one or more residues includes a substitution at a solvent-inaccessible residue. In some embodiments, substitutions (conservative or non-conservative) may optionally be made to both solvent accessible and non-solvent accessible residues. In several embodiments, the polypeptide agent has an amino acid sequence that differs from SEQ ID NO: 1 by deleting the amino acid at one or more residues.

In several embodiments, a method of producing an affinity resin is provided, comprising attaching to a solid support a polypeptide agent having an amino acid sequence

- 8 043883 activity, including a sequence selected from the group consisting of:

MGSWX ₅ X ₆ FKX ₉ X _lo LAX ₁₃ IKX ₁₆ X ₁₇ LEALGGSEAELAX ₃ oFEX ₃₃ X ₃ 4 IAX ₃₇ FEX4oX ₄ iLQX44YKGK

GNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2),

MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX ₃ 2X ₃ 3EIX36AFX ₃ 9X4oELX ₄₃ AYKGKGNPEVEAL

X3 ₇ X ₅ 8EAX ₆₁ AIX ₆ 4X65ELX ₆ gAYRHN (SEQ ID NO:3),

MGSWXsEFXgXgRLXisAIXisXieRLXigALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEXssL RX ₅₈ X ₅ 9AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO:4),

MGSWX ₅ X ₆ FKXgXioLAXi ₃ IKX ₁₆ Xi ₇ LEALGGSEAELAAFX ₃₂ X ₃₃ EIX ₃₆ AFX ₃₉ X4 _O ELX43AYKGKG NPEVEX ₅₅ LRX ₅₈ X5 ₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO:5),

MGSWX ₅ EFX8X ₉ RLX ₁₂ AIX ₁ 5X ₁₆ RLX ₁₉ ALGGSEAELAX ₃ oFEX ₃₃ X ₃ 4lAX ₃₇ FEX4oX4iLQX ₄ 4YKGK GNPEVEALX ₅₇ X ₅₈ EAX ₆ iAIX ₆ 4X65ELX ₆₈ AYRHN (SEQ ID NO:6),

MGSWX ₅ X ₆ FKX9XioLAXi3lKX ₁₆ Xi ₇ LEALZiEAELAX2 ₈ FEX3iX32lAX ₃ 5FEX38X3 ₉ LQX ₄ 2YZ ₂ NP EVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7),

MGSWAEFKQRLAAIKTRLEALZ2EAELAAFX30X31EIX34AFX37X38ELX42AYZ2NPEVEALX52 X ₅ 3EAX5 ₆ AIX ₅ 9X ₆ oELX ₆₃ AYRHN (SEQ ID NO:8),

MGSWX ₅ EFX8X9RLXi2AIXi5X ₁₆ RLXi ₉ ALZiEAELAAFEKEIAAFESELQAYZ ₂ NPEVEX ₅ oLRX ₅ 3 X ₅ 4AAX ₅₇ IRX ₆ oX ₆ iLQAYRHN (SEQ ID NO:9),

MGSWX ₅ X ₆ FKX9XioLAXi ₃ IKX ₁₆ Xi ₇ LEALZiEAELAAFX3oX ₃₁ EIX34AFX ₃₇ X38ELX4iAYZ2NPEV EX ₅ oLRX ₅₃ X54AAX57IRX6oX61LQAYRHN (SEQ ID NO: 10) and MGSWX ₅ EFX ₈ X9RLXi2 AIX ₁₅ Xi ₆ RLXi9ALZ ₁ EAELAX28FEX3iX32lAX3 ₅ FEX38X39LQX42YZ ₂ NPEVEALX52X53 EAX ₅ 6AIX ₅ 9X ₆ oELX ₆₃ AYRHN (SEQ ID NO: 11), and combinations thereof, where the amino acid sequence is not SEQ ID NO: 1. In several embodiments, X sequence positions (eg, Xn) may include a natural or unnatural amino acid; where each Xn is the same or different natural or unnatural amino acids; and/or where Z1 and/or Z2 are 2-30 natural or unnatural amino acids. In several embodiments, the polypeptide agent is attached to a solid support through covalent binding, non-covalent association, or combinations thereof. In several embodiments, the solid support includes one or more of a sphere, glass slide, chip, gelatin, or agarose.

Additionally, for protein purification, several embodiments provide a composition comprising a solid support coupled to a polypeptide agent having an amino acid sequence including a sequence selected from the group consisting of

MGSWX ₅ X ₆ FKX ₉ X ₁ oLAX ₁ 3lKX ₁₆ X ₁₇ LEALGGSEAELAX3oFEX33X ₃ 4lAX3 ₇ FEX4oX4iLQX ₄ 4YKGK

GNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2),

MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX ₃ 2X ₃₃ EIX3 ₆ AFX3 ₉ X4oELX4 ₃ AYKGKGNPEVEAL

X5 ₇ X58EAX ₆₁ AIX ₆ 4X ₆ 5ELX ₆₈ AYRHN (SEQ ID NO:3),

MGSWX ₅ EFX8X ₉ RLXi ₂ AIXi ₅ Xi ₆ RLXi ₉ ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX ₅₅ L

RX ₅ 8X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO:4),

MGSWX ₅ X ₆ FKX9XioLAXi3lKXi ₆ Xi ₇ LEALGGSEAELAAFX32X33EIX ₃₆ AFX39X4oELX43AYKGKG

NPEVEX ₅₅ LRX ₅ 8X ₅₉ AAX ₆₂ IRX65X66LQAYRHN (SEQ ID NO:5),

MGSWX ₅ EFX8X ₉ RLXi2AIXi ₅ Xi ₆ RLXi9ALGGSEAELAX3oFEX33X34lAX ₃₇ FEX4oX4iLQX44YKGK

GNPEVEALX ₅₇ X ₅ 8EAX ₆ iAIX ₆ 4X ₆₅ ELX ₆ 8AYRHN (SEQ ID NO:6),

MGSWX ₅ X ₆ FKX ₉ XioLAXi3lKXi ₆ Xi ₇ LEALZiEAELAX28FEX3iX32lAX3 ₅ FEX38X3 ₉ LQX42YZ ₂ NP

EVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7),

MGSWAEFKQRLAAIKTRLEALZiEAELAAFX3oX3iEIX34AFX3 ₇ X38ELX4iAYZ ₂ NPEVEALX ₅ 2 X ₅ 3EAX ₅₆ AIX ₅₉ X ₆ oELX ₆₃ AYRHN (SEQ ID NO:8),

MGSWX ₅ EFX8X ₉ RLXi2AIXi ₅ Xi ₆ RLXi ₉ ALZiEAELAAFEKEIAAFESELQAYZ ₂ NPEVEX ₅ oLRX ₅ 3

X ₅ 4AAX ₅₇ IRX ₆ oX6iLQAYRHN (SEQ ID NO:9),

MGSWX5X6FKX ₉ XioLAXi ₃ IKXi6Xi ₇ LEALZiEAELAAFX ₃ oX3iEIX ₃ 4AFX ₃₇ X ₃ 8ELX ₄ iAYZ ₂ NPEV

EX ₅ oLRX ₅₃ X ₅ 4AAX ₅₇ IRX ₆ oX6iLQAYRHN (SEQ ID NO: 10) and MGSWX ₅ EFX ₈

X ₉ RLXi ₂ AIXi ₅ Xi ₆ RLXi ₉ ALZiEAELAX ₂ 8FEX3iX3 ₂ IAX3 ₅ FEX38X ₃₉ LQX4 ₂ YZ ₂ NPEVEALX ₅₂ X ₅ 3

EAX ₅₆ AIX ₅₉ X ₆ oELX ₆₃ AYRHN (SEQ ID NO: 11), and combinations thereof, where the amino acid sequence is not SEQ ID NO: 1. In several embodiments, X _n is a natural or unnatural amino acid; where each X _n is the same or different natural or unnatural amino acids; and/or Z ₁ and/or Z ₂ are 2-30 natural or unnatural amino acids. In several embodiments, the polypeptide agent has an amino acid sequence that differs from SEQ ID NO:

amino acid substitution at one or more residues.

Depending on the embodiment, an amino acid substitution at one or more residues may involve a conservative substitution or may involve a non-conservative substitution. Combinations of conservative and non-conservative substitutions may also be used in some embodiments. In several embodiments, an amino acid substitution at one or more residues includes a substitution at a solvent accessible residue. In several embodiments, an amino acid substitution at one or more residues includes a substitution at a solvent-inaccessible residue. In some embodiments, substitutions are made for both solvent-accessible and solvent-inaccessible residues. In several embodiments, the polypeptide agent has an amino acid sequence that differs from SEQ ID NO: 1 by deleting the amino acid at one or more residues. Depending on embodiments, the solid support may include one or more of a sphere, glass slide, chip, gelatin, or agarose.

In several embodiments, the polypeptides disclosed in the present invention can be used in protein analysis, for example, to act as detection agents or labels. As such, the present invention, in several embodiments, provides a composition comprising a polypeptide agent conjugated to a detectable agent and/or label, wherein the polypeptide agent has an amino acid sequence including a sequence selected from the group consisting of:

MGSWX ₅ X ₆ FKX ₉ XioLAXi3lKXi ₆ Xi7LEALGGSEAELAX3oFEX33X ₃₄ IAX37FEX ₄ oX ₄ iLQX ₄₄ YKGK

GNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2),

MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX ₃ 2X33EIX3 ₆ AFX3 ₉ X ₄ oELX ₄₃ AYKGKGNPEVEAL

X ₅₇ X58EAX ₆ iAIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO:3),

MGSWX ₅ EFX ₈ X ₉ RLX1 ₂ AIX1 ₅ X1 ₆ RLX1 ₉ ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX ₅₅ L

RX ₅₈ X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO:4),

MGSWX5X ₆ FKX ₉ X ₁ oLAX ₁ 3lKX ₁₆ Xi7LEALGGSEAELAAFX3 ₂ X33EIX3 ₆ AFX3 ₉ X ₄ oELX ₄ 3AYKGKG

NPEVEX55LRX ₅₈ X5 ₉ AAX ₆₂ IRX65X66LQAYRHN (SEQ ID NO:5),

MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALGGSEAELAX3oFEX33X ₃₄ IAX37FEX ₄ oX ₄ iLQX ₄₄ YKGK GNPEVEALX5 ₇ X5 ₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO:6),

MGSWX5X ₆ FKX ₉ X ₁ oLAX ₁ 3lKX ₁₆ Xi7LEALZ ₁ EAELAX ₂₈ FEX3 ₁ X3 ₂ IAX35FEX3 ₈ X3 ₉ LQX ₄₂ YZ ₂ NP EVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7),

MGSWAEFKQRLAAIKTRLEALZ ₁ EAELAAFX ₃ oX3iEIX3 ₄ AFX37X ₃₈ ELX ₄₁ AYZ ₂ NPEVEALX ₅₂ X ₅ 3EAX5 ₆ AIX5 ₉ X ₆ oELX ₆₃ AYRHN (SEQ ID NO:8),

MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALZ ₁ EAELAAFEKEIAAFESELQAYZ ₂ NPEVEX ₅₀ LRX ₅ 3 X ₅₄ AAX ₅₇ IRX ₆ oX ₆ iLQAYRHN (SEQ ID NO:9),

MGSWX5X ₆ FKX ₉ X ₁ oLAX ₁ 3lKX ₁₆ X ₁ 7LEALZ ₁ EAELAAFX3oX3iEIX3 ₄ AFX37X3 ₈ ELX ₄₁ AYZ ₂ NPEV EX _5O LRX ₅₃ X ₅₄ AAX ₅ 7lRX ₆ oX6iLQAYRHN (SEQ ID NO: 10) and MGSWX ₅ EFX ₈ X ₉ RLXi ₂ AIXi ₅ Xi ₆ RLXi ₉ ALZiEAELAX ₂₈ FEX3iX3 ₂ IAX3 ₅ FEX3 ₈ X ₃₉ LQX ₄₂ YZ ₂ NPEVEALX ₅₂ X ₅ 3 EAX5 ₆ AIX5 ₉ X ₆₀ ELX ₆₃ AYRHN (SEQ ID NO: 11), and combinations thereof, where the amino acid sequence is not is SEQ ID NO: 1. In several embodiments, Xn is a natural or unnatural amino acid; where each Xn is the same or different natural or unnatural amino acids; and/or where Z ₁ and/or Z 2 are 2-30 natural or unnatural amino acids.

In several embodiments, the detectable agent includes a chromogen. In several embodiments, the detectable agent includes a fluorescent dye. In several embodiments, the detectable agent includes a radionuclide. In such embodiments, the detectable agent is quantifiable.

In several embodiments of the composition, the polypeptide agent is conjugated to a chromatography sphere, resin, slide, chip, gelatin, or agarose. In several embodiments, the tag includes a polyhistidyl tag, a myc tag, or a FLAG tag. Combinations of labels may also be used in several embodiments. In several embodiments, the polypeptide agent is conjugated to a detectable agent or label through covalent bonding. In several embodiments of the composition, the polypeptide agent is a fusion protein. In several embodiments, the polypeptide agent is multimeric.

Proposed are de novo binding domain (DBD)-containing polypeptides (DBDpp) that specifically bind targets of interest, as well as nucleic acids encoding the proposed DBDpp, vectors containing the nucleic acids, and host cells containing the nucleic acids and vectors. DBDpp libraries, methods for obtaining and screening such libraries, and DBDpp identified in such libraries and screens are also proposed. Also provided are DBDpps, such as DBDpp fusion proteins, as well as methods for making and using DBDpps. Such applications include, but are not limited to, affinity purification, as well as diagnostic and therapeutic applications.

In one embodiment, a DBDpp is provided whose amino acid sequence differs (e.g., as a result of amino acid modifications) from the amino acid sequence of a reference scaffold having the sequence SEQ ID NO: 1. The reference scaffold is a variant of one that does not exist in nature and has no target (e.g., to the best of our knowledge applicant, no currently known targets) containing an antiparallel triple-stranded bundle of a reference polypeptide originally designed as a protein folding experiment (see, Walsh et al., PNAS 96:5486-5491 (1999), hereby incorporated by reference in its entirety) . It has been discovered, and disclosed herein in several embodiments, that polypeptides containing modifications of an off-target reference framework having the amino acid sequence of SEQ ID NO: 1 are capable of specifically binding targets of interest. Without wishing to be bound by theory, it is believed that when creating a DBD, the structural constraints of the surface residues (which can be modified) impart the ability of the surface residues to specifically bind the target of interest.

In one embodiment, the DBDpp agent includes a polypeptide the amino acid sequence of which shows homology to SEQ ID NO: 1, but differs from SEQ ID NO: 1 by modification of one or more amino acids. In several embodiments, the target binding agents (eg, DBDpp) of the present invention specifically bind to a target of interest (such as a cancer or tumor associated marker such as CD123, CD137, PD-L1, CD19, CD22, NY- ESO, MAGE A3, as non-limiting embodiments). In several embodiments, the proposed target binding agent (e.g., DBDpp) comprises a total of 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 amino acids residues that have been modified from SEQ ID NO: 1; and where the agent specifically binds the target of interest. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 modified amino acid residues are substitutions. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 modified amino acid residues are conservative substitutions. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 modified amino acid residues are non-conservative substitutions. In another embodiment, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 of the amino acid residue modifications are conservative substitutions, and 5-15, 5-20, 5- 25, 5-30, 5-35, 5-40 or 5-45 of the amino acid residue modifications are non-conservative substitutions. In additional embodiments, 1-25, 1-30, 1-35, 5-40, 5-45, 5-50, 5-55, or 5-60 of the substitutions are made at amino acid residues SEQ ID NO: 1 selected from the group , consisting of: M1, G2, S3, W4, A5,E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72 and N73. In another embodiment, 1-20, 1-30, or 1-40 of the substitutions are made at amino acid residues of SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10 , A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59 , A61, A62, R64, D65, E66, Q68, A69 and Y70. In another optional embodiment, DBDpp optionally also includes an amino acid sequence in which 1-5, 1-10, 1-15, 5-10, or 5-15 residues corresponding to solvent-inaccessible residues in the amino acid sequence SEQ ID NO: 1 are replaced , and where DBDpp specifically binds a target of interest. In several embodiments, DBDpp comprises an amino acid sequence of about 1 to about 5, about 1 to about 10, about 1 to about 15, about 5 to about 10, about 5 to about 15 (or more) residues , which correspond to solvent-accessible or solvent-inaccessible residues in the amino acid sequence of SEQ ID NO: 1, are replaced. In several embodiments, replacement of both accessible and inaccessible residues confers a higher degree of target specificity compared to replacement of only accessible or only inaccessible residues. In another optional embodiment, the substituted moieties corresponding to the solvent-inaccessible moiety in SEQ ID NO: 1 are selected from the group consisting of: F7, L11, I14, L18, L28, F31, I35, F38, L42, V53, L56, A60 , I63 and L67, and Y70. In a further embodiment, L21 and Y45 are also included in the group of substituted solvent-inaccessible residues. In a further embodiment, DBDpp is a fusion protein (e.g., DBDpp is fused, conjugated, or otherwise linked, directly or indirectly, to another molecule, such as a therapeutic or diagnostic

- 11 043883 static agent). In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, glass slide, chip, gelatin and agarose. In a further embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, tumor specific antigen (TSA), cancer antigen (CSA) and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell displays DBDpp on its surface. In additional embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. Libraries including many DBDpps are also proposed.

In one embodiment, DBDpp comprises an amino acid sequence selected from the group consisting of: (a) MGSWX ₅ EFX ₈ X9RLXi ₂ AIXi ₅ Xi ₆ RLXi9ALGGSEAELAAFEKEIAAFE SELQAYKGKGNPEVEX ₅₅ LRX _5s X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ id NO: | , where X _{5 >} X, X, X^ X _u , X16, X19, X ₅₅ , X ₅₈ , X59, X ₆₂ , X ₆₅ and/or X ₆₆ is a natural and/or unnatural amino acid residue; (b) MGSWX ₅ X ₆ FKX ₉ X ₁ oLAX ₁₃ IKX ₁₆ X ₁₇ LEALGGSEAELAX3oFEX33X3 ₄ IAX37FEX ₄ oX4iL QX«YKGKGNPEVEALRKEMAIRDELQAYRHN (seq id NO: 2), where X _5> X _b X9, X10, X13, X16, X _n> X3o, X ₃₃ , X ₃₄ , X ₃₇ , X ₄₀ , X41 and/or X ₄₄ is a natural and/or unnatural amino acid residue; (c) MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX32X33EIX36AFX39X ₄ oELX ₄₃ AYKGKGNPEVEAL X ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO : 3), where X32, X33, X36, X39, X40, X43, X57, X58, X61, X64, X65 and/or _X68 is a natural and/or unnatural amino acid residue, and; (d) MGSWX ₅ X ₆ FKX ₉ Xi _O LAXi3IKXi ₆ X ₁₇ LEALGGSEAELAAFX32X33EIX36AFX3 ₉ X ₄ oELX ₄ 3AYKGKG NPEVEX ₅₅ LRX ₅₈ X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO: 5) where X ₅ , X6, X9, X _w , X1 3, X13, X17, X32, X33, _X36 , X39, _X40 , _X43 , _X55 , _X58 , _X59 , _X62 , _X65 and/or _X66 is a natural and/or unnatural amino acid residue _; and (e) MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALGGSEAELAX ₃ oFEX33X3 ₄ I

AX ₃₇ FEX ₄₀ X ₄₁ LQX ₄₄ YKGKGNPEVEALX ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (seq id NO: 6), where X5, X8, X ₉ , X ₁₂ , X ₁₅ , X ₁₆ , X ₁₉ , X ₃₀ , X ₃₃ , X ₃₄ , X ₃₇ , X ₄₀ , X ₄₁ , X ₄₄ , X ₅₇ , X ₅₈ , X ₆₁ , X ₆₄ , X ₆₅ and/or X ₆₈ is a natural and/or unnatural amino acid residue; and wherein DBDpp specifically binds a target of interest. In several embodiments, DBDpp includes, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In a further embodiment implementation X _n is a natural amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In other further embodiments, _Xn is a deletion of an amino acid (eg, optionally a missing position in the sequence). In a further embodiment, DBDpp is a fusion protein. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In a further embodiment, a library is provided containing a plurality of DBDpps. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte (e.g., B cell, T cell, killer T cell, helper T cell, regulatory T cell, antigen presenting cell, NK cell, etc.) that expresses one or more DBDpp fusion proteins on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: a sphere, a glass slide, other glass or polymer-based materials (e.g., a filter or filter device), a filter material (e.g., fiberglass, steel wool, polyethersulfone, and etc.), chip, gelatin and agarose, as well as combinations thereof.

Also provided is an isolated DBDpp that includes an amino acid sequence selected from the group consisting of: (a)

MGSWX ₅ EFX ₈ X ₉ RLX12AIX1 ₅ X16RLX1 ₉ ALZ1EAELAAFEKEIAAFES

ELQAYZ ₂ NPEVEX ₅ oLRX ₅ 3X ₅₄ AAX ₅₇ IRX ₆ oX ₆ iLQAYRHN (SEQ ID _N O: 9), _Where χ5, χ ₈ , χ ₉ , χ ₁₂ , χ15, χ ₁₆ , χ ₁₉ , X ₅₀ , X ₅₃ , X ₅₄ , X ₅₇ , X ₆₀ and/or X ₆₁ is a natural and/or unnatural amino acid residue, and Z1 and/or Z2 are 2-30 natural and/or unnatural amino acid residues; (b) MGSWX ₅ X ₆ FKX9XioLAXi3lKXi ₆ X ₁₇ LEALZ ₁ EAELAX28FEX3iX32lAX3 ₅ F

EX ₃₈ X39LQX ₄ 2YZ ₂ NPEVEALRKEAAAIRDELQAYRHN (seq id NO: 7), where X5, χ ₆ , χ ₉ , X _w , χ _η , χ^, χ _ρ , χ2 ₈ , X _3i , X ₃₂ , X ₃₅ , X ₃₈ , X ₃₉ and/or X ₄₂ is a natural and/or unnatural amino acid residue, and Z1 and/or Z ₂ are 2-30 natural and/or unnatural amino acid residues; (c) MGSWAEFKQRLAAIKTRLEALZiEAELAAFX ₃ oX3iEIX3 ₄ AFX3 ₇ X ₃₈ EL

X ₄₁ AYZ ₂ NPEVEALX ₅₂ X53EAX ₅₆ AIX5 ₉ X ₆₀ ELX ₆₃ AYRHN (SEQ ID NO: 8), where X ₃₀ , X ₃₁ , X ₃₄ , X ₃₇ , X ₃₈ , X _4i , X ₅₂ , X ₅₃ , X56, X ₅₉ , X ₆₀ and/or X ₆₃ is a natural and/or unnatural amino acid residue, and Z1 and/or Z ₂ are 2-30 natural and/or unnatural amino acid residues; (d) MGSWX ₅ X ₆ FKX ₉ XioLAXi3lKXi ₆ Xi ₇ LEALZiEAELAAFX3oX3iEIX ₃₄ A

FX37X3 ₈ ELX ₄ iAYZ ₂ NPEVEX5oLRX53X5 ₄ AAX ₅₇ IRX ₆ oX ₆ iLQAYRHN (SEQ ID NO: 10), where X ₅ , X ₆ , X ₉ , X ₁₀ , X _i3 , Xi6, Xi7, X30, X31, X34, X37 , X38, X41, X50, X53, X54, X57, X60 and/or X6i is a natural and/or unnatural amino acid residue, and Z1 and/or Z ₂ are 2-30 natural and/or unnatural amino acid residues; and (e)

MGSWX ₅ EFX ₈ X ₉ RLX ₁ 2AIX ₁ 5X ₁₆ RLX ₁₉ ALZ ₁ EAELAX ₂₈ FEX3 ₁ X32lAX3 ₅ FEX3 ₈ X3 ₉ LQX ₄₂ YZ ₂ NP EVEALX ₅₂ X ₅₃ EAX ₅₆ AIX ₅₉ X ₆₀ ELX ₆₃ AYRHN (seq id NO:ii ), where X5, X^ X9, X12, X15, Xi6, X19, X28, X31, X32, X ₃₅ , X ₃₈ , X ₃₉ , X ₄₂ , X ₅₂ , X ₅₃ , X ₅₆ , X ₅₉ , X ₆₀ and /or X ₆₃ is a natural and/or unnatural amino acid residue, and Zi and/or Z ₂ are 2-30 natural and/or unnatural amino acid residues; and wherein DBDpp specifically binds a target of interest. In several embodiments, DBDpp includes, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO :ii. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In other further embodiments, _Xn is a deletion of an amino acid (eg, optionally a missing position in the sequence). In other further embodiments, Z1 and/or _Z2 are amino acid deletions (eg, optionally missing positions in the sequence). In a further embodiment, DBDpp is a fusion protein. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In a further embodiment, a library is provided containing a plurality of DBDpps. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells, including viral particles containing nucleic acids, are also provided. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In additional embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, slide, chip, gelatin and agarose.

Also provided are nucleic acids encoding DBDpp, such as a DBDpp fusion protein. Additionally provided are vectors containing nucleic acids encoding DBDpp (eg, DBDpp fusion proteins) and host cells containing the nucleic acids and vectors. In some embodiments, the host cell is a viral particle or a bacterial, yeast, fungal, or plant cell. In a particular embodiment, the host cell is a mammalian cell. In another embodiment, the mammalian cell is an immunocyte. In another embodiment, the host cell is a human immunocyte. In some embodiments, the host cell displays DBDpp as a fusion protein on the surface of the cell. In another embodiment, the host cell is a human immunocyte that exhibits

- 13 043883

DBDpp on the cell surface. Additionally, the present invention provides vector libraries comprising nucleic acids encoding multiple DBDpps.

A library containing multiple DBDpps is also proposed. In one embodiment, the DBDpp library includes a plurality of DBDpps containing different amino acid sequences, and which include the amino acid sequence of SEQ ID NO: 1, in which a total of 5-25, 5-30, 5-35, 5-40, 5-45 , 5-50, 5-55 or 5-60 amino acid residues (including any number in between) have been modified; and wherein DBDpp specifically binds a target of interest. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 560 (including any number in between) modified amino acid residues are substitutions. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 (including any number in between) modified amino acid residues are conservative substitutions. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 (including any number therebetween) modified amino acid residues are non-conservative substitutions. In another embodiment, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 (including any number in between) amino acid residue modifications are conservative substitutions, and 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 (including any number in between) amino acid residue modifications are non-conservative substitutions. In additional embodiments, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 (including any number in between) substitutions are made at one or more amino acid residues in SEQ ID NO: 1, selected from the group consisting of: M1, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72 and N73. In another embodiment, 1-20, 1-30, or 1-40 (including any number in between) substitutions are made at one or more amino acid residues in SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69 and Y70. In another embodiment, the library includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 (including any range between the listed values, such as 2-10, 5-25, 50-100, 250-1000, etc.) different DBDpps that specifically bind different targets (or DBDpps that have distinctive specificity for a given target). In another embodiment, the various targets bound by DBDpp in the library are selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 (including any range between the listed values, such as 2-10, 5-25, 50-100, 250-1000, etc.) different DBDpp that specifically bind a protein target selected from the group consisting of: immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, the library includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 (including any range between the listed values, such as 2-10, 5-25, 50-100, 250-1000, etc.) different DBDpp that specifically bind the target disclosed in the present invention. In a further embodiment, the library is a vector library or a host cell library. In a further embodiment, the vector library is a host cell library. In another embodiment, the host cell library includes a plurality of host cells that display DBDpp on their surface. In another embodiment, the host cells are a phage that displays DBDpp on its surface. In some embodiments, the vector library includes: (a) nucleic acids encoding 3 DBDpp that specifically bind to various targets; (b) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to the same target; (c) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to the same target epitope; (d) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to different epitopes of the target; (e) nucleic acids encoding 3 DBDpp having different sequences that compete for binding to the same target; or (f) 3 different nucleic acid sequences encoding the same DBDpp sequence. Host cells containing vectors are also provided.

Also provided is a vector library comprising a variety of different nucleic acid sequences encoding DBDpp, which include the amino acid sequence SEQ ID NO: 1, in which a total of 1-5, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55 or 5-60 amino acid residues (or any number in between) have been modified; and wherein DBDpp specifically binds a target of interest. In another embodiment, 1-5, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 modified amino acid residues

- 14 043883 (or any number in between) encoded by nucleic acid sequences are replacements. In another embodiment, 1-5, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 modified amino acid residues (or any number in between) are conservative substitutions. In another embodiment, 1-5, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 encoded modified amino acid residues (or any number in between) are non-conservative substitutions. In another embodiment, 1-5, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 encoded amino acid residue modifications (or any number in between) are conservative substitutions , and 1-5, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 encoded amino acid residue modifications (or any number in between) are non-conservative substitutions. In additional embodiments, 1-5, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 coded substitutions (or any number in between) are made by amino acid residues in SEQ ID NO: 1 selected from the group consisting of one or more of the following: M1, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19 , A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49 , N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72 and N73. In another embodiment, 120, 1-30 or 1-40 encoded substitutions (or any number in between) are made at amino acid residues in SEQ ID NO: 1 selected from the group consisting of one or more of the following: G2, S3, W4 , A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52 , E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69 and Y70. In another embodiment, the nucleic acids optionally encode a DBDpp, which further includes an amino acid sequence wherein 1-5, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 ( or any number therebetween) residues corresponding to solvent-inaccessible residues in the amino acid sequence of SEQ ID NO: 1 are replaced, and wherein DBDpp specifically binds the target of interest. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind different targets (or have different affinities for same target). In another embodiment, the various targets bound by DBDpp in the library are selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a protein target selected from the group consisting of from: immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and peptide tag protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a target disclosed in the present invention. In a further embodiment, the vector library is contained in host cells (eg, viral particles). In another embodiment, the library includes a plurality of host cells that display DBDpp on their surface. In another embodiment, the host cells are a phage that displays DBDpp on its surface. In some embodiments, the vector library includes: (a) nucleic acids encoding 3 DBDpp that specifically bind to various targets; (b) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to the same target; (c) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to the same target epitope; (d) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to different epitopes of the target; (e) nucleic acids encoding 3 DBDpp having different sequences that compete for binding to the same target; or (f) 3 different nucleic acid sequences encoding the same DBDpp sequence. Host cells containing vectors are also provided.

In one embodiment, the vector library includes a plurality of different nucleic acids encoding a DBDpp, wherein the encoded DBDpp includes an amino acid sequence selected from the group consisting of: (a) MGSWXsEFXsXgRLX^AIXisXifjRLXigALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEXssL RX5 ₈ X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQ AYRHN (SEQ ID NO : 4), where X5, X8, X9, X12, X,, _X16 , X„, X55, X58, X59, X62, X65 and/or _X66 is a natural and/or unnatural amino acid residue; (b) MGSWX5X ₆ FKX9XioLAX ₁ 3lKXi ₆ X ₁₇ LEALGGSEAEL7\X3oFEX33X3 ₄ I7\X3 ₇ FEX4oX ₄ iLQX ₄₄ YKGK GNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 2) where X5, X6, X9, X _w , X ₁₃ , X ₁₆ , X ₁₇ , X _M , X55, X54, X37, X40, X ₄₁ and/or X ₄₄ is a natural and/or unnatural amino acid residue; (c) MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX ₃ 2X33EIX36AFX39X ₄₀ ELX ₄₃ AYKGKGNPEVEAL X ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO: 3) where X32, X33, X36, X39, X40, X4 3, X57, X58, X61, X64, X65

- 15 043883 and/or X ₆₈ is a natural and/or unnatural amino acid residue, and; (d)

MGSWX ₅ X ₆ FKX9XioLAX ₁ 3lKX ₁₆ Xi ₇ LEALGGSEAELAAFX32X33EIX36AFX ₃₉ X ₄ oELX43AYKGKG

NPEVEX ₅₅ LRX ₅₈ X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (seq id no: 5), where X5, χ6, χ9, x _w , x ₁₃ , χ^, χ _π , χ^, χ ₃₃ , χ36,

X ₃₉ , X ₄₀ , X ₄₃ , X ₅₅ , X ₅₈ , X ₅₉ , X ₆₂ , X ₆₅ and/or X ₆₆ is a natural and/or unnatural amino acid residue; and (e) MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALGGSEAELAX ₃ oFEX ₃₃ X ₃₄ IA

X ₃₇ FEX ₄ oX ₄₁ LQX ₄₄ YKGKGNPEVEALX ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN ( _S eq id NO: 6), where X5, X ₈ , X9, ^X i2, ^X i5, ^X i6, X19, X30 , X33, X34, X37, X40, ^X4i , X44, X57, X58, ^X6i , X64, ^{X65 and/or} X68 is a natural and/or unnatural amino acid residue; and wherein DBDpp specifically binds a target of interest. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In other further embodiments, _Xn is a deletion of an amino acid (eg, optionally a missing position in the sequence). In a further embodiment, a plurality of vectors in the library encode a DBDpp fusion protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind different targets. In another embodiment, the various targets bound by the DBDpp encoded by the nucleic acids in the library are selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a protein target selected from the group consisting of from: immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and peptide tag protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a target disclosed in the present invention. In a further embodiment, a plurality of vector library vectors are contained in host cells (eg, viral particles such as phage), E. coli, yeast and mammalian cells. In another embodiment, host cells display DBDpp on their surface. In another embodiment, the host cells are a phage that displays DBDpp on its surface. In some embodiments, the vector library includes: (a) nucleic acids encoding 3 DBDpp that specifically bind to various targets; (b) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to the same target; (c) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to the same target epitope; (d) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to different epitopes of the target; (e) nucleic acids encoding 3 DBDpp having different sequences that compete for binding to the same target; or (f) 3 different nucleic acid sequences encoding the same DBDpp sequence. Host cells containing vectors are also provided.

In one embodiment, the vector library includes a plurality of nucleic acids encoding DBDpp, comprising an amino acid sequence selected from the group consisting of: (a) MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALZ ₁ EAELAA

FEKEIAAFESELQAYZ ₂ NPEVEX ₅ oLRX ₅₃ X ₅₄ AAX ₅₇ IRX ₆ oX ₆₁ LQAYRHN (seq id NO: 9), where X5, X ₈ , X9, X ₁₂ , X15, Xi6, X _i9 , X ₅₀ , X ₅₃ , X ₅₄ , X ₅₇ , X ₆₀ , and/or X _6i is a natural and/or unnatural amino acid residue, and Zi and Z2 are 2-30 natural and/or unnatural amino acid residues; (b) MGSWX ₅ X ₆ FKX ₉ XioLAXi ₃ IKXi ₆ Xi ₇ LEALZiEA

ELAX ₂₈ FEX ₃₁ X ₃₂ IAX ₃₅ FEX ₃₈ X ₃₉ LQX4 ₂ YZ ₂ NPEVEALRKEAAAIRDELQAYRHN (seq id NO: 7), where X ₅ , X6, X9, X10, Xi3, X _i6 , X17, X ₂₈ , X31, X ₃₂ , X ₃₅ , X ₃₈ , X ₃₉ and/or X ₄₂ is a natural and/or non-natural amino acid residue, and Zi and Z2 are 2-30 natural and/or non-natural amino acid residues; (c) MGSWAEFKQRLAAIKTRLEALZ ₁ EAELAAF

X3 ₀ X ₃₁ EIX34AFX3 ₇ X3 ₈ ELX ₄₁ AYZ ₂ NPEVEALX ₅₂ X ₅ 3EAX ₅₆ AIX ₅₉ X ₆₀ ELX ₆ 3AYRHN (seq id NO: 8), where X30, x ₃₁ , X ₃₄ , X ₃₇ , X ₃₈ , X ₄₁ , X ₅₂ , X ₅₃ , X ₅₆ , X ₅₉ , X ₆₀ and/or X ₆₃ is a natural and/or unnatural amino acid residue, and Zi and Z2 are 2-30 natural and/or unnatural amino acid residues; (d) MGSWX ₅ X _e FKX ₉ X ₁₀ IAX ₁₃ IKX _l(i X ₁ ,LEALZ ₁ EA

ELAAFX_thirtyX₃₁EIX_MAFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₁AAX₅₇IRX_e0X₆₁LQAYRHN (Seq id NO: 10),^Where X5, X6, X9, X_W,^Xi3,^Xi6, X17, X30, X31, X34, X37, X38, X41, X50, X53, X54, X57, X60 and/or^X61 Appears at a native and/or unnatural amino acid residue, and Zi and Z2 are 2-30 natural and/or unnatural amino acid residues; and (e)

MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁ 5X ₁₆ RLX ₁ 9ALZ ₁ EAELAX ₂₈ FEX3 ₁ X3 ₂ IAX35FEX3gX3 ₉ LQ X ₄₂ YZ ₂ NPEVEALX ₅₂ X ₅ 3EAX5 ₆ AIX ₅₉ X ₆ oELX ₆₃ AYRHN (seq ID NO: ii), where X ₅ , X ₈ , X ₉ , X ₁₂ , X ₁₅ , X ₁₆ , X ₁₉ , X _{28 , X 3i ,} X 32 , X ₃₅ , X ₃₈ , X ₃₉ , _X42 , X ₅₂ , X ₅₃ , X ₅₆ , X ₅₉ , X ₆₀ and/or X ₆₃ is natural and/or non-natural

- 16 043883 amino acid residue, and Z ₁ and Z2 are 2-30 natural and/or unnatural amino acid residues; and wherein DBDpp specifically binds a target of interest. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In other further embodiments, _Xn is a deletion of an amino acid (eg, optionally a missing position in the sequence). In a further embodiment, a plurality of vectors in the library encode a DBDpp fusion protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind different targets. In another embodiment, the various targets bound by the DBDpp encoded by the nucleic acids in the library are selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a protein target selected from the group consisting of from: immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and peptide tag protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a target disclosed in the present invention. In a further embodiment, a plurality of vector library vectors are contained in host cells. In another embodiment, host cells (eg, viral particles) display DBDpp on their surface. In another embodiment, the host cells are a phage that displays DBDpp on its surface. In some embodiments, the host cells are mammalian cells. In some embodiments, the vector library includes: (a) nucleic acids encoding 3 DBDpp that specifically bind to various targets; (b) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to the same target; (c) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to the same target epitope; (d) nucleic acids encoding 3 DBDpp having different sequences that specifically bind to different epitopes of the target; (e) nucleic acids encoding 3 DBDpp having different sequences that compete for binding to the same target; or (f) 3 different nucleic acid sequences encoding the same DBDpp sequence. Host cells containing vectors are also provided.

DBDpp, according to several embodiments provided herein, have activities that include, but are not limited to, target binding, the ability to bind, bind, and/or otherwise associate with a target of interest (e.g., a purifiable target, a therapeutic target, a diagnostic target, a peptide tag and a serum protein such as human serum albumin (HSA) or immunoglobulin) in vitro or in vivo and the ability to serve as a reactive site for binding or association with proteins such as DBDpp fusion proteins with additional structures (eg, solid support) , and/or other modifications. The DBDpp of the present invention may also have additional desirable properties and/or functions useful in manufacturing, purification, formulation, and biological, diagnostic, and therapeutic applications.

In some embodiments, DBDpp is used to bind, detect, quantify, remove, and/or purify a target of interest in a sample containing the target.

In one non-limiting embodiment, a method is provided for detecting a target of interest in a sample, comprising: (a) contacting the sample with a DBDpp that specifically binds the target, under conditions suitable for DBDpp to specifically bind to the target, to form a target/DBDpp complex, and (b ) detection of the presence of a complex and/or a captured target. In one embodiment, DBDpp is immobilized on a solid support.

Also provided is a method for quantifying a target of interest in a sample containing the target, comprising: (a) contacting the sample with DBDpp, which specifically binds the target and which is immobilized on a solid support, under conditions suitable for specific binding of DBDpp to the target, forming a target complex /DBDpp, and (b) detecting the presence of a target/DBDpp complex and/or captured target, wherein the quantitative detection of the product indicates or can otherwise be correlated with the amount of target in the sample.

In some embodiments, methods are provided for purifying a target of interest from a sample containing the target, which comprise: (a) contacting a sample containing the target of interest with a DBDpp that specifically binds the target, under conditions appropriate for the DBDpp to specifically bind the target, and (b) isolating the associated target. Somehow

- 17 043883 In some embodiments, the target is isolated during elution. In one embodiment, DBDpp is immobilized on a solid support. In another embodiment, the elution of the bound target is monitored by ultraviolet absorption or other imaging or chemical detection method. In some embodiments, methods are provided for removing an unwanted target of interest from a sample, and wherein the associated unwanted target is removed directly or eluted (or collected or otherwise isolated) and then removed.

In a further embodiment, a method of screening a DBDpp library for a DBDpp that specifically binds a target of interest is provided, which comprises: (a) obtaining a plurality of host cells (e.g., viral particles, phage, bacterial and/or mammalian cells) displaying the DBDpp libraries on its surface; (b) contacting a plurality of host cells with a target of interest under conditions suitable for specific binding of the target to DBDpp; and (c), determining target binding to DBDpp. In one embodiment, the host cell is a phage that displays DBDpp on its surface.

Methods of using DBDpp in diagnostic and therapeutic applications are also provided in several embodiments. In one embodiment, a method of treating a disease or disorder is provided, comprising administering a therapeutically effective amount of DBDpp (eg, a DBDpp fusion protein) that specifically binds a therapeutic target of interest to a subject in need thereof. In some embodiments, the disease or disorder is cancer, an immune system disease or disorder, or an infection. Also provided are methods of treating a disease or disorder that include co-administration of an additional therapeutic agent with DBDpp.

Additionally provided are methods of treating or preventing cancer, comprising administering a DBDpp-CAR T lymphocyte to a patient (eg, predisposed to or having cancer) that expresses a tumor antigen on the surface of target cells, and wherein the DBDpp specifically binds the antigen.

Some of the methods, briefly described above and presented in more detail below, describe some of the actions taken by the practitioner; however, it should be understood that they may also include instructions for such actions to be performed by another party. Thus, acts such as administering a T cell comprising a target-specific binding polypeptide-CAR include instructions for use of a T cell comprising a target-specific binding polypeptide-CAR.

Brief description of drawings

Fig. 1A-B. Schematic representation of the homologous model DBDpp derived from SEQ ID NO: 1. Transverse view illustrating the helices and three planes of the domain (Figure 1A). Longitudinal image illustrating the position of residue E19, N-terminus and C-terminus (Fig. 1B).

Fig. 2A-J. Schematic representation of the various homologous DBDpp models based on the reference framework SEQ ID NO: 1. Residues targeted for modification in the Plane Libraries (F1, F2 and F3) and Combined DBDpp Libraries (C1 and C2) are shaded dark. Longitudinal and transverse perspective views of Library F1 are shown in FIG. 2A and FIG. 2B respectively. Longitudinal and transverse perspective views of Library F2 are shown in FIG. 2C and FIG. 2D respectively. Longitudinal and transverse perspective views of Library F3 are shown in FIG. 2E and FIG. 2F respectively. Longitudinal and transverse perspective views of Library C1 are shown in FIG. 2G and fig. 2H respectively. Longitudinal and transverse perspective views of Library C2 are shown in FIG. 2I and fig. 2J respectively. The N-terminus and C-terminus for each model are indicated.

Fig. 3A-D. Fig. 3A. Schematic description of a phage display construct for use in accordance with several embodiments disclosed in the present invention. Fig. 3B. Shown is a linear vector map for the pComb phagemid vector used to generate the libraries disclosed in the present invention. Libraries were generated by Kunkel mutagenesis using oliages containing NNK or trimeric codons. The peptide sequences of the DBDpp variant were expressed in frame between the FLAG peptide tag sequence and the pIII M13 gene. DBDpp was expressed as a construct fused at the N-terminus to the pIII gene under the control of the DsbA signal peptide. Fig. 3C. Shown is a linear vector map for the pComb phagemid vector used to generate the DBDpp libraries described in the examples. DBDpp were expressed in frame between the signal peptide DsbA and the pIII gene M13. The modified phagemid vector pComb is the same as the vector shown in FIG. 3B, but does not contain a FLAG tag peptide sequence, consistent with some embodiments disclosed in the present invention (where the FLAG tag is optionally removed or replaced with a different type of tag). In fig. 3D shows the data obtained from the comparative binding assay. N-terminally FLAG-tagged fusion constructs were expressed and purified from E. coli cultures. ELISA-based binding assessment demonstrated that purified FLAG-pb04 (PD-L1 targeting) dose-dependently bound to PD-L1-Fc-coated wells in a microtiter plate, whereas FLAG-a3D (reference sequence SEQ ID 49, with N-terminal tag

FLAG) shows no changes in binding.

Fig. 4A-D. DBDpp has new binding specificities and imparts such new binding specificities to another molecule (eg, an antibody) as part of a fusion protein (eg, an antibody-DBDpp fusion protein). Schematic representation of a recombinant DBDpp fusion construct (shown as a circle) with the C terminus (Figure 4A) and N terminus (Figure 4B) of the antibody heavy chain. DBDpp-antibody fusion constructs were generated using an RSV-specific antibody (SYN) and either a non-targeted peptide of SEQ ID NO: 1 (DBD) or a CD137-specific DBDpp (bb10). DBDpp are fused to the N-terminus (bb10-SYN and DBD-SYN) or the C-terminus (SYN-bb10 and SYN-DBD). All four antibody fusion constructs bind to RSV (Fig. 4C). However, a fusion construct of bb10 to the N-terminus (bb10-SYN) or the C-terminus (SYN-bb10) of the antibody heavy chain confers a novel CD137 binding specificity to an otherwise monospecific antibody ( Fig. 4D ).

Fig. 5A-5C. Fig. 5A. Presented is a schematic representation of DBDpp-CAR fusion proteins according to several embodiments disclosed in the present invention. The six different DBDpp-CAR formats are provided by way of example and are intended to be illustrative and non-limiting. The extracellular domains of DBDpp may be specific for a single target (eg, DBDpp A) or more than one target or epitope (eg, DBDpp A and DBDpp B). Non-limiting examples of transmembrane (TM) domains are shown, as well as non-limiting examples of intracellular domains derived from CD3, CD28 and 41BB. The domains are optionally linked via peptide linkers (shown in shading). Fig. 5B. Another diagram of a membrane-bound (eg, extracellular) DBDpp-CAR fusion construct is shown. Fig. 5C. A diagram of soluble DBDpp is shown.

Fig. 6A-C. Multispecific DBDpp fusion constructs recognize cell surface targets. FACS analysis shows that the bispecific antibodies bb10-SYN and SYNbb10 bind (shaded histogram) to activated CEM cell at higher levels than SYN alone (black outline). The weaker binding observed with the bb10 N-terminal fusion construct (Fig. 6A) compared to the C-terminal fusion construct (Fig. 6B) is consistent with the above ELISA data. URE1 is a recombinant antibody construct formed from the variable domains of CD137-targeted urelumab fused to an IgG backbone. Binding of CEM cells was performed after activation with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 48 h. Detection of bound antibody was performed using Fc against IgG1 (FITC-A).

Fig. 7A-D. DBDpp confers novel biological activity to the antibody-DBDpp fusion protein. Activation of CD137 by ligand or agonistic antibodies such as urelumab induces a signaling cascade that leads to the production of cytokines, expression of anti-apoptotic molecules, and enhanced immune responses. The agonistic potential of the CD137-targeted DBDpp, bb10, was assessed by measuring the ability of bb10-SYN and SYN-bb10 to induce cytokine release from PBMCs. The bb10 fusion constructs were tested in both soluble and coated plastic well formats. PBMCs in complete RPMI medium were added to the plates and incubated overnight. Cell culture supernatants were then tested for TNFα and IL8 using ELISA. In two populations of donor PBMCs, bb10 fusion constructs induced secretion of IL8 and TNF-alpha at levels greater than or equal to that of the agonistic anti-CD137 monoclonal antibody, URE1.

Fig. 8. In vivo stability is critical to the clinical efficacy of most biotherapeutics. Pharmacokinetic measurements of bb10 fusion constructs were performed to evaluate the relative stability of DBDpp compared to the antibody used as a partner in the fusion construct. In vivo stability was determined using an RSV and CD137 binding assay with a bispecific antibody present in the serum of CD1 mice that received a single intravenous injection (1 mg/kg) of the fusion construct. Serum samples were collected at 15 min and 48 h and tested by ELISA. Both N-terminal and C-terminal DBDpp fusion proteins exhibit long-term stability in vivo. As discussed in more detail below, several embodiments include DBDpp fusion constructs with increased stability (e.g., on the order of 24 hours, 48 hours, 72 hours, 96 hours, 6 days, 8 days, 10 days or more, including time periods in between). .

Fig. 9. In FIG. 9 shows HPLC purification of DBDpp prepared according to several embodiments disclosed in the present invention.

Fig. 10. In FIG. 10 shows SDS-PAGE analysis of purified DBDpp prepared according to several embodiments disclosed in the present invention. Lane 1 is a molecular weight marker, Lane 2 corresponds to purified DBDpp SEQ ID NO: 58, and lanes 3-9 correspond to purified DBDpp SEQ ID NO: 51-57, respectively.

Fig. 11. In FIG. 11 shows an expanded electrospray ionization mass spectrum (ESI-MS) SEQ ID NO. 54.

Fig. 12A-12P. In fig. 12A-12P show data that relate to the binding of CD137- 19 043883 targeted DBDpp to CD137 that was immobilized on a solid surface. Fig. 12A, 12C,

12E, 12G, 12I, 12K, 12M and 12O are sensorgrams for DBDpp corresponding to SEQ ID NO: 51 (12A), 52 (12C), 53 (12E), 54 (12G), 55 (12I), 56 (12K) , 57 (12M) and 58 (12O). In fig. 12B, 12D, 12F, 12H,

12J, 12L, 12H and 12P show the corresponding equilibrium binding data for DBDpp corresponding to SEQ ID 51 (12B), 52 (12D), 53 (12F), 54 (12H), 55 (12J), 56 (12L), 57 ( 12H) and 58 (12P).

Fig. 13. In FIG. 13 shows chromatographic data for the purification of CD137 protein from the supernatant of Chinese hamster ovary (CHO) cells.

Fig. 14A-14B. Analysis of proteins purified using DBDpp. In fig. 14A shows a Coomassie-stained gel loaded with purified fractions from the DBDpp purification columns. Lane 1 is a molecular weight marker. Lane 2 is IMAC purified CD137 protein, and Lanes 3-8 are column eluates with various DBDpps according to several embodiments of the present invention. Fig. 14B is a Western blot analysis with samples corresponding to those shown in FIG. 14A.

Fig. 15A-15D. Thermal stability DBDpp. In fig. 15A shows an assessment of DR5 scFv binding to PD-L1PD-L1-Fc-coated microtiter plate wells after exposure to various elevated temperatures. In fig. 15B shows data demonstrating a correlation between elevated temperature and decreased PDL1 scFv binding directed against PD-L1PD-L1. In fig. 15C shows a DBDpp, according to one embodiment disclosed in the present invention (pb04 DBDpp), retaining binding affinity for PD-L1PD-L1 after exposure to elevated temperatures up to 100°C. In fig. 15D shows an additional DBDpp (pb06 DBDpp) that also exhibits thermal stability and can bind PD-L1PD-L1 after exposure to temperatures up to 100°C.

Fig. 16A-16B. Cross-reactivity DBDpp. In fig. 16A provides data regarding the ability of DBDpp to bind targets from different species. In particular, in FIG. 16A demonstrates that soluble DBDpp directed against PD-L1 can bind to human PD-L1 (upper trace) as well as cynomolgus PD-L1 (lower trace) with similar binding affinities. In fig. 16B shows flow cytometry data confirming that when expressed in a T cell, particularly a chimeric antigen receptor T cell, the T cell can recognize and bind to both human and cynomolgus PD-L1.

Fig. 17. Assessment of DBDpp-CAR expression and target binding. In fig. 17 presents data that relates to DBDpp-CAR expression and CD-123-Fc binding of various DBDppCAR candidate HEK-293T cells.

Fig. 18. DBDpp mediates signal transduction. In fig. 18 presents data that relate to the expression and ability of Jurkat cell DBDpp-CAR to function through an intracellular signaling pathway.

Fig. 19A-19B. CD123-DBDpp-CAR T cells produce cytokines in response to target binding. In fig. 19A shows data relating to the production of interferon gamma (IFN) by T cells expressing DBDpp-CAR receptors directed against CD123. In fig. 19B presents similar data measuring interleukin 2 (IL2) production by CD123-targeted DBDpp-CAR T cells.

Fig. 20A-20B. PD-L1-DBDpp-CAR T cells produce cytokines in response to target binding. In fig. 20A shows data relating to interferon (IFN) production by T cells expressing DBDpp-CAR receptors directed against PD-L1. In fig. 20B shows similar data measuring interleukin 2 (IL2) production by PD-L1-targeted DBDpp-CAR T cells.

Fig. 21. CD123-DBDpp-CAR T cells proliferate in response to target binding. In fig. 21 presents data that relate to the proliferation of CD123-targeted DBDpp-CAR T cells compared to control and CD123-targeted scFv.

Fig. 22. PD-L1-DBDpp-CAR T cells proliferate in response to target binding. In fig. 22 presents data that relates to the proliferation of PD-L1-targeted DBDpp-CAR T cells compared to control conditions.

Fig. 23A-23B. T cells expressing DBDpp-CAR receptors are not overdepleted to a greater extent than scFv. In fig. 23A shows the expression of three exhaustion markers (LAG-3, PD-1 and TIM3) on T cells expressing various DBDpp-CAR receptors with similar levels of expression of such markers on the scFv. In fig. 23B shows flow cytometry data depicting similar expression of exhaustion markers on DBDpp-CAR T cells (expressing the CD123-targeted cg06 DBDpp) compared to a CAR T cell expressing the CD123-specific scFv (32716).

Fig. 24A-24D. T cells expressing DBDpp-CAR receptors degranulate in response to target binding. In fig. 24A shows the production of CD107a (as a marker of DBDpp-CAR T cell degranulation) when CD123-targeted DBDpp-CAR T cells are separately cultured. In fig. 24B shows CD107a production when DBDpp-CAR T cells are cocultured with CD123-20043883 negative K562 tumor cells. In fig. 24C shows CD107a in co-culture

CD123-targeted DBDpp-CAR T cells with CD123-positive BDCM cells. In fig. 24D shows data from replicate coculture experiments of CD123-targeted DBDpp-CAR Ts with CD123-positive BDCM cells.

Fig. 25A-25D. T cells expressing PD-L1-DBDpp-CAR receptors degranulate in response to target binding. In fig. 25A shows the expression of CD107a (as a marker of DBDppCAR T cell degranulation) when PD-L1-targeted DBDpp-CAR T cells are separately cultured, eg, non-activated. In fig. 25B shows measurement of CD107a when DBDppCAR T cells are co-cultured with PD-L1 negative K562 tumor cells. In fig. 25C shows increased CD107a when PD-L1-targeted DBDpp-CAR T cells are co-cultured with PD-L1-positive SUDHL1 cells. In fig. 25D shows data from replicate coculture experiments of PD-L1-targeted DBDpp-CAR Ts with PD-L1-positive SUDHL1 cells.

Fig. 26A-26D. T cells expressing DBDpp-CAR receptors mediate target-specific antitumor cytotoxicity. In fig. 26A shows data that relates to the percentage of K562 tumor cells killed by CD123-directed DBDpp-CAR T cells that are CD123 negative. In fig. 26B shows the percentages of cells killed when CD123-targeted DBDpp-CAR T cells were co-cultured with CD123-positive BDCM cells. Data in Fig. 26A and 26B were obtained using T cells from a blood sample from the first donor. In fig. 26C and 26D show similar data for T cells collected from a second donor.

Fig. 27A-27F. T cells expressing DBDpp-CAR receptors mediate target-specific antitumor cytotoxicity. In fig. 27A shows data that relates to the percentage of K562 tumor cells killed by PD-L1-directed DBDpp-CAR T cells that are PD-L1 negative. CAR T cells expressing various PD-L1-targeted DBDpps exhibited lower rates of killed cells than mock controls. Similar data is shown in Fig. 27C and 27E for two additional donors. In fig. 27B shows increased percentages of cells killed when PD-L1-targeted DBDppCAR T cells were co-cultured with PD-L1-positive SUDHL1 cells. Similar data is shown in Fig. 27D and 27F for two additional donors.

Fig. 28A-28D. DBDpp has reduced immunogenic potential. Since DBDpp, as disclosed herein, is synthetic, an assay was performed to identify potentially immunogenic epitopes. The 3D model of DBDpp (cg06) is shown in Fig. 28A. In fig. 28B shows cg06 with one of the (three) potentially immunogenic epitopes modified to make them less potentially immunogenic. In fig. 28C shows cg06 with two of the (three) potentially immunogenic modified epitopes. In fig. 28D shows cg06 with all three potentially immunogenic modified epitopes.

Fig. 29A-29B. DBDpp with modified epitopes retains functionality. In fig. 29A presents data that relates to CAR T cells expressing variants of the CD123-directed DBDpp (cg06). Even when all three potentially immunogenic epitopes are removed from the DBDpp sequence, the variants retain the ability to mediate signal transduction (activating recombinant Jurkat cells expressing luciferase) upon binding to CD123-positive BDCM target cells (unmodified cg06 is indicated by an arrow). In fig. 29B shows similar efficacy in binding of the modified variants to CD123 positive KG-1a cells (unmodified cg06 is indicated by an arrow).

Fig. 30A-30B. Dual expression of markers on tumor cells. In fig. 30A shows flow cytometry data for CD123 expression on K562 cells, KG1a cells, BDCM cells, SUDHL cells or H460 cells. In fig. 30B shows flow cytometry data for PD-L1 expression on the same cell lines.

Fig. 31A-31E. Bispecific DBDpp-CAR T cells. In fig. 31A shows the percentage of T cells expressing CD123-targeting DBDpp-CAR receptors. In fig. 31B shows the percentage of T cells expressing PD-L1-targeting DBDpp-CAR receptors. In fig. 31C shows the percentage of T cells expressing bispecific CD123-PD-L1-targeting DBDpp-CAR receptors (expressed with cg06 DBDpp distal to the T cell membrane versus pb04 DBDpp). In fig. 31D shows the percentage of T cells expressing bispecific PD-L1-CD123-targeting DBDpp-CAR receptors (expressed with pb04 DBDpp distal to the T cell membrane versus cg06 DBDpp). In fig. 31E presents data associated with increased intracellular signaling of bispecific DBDpp.

Fig. 32. DBDpp competitive binding assay. In fig. 32 demonstrates one embodiment of a competitive binding assay that can be used to identify DBDpps that exhibit binding to common epitopes even though the DBDpps tested have different primary amino acid sequences.

- 21 043883

Detailed description

The section headings used herein are for organizational purposes only and should not be construed as any limitation of the subject matter described.

Definition of terms.

It should be understood that whenever embodiments are described herein with the language including, other similar embodiments described in the sense of consisting of and/or consisting essentially of are also contemplated. However, when used as transitional phrases in claims, each should be interpreted separately and in the appropriate legal and factual context (for example, including is considered more of an open phrase, while consisting of is more exclusive, and essentially consisting of is an intermediate concept).

As used herein, the singular form includes plural references unless otherwise indicated.

The term and/or, when used in a phrase such as A and/or B as used herein, is intended to include both A and B; A or B; (only) A; and (only) B. Likewise, the term and/or, as in a phrase such as A, B and/or C, is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; (only) A; (only) B; and (only) C.

The terms protein and polypeptide are used interchangeably herein to refer to a biological polymer comprising units derived from amino acids linked by peptide bonds; a protein may consist of two or more polypeptide chains.

The terms antibody or immunoglobulin, as used interchangeably herein, include whole antibodies and antibody fragments comprising any functional domain of an antibody, such as an antigen-binding fragment or single chains thereof, an effector domain, a salvage receptor binding epitope, or a portion thereof. A typical antibody includes at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, C1. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), which alternate with regions that are more conserved, called framework regions (FW). Each VH and VL consists of three CDRs and four FWs, arranged from N-terminus to C-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to tissues or factors in the body, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system. Non-limiting types of antibodies herein include typical antibodies, scFvs, and combinations thereof, where, for example, DBDpp is covalently linked (e.g., via peptide bonds or a chemical linker) to the N-terminus of the heavy chain and/or light chain of a typical full-length antibody, or integrated into the H chain and/or L chain of a whole antibody.

The term antibody fragment refers to a portion of an intact antibody and refers to any functional domain of an antibody, such as an antigen binding fragment or single chains thereof, an effector domain or a portion thereof, and a salvage receptor binding epitope or a portion thereof. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. An antibody fragment as used herein includes an antigen binding region or an epitope binding region. In one embodiment, the DBDpp fusion protein includes an effector domain or a portion thereof. In one embodiment, the DBDpp fusion protein includes a recycling receptor binding epitope or a portion thereof.

As used herein, the term Fc region or simply Fc is intended to mean the C-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof. For example, the Fc region of an immunoglobulin may include: (1) a CH1 domain, a CH2 domain, and a CH3 domain, (2) a CH1 domain and a CH2 domain, (3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or (5 ) a combination of two or more domains and a hinge region of an immunoglobulin. In a preferred embodiment, the immunoglobulin Fc region includes at least an immunoglobulin hinge region, a CH2 domain and a CH3 domain, and preferably does not contain a CH1 domain. In one embodiment, the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igy) (γ subclasses 1, 2, 3 or 4). Other classes of immunoglobulin that may be used are IgA (Iga), IgD (Igδ), IgE (Igε), and IgM (Igμ). The choice of the appropriate constant region of the immunoglobulin heavy chain is discussed in detail in US Pat. US 5,541,087 and 5,726,044, each of which is hereby incorporated by reference in its entirety. The selection of specific immunoglobulin heavy chain constant region sequences from certain classes and subclasses of immunoglobulins to obtain a particular result is believed to be known in the art. The portion of the DNA construct encoding the immunoglobulin Fc region preferably includes at least a portion of the hinge domain and preferably at least a portion of the CH3 domain of Fc-gamma or homologous domains in any of IgA, IgD, IgE, or IgM. It is further contemplated that substitution or deletion of amino acids in immunoglobulin heavy chain constant regions may be beneficial in the practice of the methods and compositions disclosed herein. One example is to introduce amino acid substitutions in the upper CH2 region to produce an Fc variant with reduced affinity for Fc receptors (Cole, J. Immunol. 159:3613 (1997)).

Antibody-dependent cell-mediated cytotoxicity or ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on the target cell and then cause lysis (or other cytotoxic effects) of the target cell. To assess the ADCC activity of a molecule of interest, any in vitro ADCC assay known in the art, such as that described in US Pat. US 5,500,362 or 5,821,337. Useful effector cells for such assays include, but are not limited to, peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as disclosed in Clynes et al. PNAS 95:652–656 (1998).

The terms single chain variable fragment(s) or scFv antibodies as used herein refer to forms of antibodies (eg, antibody fragments) comprising variable regions of only the heavy and light chains linked by a peptide linker. In one embodiment, the DBDpp fusion protein includes DBDpp and scFv.

The term linker refers to a peptide or other chemical bond located between DBDpp and another DBDpp fusion protein polypeptide. Suitable linkers for joining two or more linked DBDpp will be known to those skilled in the art, with non-limiting examples described herein.

The term operably linked as used herein indicates that two molecules are connected, with each molecule retaining at least some level of functional activity that each molecule had separately (this assumes that each molecule has functional activity). In embodiments, where one molecule has no functional activity, it is operably linked to another molecule if the other molecule retains at least some level of its functional activity. Functionally linked can also refer to the connection of two molecules that do not have a function. Two molecules can be operably linked whether they are connected directly or indirectly (eg, through a linker).

The terms specifically bind or have selective affinity for mean that a binder, such as DBDpp, reacts or binds more frequently, more rapidly, for a longer duration, with greater affinity, or some combination of the above, with an epitope, protein, or target molecule than with alternative substances, including proteins that are not related to the target epitope. Due to the sequence identity of homologous proteins in different biological species, specific binding, in several embodiments, may include a binding agent that recognizes a protein or target in more than one biological species. Likewise, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding may involve a binding agent that recognizes more than one protein or target. It should be understood that in some embodiments, a binding agent that specifically binds a first target may or may not specifically bind a second target. As such, specific binding does not necessarily require (although it may include) exclusive binding, such as binding to a single target. Thus, the binding agent in some embodiments can specifically bind more than one target. In some embodiments, multiple targets may be bound by the same antigen binding site on the binding agent.

A target refers to any molecule or combination of molecules that can be bound by a DBDpp, such as a DBDpp fusion protein or another component of a DBDpp fusion protein, such as an antibody or antibody variable domain fragment.

The terms epitope and antigenic determinant are used interchangeably herein and refer to that portion of any molecule (e.g., target of interest) that may

- 23 043883 can recognize and specifically bind a particular binding agent (eg DBDpp or antibody). In the case where the recognition molecule is a polypeptide, epitopes can be formed from contiguous amino acids and non-contiguous amino acids, and/or other reactive surface groups of molecules (such as carbohydrates) brought into contact by tertiary folding of the protein. Epitopes formed from contiguous amino acids tend to be retained after protein denaturation, whereas epitopes formed by tertiary folding tend to be lost after protein denaturation. An epitope typically comprises at least 3 amino acids, and more often at least 5 or 8-10 amino acids, in a unique spatial conformation.

A peptide tag as used herein refers to a peptide sequence that is part of or attached (eg, by genetic engineering) to another protein, conferring the function of the resulting fusion construct. Peptide tags are usually relatively short compared to the protein to which they are fused; By way of example, peptide tags in several embodiments are four or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, or 25, or more amino acids. In some embodiments, DBDpp is a fusion protein that contains a peptide tag. In other embodiments, DBDpp specifically binds a peptide tag. Many peptide tags that have the uses proposed in the present invention are known in the art. Examples of peptide tags that may be a component of a DBDpp fusion protein or a target bound by DBDpp (e.g., a DBDpp fusion protein) include, but are not limited to, HA (hemagglutinin), c-myc, herpes simplex virus glycoprotein D (gD), T7, GST, GFP, MBP, Strep tags, His tags, Myc tags, TAP tags, and FLAG® tags (Eastman Kodak, Rochester, NY). Likewise, antibodies to an epitope tag provide detection and localization of the fusion protein, for example, in affinity purification, Western blotting, ELISA, and cell immunostaining.

The term naturally occurring, when used to refer to biological materials such as nucleic acid molecules, polypeptides and host cells, refers to those that are naturally occurring and unaltered by man. On the other hand, non-natural or synthetic when used in relation to biological materials refers to those that are not present in nature and have been altered by man.

As used herein, modifications with respect to the reference framework sequence SEQ ID NO: 1 (or with respect to other sequences) include substitutions, deletions, insertions and/or additions of the sequence of the corresponding amino acid position of SEQ ID NO: 1 (or with respect to the corresponding position of another sequences).

Substitution with respect to the reference framework sequence SEQ ID NO: 1 (or with respect to other sequences) refers to the replacement of a particular amino acid residue with another amino acid residue at the corresponding amino acid position of SEQ ID NO: 1 (or with respect to the corresponding position of another sequence).

A conservative amino acid substitution is one in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine (K), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E)), uncharged polar side chains (e.g. glycine (G), asparagine (N), glutamine (Q), series (S), threonine (T), tyrosine (Y), cysteine (C)), non-polar side chains (e.g. alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W), beta-branched side chains chains (e.g. threonine (T), valine (V), isoleucine (I)) and aromatic side chains (e.g. tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). tyrosine phenylalanine is a conservative substitution.In one embodiment, conservative substitutions in DBDpp sequences result in specific binding of the DBDpp containing the substitution to the target of interest to which it binds. In one embodiment, conservative substitutions in DBDpp sequences do not abolish binding of the DBDpp containing the substitution to the target of interest to which it binds. Methods for identifying nucleotide and amino acid conservative substitutions and non-conservative substitutions that impart, alter or maintain selective binding affinity are known in the art (see, for example, Brummell, Biochem. 32:1180-1187 (1993); Kobayashi, Protein Eng. 12 (10):879-884 (1999); and Burks, PNAS 94:412-417 94:412-417 (1997)).

A non-conservative amino acid substitution is one in which one amino acid residue is replaced by another amino acid residue having a different side chain. In one embodiment, non-conservative substitutions in DBDpp sequences result in specific binding of the DBDpp containing the substitution to the target of interest to which it binds. In one embodiment, non-conservative substitutions in DBDpp sequences do not abolish binding of the DBDpp containing the substitution to the target of interest to which it binds.

Unnatural amino acids, amino acid analogues and non-standard amino acid residues

- 24 043883 are used interchangeably in the present description. Unnatural amino acids that can be substituted in DBDpp as proposed in the present invention are known in the art. In one embodiment, the unnatural amino acid is 4-hydroxyproline, which can replace proline; 5-hydroxylysine, which can replace lysine; 3-methylhistidine, which can replace histidine; homoserine, which can replace serine; and ornithine, which can replace lysine. Additional examples of unnatural amino acids that can be used for substitution in DBDpp include, but are not limited to, molecules such as: D-isomers of common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, A-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, lanthionine, dehydroalanine, γ-aminobutyric acid, selenocysteine and pyrrolysine, fluoroamino acids, artificial amino acids such as betamethylamino acids, C-alpha-methylamino acids and N-alpha-methylamino acids or combinations of unnatural amino acids. Other additional unnatural amino acids may include 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6methylheptanoic acid, 2-thienyl-alanine and/or D-amino acid isomers. As discussed herein, in several embodiments, unnatural amino acids or amino acid analogues may include the deletion of one or more amino acids from a sequence.

The terms polynucleotide and nucleic acid, used interchangeably herein, refer to the polymeric form of nucleotides of any length, ribonucleotides or deoxyribonucleotides. These terms include, but are not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snRNA (small nucleolar RNA). , microRNA (miRNA), genomic DNA, synthetic DNA, synthetic RNA and/or tRNA.

The term naked DNA as used herein refers to DNA (e.g., non-histone DNA) encoding a protein, such as DBDpp (e.g., CAR), and is DNA cloned into a suitable expression vector in the desired orientation for expression (e.g., plasmid). Viral vectors that may be used include, but are not limited to, SIN lentiviral vectors, retroviral vectors, foam virus vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, hybrid vectors, and/or plasmid transposons (e.g., the transposon system Sleeping beauty) or integrase-based vector systems. Other vectors that can be used in combination with the production and use of DBDpp are described in the present invention or otherwise known in the art.

The terms vector, cloning vector, and expression vector as used herein refer to a carrier by which a nucleic acid sequence (eg, a DBDpp coding sequence) can be maintained or amplified in a host cell (eg, a cloning vector), or can be introduced into a cell. host to transform the host and cause expression (eg, transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.

A host cell includes a single cell or culture of cells that can be or has been the recipient of nucleic acids encoding DBDpp. Host cells include, but are not limited to, viral particles, phagemids, bacterial, yeast, plant, animal and mammalian cells. Host cells include the progeny of a single host cell, where the progeny may not necessarily be completely identical (in morphology or genomic DNA) to the original parent cell due to natural, accidental or intentional mutation and/or change. A host cell includes cells transfected or infected in vivo, in vitro or ex vivo with nucleic acids encoding DBDpp. In some examples, the host cell is capable of expressing and displaying DBDpp on its surface, such as in phage display. Expression includes transcription and/or translation.

A DBDpp library refers to multiple unique DBDpps and does not necessarily include multiple DBDpps that bind to the same target but have different binding sites and/or specificities.

A DBDpp vector library refers to a plurality of unique DBDpp-encoding nucleic acids (as defined above, optionally including DBDpp-encoding nucleic acids that bind to the same target but have different binding sites and/or specificities).

As used herein, the terms solid support, support, matrices, and resins are used interchangeably and refer, without limitation, to any column (or column material), bead, tube, microtiter plate, solid particle (e.g., agarose or sepharose), microchip ( e.g., a silicon, silica-glass, or gold chip) or membrane (e.g., biological or filtration membrane) to which a DBDpp, antibody, or other protein may be attached (e.g., cross-linked, bound, or coupled), directly or indirectly (eg, through other binding intermediate partners, such as other antibodies or protein A), or into which DBDpp or the antibody may be incorporated (eg, through a receptor or channel). Reagents and methods for attaching polypeptides to solid supports (eg, matrices, resins, plastics, etc.) are known in the art. Suitable solid supports include, but are not limited to, a chromatography resin or matrix (e.g., SEPHAROSE-4 FF agarose beads), the wall or bottom of a well in a plastic microtiter plate, a silica biochip, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal and their combinations. DBDpp and other compositions can be attached to a carrier material by non-covalent association or covalent bonding using reagents and methods known in the art. In one embodiment, DBDpp is connected to the chromatographic material using a linker.

As used herein, the terms pharmaceutically acceptable or physiologically tolerable and their grammatical variations, when used in relation to compositions, carriers, diluents and regents, are used interchangeably and mean that the materials can be administered or applied to a person without the development of therapeutically unacceptable adverse physiological effects such as such as nausea, dizziness, stomach upset, etc.

To modulate means to correct or regulate amplitude, frequency, degree or activity. In another suitable aspect, such modulation may be positive modulation (eg, increasing frequency, extent, or activity) or negative modulation (eg, decreasing frequency, extent, or activity). In several embodiments, modulation in a positive or negative direction is indicated in comparison to cell, tissue, or organ function prior to administration of the therapeutic agent. In further embodiments, modulation in a positive or negative direction is in relation to a normal, healthy cell, tissue or organ.

An effective amount of a DBDpp, such as a DBDpp fusion protein of the present invention, is an amount sufficient to accomplish its specifically stated purpose, e.g., to cause a marked change in the level of one or more biological activities associated with the target to which the DBDpp binds (e.g., a DBDpp fusion protein protein DBDpp). In some embodiments, the change increases the level of target activity. In other embodiments, the change reduces the level of target activity. The effective amount can be determined empirically and in a routine manner for the intended purpose. The term "therapeutically effective amount" refers to an amount of DBDpp, such as a DBDpp fusion protein, or other therapeutic agent effective for treating (eg, reducing symptoms) a disease or disorder in a subject (mammalian). A prophylactically effective amount refers to the effective amount, in doses and for periods of time, necessary to achieve the desired prophylactic result.

Patient, subject, animal and mammal are used interchangeably and refer to mammals such as diseased humans and non-human primates, as well as experimental animals such as rabbits, rats and mice, and other animals. Animals include all vertebrates, such as mammals and non-mammals such as chickens, amphibians and reptiles. Mammal, as used herein, refers to any member of the class Mammal, including, without limitation, humans and non-human primates such as chimpanzees and other species of apes and apes; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs, etc. In a particular embodiment, the patient is a human. The term does not indicate a specific age or gender. Thus, the term is intended to include adult and newborn subjects, as well as embryos and fetuses, male or female.

The terms treat and cure as used herein refer to both therapeutic treatment and prophylactic or preventive measures where the goal is to prevent or slow (reduce or delay) the symptoms, complications or biochemical manifestations of a disease, condition or disorder. relieve symptoms or stop or slow the further progression of a disease, condition or disorder. Treatment may be prophylactic (to prevent or delay the onset of a disease or prevent the manifestation of its clinical or subclinical symptoms) or therapeutic suppression or relief of symptoms after the onset of a disease, condition or disorder, target pathological condition, with the aim of preventing the pathological condition, achieving or obtaining beneficial results or reducing the likelihood of an individual developing the condition, even if treatment is ultimately ineffective. Those in need of treatment include persons who already have the condition, those who are at risk of developing the condition, or those in whom the condition is to be prevented. Treatment can be carried out with the DBDpp fusion protein alone or in combination with an additional

- 26 043883 new therapeutic agent.

Cancer, tumor, or malignancy are used as synonymous terms and refer to any of a number of diseases that are characterized by uncontrolled, abnormal cell proliferation, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (metastasize), and any of many characteristic structural and/or molecular features. Tumor as used herein refers to the growth and proliferation of all neoplastic cells, malignant or benign, as well as all precancerous and cancerous cells and tissues. A malignant tumor or malignant cell should be understood as a cell that has certain structural properties, is undifferentiated and is capable of invasion and metastasis. Cancers that can be treated using the DBDpp fusion proteins provided herein include, but are not limited to, breast, lung, brain, bone, liver, kidney, colon, head and neck, ovarian, hematopoietic (eg, leukemia) ) and prostate gland. Other types of cancers and tumors that can be treated using DBDpp-containing antibodies are described in the present invention or otherwise known in the art.

The terms tumor antigen or cancer antigen are used interchangeably herein. Tumor and cancer antigens can be tumor-specific antigen (TSA), cancer-associated antigen (CSA), tumor-associated antigen (TAA), or cancer-associated antigens (CAA). TSA is an antigen that is unique to tumor cells and is not present on other cells in the body. TAA is an antigen that is present on both tumor cells and some normal cells. Due to the dynamic nature of tumors, in some cases tumor cells may express unique antigens at some stages, and at other stages they may also express antigens that are also expressed on non-tumor cells. Thus, the inclusion of a particular marker such as TAA does not preclude it from being considered a TSA. Examples of TAAs and TSAs that may be specifically bound by DBDpp include, but are not limited to: CD19, CD20, CD22, ROR 1, mesothelin, CD33/lL3Ra, cMet, PSMA, glycolipid F77, EGFRvIII, GD2, NY-SO-1TCR, MAGE A3 TCR MARTI, gp100 (Pmel 17), tyrosinase, TRP1, TRP2, MAGE1, MAGE3, BAGE, GAGE1, GAGE2, pi5, CEA; p53, Ras, HER-2/neu; BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; EBVA, HPV E6 and E7 antigens, TSP-180, MAGE4, MAGE5, MAGE6, RAGE, NYESO, pl85erbB2, pl80erbB3, nm-23Hl, PSA, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4 (791Tgp72) alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA 15-3\CA 27.29\BCAA, CA195, CA242, CA50, CAM43, CD68\I, CO-029, FGF5, G250, Ga733VEpCAM, HTgp-175, M344, MA50, MG7-Ag, MOV 18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA90\Mac -2, TAAL6, TAG72, TLP and TPS.

The term target cell as used herein refers to cells that are involved in disease and may be the target of DBDpp-containing compositions. Other target cells include any cell in a subject (eg, human or animal) that may be a target of DBDpp according to the invention. The target cell may be a cell expressing or overexpressing a target specifically bound by the DBDpp fusion protein.

The term effector cells are leukocytes that express one or more FcRs and perform effector functions. Preferably the cells express at least FcRIII and have ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; with PBMCs and NK cells being preferred in some embodiments. Effector cells can be isolated from their native source, for example, from blood or PBMC, as described in the present invention or otherwise known in the art. In a certain embodiment, the effector cells are human effector cells.

The term effector function refers to the specialized immune function of a differentiated cell. The effector function of a T cell, for example, may be cytolytic activity or helper activity, including the secretion of cytokines.

The terms T cell and T lymphocyte are interchangeable and are used synonymously herein. Examples include, but are not limited to, naïve T cells, central memory T cells, effector memory T cells, or combinations thereof.

The term immunocyte as used herein refers to cells of the mammalian immune system including, but not limited to, antigen presenting cells, B cells, basophils, cytotoxic T cells, dendritic cells, eosinophils, granulocytes, helper T cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, NK cells, neutrophils, phagocytes, plasma cells and T cells.

The term immune response as used herein refers to immunity including, but not limited to, innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, and

- 27 043883 immunity and/or hyperactive immunity.

The term transduction as used herein refers to the introduction of a foreign nucleic acid into a cell using a viral vector. Transfection as used herein refers to the introduction of a foreign nucleic acid into a cell using recombinant DNA technology. The term transformation means the introduction of a foreign (e.g., external, extracellular, or otherwise non-endogenous) nucleic acid sequence (DNA or RNA) into a host cell such that the host cell expresses the introduced nucleic acid to produce a desired substance, such as a protein or enzyme encoded by entered coding sequence. The introduced nucleic acid sequence may also be referred to as a cloned or foreign gene or sequence and may include regulatory or control sequences such as start, stop, promoter, signal, secretion or other sequences used by the cell's genetic apparatus. The nucleic acid sequence may include non-functional sequences or sequences having no known function. The host cell that receives and expresses the introduced nucleic acid (eg, DNA or RNA) has been transformed and is a transformant or clone. The DNA or RNA introduced into a host cell may come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species, or may not exist in nature.

A cell surface receptor refers to molecules and complexes of molecules capable of receiving a signal and transmitting such a signal across the plasma membrane of a cell. An example of a cell surface receptor provided herein is an activated integrin receptor, for example, the activated integrin receptor ave3 on a metastatic cell. As used herein, a cell surface receptor also includes a molecule expressed on the surface of cells that contains DBDpp capable of binding a target of interest. The term receptor refers to a cell-associated protein that binds or otherwise interacts with a molecule (eg, a ligand) and mediates the effect of the ligand on the cell. In several embodiments, the molecule that interacts with the receptor is a bioactive molecule. Membrane-bound cell surface receptors are characterized by a multidomain structure, including an extracellular ligand-binding domain, a transmembrane domain, and an intracellular effector domain, which is typically involved in signal transduction.

Chimeric antigen receptor or CAR or CAR receptors as used herein refer to engineered receptors that allow antigen or target specificity to be imparted to cells (e.g., T cells such as naïve T cells, central memory T cells, effector T cells memory cells, NK cells, NKT cells, or a combination thereof). CAR receptors are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors.

Polypeptides with a de novo binding domain.

The terms de novo binding domain and DBD are used interchangeably herein to describe a target binding sequence that shares some common sequence and some common structural features of the reference framework sequence: MGSWAEFKQRLAAIKTRLEALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEALRKEAAAIR

DELQAYRHN (SEQ ID NO: 1). The terms DBDpp and DBD polypeptides include single (i.e., DBD polypeptide) and multiple (i.e., DBD polypeptides) references unless otherwise expressly indicated or as context requires. DBDpp is a polypeptide that can specifically (non-randomly) bind to a target molecule.

It has been discovered and disclosed in the present invention in several embodiments that a non-naturally occurring and target-less (applicant is not aware of a target that can be bound) anti-parallel triple-stranded bundle having the amino acid sequence SEQ ID NO: 1 can be used as a reference framework platform for producing de novo binding domain (DBD)-containing polypeptides (DBDpp) that bind to a target of interest, and for generating DBDpp libraries that can be screened for DBDpps having the desired functional and/or biological activities. Thus, in some aspects, the present disclosure relates to the use of DBDpp in methods for producing DBDpp having desired properties, such as the ability to bind a target of interest; methods for obtaining DBDpp libraries; DBDpp libraries obtained using such methods; methods for screening such DBDpp libraries for desired biological activity; and DBDpp identified in such libraries.

Unless otherwise indicated, the practice of the disclosed compositions and methods utilizes standard techniques of molecular biology (including recombinant technologies, tissue culture, and cell transformation), microbiology, cell biology, biochemistry, and immunology as are known in the art. Such methods are usually performed according to technical

- 28 043883 to the manufacturer's requirements or as they are usually applied, using or with the usual modifications of known techniques such as those given in

Sambrook et al.

(Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)); PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Freshney, ed., 1987); Handbook of Experimental Immunology (Weir et al., eds.; Gene Transfer Vectors for Mammalian Cells (Miller, ed., 1987); Current Protocols in Molecular Biology (Ausubel., ed., 1987); PCR Protocols: A Guide to Methods and Applications (Innis, ed., Academic Press, San Diego, Calif., 1990); Mattila, et al., Nucleic Acids Res. 19:967 (1991); Eckert, et al., PCR Methods and Applications 1:17 (1991); PCR (McPherson, ed., IRL Press, Oxford); PCR: The Polymerase Chain Reaction, (Mullis, ed., 1994); Harlow, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed . 1988) and Kontermann, ed., The Antibody Engineering Lab Manual (Springer Verlag, Heidelberg/New York, 2000), Current Protocols in Immunology (Coligan, ed., 1991), The Immunoassay Handbook (Wild, ed., Stockton Press NY, 1994); and Methods of Immunological Analysis (Masseyeff., ed., Weinheim: VCH Verlags gesellschaft mbH, 1993); and Gennaro, et al. 2000, Remington: the Science and Practice of Pharmacy, 20th Ed. Lipincott Williams and Wilkins: Baltimore, Md. , or as described herein. Unless specific definitions are provided, the nomenclature used in this regard, as well as the laboratory techniques and methods of analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry described herein, are known and used in the prior art. In addition, standard methods can be used for chemical syntheses, chemical research, recombinant production, purification, pharmaceutical formulation, formulation, delivery and treatment of patients.

In one embodiment, DBDpp is not derived from a naturally occurring cellular ligand from a public domain document (as of the filing date of US Provisional Patent Application 62/143,772, filed April 6, 2015, and to the knowledge of the present applicant). In another embodiment, the DBDpp is not derived from an immunoglobulin-derived antigen binding domain or another antibody domain such as a constant region, variable region, complementarity determining region (CDR), framework region, Fc domain, or hinge region. In another embodiment, DBDpp does not contain three CDR regions. In another embodiment, DBDpp does not contain CDR1 and CDR2. In yet another embodiment, DBDpp does not contain CDR1. In yet another embodiment, DBDpp does not contain CDR2. In another embodiment, DBDpp is not derived from protein A. In another embodiment, DBDpp is not derived from a naturally occurring bacterial receptor. In another embodiment, DBDpp is not derived from fibronectin. In another embodiment, DBDpp is not derived from the fibronectin type III domain. In yet another embodiment, DBDpp is not derived from the nottin protein. In yet another embodiment, DBDpp is not derived from lipocalin. In yet another embodiment, DBDpp is not derived from the affibody.

Properties of a sequence.

As stated above, the reference framework polypeptide SEQ ID NO: 1 contains three antiparallel alpha helices and is a variant of a non-naturally occurring and untargeted polypeptide sequence originally constructed as a protein folding experiment. Provided herein is DBDpp containing certain modifications of amino acid residues in the sequence of the reference framework polypeptide SEQ ID NO: 1 that impart the ability of DBDpp to bind a target of interest, and the use of DBDpp as a target-binding and targeting agent.

In one embodiment, a single DBDpp is about 65-150 amino acids in length, about 65-125 amino acids, about 65-100 amino acids, about 6590 amino acids, about 65-80 amino acids, about 65-70 amino acids. Also in some embodiments, the DBDpp is provided to be about 75-150 amino acids in length, about 75-125 amino acids, about 75-100 amino acids, about 75-29043883 amino acids, about 75-80 amino acids. DBDpp may be naked or conjugated to other molecules, including, but not limited to, toxins and radioisotopes. In other further embodiments, a DBDpp of longer length is used, for example, a DBDpp having a length ranging from about 150 to about 160 amino acids, from about 160 to about 170 amino acids, from about 170 to about 180 amino acids, from about 180 to about 190 amino acids amino acids, from about 190 to about 200 amino acids, or any length between the values listed (inclusive).

For known binding proteins, the specific residues that constitute the binding region of the molecule have either been, or theoretically can be, experimentally determined. Natural binding proteins (eg, antibodies or protein A) have identifiable residues that facilitate binding to their known targets. However, unlike natural ligands and binding proteins, the engineered a3d protein (SEQ ID NO: 49) or the reference framework sequence SEQ ID NO: 1 is not known to bind specifically to another protein (eg, target). Therefore, endogenous binding residues cannot be used as a guide to design new binding specificities. In DBDpp designs that bind to targets, residues were considered for mutation (e.g., library randomization) if they were superficial—showing significant solvent accessibility. The relative accessibility of a residue in a domain (region D) compared to the isolated state (region I) is expressed as a percentage (%A). Amino acid residues in SEQ ID NO: 1 that have %A values less than about 10-11% (e.g., residues corresponding to F7, L11, I14, L18, L21, S24, L28, F31, I35, F38, L42, Y45 , G49, V53, L56, A60, I63 and L67 in SEQ ID NO: 1) are assumed to be inaccessible to external solvent and are considered internal core residues of the structure of SEQ ID NO: 1. On the other hand, the amino acid residues in SEQ ID NO: 1 with %A values greater than approximately 1011% are assumed to occupy positions that have greater potential for interaction with the target of interest. Protein binding surfaces typically consist of multiple amino acid residues that are either contiguous or located in close proximity to each other in three-dimensional space. Thus, a secondary criterion for library construction, according to several embodiments of the present invention, was the relative proximity of these selected residues in the predicted secondary and tertiary structure of DBDpp.

Protein secondary structure, such as alpha helices, can change depending on external variables such as temperature, matrix or buffer composition, and concentration. The alpha-helical secondary structures of the reference polypeptide sequence SEQ ID NO: 1 are predicted to consist of residues G2-A20 in helix 1, residues L28-A44 in helix 2, and residue E52Y70 in helix 3. In further embodiments, the alpha-helical secondary structures of the reference polypeptide sequences of SEQ ID NO: 1 are predicted to consist of residues W4-L21 in helix 1, residues E25-Y45 in helix 2, and residues P51-Y70 in helix 3. Amino acid positions of the reference framework corresponding to residues in alpha helices having low solvent accessibility are as follows: F7, L11, I14, L18, L21, L28, F31, I35, F38, L42, Y45, V53, L56, A60, 163 and L67 in SEQ ID NO: 1. Amino acid positions of the reference framework, corresponding to solvent accessible residues in alpha helices are as follows: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33 , E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69 and Y70 in SEQ ID NO: 1 The amino acid positions of the reference framework corresponding to non-alpha helical residues are as follows: M1, G22, G23, S24, E25, A26, E27, K46, G47, K48, G49, N50, P51, R71, H72 and N73 in SEQ ID NO : 1.

In one embodiment, DBDpp are defined as target-binding polypeptides consisting of SEQ ID NO: 1 with one or more amino acid substitutions. In one embodiment, a sequence alignment of DBDpp to SEQ ID NO: 1 may show greater than 90% sequence identity. In other embodiments, a sequence alignment of DBDpp to SEQ ID NO: 1 may show greater than 80% sequence identity. In other embodiments, a sequence alignment of DBDpp to SEQ ID NO: 1 may show greater than 70% sequence identity. In other embodiments, a sequence alignment of DBDpp to SEQ ID NO: 1 may show greater than 60% sequence identity. In other embodiments, a sequence alignment of DBDpp to SEQ ID NO: 1 may show greater than 50% sequence identity.

In some embodiments, DBDpp residues with %A values that are less than 10% may remain constant or may be replaced with a conservative amino acid change. In certain embodiments, the solvent accessible (i.e., %A greater than 10) DBDpp residue has an amino acid sequence that is modified by mutagenesis, may be located in regions of the polypeptide associated with the alpha helical auto

- 30 043883 rich structure. Alpha helical positions in SEQ ID NO: 1 containing solvent inaccessible residues correspond to F7, L11, I14, L18, L21, L28, F31, I35, F38, L42, Y45, V53, L56, A60, I63 and L67 in SEQ ID NO: 1. Amino acid substitutions at these positions are preferably conservative in nature and may include non-standard or unnatural amino acids. In some embodiments, the selection of natural amino acid substitutions includes L, I, V, A, and F (and W, Y, M). In some DBDpps, the solvent-accessible DBD residues contained in the DBDpp are more than 60%, 70%, 80% or 90%, or 100% identical to the corresponding residues in SEQ ID NO: 1. F7, L11, I14, L18, L21, L28, F31, I35, F38, L42, Y45, V53, L56, A60, I63 and L67 in SEQ ID NO: 1.

In one embodiment, DBDpp includes the amino acid sequence of SEQ ID NO: 1, wherein a total of 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5- 60 amino acid residues; and wherein DBDpp specifically binds a target of interest. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 modified amino acid residues are substitutions. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 modified amino acid residues are conservative substitutions. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 modified amino acid residues are non-conservative substitutions. In another embodiment, 5-15, 5-20, 5-25, 5-30, 535, 5-40, or 5-45 amino acid residue modifications are conservative substitutions, and 5-15, 5-20, 5-25, 5 -30, 5-35, 5-40 or 5-45 amino acid residue modifications are non-conservative substitutions. In additional embodiments, 1-25, 1-30, 1-35, 5-40, 5-45, 5-50, 5-55, or 5-60 substitutions are made at amino acid residues in SEQ ID NO: 1 selected from the group , consisting of: M1, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72 and N73. In additional embodiments, 1-25, 1-30, 1-35, 5-40, 5-45, 5-50, 5-55, or 5-60 substitutions are made at amino acid residues in SEQ ID NO: 1 selected from the group , consisting of: M1, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72 and N73. In another embodiment, 1-20, 1-30, or 1-40 substitutions are made at amino acid residues in SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69 and Y70. In another optional embodiment, DBDpp optionally further includes an amino acid sequence in which 1-5, 1-10, 1-15, 5-10, or 5-15 residues corresponding to solvent-inaccessible residues in the amino acid sequence SEQ ID NO: 1 are replaced , and where DBDpp specifically binds a target of interest. In another optional embodiment, the substituted moieties corresponding to the solvent-inaccessible moiety in SEQ ID NO: 1 are selected from the group consisting of: F7, L11, I14, L18, L28, F31, I35, F38, L42, V53, L56, A60 , I63 and L67, and Y70. In some embodiments, the substituted moieties corresponding to the solvent-inaccessible moiety in SEQ ID NO: 1 are selected from the group consisting of: F7, L11, I14, L18, L21, L28, F31, I35, F38, L42, Y45, V53, L56, A60, I63 and L67, and Y70. In a further embodiment, DBDpp is a fusion protein. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, glass slide, chip, gelatin and agarose. In a further embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell displays DBDpp on its surface. In additional embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. Libraries including many DBDpps are also proposed.

In one embodiment, the isolated DBDpp includes a variation of the amino acid sequence of SEQ ID NO: 1, where the 5-15, 5-20, 5-25, 5-30, 5-35, 5-40 solvent accessible amino acid residues in SEQ ID NO are replaced : 1, and where 1-5, 1-10, 1-15, 5-10, or 5-15 solvent-accessible residues in SEQ ID NO: 1 are optionally replaced by a conservative amino acid substitution, and where

- 31 043883

DBDpp specifically binds the target of interest. In some embodiments, the solvent accessible substituted amino acid residues in SEQ ID NO: 1 have an %A greater than 10. In some embodiments, the solvent accessible substituted amino acid residues in SEQ ID NO: 1 have an %A less than 10. In one embodiment, the solvent accessible substituted amino acid residues in SEQ ID NO: 1 have an %A less than 10. solvent substituted amino acid residues in SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30 , E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69 and Y70. In some embodiments, at least 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 solvent accessible amino acid residues are replaced. In some embodiments, at least 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 solvent accessible amino acid residues in SEQ ID NO: 1 are replaced by conservative amino acid residue substitutions. In some embodiments, at least 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 solvent accessible amino acid residues in SEQ ID NO: 1 are replaced by non-conservative amino acid residue substitutions. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine or proline. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1-5, 1-10, 5-15, 5-20, 5-25, 5-30, or 5-35 solvent accessible amino acid residues are replaced. In some embodiments, 5-41, 10-41, 15-41, 20-41, 25-41, 30-41, or 35-41 solvent accessible amino acid residues are replaced. In some amino acid embodiments, 5-35 solvent-accessible acidic residues are replaced by conservative substitutions and 5-35 solvent-accessible amino acid residues are replaced by non-conservative substitutions, or 5-25 solvent-accessible amino acid residues are replaced by conservative substitutions and 5-25 solvent-accessible amino acid residues residues are replaced by non-conservative substitutions. In another optional embodiment, DBDpp optionally further comprises an amino acid sequence wherein 1-5, 1-10, 1-15, 5-10, or 5-15 residues corresponding to solvent-inaccessible residues in the amino acid sequence SEQ ID NO: 1 are replaced, and wherein DBDpp specifically binds a target of interest. In another optional embodiment, the substituted moieties corresponding to the solvent-inaccessible moiety in SEQ ID NO: 1 are selected from the group consisting of: F7, L11, I14, L18, L28, F31, I35, F38, L42, V53, L56, A60 , I63 and L67, and Y70. In another optional embodiment, the substituted moieties corresponding to the solvent-inaccessible moiety in SEQ ID NO: 1 are selected from the group consisting of: F7, L11, I14, L18, L21, L28, F31, I35, F38, L42, Y45, V53 , L56, A60, I63 and L67, and Y70. In a further embodiment, DBDpp is a fusion protein. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, glass slide, chip, gelatin and agarose. In a further embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell displays DBDpp on its surface. In additional embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. Libraries including many DBDpps are also proposed.

The term loop refers to sequences in the DBD corresponding to a loop located, for example, between helix 1 and helix 2 of the reference framework SEQ ID NO: 1 (for example, positions 22-24 SEQ ID NO: 2-6 and Z1 SEQ ID NO: 7- 11) and/or a loop located between helix 2 and helix 3 of the reference framework SEQ ID NO: 1, for example, positions 46-48 SEQ ID NO: 2-6 and Z ₂ SEQ ID NO: 711). In certain embodiments, one or both loops Z1 and _Z2 are amino acid sequences consisting of 2-5, 2-10, 2-15, 2-20, 2-25, or 2-30 amino acid residues (inclusive, as well as any number between those listed). In some embodiments, one or both loops Z ₁ and Z ₂ are amino acid sequences consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20 or more than 20 amino acid residues (inclusive, as well as any number in between). In another embodiment, at least 50, 60, 70, 80, 90 or 95% of the amino acid residues of the Z1 and/or Z ₂ loop are glycine or serine. In additional embodiments, at least 50, 60, 70, 80, 90, or 95% of the amino acid residues of the Z1 and/or Z ₂ loop are selected from the group consisting of glycine, serine, threonine, alanine,

- 32 043883 proline, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine and arginine. In one embodiment, the Z1 loop has the amino acid sequence GGS. In one embodiment, the Z2 loop has the amino acid sequence KGKG.

In one embodiment, DBDpp includes the amino acid sequence

MGSWX ₅ X ₆ FKX ₉ Xi ₀ LAXi3lKXi ₆ Xi ₇ LEALGG

SEAELAX ₃₀ FEX ₃₃ X ₃₄ IAX ₃₇ FEX ₄₀ X ₄₁ LQX ₄₄ YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ id _n0; 2), where X ₅ , X6, X ₉ , X ₁₀ , Xi3, X ₁₆ , Xi7, X ₃₀ , X ₃₃ , X ₃₄ , X ₃₇ , X ₄₀ , X41 and X ₄₄ is a natural and/or unnatural amino acid residue, and where DBDpp specifically binds a target of interest. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In a particular embodiment, DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO: 50). In a further embodiment, DBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions in SEQ ID NO: 1 are replaced by conservative amino acid residue substitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues in SEQ ID NO: 1 are replaced by non-conservative amino acid residue substitutions. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine. In some embodiments, neither proline nor cysteine is included in the amino acid substitutions. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1-12 solvent accessible amino acid residues are replaced with conservative substitutions and 1-12 solvent accessible amino acid residues are replaced with non-conservative substitutions, or 5-12 solvent accessible amino acid residues are replaced with conservative substitutions and 5-12 solvent accessible amino acid residues residues are replaced by non-conservative substitutions. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In a further embodiment, a library is provided containing a plurality of DBDpps. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, glass slide, chip, gelatin and agarose.

In one embodiment, DBDpp includes the amino acid sequence MGSWAEFKQRLAAIKTRLEALGGSEAELAA

FX3 ₂ X3 ₃ EIX3 ₆ AFX ₃₉ X4qELX43AYKGKGNPEVEALX ₅₇ X ₅₈ E^ ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (seq id NO: 3), where X ₃₂ , X ₃₃ , X ₃₆ , X ₃₉ , X ₄₀ , X ₄₃ , X ₅₇ , _X58 , _X61 , _X64 , _X65 and _X68 is a natural and/or unnatural amino acid residue, and where DBDpp specifically binds a target of interest. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In a particular embodiment, DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO: 50). In a further embodiment, DBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues replaced in SEQ ID NO: 1 are replaced by conservative amino acid residue substitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues in SEQ ID NO: 1 are replaced by non-conservative amino acid residue substitutions. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine. In some embodiments, neither proline nor cysteine is included in the amino acid substitutions. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1-12 solvent accessible amino acid residues are replaced with conservative substitutions, and 1-12 solvent accessible amino acid residues are replaced with non-conservative substitutions, or 5-12 solvent accessible amino acid residues are replaced with conservative substitutions, and 5-12 solvent-accessible amino acid residues are replaced by non-conservative substitutions. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In an additional embodiment, a library is provided containing a plurality of DBDpps. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, slide, chip, gelatin and agarose.

In one embodiment, DBDpp includes the amino acid sequence MGSWX ₅ EFX ₈ X9RLX12AIX1 ₅ X1 ₆ RLX19ALG

GSEAELAAFEKEIAAFESELQAYKGKGNPEVEX ₅₅ LRX ₅₈ X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO: 4), where X ₅ , X ₈ , X ₉ , X ₁₂ , X ₁₅ , X ₁₆ , X ₁₉ , X ₅₅ , X ₅₈ , X ₅₉ , _X62 , _X65 and _X66 is a natural and/or unnatural amino acid residue, and where DBDpp specifically binds a target of interest. In a further embodiment, Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In a particular embodiment, DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO: 50). In a further embodiment, DBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues replaced in SEQ ID NO: 1 are replaced by conservative amino acid residue substitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues in SEQ ID NO: 1 are replaced by non-conservative amino acid residue substitutions. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine. In some embodiments, neither proline nor cysteine is included in the amino acid substitutions. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1-12 solvent accessible amino acid residues are replaced with conservative substitutions and 1-12 solvent accessible amino acid residues are replaced with non-conservative substitutions, or 5-12 solvent accessible amino acid residues are replaced with conservative substitutions and 5-12 solvent accessible amino acid residues residues are replaced by non-conservative substitutions. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In a further embodiment, a library is provided containing a plurality of DBDpps. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, glass slide, chip, gelatin and agarose.

In one embodiment, DBDpp includes the amino acid sequence MGSWX ₅ X ₆ FKX ₉ X ₁₀ LAX ₁₃ IKX ₁₆ X ₁₇ LEALGG

SEAELAAFX₃₂X33EIX36AFX39X4oELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:⁵⁾,^{where X}5,^X6,^X9, X_W,X13,^X16,^X17,^X32,^X33,^X36,^X39,^X40,^X43, X55,^X58,^X59,^X62,^X65^{and X}66 is PriI native and/or non-natural amino acid residue, and where DBDpp specifically binds the target of interest. In a further embodiment, Xn is a natural amino

- 34 043883 acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In a particular embodiment, DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO: 50). In a further embodiment, DBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues replaced in SEQ ID NO: 1 are replaced by conservative amino acid residue substitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues in SEQ ID NO: 1 are replaced by non-conservative amino acid residue substitutions. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine. In some embodiments, neither proline nor cysteine is included in the amino acid substitutions. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1-12 solvent accessible amino acid residues are replaced with conservative substitutions and 1-12 solvent accessible amino acid residues are replaced with non-conservative substitutions, or 5-12 solvent accessible amino acid residues are replaced with conservative substitutions and 5-12 solvent accessible amino acid residues residues are replaced by non-conservative substitutions. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In a further embodiment, a library is provided containing a plurality of DBDpps. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, slide, chip, gelatin and agarose.

In one embodiment, DBDpp includes the amino acid sequence MGSWX ₅ EFX ₈ X ₉ RLX12AIX1 ₅ X1 ₆ RLX19ALGG

SEAEL^30FEX33X3₄IAX37FEX₄₀X₄iLQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO: 6),^{where χ}5^{, χ}8^{, χ}9^, X^^χ15^{, χ}16^{, X}19^, X30, X33, X34, X37, X40, X41, X44, X57, X58, X61, X64, X65^And X68 Is at native and/or non-natural amino acid residue, and where DBDpp specifically binds the target of interest. In a further embodiment X_n is a natural amino acid residue. In another embodiment, X_n is a naturally occurring amino acid residue other than cysteine or proline. In a particular embodiment, DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO: 50). In a further embodiment, DBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues replaced in SEQ ID NO: 1 are replaced by conservative amino acid residue substitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues in SEQ ID NO: 1 are replaced by non-conservative amino acid residue substitutions. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine. In some embodiments, neither proline nor cysteine is included in the amino acid substitutions. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1-12 solvent accessible amino acid residues are replaced with conservative substitutions and 1-12 solvent accessible amino acid residues are replaced with non-conservative substitutions, or 5-12 solvent accessible amino acid residues are replaced with conservative substitutions and 5-12 solvent accessible amino acid residues residues are replaced by non-conservative substitutions. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In a further embodiment, a library is provided containing a plurality of DBDpps. Nuclei have also been proposed

- 35 043883 new acids encoding DBDpp, and vectors containing such nucleic acids. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, glass slide, chip, gelatin and agarose.

A dedicated DBDpp is also proposed, which includes the amino acid sequence: MGSWX ₅ X ₆ FKX ₉ XioLAXi3lKXi ₆ Xi7LEAL

ZiEAELAX ₂ 8FEX ₃ iX ₃₂ IAX ₃ 5FEX ₃ 8X ₃₉ LQX4 ₂ YZ ₂ NPEVEALRKEAAAIRDELQAYRHN (seq id NO: 7), where X ₅ , X6, X ₉ , X ₁₀ , X ₁₃ , X ₁₆ , X ₁₇ , X ₂₈ , X ₃₁ , X ₃₂ , X ₃₅ , X ₃₈ , X ₃₉ and X ₄₂ is a natural and/or unnatural amino acid residue, where Z1 and Z2 are 2-30 natural and/or unnatural amino acid residues, and where DBDpp specifically binds the target of interest. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In a particular embodiment, DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO: 50). In a further embodiment, DBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues replaced in SEQ ID NO: 1 are replaced by conservative amino acid residue substitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues in SEQ ID NO: 1 are replaced by non-conservative amino acid residue substitutions. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine. In some embodiments, neither proline nor cysteine is included in the amino acid substitutions. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1-12 solvent accessible amino acid residues are replaced with conservative substitutions and 1-12 solvent accessible amino acid residues are replaced with non-conservative substitutions, or 5-12 solvent accessible amino acid residues are replaced with conservative substitutions and 5-12 solvent accessible amino acid residues residues are replaced by non-conservative substitutions. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In a further embodiment, a library is provided containing a plurality of DBDpps. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, glass slide, chip, gelatin and agarose.

An isolated DBDpp has also been proposed, which includes the amino acid sequence: MGSWAEFKQRLAAIKTRLEALZiEAELAAF

X ₃₀ X ₃₁ EIX ₃ 4AFX ₃₇ X3 ₈ ELX ₄₁ AYZ ₂ NPEVE ALX ₅₂ X ₅₃ EAX ₅₆ AIX ₅₉ X ₆₀ ELX ₆₃ AYRHN (seq id NO: 8), where X30, X ₃₁ , X ₃₄ , X ₃₇ , X ₃₈ , X ₄₁ , X ₅₂ , X ₅₃ , X ₅₆ , X ₅₉ , X ₆₀ and X ₆₃ is a natural and/or unnatural amino acid residue, where Z ₁ and Z ₂ are 2-30 natural and/or unnatural amino acid residues, and where DBDpp specifically binds the target of interest. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In a particular embodiment, DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO: 50). In a further embodiment, DBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues replaced in SEQ ID NO: 1 are replaced by conservative amino acid residue substitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues in SEQ ID NO: 1

- 36 043883 replaced by non-conservative substitutions of amino acid residues. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine. In some embodiments, neither proline nor cysteine is included in the amino acid substitutions. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1-12 solvent accessible amino acid residues are replaced with conservative substitutions and 1-12 solvent accessible amino acid residues are replaced with non-conservative substitutions, or 5-12 solvent accessible amino acid residues are replaced with conservative substitutions and 5-12 solvent accessible amino acid residues residues are replaced by non-conservative substitutions. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In a further embodiment, a library is provided containing a plurality of DBDpps. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, glass slide, chip, gelatin and agarose.

An isolated DBDpp has also been proposed, which includes the amino acid sequence MGSWX ₅ X ₆ FKX ₉ XioLAXi3lKXi ₆ Xi7LEAL

Z ₁ EAELAAFX30X3 ₁ EIX34AFX ₃₇ X ₃₈ ELX4 ₁ AYZ ₂ NPEVEX ₅₀ LRX ₅₃ X54AAX ₅₇ IRX ₆₀ X ₆₁ LQAYRHN (seq id ^{NO: 10} X ^{where X} 5 ^{, X} 6 ^{, X} 9 ^{, X} 10, ^X 13 ^{, X} 16 ^{, X} 17, ^X 30 ^{, X} 31, ^X 34, ^X 37, ^X 38, X41, ^X 50, ^X 53, ^{X 54, X 57} , X60 ^{and X} 61 is a natural and/or unnatural amino acid residue, where Z ₁ and Z2 are 2-30 natural and/or unnatural amino acid residues, and wherein DBDpp specifically binds a target of interest. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, _Xn is a naturally occurring amino acid residue other than cysteine or proline. In a particular embodiment, DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO: 50). In a further embodiment, DBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are replaced residues in SEQ ID NO: 1 are replaced by conservative amino acid residue substitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues in SEQ ID NO: 1 are replaced by non-conservative amino acid residue substitutions. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine. In some embodiments, neither proline nor cysteine is included in the amino acid substitutions. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1-12 solvent accessible amino acid residues are replaced with conservative substitutions and 1-12 solvent accessible amino acid residues are replaced with non-conservative substitutions, or 5-12 solvent accessible amino acid residues are replaced with conservative substitutions and 5-12 solvent accessible amino acid residues residues are replaced by non-conservative substitutions. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In a further embodiment, a library is provided containing a plurality of DBDpps. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, glass slide, chip, gelatin and agarose.

An isolated DBDpp has also been proposed, which includes the amino acid sequence

MGSWX ₅ EFX ₈ X9RLX ₁₂ AIX15X1 ₆ RLX19AL

Z ₁ EAELAX ₂₈ FEX ₃₁ X ₃₂ IAX ₃₅ FEX ₃₈ X ₃ 9LQX ₄₂ YZ ₂ NPEVEALX ₅₂ X ₅₃ EAX ₅₆ AIX ₅₉ X ₆ oELX ₆₃ AYRHN id

NO: 1 1), where X5, X8, X9, X12, X15, X16, X19, X28, X31, X32, X35, X38, X39, X42, X52, X53, X56, X59, X60 ^and X63 ^is natural and /or a non-natural amino acid residue, where Z ₁ and Z 2 are 2-30 natural and/or non-natural amino acid residues, and where DBDpp specifically binds the target of interest. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In a particular embodiment, DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ ID NO: 50). In a further embodiment, DBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues replaced in SEQ ID NO: 1 are replaced by conservative amino acid residue substitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residues in SEQ ID NO: 1 are replaced by non-conservative amino acid residue substitutions. In some embodiments, the amino acid substitutions do not contain proline. In some embodiments, the amino acid substitutions do not contain cysteine. In some embodiments, neither proline nor cysteine is included in the amino acid substitutions. In some embodiments, the amino acid residue substitutions include no more than one cysteine. In some embodiments, 1-12 solvent accessible amino acid residues are replaced with conservative substitutions and 1-12 solvent accessible amino acid residues are replaced with non-conservative substitutions, or 5-12 solvent accessible amino acid residues are replaced with conservative substitutions and 5-12 solvent accessible amino acid residues residues are replaced by non-conservative substitutions. In another embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, DBDpp specifically binds a protein selected from the group consisting of: immunoglobulin, enzyme, hormone, serum protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a peptide tag. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. In a further embodiment, a library is provided containing a plurality of DBDpps. Nucleic acids encoding DBDpp and vectors containing such nucleic acids are also provided. Host cells (including viral particles) containing nucleic acids and vectors are also provided. In some embodiments, the host cell is a prokaryote or eukaryote that displays DBDpp on its surface. In some embodiments, the host cell displays DBDpp on its surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface. In another embodiment, the host cell is a human immunocyte that expresses a DBDpp fusion protein on its surface. In one embodiment, the DBDpp is attached to a solid support. In another embodiment, the solid support is selected from the group consisting of: sphere, glass slide, chip, gelatin and agarose.

In some embodiments, DBDpp includes a substitution at a corresponding position in the sequence SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17 , E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66 , Q68, A69 and Y70. In further embodiments, DBDpp includes substitutions at at least 1, 5, 10, 15, 20, or 30 of the above positions in the sequence SEQ ID NO: 1. These substitutions may be conservative, non-conservative, or a combination of conservative and non-conservative substitutions. In some embodiments, the substitutions do not include the addition of proline or cysteine. In some embodiments, the substitutions include no more than one cysteine. In some DBDpp, the specified residues may be more than 90% identical to SEQ ID NO: 1. In another DBDpp, the specified residues may be more than 80% identical to SEQ ID NO: 1. In another DBDpp, the specified residues may be more than 70% identical identical to SEQ ID NO: 1. In another DBDpp, the specified residues may be more than 60% identical to SEQ ID NO: 1. In another DBDpp, the specified residues may be more than 50% identical to SEQ ID NO: 1. In another DBDpp, the specified residues may be more than 40% identical to SEQ ID NO: 1. In another DBDpp, the specified residues may be more than 30% identical to SEQ ID NO: 1. In another DBDpp, the specified residues may be more than 20% identical to SEQ ID NO: 1. In another DBDpp, the indicated residues are more than 10% identical to SEQ ID NO: 1.

In some embodiments, DBDpp includes a substitution at a sequence position of SEQ ID NO: 1 selected from the group consisting of: M1, L21, G22, G23, S24, E25, A26, E27, Y45, K46,

- 38 043883

G47, K48, G49, N50, P51, R71, H72 and N73.

Additionally provided herein is a DBDpp in which amino acid residues have been deleted from the N-terminus, the C-terminus, or both the N-terminus and the C-terminus of the corresponding sequence SEQ ID NO: 1. In some embodiments, the DBDpp contains a sequence in which 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues are removed from the N-terminus of the DBDpp sequence corresponding to SEQ ID NO: 1. In some embodiments, DBDpp comprises a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues are deleted from the C-terminus of the DBDpp sequence corresponding to SEQ ID NO: 1. In some embodiments, DBDpp comprises a sequence in in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11 or 12 amino acid residues are deleted from the N-terminus of the corresponding sequence SEQ ID NO: 1, and a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues are deleted from the C-terminus corresponding to SEQ ID NO: 1. In further embodiments, DBDpp comprises a sequence in which 1-5, 1 -10 or 1-15 amino acid residues are deleted from the C-terminus of the sequence corresponding to SEQ ID NO: 1. In some embodiments, DBDpp comprises a sequence in which 1-5, 1-10, or 1-15 amino acid residues are deleted from the N-terminus the sequence corresponding to SEQ ID NO: 1, and a sequence in which 1-5, 1-10 or 1-15 amino acid residues are deleted from the C-terminus of the sequence corresponding to SEQ ID NO: 1.

In some embodiments, DBDpp contains a sequence that differs from the corresponding sequence in reference SEQ ID NO: 1 in 2 or more categories of sequence modifications (ie, substitutions, deletions, insertions, and additions). For example, DBDpp may include combinations of amino acid deletions, insertions and substitutions compared to the corresponding sequence in the reference polypeptide sequence. In some embodiments, DBDpp contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acid deletions in the reference sequence sequence shown in SEQ ID NO: 1. In some embodiments, DBDpp contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 amino acid insertions in the reference sequence shown in SEQ ID NO: 1.

DBDpp binds to targets of interest.

In some embodiments, DBDpp can bind to a target of interest, and in some embodiments, does not significantly affect the function of the target. Alternatively, in several embodiments, DBDpp can bind to a target of interest and completely or partially inhibit, antagonize, agonize, block, increase, stimulate, or disrupt the biological activity of that target. Binding may be identified as agonistic or antagonistic and determined using or by standard modification of assays, bioassays and/or animal models known in the art for assessing such activity.

A DBDpp agonist refers to a DBDpp that in any way increases or enhances the biological activity of a DBDpp target or has biological activity comparable to a known DBDpp target agonist. In another embodiment, DBDpp is an antagonist of the target that it binds. DBDpp antagonist refers to a DBDpp that completely or partially blocks or otherwise impairs the biological activity of a DBDpp target protein, or has biological activity comparable to a known antagonist or inhibitor of a DBDpp target protein.

Expressions such as target binding affinity, target binding, etc. refers to a property of a polypeptide that can be directly measured by determining affinity constants, such as the amount of DBDpp that associates and dissociates at a given antigen concentration. Various methods can be used to characterize molecular interactions, such as, but not limited to, competitive analysis, equilibrium analysis, microcalorimetric analysis, and real-time interaction analysis based on surface plasmon resonance interaction (eg, using a Biacore® instrument). Such methods are known to the person skilled in the art and are described, for example, in Neri D et al. (1996) Tibtech 14:465-470, and Jansson M et al. (1997) J Biol Chem 272:8189–8197.

The affinity requirements for a given DBDpp binding event depend on multiple factors including, but not limited to: the composition and complexity of the binding matrix, the valency and density of the DBDpp and target molecules, and the functional application of the DBDpp. In one embodiment, DBDpp binds a target of interest with a dissociation constant (KD) less than or equal to 5x10'3 M, 10 ^-3 M, ^5x10'4 M, 10 ^-4 M, 5x10'5 M, or 10'5 M. In an additional embodiment In an embodiment, DBDpp binds a target of interest with a KD of less than or equal to 5x10'6 M, ^10'6 M, ^5x10'7 M, ^10'7 M, 5x10'8 M, or ^10'8 M. In further embodiments, DBDpp binds a target of interest target with KD less than or equal to 5x10'9 M, 10' ⁹ M, 5x10' ¹⁰ M, 10' ¹⁰ M, 5x10'11 M, 10' ¹¹ M, 5x10' ¹² M, 10' ¹² M, 5x10' ¹³ M , ^10'13M , ^5x10'14M , ^10'14M , 5x10'15M, or ^10'15M . In several embodiments, DBDpp produced by the methods disclosed herein have a dissociation constant selected from group consisting of 10 ^-4 -10 ^-5 M, 10-5-10 ^-6 M, 10 ^-6 -10 ^-7

M, 10 ^-7 -10 ^-8 M, 10 ^-8 -10 ^-9 M, W ⁹ -W ¹⁰ M, W ¹⁰ -10' ⁿ M and NG-NG M.

In one embodiment, DBDpp binds the target of interest in an active form. In one embodiment, DBDpp reversibly binds a target of interest in an active form and also releases the bound target in an active form. In one embodiment, DBDpp binds the target of interest in its native form. In certain embodiments, DBDpp binds a target of interest with a dissociation rate or Koff greater than or equal to 10 ^-10 s ^-1 , 5x10 ^-9 s ^-1 , 10 ^-9 s ^-1 , 5x10 ^-8 s ^-1 , 10 ^-8 s ^{- 1} , 5x10 ^-7 s ^-1 , 10 ^-7 s ^-1 , 5x10 ^-6 s ^-1 , 10 ^-6 s ^-1 , 5x10 ^-5 s ^-1 , 10 ^-5 s ^-1 , 5x10 ^-4 s ^-1 , 10 ^-4 s ^-1 , 5x10 ^-3 s ^-1 , 10 ^-3 s ^-1 , 5x10 ^-2 s ^-1 , 10 ^-2 s ^-1 , 5x10 ^-1 s ^-1 or 10 ^-1 s ^-1 .

Binding experiments to determine KD and dissociation rate can be performed under many conditions including, but not limited to, [pH 6.0, 0.01% Tween 20], [pH 6.0, 0.1% gelatin], [pH 5.0, 0.01% Tween 20], [pH 9.0, 0.1% Tween 20], [pH 6.0, 15% ethylene glycol, 0.01% Tween 20], [pH 5.0, 15% ethylene glycol, 0.01% Tween 20] and [pH 9.0, 15% ethylene glycol, 0.01% Tween 20]. Buffers in which these solutions can be prepared can be easily determined by one skilled in the art and depend mainly on the required pH of the final solution. Low pH solutions (<pH 5.5) can be prepared, for example, in citrate buffer, glycine-HCl buffer, or succinate buffer. High pH solutions can be prepared, for example, in Tris-HCl, phosphate buffers or sodium bicarbonate buffers. A number of conditions can be used to determine KD and dissociation rate to determine, for example, optimal pH and/or salt concentrations.

In one embodiment, DBDpp specifically binds a target of interest with a K _Off ranging from 0.1 to 10 ^-7 s ^-1 , from 10 ^-2 to 10 ^-7 s ^-1 , or from 0.5x10 ^-2 to 10 ^-7 s ^-1 . In a certain embodiment, DBDpp (eg, a DBDpp fusion protein) binds a target of interest with a dissociation rate (KOff) of less than 5x10 ^-2 s ^-1 , 10 ^-2 s ^-1 , 5x10 ^-3 s ^-1 or 10 ^-3 s ^-1 . In a further embodiment, DBDpp binds a target of interest with a dissociation rate (KOff) of less than 5x10 ^-4 s ^-1 , 10 ^-4 s ^-1 , 5x10 ^-5 s ^-1 or 10 ^-5 s ^-1 , 5x10 ^-6 s ^-1 , 10 ^-6 s ^-1 , 5x10 ^-7 s ^-1 or 10 ^-7 s ^-1 .

In one embodiment, DBDpp specifically binds a target of interest to a K _On ranging from 10 ³ to 10 ⁷ M ^-1 s ^-1 , 10 ³ to 10 ⁶ M ^-1 s ^-1 , or 10 ³ to 10 ⁵ M ^-1 s ^-1 . In other certain embodiments, DBDpp (eg, a DBDpp fusion protein) binds a target of interest, its target of interest, with an association rate (KOn) greater than 10 ³ M ^-1 s ^-1 , 5 x 10 ³ M ^-1 s ^-1 , 10 ⁴ M ^-1 s ^-1 , or 5x10 ⁴ M ^-1 s ^-1 . In an additional embodiment, DBDpp binds the target of interest with a KOn greater than 10 ⁵ M ^-1 s ^-1 , 5x10 ⁵ M ^-1 s ^-1 , 10 ⁶ M ^-1 s ^-1 or 5x10 ⁶ M ^-1 s ^-1 , or 10 ⁷ M ^-1 s ^-1 .

DBDpp targets of interest.

The target of interest specifically bound by DBDpp can be any molecule for which it is desirable to bind to DBDpp. For example, targets specifically bound by DBDpp can be any target of purification, manufacturing, formulation, therapeutic, diagnostic or prognostic relevance or significance. A number of example targets are presented herein by way of example and are intended to be illustrative and non-limiting. The target of interest may be natural or synthetic. The target of interest may be an extracellular component or an intracellular component, a soluble factor (eg, an enzyme, hormone, cytokine and growth factor, toxin, poison, pollutant, etc.) or a transmembrane protein (eg, a cell surface receptor). In one embodiment, the target of interest specifically bound by DBDpp is itself a DBDpp having a different sequence.

In one embodiment, the DBDpp fusion protein specifically binds a target of interest on the surface of a target cell. In another embodiment, the DBDpp fusion protein specifically binds a cell surface receptor. In one embodiment, the DBDpp fusion protein specifically binds a target of interest that is a member of a family selected from: growth factor receptor, receptor tyrosine kinase, TNF family receptor, G protein-coupled receptor, and chemokine receptor. In some embodiments, the DBDpp fusion protein binds multiple members of the same family (eg, TNF receptors TRAILR1 and TRAILR2). In some embodiments, the DBDpp fusion protein binds members of different families. Thus, for example, in some embodiments, a DBDpp fusion protein may bind to a growth factor receptor and a TNF receptor or a G protein-coupled receptor and a chemokine receptor.

In one embodiment, DBDpp specifically binds a serum protein or therapeutic protein, such as an antibody fragment or antibody. In some embodiments, the DBDpp-binding target of interest (eg, a DBDpp fusion protein) is a human protein. In one embodiment, DBDpp (eg, a DBDpp fusion protein) binds a human target protein of interest and a monkey (eg, cynomolgus), mouse, rabbit, hamster, and/or rabbit ortholog thereof.

In one embodiment, DBDpp specifically binds a target of interest, which is a whey protein. In one embodiment, DBDpp specifically binds a serum protein selected from: serum albumin (eg, human serum albumin (HSA)), thyroxine-binding protein, transferrin, fibrinogen, and immunoglobulin (eg, IgG, IgE, and IgM). Without being bound by theory, binding of DBDpp to a carrier protein is believed to impart to DBDpp (or a fusion construct thereof) an improved pharmacodynamic profile, which includes, but is not limited to, improved tumor targeting, tumor penetration, tumor diffusion, and improved therapeutic activity compared to a DBDpp fusion protein that lacks the carrier protein binding sequence (see, for example, WO 01/45746, the contents of which are hereby incorporated by reference in their entirety).

In one embodiment, the target of interest specifically bound by DBDpp is a disease-associated antigen. The antigen may be an antigen characteristic of a cancer and/or a specific cell type (eg, a hyperproliferative cell), and/or a pathogen (eg, a bacterial cell (eg, tuberculosis, smallpox and anthrax), a virus (eg, HIV), a parasite (eg, malaria and leishmaniasis), fungal infection, mold, mycoplasma, prion antigen, or antigen associated with an immune system disorder.

In a further embodiment, the target of interest bound by DBDpp (eg, a DBDpp fusion protein) is a bacterial antigen, viral antigen, fungal antigen, mycoplasma antigen, prion antigen, or parasite antigen (eg, a parasite infecting a mammal). In one embodiment, the target of DBDpp is anthrax, hepatitis B, rabies, Nipah virus, West Nile virus, meningitis virus, or CMV. In a further embodiment, DBDpp specifically binds a pathogen.

In one embodiment, DBDpp specifically binds a cancer target. In another embodiment, DBDpp specifically binds TSA or TAA. In some embodiments, DBDpp specifically binds a target selected from the group consisting of PTGER4, ITGA4, CD37, CD52, CD62L (L-selectin), CXCR4, CD69, EVI2B (CD361), SLC39A8, MICB, LRRC70, CLELC2B, HMHA1, LST1 and CMTM6 (CKLFSF6).

In one embodiment, DBDpp specifically binds CD19 (B-CLL, B-ALL, leukemia, lymphoma, B-NHL/CLL, post-HSCT ALL, B-lymphoid neoplasia, B-cell neoplasia), CD20 (mantle cell lymphoma/indolent B- NHL), PSMA (prostate cancer), CEA (breast cancer, colon and rectal cancer), Her2/neu (lung cancer, osteosarcoma, glioblastoma), kappa light chain (B-NHL and B-CLL).

In one embodiment, DBDpp specifically binds a target selected from the group consisting of CD47, CTLA4, DR5, KIR, LAG3, OX40, PD-L1 and TIM3.

In one embodiment, DBDpp specifically binds a target of interest that is selected from the group consisting of: PDGFRA, PDGFRB, PDGFA, PDGFB, PDGFCC, PDGFC, PDGFD, VEGFR1, VEGFR2, VEGFR3, VEGFC, VEGFD, neuropilin 2 (NRP2), betacellulin , PLGF, RET (rearranged upon transfection), TIE1, TIE2 (TEK), CA125, CD3, CD4, CD7, CD10, CD13, CD19, CD22, CD25, CD30, CD32, CD32b, CD33, CD38, FRSF5 (CD40), CD44 (eg CD44v6), CD47, CD49e (integrin alpha 5), CD52, CD54 (ICAM), CD55, CD64, CD74, CD80, CD90, CD117 (cKit), CD133, CD200, (prominin 1), CD147, CD166 , CD200, ESA, SHH, DHH, IHH, patched 1 (PTCH1), smoothened (SMO), WNT1, WNT2B, WNT3A, WNT4. WNT4A, WNT5A, WNT5B, WNT7B, WNT8A, WNT10A, WNT10B, WNT16B, LKP5, LRP5, LRP6, FZD1, FZD2, FZD4, FZD5, FZD6, FZD7, FZD8, Notch, Notch1, Notch3, Notch4, DLL4, Jagged, Jagged1, Jagged2, Jagged3, TNFSF1 (TNFb, LTa), TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFSF6 (Fas ligand), TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3), TNFSF4 (OX40 ligand), TNFSF5 (CD40 Ligand), TNFSF7 (CD27 Ligand, CD70), TNFRSF7 (CD27), TNFSF8 (CD30 Ligand), TNFSF9 (41BB Ligand), TNFRSF8 (CD30), TNFSF11 (RANKL), TNFRSF10A (TRAILR1, DR4), TNFRSF10B (TRAILR2 , DR5), TNFRSF4 (OX40), TNFRSF11A (RANK), TNFSF12 (TWEAK), TNFRSF12 (TWEAKR), TNFSF13 (APRIL), TNFSF13B (BLYS), TNFRSF 13B (TACI), TNFRSF13C (BAFFR), TNFSF15 (TL1A), TNFRSF17 (BCMA), TNFRSF19L (KELT), TNFRSF19 (TROY), TNFRSF21 (DR6), TNFRSF25 (DR3), ANG1 (ANGPT1), ANG2 (ANGPT2), ANG3 (ANGPTL1), ANG4 (ANGPT4), TIE2, IL1 alpha, IL1 beta, ILIRI, 1L1R2, IL2 IL2R, IL5, IL5R, IL6, IL6R, 1L8, 1L8R, IL10, IL10R, IL12, IL12R, IL13, IL13R, IL15, IL15R, IL18, IL18R, IL19, IL19R, IL21, IL21R, IL23, IL23R, mif XAG1, XAG3, REGIV, FGF1, FGF2, FGF3, FGF4, FGFR1, FGFR2, FGFR3, ALK, ALK1, ALK7, ALCAM, Artemin, Axl, TGFb, TGFb2, TGFb3, TGFBR1, IGFIIR, BMP2, BMP5 , BMP6, BMPRI, GDF3, GDF8, GDF9, N-cadherin, E-cadherin, VE-cadherin, EPCAM (EGP2), NCAM, LI CAM (GDI 71), ganglioside GM2, ganglioside Gd2, calcitonin, PSGR, DCC, CDCP1 , CXCR2, CXCR7, CCR3, CCR4, CCR5, CCR7, CCR10, CXCR4, CXCL1, CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL3, CCL4, CCL5, CCL11, Claudin1, Claudin2, Claudin3, Claudin4, TMEFF2, neuregulin, MCSF , CSF, CSFR (fms), GCSF, GCSFR, BCAM, HPV, hCG, SR1F, PSA, FOLR2 (folate receptor beta), BRCA1, BRCA2, HLA-DR, ABCC3, ABCB5, HM 1.24, LFA1, LYNX,

- 41 043883

S100A8, S100A9, SCF, von Willebrand factor, Lewis Y6 receptor, Lewis Y, CA G250 (CA9), CRYPTO, VLA5, CTLA4, HLA-DR, MUCl, MUCl 8, CanAg mucin, ganglioside GD3, EGFL7, PDGFRa, IL21, IGF1, IGF2, HGF, PSMA, SLAMF7, carcinoembryonic antigen (CEA), FAP, avb3 integrin, α5β integrin, activin B1 alpha, leukotriene B4 receptor (LTB4R), neurotensin NT receptor (NTR), oncofetal antigen 5T4, tenascin C, MMP , MMP2, MMP7, MMP9, MMP12, MMP14, MMP26, cathepsin G, cathepsin H, cathepsin L, SULF1, SULF2, MET, UP A, MHCL MN (CA9), TAG-72, TM4SF1, heparanase (HPSE), syndecan ( SDCl), ephrin B2, ephrin B4, T neuropilin 1 (NrP1), TEM1, mesothelin, TGF-beta 1, TGFBRII, FcRn, phosphatidylserine, folate receptor alpha (FOLR1) and relaxin2. The above targets and targets otherwise described herein are intended to be illustrative and non-limiting.

In one embodiment, DBDpp (e.g., a DBDpp fusion protein) specifically binds a target of interest selected from: VEGF, VEGFA, VEGFR1, VEGFR2, IGF1R, integrin, cMet, EGFR, ErbB2 (Her2), CD20, nerve growth factor (NGR) , hepatocyte growth factor receptor, EbrB3 (Her3), EbrB4, prostate-specific membrane antigen.

In one embodiment, the target of interest specifically bound by DBDpp (eg, a DBDpp fusion protein) is an antigen associated with an autoimmune disorder, inflammatory or other disorder of the immune system, or associated with regulation of the immune response.

In one embodiment, DBDpp specifically binds a target of interest, which is an immunoinhibitory target. In another embodiment, DBDpp specifically binds an immunoinhibitory target selected from: IL1, IL1b, IL1Ra, IL5, IL6, IL6R, CD26L, CD28, CD80, FcRn or Fc-gamma RIIB. In another embodiment, DBDpp specifically binds an immunostimulatory target selected from: CD25, CD28, CTLA4, PD1, B7-H1 (PD-L1), B7-H-4, IL10, TGF-beta, TNFSF4 (OX40 ligand), TNFRSF4 ( OX40), TNFSF5 (CD40 ligand), TNFRSF5 (CD40), TNFSF9 (41BB ligand), TNFRSF9 (41BB, CD137), TNFSF14 (LIGHT, HVEM ligand), TNFRSF14 (HVEM), TNFSF15 (TL1A), TNFRSF25 (DR3), TNFSF18 (GITR ligand) and TNFRSF18 (GITR).

In a further embodiment, DBDpp specifically binds a target of interest selected from: IL1Rb, IL2, IL3, IL4, IL7, IL11, IL15, IL16, IL17, IL17A, IL17F, IL18, IL19, IL25, IL32, IL33, interferon beta, SCF , BCA1/CXCL13, CXCL1, CXCL2, CXCL6, CXCL13, CXCL16, C3AR, C5AR, CXCR1, CXCR2, CCR1, CCR3, CCR7, CCR8, CCR9, CCR10, ChemR23, CCL3, CCL5, CCL11, CCL13, CCL17, CCL18, CCL19 , CCL20, CCL21, CCL22, CCL24, CCL25, CCL26, CCL27, MPL, GP130, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TREM1, TREM2, oncostatin M, lymphotoxin alpha (LTa), integrin beta subunit 7 , CD49a (integrin alpha 1), integrin a5b3, MIF, ESM1, WIF1, cathepsin B, cathepsin D, cathepsin K, cathepsin S, TNFSF2 (TNFa), TNFSF3 (LTb), TNFRSF3 (LTBR), TNFSF6 (Fas ligand), TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3), TNFSF8 (CD30 ligand), TNFRSF8 (CD30), TNFSF11 (RANKL), TNFRSF11A (RANK), TNFRSF16 (NGFR), TNFRSF19L (RELT), TNFRSF19 (TROY), TNFRSF21 ( DR6), CD14, CD23 CD36, CD36L, CD39, CD52, CD91, CD137, CD153, CD164, CD200, CD200R, BTLA, B7-1 (CD80), B7-2 (CD86), B7h, B7-DC (PDL2) , ICOS, ICOSL, MHC, CD, B7-H2, B7-H3, B7x, SLAM, KIM-1, SLAMF2, SLAMF3, SLAMF4, SLAMF5, SLAMF6 and SLAMF7, TNFSF1A (TNF alpha), TNFRSF1A (TNFR1, p55, p60 ), TNFRSF1B (TNFR2), TNFSF7 (ligand CD27, CD70), TNFRSF7 (CD27), TNFSF13B (BLYS), TNFSF13 (APRIL), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFRSF17 (BCMA), TNFSF12 (TWEAK), TNFRSF12 (TWEAKR), TNFRSF5 (CD40), IL1, IL1b, IL1R, IL2R, IL4-Pa, IL5, IL5R, IL6, IL6R, IL9, IL12, IL13, IL14, IL15, IL15R, IL17f, IL17R, IL17Rb, IL17RC, IL20, IL21, IL22RA, IL23, IL23R, IL31, TSLP, TSLPR, interferon alpha, interferon, B7RP1, cKit, GMCSF, GMCSFR, CTLA4, CD2, CD3, CD4, CD11a, CD18, CD20, CD22, CD30, CD40, CD86 , CXCR3, CXCR4, CCR2, CCR4, CCR5, CCR8, CCL2, CXCL10, PlGF, alpha4 integrin subunits, A4B7 integrin, C5, RhD, IgE and Rh.

In another embodiment, DBDpp specifically binds a target of interest selected from: amyloid beta (Abeta), amyloid beta, complement factor D, PLP, ROBO4, ROBO, GDNF, NGF, LINGO, myostatin, oxidized LDL, gpIIB, gpIIIa, PCSK9 , factor VIII, integrin a2bB3, AOC3, mesothelin, DKK1, osteopontin, cathepsin K, TNFRSF19L (RELT), TNFRSF19 (TROY) and sclerostin.

In one embodiment, DBDpp specifically binds a target of interest selected from the group consisting of: CD137, CD47, CTLA4, DR5, KIR, PD-L1, PD1 and TIM3.

In one embodiment, DBDpp specifically binds CD137. In another embodiment, DBDpp specifically binds CD137 and includes an amino acid sequence selected from:

- 42 043883 (a) MGSWVEFGHRLWAIDQRLYALGGSE

AELAAFEKEIAAFESELQAYKGKGNPEVEKLRQRAAFIRRLQAYRHN (SEQ ID NO:

12), (b) MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEH

LRDQAAFIRHKLQAYRHN (SEQ ID NO: 13), (c) MGSWYEFRHRLWAIDQRLYALGGS

EAELAAFEKEIAAFESELQAYKGKGNPEVEGLREAAAFIRAKLQAYRHN (SEQ ID NO:

14), (d) MGSWYEFSMRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEA

LRAKAAYIRWKLQAYRHN (SEQ ID NO: 15), (e) MGSWFEFNHRLWAINERLYALGG

SEAELAAFEKEIAAFESELQAYKGKGNPEVERLRSMAAFIRYKLQAYRHN (SEQ ID NO:

16), (f) MGSWYEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPE

VEYLRETAAHIRTRLQAYRHN (SEQ ID NO: 17), (g) MGSWYEFHYRLHAIDQ

RLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEELRIKAAFIRRDRLQAYRHN (SEQ

ID NO: 18) and (h) MGSWAEFKQRLAAIKTRLEALGGSEAELAAFLGEIWAFEME

LAAYKGKGNPEVEALGREAAAIRMELQAYRHN (SEQ ID NO: 19).

Other DBDpp and polypeptides are provided that bind in whole or in part (eg, epitope overlap) to the same CD137 epitope as the above DBDpp. In addition, DBDpp and polypeptides that compete in whole or in part with the above DBDpp for binding to CD137 are also provided. Nucleic acids encoding DBDpp are also provided, as are vectors containing the nucleic acids, and host cells containing the nucleic acids and vectors.

In one embodiment, DBDpp specifically binds CD47. In another embodiment, DBDpp specifically binds CD47 and includes an amino acid sequence selected from (a) MGSWYEFDLRLHAIYDRLVALGGSEAELAAFEKEIAAFESELQAYKG

KGNPEVEILRDNAAYIRQMLQAYRHN (SEQ ID NO: 20), (b)

MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEHLRDQAAFIR

HKLQAYRHN (SEQ ID NO: 21), (c) MGSWTEFTYRLSAIEWRLWALGGSEAE

LAWFEQKIAFFEDFLQYYKGKGNPEVEALKHEAGAILNELMAYRHN (SEQ ID NO: 22), (d) MGSWAEFDHRLHAIRERLHALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEI

LRGNAAYIRALLQAYRHN (SEQ ID NO: 23) and (e) MGSWTEFVGRLAAIEFRL

WALGGSEAELAWFEAHIAFFEDYLQWYKGKGNPEVEALREEAGAIMEELKAYRHN (SEQ ID NO: 24).

Other DBDpp and polypeptides are provided that bind in whole or in part to the same CD47 epitope as the above DBDpp. In addition, DBDpp and polypeptides that compete in whole or in part with the above DBDpp for binding to CD47 are also provided. Nucleic acids encoding DBDpp are also provided, as are vectors containing the nucleic acids, and host cells containing the nucleic acids and vectors.

In one embodiment, DBDpp specifically binds CTLA4. In another embodiment, DBDpp specifically binds CTLA4 and includes the amino acid sequence MGSWHEFHDRLQAIHERLYALGGSEAELAAFEKEIAAFESELQAY

KGKGNPEVESLRIAAAHIRQVLQAYRHN (SEQ ID NO: 25). Other DBDpp and polypeptides are provided that bind in whole or in part to the same CTLA4 epitope as the above DBDpp. In addition, DBDpp and polypeptides that compete in whole or in part with the above DBDpp for binding to CTLA4 are also provided. Nucleic acids encoding DBDpp are also provided, as are vectors containing the nucleic acids, and host cells containing the nucleic acids and vectors.

In one embodiment, DBDpp specifically binds DR5. In another embodiment, DBDpp specifically binds DR5 and includes an amino acid sequence selected from:

- 43 043883 (a) MGSWNYFKDHLAWIKNSLEALGGSEAELAHFETAIASFERQLQEYK GKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 26), (b)

MGSWLYFKEHLAHIKAWLEALGGSEAELAHFELAIADFEYHLQEYKGKGNPEVEALRKEAAAIR DELQAYRHN (SEQ ID NO: 27), (c) MGSWTEFTYRLSAIEWRL

WALGGSEAELAWFEQKIAFFEDFLQYYKGKGNPEVEALKHEAGAILNELMAYRHN (SEQ ID NO: 28), (d) MGSWFYFKQHLAWIKSYLEALGGSEAELAHFERAIAAFEQHLQMYKGKG

NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 29), (e)

MGSWHYFKDHLAEIKGLLEALGGSEAELAHFEMAIADFEHNLQYYKGKGNPEVEALRKEAAAIR DELQAYRHN (SEQ ID NO: 30), (f) MGSWHYFKGHLAEIKNHLEALG

GSEAELAHFERAIAAFERSLQWYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 31), (g) MGSWIYFKEHLAYIKKELEALGGSEAELAHFESAIAVFESTLQYYKGKGNPE

VEALRKEAAAIRDELQAYRHN (SEQ ID NO: 32), (h) MGSWTYFKEHLAEIKYM

LEALGGSEAELAHFEVAIADFEKMLQYYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 33) and (i) MGSWWLFKDHLAEIKTALEALGGSEAELAHFEMAIAAFEKQLQYYKG KGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 34) .

Other DBDpp and polypeptides are provided that bind in whole or in part to the same DR5 epitope as the above DBDpp. In addition, DBDpp and polypeptides that compete in whole or in part with the above DBDpp for binding to DR5 are also provided. Nucleic acids encoding DBDpp are also provided, as are vectors containing the nucleic acids, and host cells containing the nucleic acids and vectors.

In one embodiment, DBDpp specifically binds KIR. In another embodiment, DBDpp specifically binds KIR and includes an amino acid sequence selected from:

(a) MGSWSEFYNRLDAIES RL LAL GGSEAELALFEIQIARFEKVL QAYK

GKGNPEVEALRGEARAIFAELYAYRHN (SEQ ID NO: 35), (b) MGSWYEFYNRLYAIE IRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVERLRVRAAKIRVILQAYRHN (SEQ ID NO: 36) and (c) MGSWLWFKIFLAEIKYFLEALGGSEAELAAFDFEIHAFHVELFA YK GKGNPEVEVLREVAAEIRWDLQAYRHN (SEQ ID NO: 37) .

Other DBDpp and polypeptides are provided that bind in whole or in part to the same KIR epitope as the above DBDpp. In addition, DBDpp and polypeptides that compete in whole or in part with the above DBDpp for binding to KIR are also provided. Nucleic acids encoding DBDpp are also provided, as are vectors containing the nucleic acids, and host cells containing the nucleic acids and vectors.

In one embodiment, DBDpp specifically binds PD-L1. In another embodiment, DBDpp specifically binds PD-L1 and includes an amino acid sequence selected from:

(a) MGSWTEFQSRLDAIHSRLLRALGGSEA ELAAFEKEIAAFESELQAYKGKGNPEVELLRDDAAFIRHFLQAYRHN (SEQ ID NO: 38), (b) MGSWQEFDDRLNAIKARLQALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEDLRDDA AFIRRFLQAYRHN (SEQ ID NO: 39), (c) MG SWYEFQNRLHAIHERLNAL

GGSEAELAAFEKEIAAFESELQAYKGKGNPEVELLRDDAAFIRHFLQAYRHN (SEQ ID NO: 40), (d) MGSWFEFQDRLTAINERLSALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVET

LRSDAAFIRRFLQAYRHN (SEQ ID NO: 41), (e) MGSWYEFESRLDAIHERLHALGGS EAELAAFEKEIAAFESELQAYKGKGNPEVENLRGDAAFIRHFLQAYRHN (SEQ ID NO: 42), (f) MGSWYEFNHRLDAISKRLNALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEE

LRGDAAFIRHFLQAYRHN (SEQ ID NO: 43) and (g) MGSWFEFENRLHAIVHRLG ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVETLRADAAFIRHYLQAYRHN (SEQ ID NO: 44).

Other DBDpp and polypeptides are provided that bind in whole or in part to the same PD-L1 epitope as the above DBDpp. In addition, DBDpp and polypeptides that compete in whole or in part with DBDpp for binding to PD-L1 are also proposed. Nucleic acids encoding DBDpp are also provided, as are vectors containing nucleic acids, and host cells containing nucleic acids and vectors.

- 44 043883

In one embodiment, DBDpp specifically binds PD1. In another embodiment, DBDpp specifically binds PD1 and includes an amino acid sequence selected from:

(a) MGSWTIFKEWLAFIKTDLEALGGSEAELAFFEGWIASFEMELQKYK

IGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 46), (b) MGSWVMFKWLLAD

IKSHLEALGGSEAELAFFEGFIAAFETHLQVYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 47) and (c) MGSWYAFKDYLADIKGWLEALGGSEAELAFFEIFIARFELE

LQAYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 48).

Other DBDpp and polypeptides are provided that bind in whole or in part to the same PD1 epitope as the above DBDpp. In addition, DBDpp and polypeptides that compete in whole or in part with the above DBDpp for binding to PD1 are also provided. Nucleic acids encoding DBDpp are also provided, as are vectors containing the nucleic acids, and host cells containing the nucleic acids and vectors.

In one embodiment, DBDpp specifically binds TIM3. In another embodiment, DBDpp specifically binds TIM3 and includes the amino acid sequence MGSWHEFHDRLQAIHERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVESLRIAAAHIR QVLQAYRHN (SEQ ID NO: 45). Other DBDpp and polypeptides have been proposed that bind in whole or in part to the same TIM3 epitope as the above DBDpp. In addition, DBDpp and polypeptides that compete in whole or in part with DBDpp for binding to TIM3 have also been proposed. Nucleic acids encoding DBDpp are also provided, as are vectors containing the nucleic acids, and host cells containing the nucleic acids and vectors.

In another embodiment, DBDpp binds a peptide tag present on a target of interest. Such peptide tags provide a useful means by which targets of interest containing such peptide tags can be purified, detected and/or attached. In one embodiment, DBDpp specifically binds a peptide tag selected from the group of a hexahistidyl (His6) tag, a myc tag, or a FLAG tag. Other peptide tags are described in the present invention or otherwise known in the art.

In another embodiment, the target to which DBDpp binds is a purification target from a mixture of contaminants. In one embodiment, the target may be a naturally occurring or recombinantly expressed protein that requires selective isolation from a cell lysate or cell culture supernatant.

DBDpp fusion proteins.

A fusion polypeptide, fusion protein, chimeric polypeptide, chimeric protein, chimeric antigen is a polypeptide consisting of at least two polypeptides and, optionally, a linker for operably linking the two polypeptides into one contiguous polypeptide, produced, for example, by recombinant methods. The two polypeptides may be operably linked, directly or indirectly.

A DBDpp fusion protein includes at least one DBDpp that specifically binds a target of interest. In one embodiment, DBDpp fusion proteins include more than one DBDpp, where two or more DBDpps have the same or different specificities. In additional embodiments, the DBDpp fusion protein consists of a tandem repeat of the same or different DBDpps, which allows the DBDpp fusion protein to bind multiple targets and/or repeated epitopes, or different epitopes on a single target. In additional embodiments, the DBDpp fusion protein includes DBDpp and a polypeptide sequence comprising an additional domain. In some embodiments, the DBDpp fusion protein includes DBDpp and a component selected from: an antibody, an antibody fragment (e.g., an antigen binding domain or part thereof (e.g., ScFv), an effector domain or part thereof, an FcRn binding domain or part thereof, and an Fc or part thereof part), whey protein (e.g., protein or part thereof), cytokine, growth factor, hormone, imaging agent, tag, and peptide tag. In some embodiments, the DBDpp fusion protein includes an immunoglobulin Fc domain (eg, a human Fc domain) or a portion thereof. In other embodiments, the Fc domain is a variant of the human Fc domain.

DBDpp as provided herein includes DBDpp fusion proteins. DBDpp and any polypeptide of interest may be operably linked to form a DBDpp fusion protein. Thus, in some embodiments, DBDpp is included in a larger multidomain molecular complex (eg, a monomeric or multimeric DBDpp fusion protein) and thereby imparts the functional attributes of the included DBDpp to the resulting fusion protein. In some embodiments, the DBDpp fusion proteins comprise DBDpp and a polypeptide sequence from an antibody, an antibody fragment, a serum protein (e.g., human serum albumin), or a serum protein fragment, or a cell receptor

- 45 043883 surface, T-cell receptor (TCR) alpha chain, T-cell receptor beta chain, cytokine, growth factor, hormone or enzyme, or a fragment thereof. Incorporation of DBDs into multidomain and/or multifunctional complexes can be readily accomplished by recombinant fusion with another polypeptide, coupling with another chemical, and covalent chemical coupling with another polypeptide (or other desired chemical) using methods known in the art. DBDpp fusion proteins may further contain other optional components, such as linkers and other components described in the present invention.

DBDpp multimers.

In some embodiments, the DBDpp fusion protein comprises one DBDpp. In some embodiments, the DBDpp fusion protein includes at least 2, 3, 4, or 5, or more than 5 DBDpp. In some embodiments, the DBDpp fusion protein contains 1-3, 1-4, 1-5, or more than 5 different DBDpps. In some embodiments, the DBDpp fusion protein contains at least 2, 3, 4, or 5, or more than 5 different DBDpps. Thus, the DBDpp fusion protein can be a monomeric DBDpp (ie, containing one DBDpp) or a multimeric DBDpp (ie, containing more than one DBDpp in tandem, not necessarily operably linked by a linker). Non-limiting embodiments of such a multimeric DBDpp are shown in FIG. 5A. In several embodiments, the use of multimeric DBDpp provides improved (eg, synergistic) target binding. In additional embodiments, the multimeric DBDpp provides targeted interaction with more than one target using a single DBDpp construct (eg, bi-, tri-specific, etc.).

A multimeric DBDpp fusion protein may be a DBDpp homo-multimeric (i.e., containing more than one identical DBDpp in tandem, optionally linked by linker(s) (e.g., homodimers, homotrimers, homotetramers, etc.)) or a hetero-multimeric DBDpp ( i.e., containing two or more DBDpps, among which at least two different DBDpp proteins are present). The amount of monomeric DBDpp included in the multimer composition may vary depending on the embodiment and may be determined, at least in part, by the expression system in which the DBDpp is produced. In several embodiments, however, the fusion proteins may comprise multimers of about 5 to about 10 DBDpp subunits, about 10 to about 15 subunits, about 15 to about 20 subunits, about 20 to about 25 subunits, or about 25 to approximately 30 subunits (including numbers in between as well as cutoffs). In addition, multiple tandem DBDpp fusion protein components may contain the same or different DBDpps. In some DBDpp fusion constructs, DBDpp is present as a monomer or homomultimers or heteromers, such as homodimers or heterodimers, homotrimers or heterotrimers, homotetramers or heterotetramers.

In one embodiment, two or more DBDpp are operably fused to form a DBDpp fusion protein. In one embodiment, the merge partner of the DBDpp is an identical DBDpp. The linkage of two or more identical DBDpps results in a multivalent molecule, which provides distinct advantages (eg, increased binding avidity, target clustering, and receptor activation) over monomer compositions. In another embodiment, the merge partner of DBDpp is a non-identical DBDpp. The binding of two or more non-identical DBDpps results in a multivalent and multispecific molecule that has the potential to bind more than one target antigen, independently or simultaneously.

The DBDpp fusion protein can be monospecific or multispecific. A DBDpp fusion protein that is multispecific (e.g., bispecific, trispecific, or more multispecific) recognizes and binds to two or more different epitopes present on one or more different molecules (e.g., proteins, solid scaffolds, etc.). ).

In one embodiment, the multispecific DBDpp fusion protein contains at least two DBDpps that bind to at least two different epitopes on a single target of interest. In additional embodiments, the multispecific DBDpp fusion protein includes at least one DBDpp that specifically binds one epitope on a target of interest, and at least one other function-conferring domain or sequence (e.g., an antibody fragment or domain, such as scFv), that specifically bind to a different epitope on the same target of interest. In one embodiment, a multispecific DBDpp fusion protein includes at least one DBDpp that specifically binds to an epitope on a target of interest, and at least one function-conferring domain or sequence, such as an antibody fragment or domain (e.g., scFv), that bind specifically to an epitope on another target of interest. In other embodiments, the DBDpp fusion protein includes at least one DBDpp and at least one other DBDpp or domain sequence conferring a function, such as an antibody fragment or domain, that specifically binds to a solid support.

- 46 043883

In another embodiment, a multimeric DBDpp fusion construct comprising 2 or more DBDpps is in turn fused to other heterologous proteins (or subdomains thereof) and thereby imparts multivalent and multispecific properties to the fusion partner. Examples of DBDpp fusion partners include, but are not limited to, antibodies, antibody subdomains (e.g., scFv or Fc domains), serum albumin, serum albumin subdomains, cell surface receptors, T cell receptor (TCR) alpha chain, T beta chain -cell receptor, cell surface receptor subdomains, peptides, peptide tags (eg, FLAG or myc), fibronectin type III repeats, z-domains, elastin-like polypeptides. The number and arrangement of DBDpp and their corresponding positions in the fusion protein may vary. For example, DBDpp may be located at one or all ends of the fusion partner and/or distributed among heterologous subunits in the DBDpp fusion partner.

In one embodiment, the DBDpp fusion construct is bispecific and specifically binds to two different targets expressed on the surface of two different cell types. In one embodiment, the bispecific DBDpp fusion protein specifically binds to a target cancer cell and a target immune effector cell. In one embodiment, the DBDpp bispecific fusion protein specifically binds a target expressed on a cancer cell (eg, CD19) and a target expressed on the surface of a T lymphocyte (eg, CD3).

DBDpp as fusion constructs with antibodies and antibody fragments.

In one embodiment, the DBDpp fusion protein includes a whole antibody or antibody fragment or domain (eg, IgG1 antibody, IgG3 antibody, antibody variable region, CDR3, ScFv, Fc, FcRn binding domain, and other antibody domains). DBDpp and DBDpp fusion proteins can be operably linked to each other and/or to one or more ends of an antibody, antibody chain, antibody fragment, or antibody domain.

The immunoglobulin component of the DBDpp fusion protein can be any suitable whole immunoglobulin or antibody fragment (eg, antigen binding domain and/or effector domain) or fragment thereof. In one embodiment, the DBDpp-antibody fusion protein retains the structural and functional properties of a conventional monoclonal antibody. Thus, in some embodiments, the DBDpp-antibody fusion protein retains epitope-binding properties but advantageously also includes, through the DBDpp fusion, one or more additional target-binding specificities. Antibodies that can be used in DBDpp fusion constructs include, but are not limited to, monoclonal, multispecific, human, humanized, primatized, and chimeric antibodies. The immunoglobulin molecules or antibodies provided by the present invention may be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of the molecule immunoglobulin. In certain embodiments, the antibodies are Fc optimized antibodies. Antibodies can originate or be obtained from any animal source, including birds and mammals, or are artificially produced. The immunoglobulin component of a DBDpp-antibody fusion protein may be natural or the result of recombinant engineering (eg, phage display, XenoMouse, and synthetic). In some embodiments, the immunoglobulin component of the antibody-DBDpp fusion construct increases the half-life, and increases or decreases antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) activity. In some embodiments, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken. In certain embodiments, the antibodies are human.

In one embodiment, DBDpp is operably linked to an antibody fragment or subdomain (e.g., ScFv, diabody, EP 404,097; WO 93/111161; WO 2014/028776; and Holliger et al., PNAS 90:6444-6448 (1993), each of which are hereby incorporated by reference in their entirety). An antibody fragment or subdomain can be any fragment or domain of an antibody. See, for example, WO 04/058820, WO 99/42077 and WO 05/017148, each of which is hereby incorporated by reference in its entirety. For example, a DBDpp fusion protein may comprise an antibody effector domain or a derivative of an antibody effector domain that confer one or more effector functions on DBDpp and/or confer on the DBDpp fusion protein the ability to bind to one or more Fc receptors. In some embodiments, the DBDpp-antibody fusion protein comprises an antigen-binding antibody fragment or fragment thereof. In additional embodiments, the DBDpp-antibody fusion protein comprises an immunoglobulin effector domain that includes one or more CH2 and/or CH3 domains of the antibody, and has effector function provided by the CH2 and CH3 domains. Other sequences in the DBDpp fusion that provide effector function and which are encompassed by the present invention will be apparent to those skilled in the art and can be readily selected and incorporated into the DBDpp fusion protein included herein depending on the desired effector function(s).

- 47 043883

In one embodiment, the immunoglobulin antibody component of the antibody-DBDpp fusion construct provided herein has been modified to enhance antibody-dependent cellular cytotoxicity (ADCC) (see, e.g., Bruhns et al., Blood 113:3716-3725 (2009); Shields et al., J. Biol. Chem. 276:6591-6604 (2001); Lazar et al., PNAS 103:4005-4010 (2006); Stavenhagen et al., Cancer Res., 67:8882-8890 ( 2007); Horton et al., Cancer Res. 68:8049-8057 (2008); Zalevsky et al., Blood 113:3735-3743 (2009); Bruckheimer, Neoplasia 11:509-517 (2009); WO2006/0201 14; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and WO2004/074455, each of which is hereby incorporated by reference in its entirety). Examples of genetically engineered Fc sequence modifications contained in the immunoglobulin component of DBDpp-antibody fusion proteins that increase ADCC include one or more modifications corresponding to the following: IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I, P396L; where the numbering of residues in the Fc region corresponds to the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition).

In one embodiment, the DBDpp fusion construct comprises a whole antibody or antibody fragment that is an antigen binding fragment. In another embodiment, the antibody or antibody fragment binds an antigen associated with a disease. In one embodiment, the DBDpp fusion protein includes an antibody or antibody fragment that specifically binds a cancer antigen. In another embodiment, the DBDpp fusion protein includes an antibody or antibody fragment that specifically binds an antigen of a particular pathogen (e.g., a bacterial cell (e.g., tuberculosis, smallpox, anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria, leishmaniasis), fungal infection, mold, mycoplasma, prion. In another embodiment, the DBDpp fusion protein includes an antibody or antibody fragment that specifically binds an antigen of a particular pathogen (e.g., a bacterial cell (e.g., tuberculosis, smallpox, anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria, leishmaniasis), fungal infection, mold, mycoplasma or prion. In another embodiment, the DBDpp fusion protein includes an antibody or antibody fragment that specifically binds an antigen associated with a disease or disorder of the immune system.

In preferred embodiments, the DBDpp fusion protein comprising an antibody fragment or domain retains the activities of the parent antibody. Thus, in some embodiments, a DBDpp fusion protein comprising an antibody fragment or domain is capable of inducing complement-dependent cytotoxicity. In some embodiments, a DBDpp fusion protein comprising an antibody fragment or domain is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).

Thus, in some embodiments, the DBDpp fusion protein includes an antibody fragment that imparts the biological or biochemical property of an immunoglobulin to the DBDpp fusion protein. In some embodiments, the antibody fragment imparts a property selected from: the ability to non-covalently dimerize, the ability to localize to a tumor site, and an increased serum half-life compared to a DBDpp fusion protein in which the one or more DBDpps have been deleted. In some embodiments, the DBDpp fusion protein is at least as stable as the corresponding antibody without the DBDpp attached. In some embodiments, the DBDpp fusion protein is more stable than the corresponding antibody without the DBDpp attached. The stability of the DBDpp fusion protein can be measured using known methods, including, for example, ELISA techniques. In some embodiments, the DBDpp fusion protein is stable in whole blood (in vivo or ex vivo) at 37°C for at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 48 hours, at least about 50 hours , at least about 55 hours, at least about 60 hours, at least about 65 hours, at least about 70 hours, at least about 72 hours, at least about 75 hours, at least about 80 hours, at least about 85 hours, at least about 90 hours, at least about 95 hours, or at least about 100 hours (including any time therebetween). In one embodiment, the DBDpp fusion construct comprises an immunoglobulin effector domain or half-life domain that corresponds to an immunoglobulin domain or fragment in which at least a portion of one or more constant region domains has been altered to obtain the desired biochemical properties, such as decreased or increased effector functions, ability to non-covalently dimerize, increased ability to localize to a tumor site, decreased half-life in serum, or increased half-life in serum, compared to an immunoglobulin fragment having a corresponding unchanged immunoglobulin sequence. Such constant domain changes

- 48 043883 of the region may be amino acid substitutions, insertions or deletions.

In one embodiment, the DBDpp fusion protein includes an immunoglobulin effector domain amino acid sequence or an immunoglobulin effector domain derivative that confers antibody-dependent cellular cytotoxicity (ADCC) to the DBDpp fusion protein. In additional embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain sequence that has been altered to increase ADCC (see, e.g., Bruhns, Blood 113:3716-3725 (2009); Shields, J. Biol. Chem. 276:6591- 6604 (2001); Lazar, PNAS 103:40054010 (2006); Stavenhagen, Cancer Res. 67:8882-8890 (2007); Horton, Cancer Res. 68:8049-8057 (2008); Zalevsky, Blood 113:3735- 3743 (2009); Bruckheimer, Neoplasia 11:509-517 (2009); WO 06/020114; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and WO 04/074455, the contents of each of which are hereby incorporated by reference in its entirety). Examples of genetically engineered modifications of immunoglobulin fragments contained in the amino acid sequence of a DBDpp fusion protein that increase ADCC include immunoglobulin effector domain sequences containing one or more modifications corresponding to the following: IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I, P396L; where the numbering of residues in the Fc region corresponds to the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).

In other embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain sequence that has been altered to reduce ADCC (see, e.g., Idusogie et al., J. Immunol. 166:2571-2575 (2001); Sazinsky et al., PNAS 105:20167-20172 (2008); Davis et al., J. Rheumatol. 34:2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23:403-411 (1993); Alegre et al. ., Transplantation 57:1537-1543 (1994); Xu et al., Cell Immunol. 200:16-26 (2000); Cole et al., Transplantation 68:563-571 (1999); Hutchins et al., PNAS 92:11980-11984 (1995); Reddy et al., J. Immunol. 164:1925-1933 (2000); WO 97/11971; WO 07/106585; US 2007/0148167A1; McEarchern et al., Blood 109: 1185-1192 (2007); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Kumagai et al., J. Clin. Pharmacol. 47:1489-1497 (2007), the contents of each of which are hereby incorporated by reference in its entirety). Examples of genetically engineered sequence modifications of immunoglobulin fragments contained in the amino acid sequence of the DBDpp fusion protein that reduce ADCC include immunoglobulin effector domain sequences containing one or more modifications corresponding to the following: IgG1-K326W, E333S; IgG2-E333S; IgG1-N297A; IgG1-L234A, L235A; IgG2V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E; IgG2-118-260; IgG4-261-447; IgG2H268Q, V309L, A330S, A331S; IgG1-C220S, C226S, C229S, P238S; IgG1-C226S, C229S, E233P, L234V, L235A; or IgG1-L234F, L235E, P331S; where the residue numbering corresponds to the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).

In additional embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain amino acid sequence or an immunoglobulin effector domain derivative that confers antibody-dependent cellular phagocytosis (ADCP) to the DBDpp fusion protein. In additional embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain sequence that has been altered to enhance antibody-dependent cellular phagocytosis (ADCP); (See, for example, Shields et al., J. Biol. Chem. 276:6591-6604 (2001); Lazar et al., PNAS 103:4005-4010 (2006); Stavenhagen et al., Cancer Res., 67:8882-8890 (2007), Richards et al., Mol. Cancer Ther. 7:2517-2527 (2008), Horton et al., Cancer Res. 68:8049-8057 (2008), Zalevsky et al., Blood 113:3735-3743 (2009); Bruckheimer et al., Neoplasia 11:509-517 (2009); WO 06/020114; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and WO 04 /074455, the contents of each of which are hereby incorporated by reference in their entirety). Examples of genetically engineered modifications of immunoglobulin fragments contained in the amino acid sequence of a DBDpp fusion protein that increase ADCP include immunoglobulin effector domain sequences containing one or more modifications corresponding to the following: IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; IgG1-F243L, R292P, Y300L, V305I, P396L; and IgG1-G236A, S239D, I332E; where the residue numbering corresponds to the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).

In other embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain sequence that has been altered to reduce ADCP (see, e.g., Sazinsky et al., PNAS 105:20167-20172 (2008); Davis et al., J. Rheumatol. 34:2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23:403-411 (1993); Alegre et al., Transplantation 57:1537-1543 (1994); Xu et al., Cell Immunol. 200:1620 (2000); Cole et al., Transplantation 68:563-571 (1999); Hutchins et al., PNAS 92:11980-11984 (1995); Reddy et al., J. Immunol. 164: 1925-1933 (2000); WO 97/11971; WO 07/106585; US 2007/0148167A1; McEarchern

- 49 043883 et al., Blood 109:1185-1192 (2007); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Kumagai et al., J. Clin. Pharmacol. 47:1489-1497 (2007), the contents of each of which are hereby incorporated by reference in their entirety). As an example, DBDpp fusion proteins may contain an antibody fragment or domain that contains one or more of the following modifications that reduce ADCC: IgG1N297A; IgG1-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E; IgG2-EU sequence 118-260; IgG4-EU sequence 261-447; IgG2-H268Q, V309L, A330S, A331S; IgG1-C220S, C226S, C229S, P238S; IgG1-C226S, C229S, E233P, L234V, L235A; and IgG1L234F, L235E, P331S; where the residue numbering corresponds to the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).

In further embodiments, the DBDpp fusion protein comprises an immunoglobulin effector domain amino acid sequence or an immunoglobulin effector domain derivative that confers complement dependent cytotoxicity (CDC) to the DBDpp fusion protein. In additional embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain sequence that has been altered to enhance complement-dependent cytotoxicity (CDC) (see, e.g., Idusogie et al., J. Immunol. 166:2571-2575 (2001); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Natsume et al., Cancer Res. 68:3863-3872 (2008), the contents of each of which are hereby incorporated by reference in their entirety). As an example, DBDpp fusion proteins may contain an antibody fragment or domain that contains one or more of the following modifications that increase CDC: IgG1-K326A, E333A; IgG1-K326W, E333S, IgG2-E333S; where the residue numbering corresponds to the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).

In additional embodiments, the DBDpp fusion protein comprises an immunoglobulin effector domain amino acid sequence or an immunoglobulin effector domain derivative that confers the DBDpp fusion construct with the ability to bind the Fc-gamma-RIIb receptor. In additional embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain sequence that has been altered to increase inhibitory binding to the Fc-gamma-RIIb receptor (see, e.g., Chu et al., Mol. Immunol. 45:3926-3933 (2008 )). An example of genetic engineering modification of immunoglobulin fragments contained in the amino acid sequence in the DBDpp fusion protein, which increase binding to the inhibitory Fc-gamma-RIIb receptor, are IgG1-S267E, L328F.

In other embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain sequence that has been altered to reduce CDC (see, e.g., WO 97/11971; WO 07/106585; US 2007/0148167A1; McEarchern et al., Blood 109:1185 -1192 (2007); Hayden Ledbetter et al., Clin. Cancer 15:2739-2746 (2009); Lazar et al., PNAS 103:4005-4010 (2006); Bruckheimer et al., Neoplasia 11:509-517 ( 2009); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Sazinsky et al., PNAS 105:20167-20172 (2008); the contents of each are hereby incorporated by reference in their entirety). As an example, DBDpp fusion proteins may contain an antibody fragment or domain that contains one or more of the following modifications that reduce CDC: IgG1-S239D, A330L, I332E; IgG2-118-260; IgG4-261-447; IgG2-H268Q, V309L, A330S, A331S; IgG1-C226S, C229S, E233P, L234V, L235A; IgG1-L234F, L235E, P331S; and IgG1-C226S, P230S; where the residue numbering corresponds to the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).

The half-life of IgG is mediated by its pH-dependent binding to the neonatal FcRn receptor. In some embodiments, the DBDpp fusion protein comprises an immunoglobulin effector domain amino acid sequence or an immunoglobulin effector domain derivative that confers the neonatal FcRn receptor binding ability of the DBDpp fusion construct. In some embodiments, the DBDpp fusion protein includes an immunoglobulin FcRn binding domain sequence that has been altered to increase binding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18:1759-1769 (2006); Dall' Acqua et al., J. Immunol. 169:5171-5180 (2002); Oganesyan et al., Mol. Immunol. 46:1750-1755 (2009); Dall'Acqua et al., J. Biol. Chem. 281 :23514-23524 (2006), Hinton et al., J. Immunol. 176:346-356 (2006); Datta-Mannan et al., Drug Metab. Dispos. 35:86-94 (2007); Datta-Mannan. et al., J. Biol. Chem. 282:1709-1717 (2007); WO 06/130834; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Yeung et al., J. Immunol. 182:7663-7671 (2009), the contents of each of which are hereby incorporated by reference in their entirety).

In additional embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain sequence that has been altered to confer selective affinity for FcRn at pH 6.0 but not at pH 7.4. As an example, DBDpp fusion proteins may contain an antibody fragment or domain that contains one or more of the following modifications that increase the half-life: IgG1-M252Y, S254T, T256E; IgG1-T250Q, M428L; IgG1H433K, N434Y; IgG1-N434A; and IgG1-T307A, E380A, N434A; where the residue numbering corresponds to the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition,

- 50 043883 incorporated into this application by reference).

In other embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain sequence that has been altered to reduce binding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18:1759-1769 (2006); Datta-Mannan et al. al., Drug Metab. Dispos. 35:86-94 (2007); Datta-Mannan et al., J. Biol. Chem. 282:1709-1717 (2007); Strohl, Curr. Op. Biotechnol. 20:685 -691 (2009); and Vaccaro et al., Nat. Biotechnol. 23:1283-1288 (2005), the contents of each of which are hereby incorporated by reference in their entirety). As an example, DBDpp fusion proteins may contain an antibody fragment or domain that contains one or more of the following modifications that reduce the half-life: IgG1-M252Y, S254T, T256E; H433K, N434F, 436H; IgG1-I253A; and IgG1-P257I, N434H and D376V, N434H; where the residue numbering corresponds to the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).

In another embodiment, the DBDpp fusion protein includes an amino acid sequence corresponding to an immunoglobulin effector domain that has been altered to include at least one substitution in its sequence corresponding to a position in the Fc region (e.g., Fc gamma) selected from the group consisting of : 238, 239, 246, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292 , 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337 , 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 and 439, where the numbering of residues in the Fc region follows the EU numbering system Kabata et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference). In a certain embodiment, the DBDpp fusion protein comprises an immunoglobulin effector domain-derived sequence, wherein at least one residue corresponding to position 434 is a residue selected from the group consisting of: A, W, Y, F, and H. In another embodiment, the fusion the DBDpp protein includes an immunoglobulin effector fragment derivative sequence having the following corresponding substitutions S298A/E333A/K334A. In a further embodiment, the DBDpp fusion protein includes an immunoglobulin effector domain derivative containing a substitution corresponding to K322A. In another embodiment, the DBDpp fusion protein includes an immunoglobulin effector domain-derived sequence containing one or any combination of the following substitutions K246H, H268D, E283L, S324G, S239D, and I332E. In yet another embodiment, the DBDpp fusion protein includes an immunoglobulin effector domain derivative sequence containing substitutions corresponding to D265A/N297A.

In some embodiments, the DBDpp fusion protein includes an immunoglobulin effector domain sequence that has been glycoengineered or mutated to enhance effector function using methods known in the art. For example, inactivation (via point mutations or other means) of the constant region domain sequence contained in DBDpp can reduce Fc receptor binding of the circulating DBDpp fusion protein, thereby increasing tumor localization. In other cases, it may be the case that constant region modifications consistent with some embodiments of the present invention limit complement fixation and thereby reduce the serum half-life and nonspecific association of the conjugated cytotoxin. Other constant region modifications can be used to modify disulfide bonds or oligosaccharide groups, which provides improved localization due to increased antigen specificity or flexibility of the antibody. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor localization, biodistribution and serum half-life, can be easily measured and quantified using known immunological methods without undue experimentation.

In some embodiments, the immune effector cell includes a cell surface receptor for an immunoglobulin or other peptide binding molecule, such as an immunoglobulin constant region receptor, and includes a class of receptors commonly referred to as Fc receptors (FcRs). Many FcR receptors have been structurally and/or functionally characterized and are known in the art, including FcRs that have the specific ability to interact with a limited subpopulation of immunoglobulin heavy chain isotypes, or that interact with Fc domains with variable affinities, and/or that can be expressed on limited subpopulations of effector immune cells under certain conditions (eg, Kijimoto Ochichai et al., Cell Mol. Life. Sci. 59:648 (2002); Davis et al., Curr. Top. Microbiol. Immunol. 266:85 (2002); Pawankar, Curr. Opin. Allerg. Clin. Immunol. 1:3 (2001); Radaev et al., Mol. Immunol. 38:1073 (2002); Wurzburg et al., Mol. Immunol. 38:1063 (2002) ; Sulica et al., Int. Rev. Immunol. 20:371 (2001); Underhill et al., Ann. Rev. Immunol. 20:825 (2002); Coggeshall, Curr. Dir. Autoimm. 5:1 (2002 ); Mimura et al., Adv. Exp. Med. Biol. 495:49 (2001); Baumann et al., Adv. Exp. Med. Biol. 495:219 (2001); Santoso et al., Ital. Heart J. 2:811

- 51 043883 (2001); Novak et al., Curr. Opin. Immunol. 13:721 (2001); Fossati et al., Eur. J. Clin. Invest. 31:821 (2001)), each of which is incorporated herein by reference in its entirety.

Cells that are capable of mediating ADCC are examples of effector immune cells. Other effector immune cells include NK cells, tumor-infiltrating T lymphocytes (TILs), cytotoxic T lymphocytes, and granulocytic cells, such as cells that trigger allergic response mechanisms. Effector immune cells thus include, but are not limited to, cells of hematopoietic origin, including cells at various stages of differentiation into the myeloid and lymphoid lineages, and which may (but should not) express one or more types of functional cell surface FcRs, such such as T lymphocytes, B lymphocytes, NK cells, monocytes, macrophages, dendritic cells, neutrophils, basophils, eosinophils, mast cells, platelets, red blood cells and progenitors, progenitor cells (for example, hematopoietic stem cells), as well as resting, activated and mature forms of such cells. Other effector immune cells may include cells of non-hematopoietic origin that are capable of mediating immune functions, such as endothelial cells, keratinocytes, fibroblasts, osteoclasts, epithelial cells and other cells. Effector immune cells may also include cells that mediate cytotoxic or cytostatic events, or endocytotic, phagocytotic or pinocytotic events, or that induce apoptosis or microbial immunity, or neutralization of microbial infection, or cells that mediate allergic, inflammatory, hypersensitivity and/or reactions. or autoimmune reactions.

DBDpp as albumin fusion constructs.

Nucleic acid molecules encoding DBDpp-albumin fusion proteins are also included herein, as are vectors containing such nucleic acids, host cells containing such nucleic acid vectors, and methods for producing DBDpp-albumin fusion proteins and using such nucleic acids , vectors and/or host cells. The invention also covers dosage forms comprising a DBDpp-albumin fusion protein and a pharmaceutically acceptable diluent or carrier. Such dosage forms may be used in methods of treating, preventing, reducing the severity of, or diagnosing a disease or disease symptom in a patient, preferably a mammal, most preferably a human, comprising the step of administering the dosage form to the patient.

DBDpp as chimeric receptors.

In addition to incorporating DBDs into soluble multidomain proteins, the present invention provides methods by which a cell-associated DBDpp can be generated consisting of at least one DBDpp designed to impart binding specificity to a membrane-bound fusion protein. DBDpp receptors can be expressed by any cell type.

In one embodiment, the DBDpp-receptor fusion protein includes a chimeric antigen receptor (CAR) or DBDpp-CAR consisting of the following elements: an extracellular targeting domain, a transmembrane domain, and a cytoplasmic domain, wherein the cytoplasmic domain includes a signaling domain. In another embodiment, the DBDpp-CAR consists of an extracellular targeting domain and a transmembrane domain. In another embodiment, a DBDpp-CAR consists of an extracellular domain consisting of one or more DBDpps, where each DBDpp constitutes a target-specific binding domain with the same or different specificities. In several embodiments, the target-specific domain is directed against one (or more) cancer or tumor antigens disclosed herein, such as CD123, CD137, PD-L1, CD19, CD22, NY-ESO, or MAGE A3, as non-limiting examples . In one embodiment, the intracellular domain (eg, cytoplasmic domain) of DBDpp-CAR includes an intracellular domain of the CD3 zeta chain. In another embodiment, the DBDpp intracellular signaling domain consists of a portion of the CD3 zeta chain intracellular domain. In another embodiment, the intracellular domain of DBDpp-CAR includes a CD3 zeta chain intracellular domain and a co-stimulatory signaling region. The co-stimulatory signaling region refers to the portion of DBDpp-CAR constituting all or part of the intracellular domain of the co-stimulatory molecule. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for an effective lymphocyte response against an antigen. Costimulatory molecules and portions of such molecules that are capable of imparting costimulatory properties to a CAR receptor are known in the art and can be incorporated into a DBDppCAR in a standard manner. In addition, truncations or mutations in such intracellular signaling and co-stimulatory domains can be included to further enhance or reduce receptor signaling. In preferred embodiments, the T cell is genetically modified to stably express DBDpp-CAR. In such embodiments, a DBDpp-CAR cytoplasmic domain may be engineered to include a CD28 and/or 4-1BB signaling domain alone or in combination with any other desired cytoplasmic domain(s) useful within the scope of the present invention. In one embodiment, the cytoplasmic domain of DBDpp-CAR may be engineered to further include a CD3-g3ema signaling domain. For example, as schematically shown in FIG. 5B, in one embodiment, the DBDpp-CAR includes an extracellular targeting domain, an extracellular protein linker with a transmembrane domain that spans the cell membrane (such as that found in T cells or NK cells), and a cytoplasmic domain, optionally including a plurality of signaling modules. In several embodiments, the DBDpp-CAR may also include an epitope tag. In several embodiments, the cytoplasmic domain of DBDpp-CAR may include, but is not limited to, CD3-zeta, 4-1BB and CD28 signaling modules and combinations thereof.

Extracellular domain.

Depending on the desired antigen to be targeted, a DBDpp-CAR can be designed comprising a suitable antigen-binding DBDpp that is specific for the desired target antigen. For example, if CD19 is the desired antigen to be targeted, one or more CD19-binding DBDpps may be included in the target-specific DBDpp-CAR binding domain. Alternatively, a DBDpp-CAR may include more than one DBDpp, conferring multispecificity or multivalency to the DBDppCAR.

The choice of DBDpp included in the extracellular domain of a DBDpp receptor (eg, DBDpp-CAR) depends on the identity of the cell or cells that will serve as targets. For example, DBDpp-CAR can specifically bind to cell surface proteins, such as a receptor on the same cell or another cell. In other embodiments, the DBDpp-CAR specifically binds to a soluble molecule, such as an immunoglobulin. In other embodiments, targets of interest bound by DBDpp-CAR include targets associated with viral, bacterial and parasitic infections, diseases and disorders of the immune system (eg, autoimmune disease).

In other embodiments, a DBDpp-CAR may be selected that recognizes a ligand that acts as a cell surface marker on cancer-associated target cells. In some embodiments, the DBDpp-CAR can specifically interact with and bind a tumor antigen (eg, TAA or other tumor antigen described in the present invention or otherwise known in the art). Thus, the present disclosure provides methods for generating DBDpp-CARs, their use in generating chimeric cells such as human T cells and NK cells, and the use of such chimeric T cells in adoptive immunotherapy.

As used herein, tumor antigen refers to antigens that are characteristic of hyperproliferative disorders such as cancer. Tumor antigens that DBDpp in DBDpp-CAR can specifically bind are disclosed herein. In one embodiment, the DBDpp in the DBDpp-CAR specifically binds a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSA is unique to tumor cells and is not present on other cells in the body. The TAA associated antigen is not unique to the tumor cell and is instead also expressed on the normal cell under conditions that do not induce a state of immunological tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. TAA antigens may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at extremely low levels on normal cells but that are expressed at much higher levels on tumor cells. Non-limiting examples of TSA or TAA antigens that DBDpp in DBDpp-CAR can specifically bind include an element selected from the following: differentiation antigen such as MART1/MelanA (MARTI), gp100 (Pmel 17), tyrosinase, TRP1, TRP2; tumor-specific antigen of mixed lineage, such as MAGE1, MAGE3, BAGE, GAGE1, GAGE2, pi5; an overexpressed fetal antigen such as CEA; and an overexpressed oncogene or mutant tumor suppressor gene such as p53, Ras, HER-2/neu; a unique tumor antigen resulting from a chromosomal translocation, such as BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; viral antigen such as Epstein-Barr virus EBVA antigens and human papillomavirus (HPV) E6 and E7 antigens; TSP-180, MAGE4, MAGE5, MAGE6, RAGE, NY-ESO, pl85erbB2, pl80erbB3, cmet, nm-23Hl, PSA, TAG72, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, beta catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4 (791Tgp72) alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA 15-3\CA 27.29\BCAA, CA195, CA242, CA50, CAM43, CD68\I, CO-029, FGF5, G250, Ga733VEpCAM, HTgp-175, M344, MA50, MG7-Ag, MOV 18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA90\Mac-2 , TAAL6, TAG72, TLP and TPS; glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, MCSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2/neu, survivin and telomere- 53 043883 for, tumor prostate cacinoma antigen-1 (PCTA1), MAGE, ELF2M, neutrophil elastase, ephrinB2, TACI (CD267), BAFF-R (CD268), BCMA (Cd269), TLR4, insulin-like growth factor (IGF) I, IGFII, IGFI receptor and mesothelin.

In a specific embodiment, the DBDpp in the antigen binding portion of the DBDpp-CAR specifically binds a target selected from: CD123, HVEM, BTLA, DR3, CD19, CD20, CD22, ROR 1, mesothelin, CD33/lL3Ra, cMet, PSMA, glycolipid F77, EGFRvDI , GD2, MY-ESO-1TCR, CD133, CD47 and MAGE A3 TCR. In another preferred embodiment, the DBDpp in the antigen binding portion of the DBDpp-CAR specifically binds all immunoglobulin classes or specific isotypes, allotypes or idiotypes.

In one embodiment, the DBDpp in the DBDpp-CAR specifically binds a tumor antigen associated with a cancer. Malignant tumors express a variety of tumor antigens, which the engineered DBDpp-CAR can bind. In one embodiment, DBDpp DBDpp-CAR binds to an antigen selected from: tissue-specific antigen such as MART-1, tyrosinase and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate specific antigen (PSA) in prostate cancer ; a transformation-associated molecule such as the HER2/Neu ErbB2 oncogene; oncofetal antigen such as carcinoembryonic antigen (CEA); B-cell lymphoma-specific immunoglobulin idiotype; B cell differentiation antigen such as CD19, CD20 and CD37; TSLPR and IL-7R on myeloid cells and testicular cancer (CT) antigens (eg, NY-ESO-1, LAGE-1a), CS-1, CD38, CD138, MUC1, HM1.24, CYP1B1, SP17, PRAME, tumors Wilms 1 (WT1) and heat shock protein gp96 on many myeloma cells.

Transmembrane domain.

A transmembrane domain (TMD) as used herein refers to the region of a cell surface expressed DBDpp fusion protein, such as DBDpp-CAR, that spans the plasma membrane. In some embodiments, the DBDpp-CAR transmembrane domain is the transmembrane region of a transmembrane protein (eg, type I transmembrane proteins), an artificial hydrophobic sequence, or a combination thereof. Other transmembrane domains will be apparent to those skilled in the art and may be used in combination with additional embodiments of the invention.

A DBDpp receptor (eg, DBDpp-CAR) can be constructed containing a transmembrane domain that is fused to the extracellular domain of the DBDpp receptor. As described above, fusion of the extracellular and transmembrane domains can be performed with or without a linker. In one embodiment, a transmembrane domain is used that is naturally associated with one of the domains in DBDpp-CAR. In a certain embodiment, the transmembrane domain in DBDppCAR is a CD8 transmembrane domain. In some cases, the transmembrane domain of DBDppCAR includes a CD8 hinge domain. In some embodiments, the transmembrane domain is selected or modified through an amino acid substitution that promotes or inhibits association with other surface membrane proteins.

The transmembrane domain can be obtained from a natural or synthetic source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions for a particular use herein may be derived from (ie may include at least transmembrane region(s)) from an element selected from the group: T cell receptor alpha, beta or zeta chain; CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Alternatively, the transmembrane domain may be synthetic, with the DBDpp-CAR transmembrane domain comprising predominantly hydrophobic residues such as leucine and valine. In other embodiments, the transmembrane domain includes a triplet of phenylalanine, tryptophan, and valine at each end of the synthetic transmembrane domain.

An extracellular spacer domain (ESD) as used herein refers to the hydrophilic region that is located between the antigen-specific targeting region and the transmembrane domain. In some embodiments, the DBDpp-CAR includes an extracellular spacer domain. In other embodiments, the DBDpp-CAR does not include an extracellular spacer domain. Extracellular spacer domains include, but are not limited to, antibody Fc regions or fragments or derivatives thereof, antibody hinge regions or fragments or derivatives thereof, antibody CH2 regions, antibody CH3 regions, artificial spacer sequences, or combinations thereof. Additional examples of extracellular spacer domains include, but are not limited to, the CD8a hinge and artificial spacers derived from polypeptides that may be no larger than, for example, Gly3 or the CH1 and CH3 domains of IgG immunoglobulins (such as human IgG4). In some embodiments, the extracellular spacer domain is any one or more of: (i) the hinge, CH2 and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 region of IgG4, (iv) the hinge region of CD8a, (v ) hinge, CH2 and CH3 region of IgG1, (vi) hinge region of IgG1, or (vi) hinge and CH2 region of IgG1. Other extracellular spacer domains will be apparent to those skilled in the art and may be used in combination with additional embodiments provided by the present invention.

In some embodiments, a short oligo- or polypeptide linker of about 1-100 amino acids in length is used to connect any of the DBDpp-CAR domains. Linkers can be composed of flexible residues such as glycine and serine (or any other amino acid), allowing adjacent protein domains to move freely relative to each other. The amino acid sequence composition of the linker can be selected to minimize the potential immunogenicity of the DBDpp-CAR or DBDpp fusion protein. Longer linkers can be used if it is necessary to ensure that two adjacent domains do not sterically hinder each other. In some embodiments, a fragment of preferably 2 to 10 amino acids in length forms a link between the transmembrane domain and the cytoplasmic signaling domain of DBDpp-CAR. In other embodiments, the linker is 10 to 15 amino acids long, or 15 to 20, or 20 to 30, or 30 to 60, or 60 to 100 amino acids long (or any length in between). In other embodiments, the linker is a glycine-serine doublet sequence. In other embodiments, a hinge region fragment derived from the human T cell surface glycoprotein CD8 alpha chain is used (eg, from amino acid positions 138 to 182 of the CD8 alpha chain; SwissProt accession number P01732). In other embodiments, a fragment of the CD8 hinge region is used that has been further modified by amino acid substitution to improve expression function or immunogenicity. In other embodiments, an extracellular region fragment derived from human CD28 is used. In other embodiments, a fragment of the extracellular region of CD28 is used that has been further modified by amino acid substitution to improve expression function or immunogenicity.

Intracellular domain.

The intracellular signaling domain (ICD), or cytoplasmic domain as used herein, refers to the portion of the DBDpp-CAR that transmits the effector function signal and directs the cell to perform its specialized function. The cytoplasmic domain (ie, intracellular signaling domain) of DBDpp-CAR is responsible for activating at least one of the normal effector functions of an immune cell genetically engineered to express DBDpp-CAR. The term effector function refers to a specialized function of a cell. T cell effector function, for example, includes cytolytic activity and helper activity, including cytokine secretion. Thus, the term intracellular signaling domain refers to the portion of the DBDpp-CAR protein that transmits the effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain corresponding to the natural receptor can generally be used, in many cases it is not necessary to use the entire chain. In cases where a fragment of an intracellular signaling domain is used, such a fragment may be used in place of the intact chain, provided that it conveys a signal to the effector function. Thus, the term intracellular signaling domain is intended to include any fragment of an intracellular signaling domain sufficient to convey a signal to effector function. In one embodiment, the intracellular signaling domain in DBDpp-CAR includes cytoplasmic T cell receptor (TCR) sequences as well as coreceptor sequences that act in concert to initiate signal transduction upon antigen receptor binding, or any derivative or variant of such sequences that have functional ability. Examples of domains that signal effector function include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologs (e.g., η chain, FcsRly and β chains, MB 1 (Iga) chain, B29 chain (Ig), etc.), human CD3 zeta chain, CD3 polypeptides (δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn etc.) and other molecules involved in T cell transduction such as CD2, CD5 and CD28.

It is known that signals generated through the TCR alone are not sufficient to fully activate a T cell, and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two different classes of cytoplasmic signal sequences: sequences that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signal sequences), and sequences that act in an antigen-independent pathway to provide a secondary or co-stimulatory signal (secondary cytoplasmic signal sequences).

Primary cytoplasmic signal sequences regulate the primary activation of the TCR complex by a stimulatory pathway or an inhibitory pathway. Primary cytoplasmic signal sequences that act in a stimulatory pathway may contain signaling motifs that are known as immunoreceptor tyrosine activating motifs (ITAMs).

Examples of ITAMs containing primary cytoplasmic signal sequences that are of particular use in the invention include ITAMs derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b and CD66d . It is particularly preferred that the cytoplasmic signal molecule in the CAR includes a cytoplasmic signal sequence derived from CD3 zeta.

A costimulatory domain (CSD) as used herein refers to the portion of a CAR or DBDpp-CAR that enhances the proliferation, survival and/or development of memory cells. DBDpp-CAR may include one or more co-stimulatory domains. Each costimulatory domain includes a costimulatory domain of any one or more of, for example, members of the TNFR superfamily selected from CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 ( CD1 la/CD18), Lck, TNFR-I, TNFR-II, Fas, CD30 and CD40 or combinations thereof. Other co-stimulatory domains (eg, from other proteins) will be apparent to those skilled in the art and may be used in combination with additional embodiments of the invention.

In a preferred embodiment, the cytoplasmic domain of DBDpp-CAR includes a CD3-zeta signaling domain, alone or combined with any other desired cytoplasmic domain(s) applicable to DBDpp-CAR. For example, the cytoplasmic domain of DBDpp-CAR may include part of the CD3 zeta chain and a co-stimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR including the intracellular domain of the costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or their ligands, that is required for an effective lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD 137), OX40, CD30, Cd4o, PD1, ICOS, lymphocyte function-associated antigen 1 (LFA1), CD2, CD7, LIGHT, NKG2C, B7H3, TIM1 and LAG- 3.

Polypeptide linkers can be located between adjacent DBDpp-CAR elements. For example, linkers can be placed between an adjacent DBDpp, or between a DBDpp and a transmembrane domain, or between a transmembrane domain and a cytoplasmic domain, or between adjacent cytoplasmic domains. The cytoplasmic signal sequences in the cytoplasmic signal portion of DBDpp-CAR may be associated with each other in a random or specific order. An optionally short linker, preferably 2 to 10 amino acids in length, can form the bond. The glycine-serine doublet represents the most suitable linker.

Epitope tag.

In some embodiments, the DBDpp fusion protein includes a peptide epitope tag. In some embodiments, the peptide tag is selected from the group consisting of a hexahistidyl (His6) tag, a myc tag, and a FLAG tag. In additional embodiments, peptide tags include, but are not limited to, AviTag (allows biotinylation of the tag and release using streptavidin), calmomodulin, E-tag, hemagglutinin (HA), S-tag, SBP tag, Softag 1, streptavidin, tetra or polycysteine, V5, VSV and Xpress tag. Additionally, polyhistidyl tags (not 6 residues) can be used. In further embodiments, etc. Covalent peptide tags and protein tags can be used. Covalent peptide tags include, but are not limited to, Isopeptag (covalently binds pilin C protein), Spytag (covalently binds SpyCatcher protein), and Snooptag (covalently binds SnoopCatcher protein). In other additional embodiments, protein tags may optionally be used, including, but not limited to, carboxybiotin transport protein (BCCP), glutathione S-transferase, green fluorescent protein (or other fluorophore), Halo tag, Nus tag, thioredoxin and Fc tags . In other additional embodiments, multiple types of labels may be used. In other further embodiments, no label is used. Any combination of extracellular, transmembrane and intracellular domains disclosed in the present invention may be used depending on the embodiment.

Linkers.

The terms linker and spacer are used interchangeably herein to refer to a peptide or other chemical bond that functions to link otherwise independent functional domains. In one embodiment, the linker in DBDpp is located between DBDpp and another polypeptide component containing an otherwise independent functional domain. Suitable linkers for joining two or more DBDpps to be linked will be apparent to those skilled in the art and generally can be any linker used in the art for linking peptides, proteins or other organic molecules. In certain embodiments, such a linker is suitable for the construction of proteins or polypeptides that are intended for pharmaceutical use.

Suitable linkers for operably linking DBDpp and an additional DBDpp fusion protein component in a single chain amino acid sequence include, but are not limited to, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine/serine linkers, glycine and serine rich linkers, or linkers consisting of

- 56 043883 mainly from polar polypeptide fragments.

In one embodiment, the linker consists of a majority of amino acids selected from glycine, alanine, proline, asparagine, glutamine and lysine. In one embodiment, the linker consists of a majority of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine and lysine. In one embodiment, the DBDpp fusion protein linker consists of one or more amino acids selected from glycine, alanine, proline, asparagine, glutamine and lysine. In one embodiment, the DBDpp fusion protein linker consists of one or more amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine and lysine. In another embodiment, the DBDpp fusion protein linker consists of a majority of amino acids that are sterically hindered. In another embodiment, a linker in which the majority of the amino acids are glycine, serine and/or alanine. In some embodiments, the peptide linker is selected from polyglycines (such as (Gly)5 and (Gly)8, poly(Gly-Ala)) and polyalanines. In some embodiments, the peptide linker comprises the sequence Gl·y-Gl·y-Gl·y-Gl·y-Thr-Gl·y-Gl·y-Gl·y-Gl·y-Ser. In some embodiments, the peptide linker comprises the sequence Gly-Gly-Gly-Gly-Asp-Gly-Gly-Gly-Gly-Ser.

In one embodiment, the DBDpp fusion construct includes DBDpp directly attached (ie, without a linker) to another component of the DBDpp fusion protein. In one embodiment, the DBDpp fused structure includes at least 2, at least 3, at least 4 DBDpps directly attached to another component of the DBDpp fused structure.

In another embodiment, DBDpp may be operably linked to another component of the DBDpp fusion protein via a linker. DBDpp fusion proteins may contain a single linker, multiple linkers, or no linkers. In one embodiment, the DBDpp fusion construct includes DBDpp operably linked to another component of the DBDpp fusion protein via a peptide linker. In one embodiment, the DBDpp fusion construct includes at least 2, 3, 4 or 5 DBDs operably linked to another component of the DBDpp fusion protein via a peptide linker.

Linkers can be of any size or composition as long as they are capable of operably linking DBDpp in a manner that allows DBDpp to bind a target of interest. In some embodiments, the linkers are from about 1 to about 100 amino acids in length, about 1-50 amino acids, about 1-20 amino acids, about 1-15 amino acids, about 1-10 amino acids, about 1-5 amino acids, about 2-20 amino acids , about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. It should be understood that the length, degree of flexibility and/or other properties of the linker(s) may have some effect on the properties of the final polypeptide of the invention, including, but not limited to, affinity, specificity or avidity for the target of interest, or in relation to one or more other target proteins of interest. When two or more linkers are used in DBDpp fusion proteins, such linkers may be the same or different. Within the context and description provided herein, one of ordinary skill in the art will be able to determine in a routine manner the optimal composition and length of the linker for the purpose of functionally linking DBDpp and other components of the DBDpp fusion protein.

The linker may also be a non-peptide linker, such as an alkyl linker or a PEG linker. For example, alkyl linkers such as -NH-(CH2)s-C(0)-, in which s=2-20, can be used. Such alkyl linkers may also be substituted with any sterically unhindering group such as lower alkyl, eg C1C6), lower acyl, halogen (eg Cl, Br), CN, NH2, phenyl, etc. An example of a non-peptide linker is a PEG linker. In some embodiments, the PEGlinker has a molecular weight of about 100-5000 kDa or about 100-500 kDa.

Suitable linkers for chemically crosslinking DBDpp and DBDpp fusion protein components include, but are not limited to, homo-bifunctional chemical crosslinkers such as glutaraldehyde, imidoesters such as dimethyl adipimidate (DMA), dimethyl suberimidate (DMS), and dimethyl pimelimidate (DMP). ) or Nhydroxysuccinimide (NHS) esters such as dithiobis(succinimidylpropionate) (DSP) and dithiobis(sulfosuccinimidylpropionate) (DTSSP). Examples of suitable linkers for joining DBDpp and DBDpp fusion protein components of heterobifunctional cross-linking reagents include, but are not limited to, cross-linkers with one amino-reactive end and a sulfhydryl-reactive group at the other end, or with an NHS ester at one end and an SH-reactive group (e.g., maleimide or pyridyl).

In additional embodiments, one or more linkers in the DBDpp fusion protein is cleavable. Examples of cleavable linkers include, but are not limited to, a peptide sequence recognized by various types of proteases (in vitro or in vivo), such as Tev, thrombin, factor Xa, plasmin (blood proteases), metalloproteases, cathepsins (e.g., GFLG, etc.). ), and proteases present in other corporal compartments.

- 57 043883

In one embodiment, the linker is a cleavable linker that facilitates the release of DBDpp or a cytotoxic agent into the cell. For example, an acid-labile linker (eg, hydrazone), protease-sensitive (eg, peptidase-sensitive) linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari, Can. Res. 52:127-131 (1992); US Pat. No. 5,208,020; US Pat. Pub. No. 20090110753; each of which is incorporated by reference in its entirety) may be used as needed, wherein the covalent bond between DBDpp or the cytotoxic agent and the fusion partner is cleaved intracellularly upon internalization of the composition into the cell. The terms intracellularly cleavable and intracellular cleavage refer to the metabolic process or reaction in a cell with a DBDpp drug conjugate that results in a covalent compound, i.e. the linker bond between DBDpp and the cytotoxic agent, DBDpp and the fusion partner, or between two DBDpps is broken, resulting in free DBDpp and/or the cytotoxic agent dissociated in the cell.

Linker optimization can be assessed using methods described in the present invention and/or otherwise known in the art. In some embodiments, the linkers do not interfere with the ability of DBDpp to bind a target molecule and/or another component of the DBDpp fusion protein, such as an antigen binding domain or antibody fragment.

DBDpp as chemical conjugates.

DBDpp, which promote specific binding to targets of interest, can be chemically conjugated to a number of compounds, such as fluorescent dyes, radioisotopes, chromatographic compositions (eg, spheres, resins, gels, etc.) and chemotherapeutic agents. DBDpp conjugates have applications that include, but are not limited to, purification, diagnostic, analytical, industrial and therapeutic applications.

The characteristic absence of cysteines in the DBD sequence allows for the introduction of unique cysteines for site-specific conjugation purposes.

In some embodiments, DBDpp (eg, a DBDpp fusion protein) contains at least one reactive residue. The reactive residues can be used, for example, as a site for the attachment of conjugates such as chemotherapeutic agents. The reactive residue may be, for example, a cysteine, lysine, or other reactive residue. Thus, a cysteine may be added to DBDpp at the N or C terminus, or within the DBDpp sequence. Cysteine can replace another amino acid in the DBDpp sequence. In addition, lysine may be added to DBDpp at both ends or within the DBDpp sequence, and/or lysine may replace another amino acid in the DBDpp sequence. In one embodiment, the reactive residue (eg, cysteine, lysine, etc.) is located in the DBD loop sequence (eg, Z ₁ and Z ₂ SEQ ID NO: 7-11). In one embodiment, the reactive residue is located between components of the DBDpp fusion construct, for example, in a linker located between DBDpp and another component of the DBDpp fusion protein. A reactive residue (eg, cysteine, lysine, etc.) may also be located in the DBDpp sequence or another component of the DBDpp fusion protein. In one embodiment, DBDpp or a DBDpp fusion protein includes at least one, at least two, at least three reactive residues. In one embodiment, DBDpp, such as a DBDpp fusion protein, includes at least one, at least two, or at least three cysteine residues.

Receiving DBDpp.

The preparation of DBDpp useful in the implementation of the proposed methods can be accomplished using a variety of standard chemical synthesis methods, semisynthetic methods and recombinant DNA techniques known in the art. Also provided is a method for producing DBDpp, alone or as part of a multi-domain fusion protein, in the form of soluble agents and cell-associated proteins.

In several embodiments, the general design of DBDpp production involves obtaining reference protein scaffolds and identifying multiple residues in the scaffolds for the purpose of modification. Depending on the embodiment, reference scaffolds may include a protein structure with one or more alpha-helical regions or other tertiary structure. Once identified, multiple residues can be changed, for example, by amino acid substitution. In some embodiments, the substitution is conservative, while in other embodiments, non-conservative substitutions are made. In some embodiments, a naturally occurring amino acid (e.g., one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine) is replaced in the reference scaffolds at the intended position for modification. In some embodiments, the modifications do not include replacement of cysteine or proline. Once modifications have been made at all specific positions required in a particular embodiment, the resulting modified polypeptides (e.g., DBDpp candidates) can be expressed recombinantly, e.g.

- 58 043883 plasmid, bacteria, phage or other vector (for example, to increase the number of each of the modified polypeptides). The modified polypeptides can then be purified and screened to identify those modified polypeptides that have specific binding to a particular target of interest. In several embodiments, certain modified polypeptides exhibit increased binding specificity to a target of interest compared to a reference scaffold, which in some embodiments may exhibit little or no binding to a given target of interest. In additional embodiments, depending on the target of interest, reference scaffolds may exhibit some interaction (e.g., nonspecific interaction) with the target of interest, while some modified polypeptides will exhibit at least about two-fold, at least about five-fold, at least at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold (or more) increase in binding specificity to the target of interest. Optionally, the reference sequence and/or modified polypeptides (eg, DBDpp) may be deimmunized. For example, residues or motifs that are potentially immunogenic can be identified and modified to reduce or eliminate potential immune responses against DBDpp. Additional detailed information regarding the various options for obtaining, selecting and isolating DBDpp is presented in more detail below.

Recombinant expression of DBDpp.

In some embodiments, the DBDpp, such as a DBDpp fusion protein, is recombinantly produced (ie, produced using recombinant DNA technology). Examples of recombinant methods available for the synthesis of DBDpp fusion proteins include, but are not limited to, polymerase chain reaction (PCR) synthesis, concatemerization, seamless cloning, and recursive directional ligation (RDL) (see, e.g., Meyer et al., Biomacromolecules 3:357-367 (2002), Kurihara et al., Biotechnol. Lett. 27:665-670 (2005), Haider et al., Mol. Pharm. 2:139-150 (2005); and McMillan et al. , 32:3643-3646 (1999), the contents of each of which are hereby incorporated by reference in their entirety).

Also proposed are nucleic acids comprising a polynucleotide sequence encoding DBDpp. Such polynucleotides optionally further include one or more expression control elements. For example, a polynucleotide may include one or more promoters or transcriptional enhancers, ribosome binding sites, transcription termination signals, and polyadenylation signals as expression regulatory elements. The polynucleotide can be inserted into any suitable vector, which can be contained in any suitable host cell for expression.

Expression of nucleic acids encoding DBDpp is typically achieved by operably linking the nucleic acid encoding DBDpp to a promoter in an expression vector. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. Methods known in the art can be used to routinely construct expression vectors containing a nucleic acid sequence encoding DBDpp, together with suitable transcription/translation regulatory signals. Such methods include, but are not limited to, in vitro recombinant DNA technologies, synthesis methods, and in vivo recombination/genetic recombination. Expression of the polynucleotide can be carried out in any suitable host organism known in the art for expression, including, but not limited to, bacterial cells, yeast cells, insect cells, plant cells or mammalian cells. In one embodiment, a nucleic acid sequence encoding DBDpp is operably linked to a suitable promoter sequence such that the nucleic acid sequence is transcribed and/or translated into DBDpp in the host. Promoters useful for expression in E. coli include, but are not limited to, the T7 promoter.

In one embodiment, a vector comprising a DBDpp-encoding nucleic acid is introduced into a host cell (eg, a phagemid) to express DBDpp. The vector may remain episomal or integrated into the chromosome, provided that the insert encoding the therapeutic agent can be transcribed. Vectors can be constructed using standard recombinant DNA technologies. Vectors can be plasmids, phages, cosmids, phagemids, viruses, or any other types known in the art that are used for replication and expression in prokaryotic or eukaryotic cells. One skilled in the art will appreciate that a variety of components known in the art (such as expression regulatory elements) may be included in such vectors, including a variety of transcription signals such as promoters and other sequences that regulate RNA polymerase binding to the promoter. Any promoter

- 59 043883 which is known or demonstrated to be effective in cells in which the vector will be expressed can be used to initiate expression of DBDpp. Suitable promoters may be inducible (eg, regulated) or constitutive. Non-limiting examples of suitable promoters include the SV40 early promoter region, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the HSV-1 (herpes simplex virus 1) thymidine kinase promoter, metallothionein gene regulatory sequences, etc., as well as the following control regions animal transcripts that show tissue specificity and have been used in transgenic animals: the elastase I gene regulatory region, which is active in pancreatic acinar cells; region of insulin gene regulation, which is active in beta cells of the pancreas, region of regulation of mouse mammary tumor virus, which is active in cells of the testes, mammary gland, lymphoid and mast cells, region of regulation of the albumin gene, which is active in the liver, region of regulation of the alpha gene -fetoprotein, which is active in the liver, the regulatory region of the alpha-1 antitrypsin gene, which is active in the liver, the regulatory region of the betaglobin gene, which is active in erythroid cells, the regulatory region of the myelin basic protein gene, which is active in oligodendrocyte cells in the brain, the regulatory region the myosin light chain 2 gene, which is active in skeletal muscle, and the gonadotropin-releasing hormone gene regulatory region, which is active in the hypothalamus. In a specific embodiment, the promoter is an immunoglobulin gene regulatory region that is active in lymphoid cells.

In one embodiment, one or more nucleic acids encoding DBDpp are expressed under the control of a constitutive promoter or, alternatively, a regulated expression system. Suitable regulated expression systems include, but are not limited to, a tetracycline-regulated expression system, an ecdysone-inducible expression system, a LacSwitch expression system, a glucocorticoid-inducible expression system, a heat-inducible promoter system, and a metallothionein metal-inducible expression system. If several different nucleic acids encoding DBDpp are contained in a host cell system, some nucleic acids may be expressed under the control of a constitutive promoter, while others may be expressed under the control of a regulated promoter. Expression levels can be determined using methods known in the art, including Western blotting and Northern blotting.

A variety of host/expression vector systems can be used to express the nucleic acid encoding DBDpp. Vectors containing nucleic acids encoding DBDpp (eg, individual DBD subunits or DBDpp fusion constructs), or parts or fragments thereof, include plasmid vectors, single- and double-stranded phage vectors, and single- and double-stranded RNA or DNA viral vectors. Phage and viral vectors can also be introduced into host cells in the form of packaged or encapsulated virus using known infection and transduction methods. In addition, viral vectors may be replication competent or, alternatively, replication defective. Alternatively, cell-free translation systems can also be used to produce protein using RNAs derived from DNA expression constructs (see, for example, WO86/05807 and WO89/01036; and US Pat. No. 5,122,464, each of which is incorporated herein by reference in its entirety ).

In general, any cell type or cultured cell line can be used to express DBDpp as provided herein. In some embodiments, the parent cell line used to generate the recombinant host cells is a phage, bacterial cell, yeast cell, or mammalian cell. A variety of host/expression vector systems can be used to express the DBDpp fusion protein coding sequence. Mammalian cells can be used as host cell systems transfected with recombinant plasmid DNA or cosmid DNA expression vectors containing the coding sequence of the target of interest and the coding sequence of the fusion polypeptide.

The cells may be primary isolates from organisms (including humans), cultures or cell lines that have been transformed or transgenic in nature. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is a human T cell. In some embodiments, the host cell is obtained from a diseased individual.

Suitable host cells include, but are not limited to, microorganisms such as bacteria (eg, E. coli, B. subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing DBDpp coding sequences; yeast (eg, Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing DBDpp coding sequences; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing DBDpp coding sequences; systems of plant cells infected with recombinant viral expression vectors (for example, color mosaic virus

- 60 043883 cabbage, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg Ti plasmid) containing DBDpp coding sequences. In certain embodiments, mammalian cell systems are used to produce DBDpp. Mammalian cell systems typically use recombinant expression constructs containing promoters derived from the mammalian cell genome (eg, metallothionein promoter) or from mammalian viruses (eg, adenovirus late promoter; vaccinia virus 7.5K promoter).

Prokaryotes useful as host cells for the production of DBDpp, such as a DBDpp fusion protein, include gram-negative or gram-positive organisms such as E. coli and B. subtilis. Expression vectors for use in prokaryotic host cells typically contain one or more phenotypic selectable marker genes (eg, genes encoding proteins that confer antibiotic resistance or confer an autotrophic requirement). Examples of useful expression vectors for prokaryotic organisms include the pKK223-3 (Pharmacia, Uppsala, Sweden), pGEM-1 (Promega, Wis., USA), pET (Novagen, Wis., USA) and pRSET (Invitrogen, Calif., USA) series of vectors. USA) (see, for example, Studier, J. Mol. Biol. 219:37 (1991) and Schoepfer, Gene 124:83 (1993)). Examples of promoter sequences frequently used in expression vectors for prokaryotic host cells include T7, (Rosenberg et al., Gene 56:125-135 (1987)), beta-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615 (1978)); and Goeddel et al., Nature 281:544 (1979)), the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, (1980)) and the tac promoter (Sambrook et al., 1990 , Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

In one embodiment, eukaryotic host cell systems are used, including yeast cells transformed with recombinant yeast expression vectors containing the DBDpp coding sequence, such as the expression systems described in the patent application. US 60/344,169 and WO03/056914 (methods for producing a human-like glycoprotein in a non-human eukaryotic host cell) (the contents of each are incorporated by reference in their entirety). Examples of yeasts that can be used to prepare the compositions of the invention, such as DBD, include yeasts from the genus Saccharomyces, Pichia, Actinomycetes and Kluyveromyces. Yeast vectors typically contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, polyadenylation sequences, transcription termination sequences, and a selectable marker gene. Examples of promoter sequences in yeast expression constructs include metallothionein promoters, 3-phosphoglycerate kinase (Hitzeman, J. Biol. Chem. 255:2073 (1980)) and other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase , phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Additional suitable vectors and promoters for use in yeast expression, as well as yeast transformation techniques, are known in the art. See, for example, Fleer, Gene 107:285-195 (1991) and Hinnen, PnAS 75:1929 (1978).

Insect and plant host cell culture systems can also be used to prepare the compositions of the invention. Such host cell systems include, for example, insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing the DBD coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the DBD coding sequence, including, but not limited to, systems expressions described in US Pat. US 6,815,184; Pat. publ. US 60/365.769 and 60/368.047; and WO2004/057002, WO2004/024927 and WO2003/078614, the contents of each of which are hereby incorporated by reference in their entirety.

In a further embodiment, host cell systems may be used, including animal cell systems infected with recombinant viral expression vectors (eg, adenoviruses, retroviruses, adeno-associated viruses, herpes viruses, lentiviruses), including recombinant cell lines containing multiple copies of DNA encoding DBDpp stably amplified (CHO/dhfr) or stably amplified on double microchromosomes (eg, mouse cell lines). In one embodiment, the vector comprising the polynucleotide(s) encoding DBDpp is polycistronic. Examples of mammalian cells useful for preparing such compositions include 293 cells (eg, 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER cells. C6 (Crucell, Netherlands) VERY, Hela cells, COS cells, MDCK cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HBT2 cells, BT20 cells, T47D cells, CRL7O30 cells, HsS78Bst cells, hybridoma cells and other mammalian cells . Additional examples of mammalian host cells that may be used in isotherapy

- 61 043883 shaving, include, but are not limited to, T cells. Some examples of expression systems and selection methods are described in the following sources and references cited therein: Borth et al., Biotechnol. Bioen. 71(4):266-73 (2000), in Werner et al., Arzneimittelforschung/Drug Res. 48(8):870-80 (1998), Andersen et al., Curr. Op. Biotechnol. 13:117-123 (2002), Chadd et al., Curr. Op, Biotechnol. 12:188-194 (2001), and Giddings, Curr. Op. Biotechnol. 12:450-454 (2001). Additional examples of expression systems and selection methods are described in Logan et al., PNAS 81:355-359 (1984), Birtner et al. Methods Enzymol. 153:51544 (1987)). Transcriptional and translational regulatory sequences for expression vectors in mammalian cells are typically derived from viral genomes. Commonly used promoter sequences and enhancer sequences in mammalian cell expression vectors include those derived from polyoma virus, adenovirus 2, simian virus 40 (SV40) and human cytomegalovirus (HCMV). Examples of commercially available expression vectors for use in mammalian host cells include pCEP4 (Invitrogen) and pcDNA3 (Invitrogen).

Physical methods for introducing a nucleic acid into a host cell (eg, a mammalian host cell) include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and/or exogenous nucleic acids are known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

Biological methods for introducing a polynucleotide of interest into a host cell include the use of RNA and DNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian (eg human) cells. Other viral vectors may be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, Pat. US 5,350,674 and 5,585,362, the contents of each of which are hereby incorporated by reference in their entirety.

Methods for introducing DNA and RNA polynucleotides of interest into a host cell include cell electroporation, where an electric field is applied to the cells to increase the permeability of the cell membrane, allowing chemicals, drugs or polynucleotides to enter the cell. DBDpp-containing DNA or RNA constructs can be introduced into mammalian or prokaryotic cells using electroporation.

In a preferred embodiment, electroporation of cells results in expression of DBDpp-CAR on the surface of T cells, NK cells, NKT cells. Such expression may be transient or stable throughout the life of the cell. Electroporation can be performed using methods known in the art, including the MaxCyte GT® and STX® transfection systems (MaxCyte, Gaithersburg, MD, USA).

Chemical methods for introducing a polynucleotide into a host cell include colloidal dispersions such as macromolecular complexes, nanocapsules, microspheres, spheres and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An example of a colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (eg, an artificial membrane vesicle). In the case where a non-viral delivery system is used, an example of a delivery vehicle is a liposome. The use of lipid dosage forms is being considered for the introduction of nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The lipid-bound nucleic acid may be encapsulated in the aqueous interior of the liposome, distributed throughout the lipid bilayer of the liposome, attached to the liposome through a binding molecule that is associated with both the liposome and the oligonucleotide enclosed in the liposome, complexed with the liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, may be suspended in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Compositions associated with a lipid, lipid/DNA or lipid/expression vector are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, in the form of micelles, or in a collapsed structure. They may also simply be distributed in solution, likely to form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic lipids. For example, lipids include lipid grains that are typically present in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl-phosphatidylcholine (DMPC) can be obtained from Sigma, St. Louis, MO; Dicetyl phosphate (DCP) can be obtained from K&K Laboratories (Plainview, NY); cholesterol (Chol) can be obtained from Calbiochem-Behring; Dimyristoylphosphatidylglycerol (DMPG) and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform/methanol can be stored at approximately -20°C. Chloroform can be used as the only solvent because it evaporates more easily than methanol. Liposome is a general term covering a variety of single and multilamellar lipid carriers.

- 62 043883 bodies formed during the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilamellar liposomes have multiple lipid layers separated by an aqueous environment. They form spontaneously when phospholipids are suspended in excess aqueous solution. Lipid components undergo self-rearrangement before forming closed structures and trap water and solutes between lipid bilayers (Ghosh et al., Glycobiology 5:505-510 (1991)). However, also included are compositions that have structures in solution other than the normal vesicular structure. For example, lipids can adopt a micellar structure or exist simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

Regardless of the method used to introduce exogenous nucleic acids into a host cell, the presence of a recombinant nucleic acid sequence in a host cell can be routinely confirmed using a variety of assays known in the art. Such assays include, for example, molecular biological assays known in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a specific peptide, for example, using immunological methods (ELISA and Western blotting), or assays described in the present invention to identify agents included in the scope of the invention.

Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present or expressed in the recipient organism, tissue, or cell, and that encodes a polypeptide whose expression results in some easily detectable property, such as enzymatic activity. Expression of the reporter gene is analyzed at an appropriate time after introduction of DNA into recipient cells. A non-limiting list of suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein gene (eg, Ui-Tei et al., FEBS Lett. 479:79-82 (2000)). Suitable expression systems are known in the art and can be prepared using known methods or obtained commercially. Typically, the construct with the minimal 5' flanking region showing the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to a reporter gene in a standard manner and used to evaluate agents for the ability to modulate promoter-driven transcription.

A number of selection systems can be used in host-vector systems for expression in mammalian cells, including, but not limited to, the herpes simplex virus thymidine kinase, hypoxanthine guanine phosphoribosyltransferase, and adenine phosphoribosyl transferase genes (Lowy et al., Cell 22:817 (1980)), which can be used in tk-, hgprt- or aprt-cells, respectively. In addition, resistance to antimetabolites can be used as a basis for selection, for example, dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine decarboxylase) and the glutamine synthase system.

Clearing DBDpp.

Once a DBDpp, such as a DBDpp fusion protein, is produced by recombinant expression, it can be purified by any method known in the art for purifying recombinant protein, for example, by chromatography (e.g., ion exchange, affinity and size exclusion column chromatography), centrifugation, differential solubility or any other standard protein purification technique. In additional embodiments, DBDpp are optionally fused to heterologous polypeptide sequences described in the present invention or otherwise known in the art to facilitate purification. In particular, it is contemplated that ligands (e.g., antibodies and other affinity matrices) for DBDpp affinity purification columns and that, optionally, DBDpp or other components of the DBDpp fusion composition that are bound by such ligands are removed from the composition prior to final production of DBDpp using methods known in the art.

Expression of cell-associated DBDpp.

In another embodiment of the invention, the production of DBDpp leads to compositions of cell-associated DBDpp. For example, expression of recombinant vectors that encode DBDpp operably linked to a cell membrane anchor or transmembrane domain has the potential to remain cell associated. Chimeric antigen receptors comprising DBDpp are intentionally cell associated and applied to the cell in which they are expressed. One specific embodiment relates to a strategy of adoptive cell transfer of T cells that have been transduced to express the DBDpp chimeric antigen receptor (CAR). Preferably, the cell can be genetically modified to stably express DBDpp on its surface, acquiring a new target specificity that is MHC independent.

A number of virus-derived vectors can be used in areas where viruses

- 63 043883 is used for transfection and integration into the genome of a mammalian cell. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses. Lentiviral vectors are particularly suitable for long-term gene transfer (eg, adoptive T-cell immunotherapy) because they provide long-term stable transgene integration and propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes. They also have the added benefit of low immunogenicity. Typically, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, suitable restriction sites, and one or more selectable markers (eg, WO 01/96584 and WO 01/29058; and US Pat. No. 6,326,193). Several vector promoter sequences are available for transgene expression. One example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of directing the expression of any polynucleotide sequence operably linked to it at high levels. Another example of a suitable promoter is EF-1a. However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, murine mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter , the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the DBDpp-CAR polypeptide or parts thereof, the expression vector introduced into the cell may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells transfected or infected with viral vectors, in other aspects the selectable marker may be present in a separate DNA fragment and used in the cotransfection procedure. Both the selectable marker and the reporter gene can be flanked by suitable regulatory sequences to allow expression in host cells. Suitable selectable markers include, for example, antibiotic resistance genes such as peo and the like.

Before expansion and genetic modification of T cells according to the invention, a source of T cells is obtained from the subject. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymic tissue, tissue from the site of infection, ascites, pleural effusion, splenic tissue, and tumors. In some embodiments of the present invention, any number of T cell lines available in the art may be used.

A detailed discussion of methods for isolating, culturing, activating and expanding T cells can be found in WO 2012079000, the contents of which are hereby incorporated by reference in their entirety.

Additionally provided is a host cell comprising nucleic acids encoding DBDpp described in the present invention. Also proposed is a composition comprising a nucleic acid sequence encoding DBDpp.

Coexpress as used herein refers to the simultaneous expression of two or more protein coding sequences. The coding sequences may be nucleic acids encoding, for example, a single protein or a chimeric protein in the form of a single polypeptide chain.

Chemical synthesis of DBDpp.

In addition to recombinant methods, the production of DBDpp can also be accomplished using organic chemical synthesis of the desired polypeptide using various liquid phase and solid phase chemical processes known in the art. Various automatic synthesizers are commercially available and can be used according to known protocols. See, for example, Tam et al., J. Am. Chem. Soc 105:6442 (1983); Merrifield, Science 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284; Barany et al., Int. J.Pep. Protein Res., 30:705 739 (1987); Kelley et al. in Genetic Engineering Principles and Methods, Setlow, J. K., ed. Plenum Press, NY. 1990, vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, 1989. One advantage of such techniques is that they allow the introduction of unnatural amino acid residues into the DBDpp sequence.

DBDpp, which are used in the methods of the present invention, can be modified during or after synthesis or translation, for example, through glycosylation, acetylation,

- 64 043883 benzylation, phosphorylation, amidation, pegylation, formylation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to antibody molecule, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation , sulfation, ubiquitinylation, etc. (see, for example, Creighton, Proteins: Structures and Molecular Properties, 2d Ed. (W.H. Freeman and Co., N.Y., 1992); Posttranslational Covalent Modification of Proteins, Johnson, ed. (Academic Press, New York, 1983), pp. 1-12 Seifter, Meth. Enzymol., 182:626646 (1990) Rattan, Ann. NY Acad. Sci., 663:48-62 (1992) In certain embodiments, the peptides are acetylated at the N-terminus and /or amidated at the C-terminus.

Any of a variety of chemical modifications can be accomplished by known methods including, but not limited to, acetylation, formylation, etc. In addition, the derivative may contain one or more non-classical amino acids.

DBDpp populations can be represented by polypeptide libraries.

The DBDpp library is one of many unique DBDpps. The DBDpp vector library refers to a variety of unique nucleic acids encoding DBDpp. Such DBDpp libraries can be used to select and identify sequences that promote binding to certain specified targets.

In one embodiment, the DBDpps are a mixed population or library of different DBDpp molecules. The DBDpp library does not imply any specific limitation on the number of unique polypeptide molecules. The library can contain as few as 3, 5, 6, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75 or 100 unique DBDpp, and can go up to more than 1020 different DBDpp. In some embodiments, the library contains up to approximately 10 ⁴ , 105, 106, 10 ⁷ or 108 unique DBDpp. In other embodiments, the library contains up to approximately 10 ¹² different DBDpp.

In one embodiment, the population of polypeptide variants is based on the sequence of parent residues and variable residues. For example, in SEQ ID NO: 3, variable residues are designated X, where X can be any amino acid residue independent of the identity of any other residue designated X in the sequence. In some embodiments, X may include a missing position (eg, there is no amino acid present at the site). In the framework amino acid sequence, various variable amino acids X can be selected from all 20 natural amino acid residues in such a way that any of the 20 natural amino acid residues can be present at the corresponding position X in one variant or another. The choice of amino acid residue at each position is more or less random, depending on the embodiment. It is also possible to limit the group from which the various variable amino acid residues are selected to 19, 18, 17, 16 or less than 20 natural amino acid residues. For example, in some embodiments, the variable residues are not replaced with cysteine and/or proline. The variability of different positions can be adjusted individually, from one, which means no randomization, to all 20 amino acids. Random introduction of a smaller subset of amino acids can be achieved by careful selection of the deoxyribonucleotide bases introduced, for example, T (A/C)C codons can be introduced to produce a random introduction of serine or tyrosine at a given position in the polypeptide chain. Likewise, codons (T/C/A/G)CC can be introduced to randomly introduce phenylalanine, leucine, alanine and valine at a given position in the polypeptide chain. As will be apparent to one of ordinary skill in the art, many alternatives for deoxyribonucleotide base combinations can be used to produce different combinations of amino acids at a given position in a polypeptide chain. The set of amino acids that may be present at a given position in a polypeptide chain can also be determined by introducing trinucleotides during oligonucleotide synthesis instead of one deoxyribonucleotide base at a time.

A library containing multiple DBDpps is also proposed. In some embodiments, the DBDpp library includes a plurality of different DBDpps that include the amino acid sequence of SEQ ID NO: 1, wherein 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 amino acid residues were modified; and wherein DBDpp specifically binds a target of interest. In some embodiments, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 modified amino acid residues are substitutions. In some embodiments, 5-25, 5-30, 535, 5-40, 5-45, or 5-50 amino acid residues modified are conservative substitutions. In some embodiments, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 amino acid residues modified are non-conservative substitutions. In another embodiment, 515, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 amino acid residue modifications are conservative substitutions, and 5-15, 5-20, 5-25, 5 -30, 5-35, 5-40 or 5-45 amino acid residue modifications are non-conservative substitutions. In additional embodiments, 5-25, 5-30, 5-35, 540, 5-45, 5-50, 5-55, or 5-60 substitutions are made at amino acid residues in SEQ ID NO: 1 selected from the group consisting of from: M1, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22,

- 65 043883

G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72 and N73. In another embodiment, 1-20, 1-30, or 1-40 substitutions are made at amino acid residues in SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69 and Y70. In another embodiment, the library includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind different targets. In another embodiment, the various targets bound by DBDpp in the library are selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a protein target selected from the group consisting of: an immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and protein containing a marker sequence. In another embodiment, the library includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a target disclosed in the present invention. In a further embodiment, the library is a vector library or a host cell library. In a further embodiment, the vector library is a host cell library. In another embodiment, the host cell library includes a plurality of host cells that display DBDpp on their surface. In another embodiment, the host cells are a phage that displays DBDpp on its surface.

In some embodiments, the DBDpp library includes: (a) 3 DBDpps that specifically bind to different targets; (b) 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to the same target; (c) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same epitope of the target of interest; (d) 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to different epitopes of the target; or (e) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that compete for binding to the same target.

A library containing multiple DBDpps is also proposed. In some embodiments, the DBDpp library includes a plurality of different DBDpps that include the amino acid sequence of SEQ ID NO: 1, wherein 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 amino acid residues were modified; and wherein DBDpp specifically binds a target of interest. In some embodiments, there are 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 modified amino acid residue substitutions. In some embodiments, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 modified amino acid residues are conservative substitutions. In some embodiments, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 amino acid residues modified are non-conservative substitutions. In another embodiment, 5-15, 5-20, 5-25, 530, 5-35, 5-40, or 5-45 amino acid residue modifications are conservative substitutions, and 5-15, 5-20, 5-25, 5 -30, 5-35, 5-40 or 5-45 amino acid residue modifications are non-conservative substitutions. In additional embodiments, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 substitutions are made at amino acid residues in SEQ ID NO: I selected from the group , consisting of: M1, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72 and N73. In another embodiment, 1-20, 1-30, or 1-40 substitutions are made at amino acid residues in SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69 and Y70. In another embodiment, the library includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind different targets. In another embodiment, the various targets bound by DBDpp in the library are selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a protein target selected from the group consisting of: an immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and peptide tag protein. In another embodiment, the library includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a target disclosed in the present invention. In a further embodiment, the library is a vector library or a host cell library [including viral particles]. In a further embodiment, the vector library is a host cell library. In another embodiment, the host cell library includes a plurality of host cells that display DBDpp on their surface. In another embodiment, the host cell is a phage that displays DBDpp on its surface.

In some embodiments, the DBDpp library includes: (a) 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10 DBDpps that specifically bind to various targets; (b) 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to the same target; (c) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same epitope of the target of interest; (d) 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to different epitopes of the target; or (e) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that compete for binding to the same target.

In a further embodiment, the DBDpp library contains a plurality of different nucleic acid sequences encoding DBDpp, which include the amino acid sequence of SEQ ID NO: 1, wherein a total of 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55 or 5-60 amino acid residues were modified; and wherein DBDpp specifically binds a target of interest. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 modified amino acid residues encoded by the nucleic acid sequences are substitutions. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 modified amino acid residues are conservative substitutions. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 of the encoded modified amino acid residues are non-conservative substitutions. In another embodiment, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 modifications to the encoded amino acid residues are conservative substitutions, and 5-15, 5-20, 5- 25, 5-30, 5-35, 5-40 or 5-45 modifications of the encoded amino acid residues are non-conservative substitutions. In additional embodiments, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 encoded substitutions are made at amino acid residues in SEQ ID NO: 1 selected from group consisting of: M1, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27 , A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59 , A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72 and N73. In another embodiment, 1-20, 1-30, or 1-40 encoded substitutions are made at amino acid residues in SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10 , A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59 , A61, A62, R64, D65, E66, Q68, A69 and Y70. In another embodiment, the nucleic acids optionally encode a DBDpp, which further includes an amino acid sequence of 1-5, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 residues, corresponding to solvent-inaccessible residues in the amino acid sequence of SEQ ID NO: 1 are replaced, and wherein DBDpp specifically binds a target of interest. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind different targets. In another embodiment, the various targets bound by DBDpp in the library are selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a protein target selected from the group consisting of from: immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and peptide tag protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a target disclosed in the present invention. In a further embodiment, the vector library is contained in host cells (eg, viral particles). In another embodiment, the library includes a plurality of host cells that display DBDpp on their surface. In another embodiment, the host cells are a phage that displays DBDpp on its surface. In some embodiments, the DBDpp library includes: (a) 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10 DBDpps that specifically bind to different targets; (b) 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to the same target; (c) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that specifically bind to the same target epitope; (d) 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to different epitopes of the target; or (e) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences that compete for binding to the same target.

Nucleic acids encoding DBDpp, such as DBDpp fusion proteins, are also provided. In some embodiments, the host cell containing the nucleic acids is a bacterial, yeast, fungal, or mammalian cell. In another embodiment, the host cell

- 67 043883 is an immunocyte. In another embodiment, the host cell is a human immunocyte. In another embodiment, the human immunocyte expresses DBDpp on its cell surface. In certain embodiments, the nucleic acid encodes a DBDpp fusion protein. In another embodiment, the host cell expresses DBDpp as a fusion protein on the cell surface. The present invention also provides vector libraries comprising a plurality of nucleic acids encoding DBDpp.

В одном варианте осуществления векторная библиотека включает множество различных нуклеиновых кислот, кодирующих DBDpp, где кодируемый DBDpp включает аминокислотную последовательность, выбранную из группы, состоящей из: (a) MGSWX ₅ EFX ₈ X9RLX1 ₂ AIX15X ₁₆ RLX19ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX5 ₅ L RX ₅₈ X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO: 4), where X ₅ , X ₈ , X ₉ , X _n , X15, Xi ₆ , X19, x ₅₅ , x 58, x ₅₉ , _{x 62 ,} x ₆₅ and X ₆₆ is natural and/or unnatural amino acid residue; (b) MGSWX ₅ X ₆ FKX9Xi ₀ LAXi ₃ IK X ₁₆ X ₁₇ LEALGGSEAELAX _3O FEX ₃₃ X ₃₄ IAX ₃₇ FEX4oX41LQX44YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 2) where X ₅ , X^, X9, X _w , X13, X ₄₆ , X17, X30, X33, X34, X37, X40, X41 and X44 is a natural and/or unnatural amino acid residue; (c) MGSWAEFKQRLAAIKTRLEALGGSEAELAA FX ₃₂ X ₃₃ EIX ₃₆ AFX ₃₉ X4 ₀ ELX4 ₃ AYKGKGNPEVEALX ₅₇ X ₅₈ EAX ₆₁ AIX ₆ 4X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO: 3) where X32, X33, X36, X39, X4 0, X43, X57, X58, X61, X64, X65 and X68 is a natural and/or unnatural amino acid residue, and; (d) MGSWX ₅ X ₆ FKX9X _1o LAX ₁₃ IKX ₁₆ X ₁₇ LEALGGSEAELAAFX ₃₂ X ₃₃ EIX ₃₆ AFX ₃ 9X4 _O E

LX ₄₃ AYKGKGNPEVEX ₅₅ LRX ₅₈ X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO: 5), where X ₅ , X6, X9, X _w , X13, X16, X17, X32, X33, X36, X39, X40, X43, X55, X58, X59, X62, X65 and X66 is a natural and/or unnatural amino acid residue; and (e) MGSWX5EFX ₈ X9RLX ₁₂ AIX ₁₅ X ₁ gRLX ₁ 9ALGGSEA

ELAX ₃₀ FEX ₃₃ X ₃₄ IAX ₃₇ FEX ₄₀ X ₄₁ LQX ₄₄ YKGKGNPEVEALX ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ^{ID NO:} 6), ^{where X} 5, ^X 8, ^X 9, ^X 12, ^X 15 , ^X 16, ^X 19, ^X 30, ^X 33, ^X 34, ^X 37, ^{X 40, X 41} , X 44, ^X 57, ^X 58, ^X 61, ^X 64, ^X 65 ^{and X 68 is} natural and/ or an unnatural amino acid residue; and wherein DBDpp specifically binds a target of interest. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In a further embodiment, a plurality of vectors in the library encode a DBDpp fusion protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind different targets. In another embodiment, the various targets bound by DBDpp encoded nucleic acids in the library are selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a protein target selected from the group consisting of from: immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and peptide tag protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a target disclosed in the present invention. In a further embodiment, a plurality of vector library vectors are contained in host cells (eg, viral particles such as phage), E. coli, yeast and mammalian cells. In another embodiment, host cells display DBDpp on their surface. In another embodiment, the host cells are a phage that displays DBDpp on its surface. In some embodiments, the vector library includes: (a) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10 DBDpp that specifically bind to various targets; (b) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 DBDpp having different sequences that specifically bind to the same target; (c) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to the same target epitope; (d) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to different epitopes of the target; (e) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 DBDpp having different sequences that compete for binding to the same target; or (f) 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 different nucleic acids encoding the same DBDpp. Host cells containing vectors are also provided.

In one embodiment, the vector library includes a plurality of nucleic acids encoding DBDpp, comprising an amino acid sequence selected from the group consisting of: (a) MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALZ ₁ EAELAAFE

KEIAAFESELQAYZ ₂ NPEVEX _5O LRX ₅₃ X ₅ 4AAX ₅₇ IRX ₆ oX ₆₁ LQAYRHN (SEQ ID NO: 9), where X5, X$, X9,

- 68 043883

_X12 , _X15 , X16, _X19 , _X50 , _X53 , _X54 , _X57 , _X60 and X61 are natural and/or unnatural amino acid residues, and Z1 and Z2 are 2-30 natural and/or unnatural amino acid residues; (b)

MGSWX ₅ X ₆ FKX ₉ X ₁ oL2\X ₁₃ IKX ₁₆ X ₁₇ LEALZ ₁ EAE

LAX ₂₈ FEX ₃₁ X32lAX35FEX3 ₈ X3 ₉ LQX ₄₂ YZ ₂ NPEVEALRKEAAAIRDELQAYRHN (seq id _{No ;} 7), where X5, χ6, X9, X1o, X13, X ₁₆ , X ₁₇ , X ₂₈ , X ₃₁ , X ₃₂ , X ₃₅ , X ₃₈ , X ₃₉ and X ₄₂ are natural and/or unnatural amino acid residues, and Z1 and Z ₂ are 2-30 natural and/or unnatural amino acid residues; (c) MGSWAEFKQRLAAIKTRLEALZjEAELAA

FX ₃₀ X _3l EIX ₃₁ AFX3 ₇ X _3e ELX _l7 lYZ ₂ NPEVEALX ₅₂ X ₅ 3EAX ₅₆ MX ₅₉ X ₆₀ ELX ₆ 3AYRHN (SEQ _ID NO; 8), where X ₃₀ , X ₃₁ , X ₃₄ , X ₃₇ , X ₃₈ , X ₄₁ , X ₅₂ , X ₅₃ , X ₅₆ , X ₅₉ , X ₆₀ and X ₆₃ are natural and/or unnatural amino acid residues, and Z1 and Z2 are 2-30 natural and/or unnatural amino acid residues; (d) MGSMX ₅ X ₆ FKX ₉ X ₁₁₎ IAX ₁ 3lKX ₁₆ X ₁₇ I,EM,Z ₁ EM:

LAAEX ₃ „X ₃₁ EIX ₃₁ AEX ₃₃ X _3B ELX ₁₁ AYZ ₂ NPEVEX ₅₀ LRX ₅ 3X _5< AAX ₅ ,IRX ₆₀ X ₆₁ LQAYRHN (SEQ ID no; 10), ^where X5, X6, X9, X10, X13, X16 , X17, X30, X31, X34, X37, X38, X41, X50, X53, X54, X57, X60 ^{and X} 61 is a natural and/or unnatural amino acid residue, and Z1 and Z2 are 2-30 natural and/or unnatural amino acid residues leftovers; and (e)

MGSWX5EFX ₈ X ₉ RLX1 ₂ AIX1 ₅ X ₁₆ RLX ₁₉ ALZ ₁ EAELAX ₂₈ FEX ₃₁ X ₃₂ IAX35FEX38X39LQX ₄ 2YZ ₂ NP EVEALX ₅ 2X53EAX5 ₆ AIX ₅ 9X6oELX ₆₃ AYRHN (SEQ ID NO; 11), where X5, X^, X9, X12, X15, X1 ₆ , X19, X28, X31, X ₃₂ , X 35, X ₃₈ , X ₃₉ , X ₄₂ , X ₅₂ , X ₅₃ , X _{56 , X 59, X 60 and X 63 is natural and} /or a non-natural amino acid residue, and Z1 and Z ₂ are 2-30 natural and/or non-natural amino acid residues; and wherein DBDpp specifically binds a target of interest. In a further embodiment, _Xn is a naturally occurring amino acid residue. In another embodiment, X _n is a naturally occurring amino acid residue other than cysteine or proline. In a further embodiment, a plurality of vectors in the library encode a DBDpp fusion protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind different targets. In another embodiment, the various targets bound by DBDpp encoded by the nucleic acids in the library are selected from the group consisting of; nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a protein target selected from the group consisting of from; immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and peptide tag protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a target disclosed in the present invention. In a further embodiment, a plurality of vector library vectors are contained in host cells. In another embodiment, host cells (eg, viral particles) display DBDpp on their surface. In another embodiment, the host cells are a phage that displays DBDpp on its surface. In some embodiments, the vector library includes; (a) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 DBDpp, which specifically bind to various targets; (b) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 DBDpp having different sequences that specifically bind to the same target; (c) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to the same target epitope; (d) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to different epitopes of the target; (e) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that compete for binding to the same target; or (f) 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 different nucleic acid sequences encoding the same DBDpp sequence. Host cells containing vectors are also provided.

In some embodiments, 4, 5, 10, or more DBDpp encoded by the nucleic acids in the library specifically bind different targets.

In one embodiment, the vector library includes a plurality of different nucleic acid sequences encoding DBDpp, which include the amino acid sequence of SEQ ID NO; 1, wherein a total of 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55 or 5-60 amino acid residues have been modified; and wherein DBDpp specifically binds a target of interest. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, or 5-60 modified amino acid residues encoded by the nucleic acid sequences are substitutions. In another embodiment, 5-25, 5-30, 5-35, 5-40, 5-45, or 5-50 modified amino acid residues are conservative substitutions. In another embodiment, 5-2, 5-30, 5-35, 5-40, 5-45, or 5-50 of the encoded modified amino acid residues are non-conservative substitutions. In another embodiment, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 modifications to the encoded amino acid residues are conservative substitutions, and 5-15, 5-20, 5- 25, 5-30, 5-35, 5-40 or 5-45 modifications of the encoded amino acid residues are non-conservative substitutions. In additional embodiments, 5-25, 5-30, 5-35, 5-40, 5-45, 550, 5-55, or 5-60 encoded substitutions are made at amino acid residues in SEQ ID NO: 1 selected from the group, consisting of: M1, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29 , A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61 , A62, R64, D65, E66, Q68, A69, Y70, R71, H72 and N73. In another embodiment, 1-20, 1-30, or 1-40 encoded substitutions are made at amino acid residues in SEQ ID NO: 1 selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10 , A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59 , A61, A62, R64, D65, E66, Q68, A69 and Y70. In another embodiment, the nucleic acids optionally encode a DBDpp, which further includes an amino acid sequence of 1-5, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, or 5-45 residues, corresponding to solvent-inaccessible residues in the amino acid sequence of SEQ ID NO: 1 are replaced, and wherein DBDpp specifically binds a target of interest. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind different targets. In another embodiment, the various targets bound by DBDpp in the library are selected from the group consisting of: nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a protein target selected from the group consisting of from: immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and peptide tag protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a target disclosed in the present invention. In a further embodiment, the vector library is contained in host cells (eg, viral particles). In another embodiment, the library includes a plurality of host cells that display DBDpp on their surface. In another embodiment, the host cells are a phage that displays DBDpp on its surface. In some embodiments, the vector library includes: (a) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10 DBDpp that specifically bind to various targets; (b) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 DBDpp having different sequences that specifically bind to the same target; (c) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to the same target epitope; (d) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to different epitopes of the target; or (e) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that compete for binding to the same target; or (f) 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 different nucleic acids encoding the same DBDpp. Host cells containing vectors are also provided.

In some embodiments, the vector library includes: (a) nucleic acids encoding 3 DBDpp that specifically bind to various targets of interest; (b) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to the same target of interest; (c) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to the same epitope of the target of interest; (d) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to different epitopes of the target of interest; (e) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that compete for binding to the same target of interest; or (f) 3 different nucleic acid sequences encoding the same DBDpp sequence.

In one embodiment, the vector library includes a plurality of nucleic acids encoding a DBDpp, including an amino acid selected from the group consisting of: (a) MGSWX5EFX ₈ X ₉ RLX ₁ 2AIX ₁ 5X ₁₆ RLX ₁₉ ALZ ₁ EAELAAFEKEIAAFESELQAYZ ₂ NPEVEX5 ₀ LRX53 X ₅ 4AAX ₅₇ IRX _6O X ₆ 1LQAYRHN (SEQ ID NO: 9), where X5, X8, X9, _Xn , _X15 , _X16 , _X19 , X50, X53, X54, X57, X60 and _X61 is natural and/or non-natural amino acid residue, and Z1 and Z ₂ are 2-30 natural and/or unnatural amino acid residues; (b) MGSWX5X ₆ FKX ₉ X ₁₀ LAX ₁₃ IKX ₁₆ X ₁₇ LEALZ ₁ EAELAX ₂₈ FEX ₃₁ X ₃₂ IAX ₃ 5FEX ₃₈ X ₃₉ LQX ₄₂ YZ ₂ NP

- 70 043883

EVEALRKEAAAIRDELQAYRHN (seq _{w N} o _; 7), _{W e} χ5, χ ₆ , χ ₉ , χ^ χ _η , χ^, χ _π , χ ₂₈ , _Хзь χ ₃₂ , χ ₃₅ , χ ₃₈ , χ ₃₉ and χ ₄₂ is a natural and/or unnatural amino acid residue, and Z ₁ and Z ₂ are 2-30 natural and/or unnatural amino acid residues; (c)

MGSWAEFKQRLAAIKTRLEALZ ₁ EAELAAFX3oX3iEIX34AFX3 ₇ X3 ₈ ELX4 ₁ AYZ ₂ NPEVEALX ₅ 2X5 ₃ EAX ₅₆ AIX ₅₉ X ₆₀ ELX ₆₃ AYRHN (SEQ ID _N O; 8), where χ3ο, χ ₃ ι, χ34, χ37, χ38, ^п, χ ₅ 2, χ ₅ 3, χ ₅ 6, χ ₅ 9, χ60 and χ63 is a natural and/or unnatural amino acid residue, and Z ₁ and Z ₂ are 2-30 natural and/or unnatural amino acid residues; (d)

MGSWX5X ₆ FKX9XioLAXi3lKXi ₆ Xi ₇ LEALZ ₁ EAELAAFX3oX3iEIX34AFX3 ₇ X3 ₈ ELX4 ₁ AYZ ₂ NPEV EX ₅₀ LRX ₅₃ X ₅₄ AAX ₅₇ IRX ₆₀ X ₆ iLQAYRHN (seq id NO; 10), where χ ₅ , χ6, χ ₉ , ^, χ _η , ^χ 16, ^χ 17, χ30, ^χ ₃ 1, χ34, χ ₃₇ , χ _{38, χ 41 ,} χ ₅₀ , χ 53, χ ₅₄ , χ ₅₇ , χ ₆₀ and _{χ 61 is} a natural and/or unnatural amino acid residue com , and Z ₁ and Z ₂ are 2-30 natural and/or unnatural amino acid residues; and (e) MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ A

LZ ₁ EAELAX ₂₈ FEX3 ₁ X32lAX3 ₅ FEX3 ₈ X39LQX42YZ ₂ NPEVEALX ₅₂ X ₅ 3EAX ₅₆ AIX ₅₉ X ₆ oELX ₆₃ AYRHN (SEQ ID ^{NO; 11) where χ} 5, ^χ 8, ^χ 9, ^χ 12, ^χ 15 , ^χ 16, ^χ 19, ^χ 28, ^χ 31, ^χ 32, ^χ 35, ^χ 38, ^χ 39, χ 42, ^χ 52, ^χ 53, ^χ 56, ^χ 59, ^χ 60 ^{and χ 63 is} natural and/or non-natural amino acid residue, and Z1 and Z2 are 2-30 natural and/or unnatural amino acid residues; and wherein DBDpp specifically binds a target of interest. In a further embodiment, _χη is a naturally occurring amino acid residue. In another embodiment, _χη is a naturally occurring amino acid residue other than cysteine or proline. In a further embodiment, a plurality of vectors in the library encode a DBDpp fusion protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind different targets. In another embodiment, the various targets bound by DBDpp encoded by the nucleic acids in the library are selected from the group consisting of; nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a protein target selected from the group consisting of from; immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and peptide tag protein. In another embodiment, the library includes nucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpps that specifically bind a target disclosed in the present invention. In a further embodiment, a plurality of vector library vectors are contained in host cells (including viral particles). In another embodiment, host cells (eg, viral particles) display DBDpp on their surface. In another embodiment, the host cells are a phage that displays DBDpp on its surface. In some embodiments, at least two, three, four, five, or ten DBDpps encoded in the vector library specifically bind different targets. In some embodiments, DBDpp binds a target of interest selected from the group consisting of; nucleic acid, oligosaccharide, peptide, protein, cell surface antigen and small organic molecule. In another embodiment, the DBDpp target of interest is a protein selected from the group consisting of; immunoglobulin, enzyme, hormone, whey protein, cell surface protein, therapeutic protein, TSA, CSA and peptide tag protein. In another embodiment, DBDpp specifically binds a target disclosed in the present invention. A library of host cells (eg, viral particles) containing a vector library is also provided. In some embodiments, the library contains a plurality of host cells (eg, viral particles) that display DBDpp on their surface. In certain embodiments, the host cell is a phage that displays DBDpp on its surface. In some embodiments, the vector library includes; (a) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 DBDpp, which specifically bind to various targets of interest; (b) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to the same target of interest; (c) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to the same epitope of the target of interest; (d) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that specifically bind to different epitopes of the target of interest; (e) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10 or more DBDpp having different sequences that compete for binding to the same target of interest; or (f) 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 different nucleic acid sequences encoding the same DBDpp sequence.

It is contemplated that DBDpp may be modified to produce polypeptides intended for specific applications without departing from the scope of the present invention. Such modifications may include additional amino acids at the N- or C-terminus of DBDpp and/or tags or therapeutics that are chemically conjugated or otherwise linked to DBDpp. The additional amino acid residues discussed above may also constitute one or more domains of a polypeptide with any desired function, such as another binding function or an enzymatic function, or a metal chelating function, or a fluorescent function, or combinations thereof.

Selection, isolation and identification of DBDpp.

Methods for selecting, isolating and identifying DBDpps that specifically bind a target of interest from a plurality of DBDpps, such as DBDpps in a library, are also provided. In one embodiment, a method of screening a DBDpp library for binding to a binding partner includes: (a) obtaining a population exhibiting the DBDpp library; (b) contact of the population with the target of interest under conditions suitable for binding; and (c) identifying those DBDpps that bind to the target. Two exemplary methods for selecting exposed DBDpp include panning and selection in cell-based screens.

In illustrative examples provided herein, DBDpp phage display libraries are generated and screened for DBDpp having desired properties, including the ability to specifically bind a variety of validated therapeutic and diagnostic targets. Representative DBDpp identified in such screens are then analyzed and determined to have the required properties useful, for example, in purification, diagnostic and therapeutic applications.

Display library.

As described in the present invention, substitutions in the reference framework SEQ ID NO: 1 provide a versatile molecular recognition platform. Such a DBDpp can be used in methods for producing DBDpp libraries that can be screened against targets of interest. Such screening methods can be used to identify DBDpp with desired properties, such as the ability to bind a target of interest. The DBDpp population used in selecting a target-specific DBDpp can take various forms and can be, but is not limited to, protein libraries, nucleic acid libraries, vector libraries, and host cell libraries.

Various methods known in the art for producing modifications to nucleic acids can be used to produce (encode) a DBDpp having a modification of one or more amino acid residues relative to another DBDpp and/or reference framework SEQ ID NO: 1. Nucleic acids encoding DBDpp , can be obtained using standard methods in the art, such as chemical synthesis, recombinant methods, and/or obtained from biological sources. The nucleic acid of interest may be placed under the control of one or more elements necessary for its expression in any particular host cell. A variety of host cells are available for propagation of nucleic acids encoding DBDpp, and display methods are known in the art and described in the present invention that can be used in displaying DBDpp on their surface. Display methods include, but are not limited to, phage display, bacterial display, yeast display, ribosomal display, and mRNA display.

In some embodiments, the creation of a (partially) randomized DBDpp library requires (partial) randomization of certain positions in the reference framework sequence SEQ ID NO: 1. In additional embodiments, other reference sequences may be used and modified according to the methods disclosed herein. In one embodiment, a DBDpp library for use in the methods of the present invention is generated using recombinant DNA techniques. In particular, libraries of nucleic acid sequences encoding DBDpp, each of which differs in sequence at certain amino acid positions, can be obtained using site-directed or non-specific mutagenesis of template sequences. Random amino acid residues can be introduced at specific positions in an amino acid sequence using methods known in the art, such as selecting (introducing) NNK' or 'NNS' codons at appropriate positions in the nucleotide sequence encoding the specified amino acid sequence. Methods for producing such libraries are known in the art, and commercial services for creating such libraries are available. The nucleotide(s) defining the corresponding amino acid residues at the desired positions are mutated in various ways, for example, to obtain a library of sequences encoding different DBDpps.

Libraries are optionally generated by selective or random mutation at specific solvent accessible positions in the DBD amino acid sequence.

In some embodiments, the number of amino acid residue positions replaced in the DBDpp libraries of the present invention ranges from 5 to 20

- 72 043883 positions of amino acid residues. Thus, the defined set of substituted amino acid residue positions in the DBDpp library proposed in the present invention includes 520 defined substituted amino acid residue positions such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19 or 20 specific amino acid residue positions replaced. In several embodiments, the replaced amino acid residues are natural or unnatural amino acids. In several embodiments, any of the 20 naturally occurring amino acids may be used. However, in some embodiments, the substitutions do not result in the replacement of any amino acids with cysteine and/or proline.

The DBDpp library may contain any suitable number of different DBDpp sequences. In some embodiments, the DBDpp library contains at least 2, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10000, at least 105, at least 106, at least 107, at least 108, at least 109 or more different DBDpp sequences (eg, DBDpp fusion proteins).

The term substituted amino acid residue position, as applied to a library of different DBDpp sequences, refers to an amino acid residue position at which at least two different types of amino acid residues are located when at least two amino acid sequences of different DBDpps from a DBDpp library are compared to each other.

In one embodiment, the present disclosure covers methods for producing a library (ie, a collection or plurality) of DBDpp that differ from each other in at least one of a defined set of 5-20 substituted amino acid residue positions. Thus, the sequences in the DBDpp library differ from each other by any one or more specific amino acid positions that are contained in a selected, specific, or random set. Thus, the term different sequences or different DBDpp sequences refers to the occurrence of sequence variation or sequence differences at a specific set of amino acid residue positions between two or more DBDpps in a library.

Display media.

A population or library of molecules is displayed on a typical display carrier (eg, phage, E. coli, ribosome), which provides linkage between phenotype and genotype.

In some embodiments, the DBDpp in the library is exposed on the surface of a phage particle, ribosome, bacterium, yeast cell, mammalian cell, or any other suitable (micro)organism to facilitate screening or selection to isolate desired DBDpp sequences having a detectable binding affinity to, or detectable in vitro activity against a target of interest. The main advantage of this technology is the linkage of genotype (i.e., encapsulated DNA encoding the exposed protein) and phenotype (i.e., exposed protein, such as DBDpp proposed in the present invention), which allows affinity-based selection from libraries with millions of or even trillions of polypeptide variants in a relatively simple in vitro assay.

Suitable methods, methods and host organisms for displaying and selecting or screening a library of DBDpp sequences with substitutions or nucleotide sequences encoding such DBDpp sequences with substitutions, and which are applicable to a DBDpp having the desired properties, are known to one skilled in the art. Such methods are described, for example, in Georgiou, Nat. Biotechnol. 15:29-34 (1997); Wittrup, Curr. Opin. Biotechnol. 12:395-399 (2001); Lipovsek and Pluckthun, J Immunol Methods 290:51-67 (2004); Reiersen, Nucl Acids Res, 33:e10, 2005; Levin, Mol BioSyst, 2:49-57 (2006); Bratkovic, Cell. Mol. Life. Sci. 67:749-767 (2010). For example, phage display library and phage display technology may be selected as a method for high-throughput identification of protein-specific binders because it is one of the most efficient and versatile selection methods available (Scott, Science 249:386-390 (1990) ); Bratkovic, Cell. Mol. Life Sci. 67:749-767 (2010))).

In addition, display technology can be used to modify, eg improve, the binding properties of DBDpp. See, for example, Scott, Science 249: 386 (1990); Devlin, Science 249: 404 (1990); Pat. US 5,223,409, 5,733,731, 5,498,530, 5,432,018, 5,338,665, and 5,922,545; WO 96/40987 and WO 98/15833, the contents of each of which are hereby incorporated by reference in their entirety. In phage display peptide libraries, natural and/or non-naturally occurring peptide sequences can be displayed when fused to filamentous phage coat proteins. The exposed peptides can be affinity eluted against the target of interest if desired. The isolated phage can be enriched in successive rounds of affinity purification and reculture. The best binding DBDpp can be sequenced to identify key residues, and mutagenesis libraries can be generated and screened to further optimize the sequence of the best binders. Lowman, Ann. Rev.

Biophys. Biomol. Struct. 26: 401-24 (1997).

Phage display.

A typical phage display protocol involves using a filamentous phage (phagemid) surface expression system, producing phage particles in a bacterial host, where each particle displays the gene product of a gene library as a fusion protein with one of its coat protein types ( proteins gIII or gVIII). The phage particle library undergoes a screening process for binding to an immobilized target molecule ('biopanning'), which includes binding of the phage library to the target, washing steps to remove unbound DBDpp phage, and elution of bound particles. Typically, several rounds of panning are required to select molecules with desired properties, including reamplification of the eluted phage in the bacterial host and selection on the immobilized target.

For example, using phagemid display (Kay et al., Phage Display of Peptides and Proteins. A Laboratory Manual, B.K. Kay et al. 1996), a given DBDpp library can be represented by a collection of phagemids, each encoding a fusion protein that includes a DBDpp library element fused to the minor coat protein pIII. Such phagemids can be introduced into suitable E. coli cells (eg, TG1) by electroporation or other means. Using helper phage infection, a phage (also packaging the phagemid genome) is produced that displays the DBDpp fusion protein. Such a phage can be used to select binding agents against a given target, and the selected phage can be propagated by infection with E. coli TG1 (Stratagene).

Thus, in certain embodiments, DBDpp libraries are provided as a phage library and the binding DBDpp is detected by contacting the phage with a labeled target of interest, and the binding phages are then isolated upon detection or selective isolation of the labeled bound target. In one embodiment, a biotinylated target is used, wherein the phage that produces DBDpp, which specifically binds to the target, is captured on a streptavidin-coated support (eg, magnetic spheres). In some embodiments, the selection steps in methods for producing one or more DBDpps having detectable binding affinity for a target of interest may include (further) enriching a DBDpp library or mixtures of DBDpp libraries into DBDpps having detectable binding affinity for a target of interest, where repeating the steps of contacting the target of interest with a DBDpp library or a mixture of DBDpp libraries (including multiple DBDpps) according to the invention and then identifying in the DBDpp library or mixture of DBDpp libraries contacting the protein one or more DBDpps having a detectable binding affinity for the target of interest target. The step of selecting a DBDpp that has detectable in vitro activity on a target of interest may include: (a) contacting a DBDpp library or a mixture of DBDpp libraries according to the invention with a cytokine or growth factor, or a cytokine or growth factor receptor of interest, and (b) identifying in the DBDpp library or mixture of DBDpp libraries one or more DBDpps having detectable in vitro activity on the target of interest.

In exemplary embodiments disclosed herein in the Examples, phage display methods are used to display and screen DBDpp for the ability to specifically bind a target of interest.

The present disclosure demonstrates that the DBD domain can be displayed and screened on the surface of a phage. Various DBDpp libraries based on the framework of SEQ ID NO: 1 and described in the present invention in the Examples were prepared and subjected to phage display methods to demonstrate the possibility of obtaining DBDpp that specifically binds to various targets of interest including CD137, CD47, CTLA4, DR5 , KIR, PDL1, PD1 and TIM3.

Cellular display.

In some embodiments, library screening methods include a cell surface display system. The cell surface display system may include prokaryotic cells such as Gram(+) cells or eukaryotic cells such as yeast cells. Numerous cell surface display systems are known in the art and can be easily adapted for screening DBDpp libraries. Prokaryotic systems, for example, are described in Francisco et al., PNAS 90:10444-10448 (1993) and Lee et al., Trends Biotechnol 21:45-52 (2003). Eukaryotic systems are described, for example, in Boder et al., Nat. Biotechnol. 15:553-557 (1997) and Gai et al., Curr. Opin. Struct. Biol. 17:467-473 (2007). E. coli display methods, such as fusion with peptidoglycan-associated lipoprotein (PAL), are also covered herein. For example, the DBDpp peptide can be fused to the C terminus of a lac repressor and expressed in E. coli.

Bacterial display and yeast display technologies known in the art allow the expression of recombinant proteins on the surface of yeast cells S. cerevisiae (Bod-74 043883er, Nat. Biotechnol. 15:553-557 (1997)) or bacteria (E. coli , Staphylococcus carnosus) (Daugherty., 1998,

Wernerus, Appl. Environ. Microbiol. 69(9):5328-5335 (2003)) as fusion proteins with the yeast agglutinin adhesion receptor or bacterial outer membrane protein (OMP), respectively.

In some embodiments, the expressed fusion proteins also contain a peptide tag allowing the surface expression of the library to be quantified by flow cytometry. When combined with indirect fluorescent ligand labeling, counter-labeling allows for FACS (fluorescence activated cell sorting) cell sorting and determination of binding affinities of interactions (Feldhaus et al., Nat. Biotechnol. 21:163-70 (2003); Wernerus et al Appl Environ Microbiol 69(9):5328-35 (2003)).

In vitro display.

In vitro display methods (also known as cell-free) can also be used to select, isolate, and identify DBDpp that bind a target of interest. In one example, translation of random RNA is stopped before release from the ribosome, resulting in a library of polypeptides with their associated RNA remaining attached. This and similar methods are collectively called ribosomal display. Other known methods use chemical binding of peptides to RNA. See, for example, Roberts et al., PNAS 94:12297-303 (1997). This and similar methods are collectively called RNA-peptide display, RNA display and mRNA display. Alternatively, in vitro display methods may use DNA as the genetic component to which the expressed polypeptide is linked. The method, known as cis-display, allows for in vitro selection of peptides from libraries of DNA-protein complexes and is described in US7842476 B2, the contents of which are hereby incorporated by reference in their entirety. Chemically derived peptide libraries have been created in which peptides are immobilized on stable, nonbiological materials such as polyethylene rods or solvent-permeable resins. Another chemically produced peptide library uses photolithography to scan peptides immobilized on glass slides. These and similar methods are collectively called chemical peptide screening. Chemical screening of peptides may have the advantage of allowing the use of D-amino acids and other non-natural analogues, as well as non-peptide elements. Biological and chemical methods are reviewed in Wells, Curr. Opin. Biotechnol. 3:355-362 (1992).

Selection DBDpp.

Biopanning is a well-known iterative selection and screening method for enriching a starting population of different molecules (such as the DBDpp library) with molecules that have affinity for a selected target. Library elements that have affinity for the target can bind. Non-specifically or weakly bound elements are washed off the substrate. The bound library elements are then isolated (eg, by elution) from the support. Isolated library elements are collected for subsequent analysis (e.g., screening) or pooled for an additional round of selection.

In one embodiment, the target is captured on a solid support after incubation with a phage library. Immobilization of the target can be accomplished by many different methods known in the art. Examples of solid supports are microtiter plates or tubes (eg, Maxisorp plates, Maxisorp tubes, Nunc) or magnetic beads (Dynabead®, Invitrogen). The target can be applied directly to plastic or spheres (surface activated Dynabeads, e.g. Dynabeads M270 Epoxy, Invitrogen) or via streptavidin if the target is biotinylated (e.g. Dynabeads MyOne Streptavidin T1, Invitrogen).

In addition, the target may be non-covalently linked to the sphere through an intermediate affinity molecule, such as an antibody or protein A directed against the target, or a target-associated peptide tag. Peptide tags, such as His tags or, alternatively, an antibody directed against the target, can also be used to capture the target on a support. Such alternative peptide tags are also compatible with Dynabeads (His-Tagged Isolation and Removal Dynabeads, Invitrogen) and Protein A-linked or Protein G-linked Dynabeads (Protein A/G Dynabeads, Invitrogen). To immobilize a target on magnetic spheres, you should follow the manufacturer's recommendations for each specific type of sphere.

The capture step could then consist of immobilizing the target on coated magnetic spheres, thereby indirectly capturing the phage bound to the target. The interaction of the target phage is carried out in solution. To allow unbound phage to be washed away, the target must be immobilized on a solid support. Target immobilization in the solution biopanning method is identical to the immobilization capabilities in direct biopanning techniques.

The classic biopanning technique consists of 2, 3-5 or more rounds of selection, depending on the type of target and library. Each round of selection usually consists of different steps: (1) immobilization of the preferred target on the substrate. This step is optional, since biopanning can also be carried out in a format where the target is not immobilized, but remains in solution (in the case of a soluble target) or remains attached to the cell (in the case of, for example, a membrane-bound target,

- 75 043883 such as a receptor), (2) incubating the library with the target, (3) washing steps to remove non-specific binding molecules, (4) optionally eluting the binders, and (5) amplifying the eluted binders from step (4) or from step (3) (in case step (4) was excluded in successive rounds of screening). Steps 1-5 will be repeated two, three, four or more times to isolate target-specific binders from the initial library. After biopanning, the target specificity of the binders isolated in the various rounds of selection is typically examined in ELISA or similar assays.

In one embodiment, a method for screening DBDpps that specifically bind a target of interest includes the following steps: (a) contacting the target of interest with a plurality of DBDpps; and (b) identifying a DBDpp that specifically binds the target of interest. The step of contacting a target of interest with a plurality of DBDpp can be performed by any method known in the art. For example, in one embodiment, a target of interest is immobilized on a solid support and a solution containing a plurality of DBDpp molecules is contacted with the immobilized target of interest. This technique is similar to the affinity chromatography process, where the affinity matrix consists of an immobilized target of interest. DBDpp having selective affinity for a target of interest can then be purified using methods known in the art, such as affinity selection. The composition of the solid support, the method of attaching the target of interest to the solid support, and the reagents, conditions and methods for screening and isolating DBDpp having selective affinity for the target of interest are generally standard and known to those of ordinary skill in the art. In some cases, it may be desirable to wash away any unbound DBDpp from a mixture of the target of interest and/or one or more DBDpps bound to the target of interest before attempting to detect or detect the presence of a selective affinity interaction. This washing step may be especially necessary when the target of interest is associated with a solid support.

It should be understood that the selection step in the methods described in the present invention may be performed by a method commonly known as a screening method or by a method commonly known as a screening method. Both methods involve the identification and subsequent selection (eg, selection step) of desired components (eg, DBDpp library elements) from an initial population that includes both desired and undesired components (eg, DBDpp library). In the case of a selection method, library elements are typically selected in a step in which the desired property is used to achieve the desired goal; in such a case, the desired property is usually limited to the property of high affinity for the given target of interest. Such a method is generally known as an affinity selection method, wherein such an affinity selection method will be applied to a DBDpp library in order to select a DBDpp having high affinity for a target of interest. In additional embodiments, the library is screened for a DBDpp that has the desired kinetic properties, such as a high rate of association for binding to a given target of interest, or a low rate of dissociation for library elements associated with the target, while controlling for appropriate selection conditions (e.g., short incubation times or long wash cycles, or other conditions as known to those skilled in the art of library screening techniques). Alternatively, in the case of a screening method, library elements are typically isolated in a step in which all library elements, or at least a substantial collection of library elements, are individually screened for a given desired property, and where elements having such desired property , are retained, while elements that do not have this required property are deleted; in such a case, and as used herein, the desired properties may be associated with either high affinity for the target of interest or functional activity, such as inhibiting, reducing and/or preventing the activity of the target of interest. Thus, it appears that the selection step in the methods can be performed using either an (affinity) selection technique or an affinity-based or activity-based functional screening technique, both methods resulting in the selection of one or more DBDpps having beneficial properties. (favorable, desirable, improved) affinity or activity properties compared to the unselected DBDpp of DBDpp.

Screening DBDpp.

Once selected, identified library elements can be individually isolated and screened.

Screening differs from selection in that screening is characterized by the analysis of library elements individually (or in pools), whereas selection is characterized by the analysis of library elements that are separated from other elements during the process (eg, isolated, eluted or washed away). In one embodiment, a collection of library items is screened directly, without a selection step. This approach, for example, can be used in affinity maturation protocols, which

- 76 043883 are known and can be used as standard.

The ability of DBDpp to specifically bind a target of interest can be determined using or standard modification of assays and other techniques described in the present invention or otherwise known in the art. For example, DBDpp-target interaction can be assayed as described in the Examples below, or alternatively using in vitro or in vivo binding assays such as Western blots, radioimmunoassays, ELISAs, sandwich immunoassays, immunoprecipitation assays , fluorescence immunoassays, protein A immunoassays, immunohistochemistry (IHC), and BiaCore assay. Likewise, the ability of DBDpp to specifically bind a target of interest and alter the biological activity of the target can be determined using or standard modification of the assays and other techniques described in the present invention or otherwise known in the art. Assays to assess the ability of DBDpp to functionally affect its target (eg, assays to measure signaling, proliferation, migration, etc.) can also be used to indirectly assess DBDpp-target interaction. Additionally, DBDpp can be identified based on its effects in assays that measure specific pathways or activities. For example, assays that measure signaling pathways (eg, phosphorylation or multimerization assays), ion channel fluxes, intracellular cAMP levels, cellular activities such as migration, adhesion, proliferation or apoptosis, and virus entry, replication, budding or integration, can be used to identify, analyze and improve the required properties of DBDpp. The ability of DBDpp to competitively inhibit another DBDpp-containing sequence can be determined using methods known in the art, including the BiaCore assay and ELISA.

Identification DBDpp.

In cases where a candidate DBDpp contained in a DBDpp library is displayed on a suitable cell or phage or particle, the coding nucleic acid sequence can be isolated and readily determined. A nucleotide sequence that encodes such a DBDpp sequence can be isolated from said cell or phage or particle. Thus, the nucleotide sequence of the selected element(s) of the DBDpp library can be determined using standard sequencing methods.

Elements of the DBDpp library that are specific for the target can be examined using nucleic acid sequencing. Sequence information is used to classify elements and remove redundant elements (i.e. elements that encode the same DBDpp). DBDpp libraries and library elements (including some elements to which epitope tags have been added) according to several embodiments include, but are not limited to, those defined in table. 1 below. Additionally included are those DBDpps that correspond to any of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, wherein one or more X _n positions are replaced by a natural or unnatural amino acid. In some embodiments, DBDpp that corresponds to any of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 does not include the cysteine and/or proline residues replaced at the Xn position.

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Table 1

Non-limiting examples of DBDpp libraries and library elements

SEQ ID NO Subsequence Target Library 2 MGSWX5X ₆ FKX ₉ X _lo LAX ₁₃ IKX ₁₆ X ₁₇ LEALGGSEAELAX ₃ oFEX ₃₃ X ₃₄ IAX ₃₇ FE X ₄₀ X ₄₁ LQX ₄₄ YKGKGNPEVEALRKEAAAIRDELQAYRHN Fl 3 mgswaefkqrlaaiktrlealggseaelaafx ₃₂ x ₃₃ eix ₃₆ afx ₃₉ x ₄₀ elx ₄₃ AYKGKGNPEVEALX5 ₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆ 5ELX ₆₈ AYRHN F2 4 MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALGGSEAELAAFEKEIAAFESELQA YKGKGNPEVEX55LRX5 ₈ X5 ₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN F3 5 MGSWX ₅ X ₆ FKX ₉ X ₁₀ LAX ₁₃ IKX ₁₆ X ₁₇ LEALGGSEAELAAFX ₃₂ X ₃₃ EIX ₃₆ AFX ₃₉ X ₄₀ ELX ₄₃ AYKGKGNPEVEX55LRX5 ₈ X5 ₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN Cl 6 mgswx ₅ efx ₈ x ₉ rlx ₁₂ aix ₁₅ x ₁₆ rlx ₁₉ alggseaelax ₃₀ fex ₃₃ x ₃₄ iax ₃₇ fe X ₄ oX ₄ iLQX ₄₄ YKGKGNPEVEALX ₅₇ X5 ₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN C2 7 MGSWXsXgFKXgX^LAX^IKX^XnLEALZ^AELAXafjFEX^XMlAX^FEX;; ₈ x ₃₉ lqx ₄₂ yz ₂ npevealrkeaaairdelqayrhn 8 MGSWAEFKQRLAAIKTRLEALZ^AELAAFX^XsiEIX^AFX^XmELX^AY Z ₂ NPEVEALX ₅₂ X5 ₃ EAX5 ₆ AIX ₅₉ X ₆ oELX ₆₃ AYRHN 9 MGSWXsEFXfjXgRLX^AIX^X^RLX^ALZ^AELAAFEKEIAAFESELQAY Z ₂ NPEVEX ₅ oLRX5 ₃ X5 ₄ AAX5 ₇ IRX ₆ oX ₆ iLQAYRHN 10 MGSWX ₅ X ₆ FKX ₉ X ₁₀ LAX ₁₃ IKX ₁₆ X ₁₇ LEALZ ₁ EAELAA.FX ₃₀ X ₃ iEIX ₃₄ AFX ₃₇ X _{3 8} ELX ₄₁ AYZ ₂ NPEVEX5oLRX5 ₃ X5 ₄ AAX ₅₇ IRX ₆ oX61LQAYRHN eleven MGSWXsEFXfjXgRLX^AIX^X^RLX^ALZ^AELAX^FEX^XMlAXssFEXs ₈ x ₃₉ lqx ₄₂ yz ₂ npeveal x ₅₂ x ₅₃ eax ₅₆ aix ₅₉ x ₆₀ elx ₆₃ ayrhn 12 MGSWVEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEKLRQRAAFIRRFRLQAYRHN CD137 F3 13 MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEHLRDQAAFIRHKLQAYRHN CD137 F3 14 MGSWYEFRHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEGLREAAAFIRAKLQAYRHN CD137 F3 15 MGSWYEFSMRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEALRAKAAYIRWKLQAYRHN CD137 F3 16 MGSWFEFNHRLWAINERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVERLRSMAAFIRYKLQAYRHN CD137 F3 17 MGSWYEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRETAAHIRTRLQAYRHN CD137 F3 18 MGSWYEFHYRLHAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEELRIKAAFIRRDRLQAYRHN CD137 F3 19 MGSWAEFKQRLAAIKTRLEALGGSEAELAAFLGEIWAFEMELAAYKGKGNP EVEALGREAAAIRMELQAYRHN CD137 F2 20 MGSWYEFDLRLHAIYDRLVALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEILRDNAAYIRQMLQAYRHN CD47 F3 21 MGSWTEFTYRLSAIEWRLWALGGSEAELAWFEQKIAFFEDFLQYYKGKGNP EVEALKHEAGAILNELMAYRHN CD47 C2 22 MGSWAEFDHRLHAIRERLHALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEILRGNAAYIRALLQAYRHN CD47 F3 23 MGSWTEFVGRLAAIEFRLWALGGSEAELAWFEAHIAFFEDYLQWYKGKGNP EVEAL RE EAGAIMEELKAYRHN CD47 C2

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24 MGSWTEFYSRLEAIWVRLQALGGSEAELAMFEDRIAHFEWFLQQYKGKGNP EVEALHEEAIAIRKELAAYRHN CD47 C2 25 MGSWHEFHDRLQAIHERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVESLRIAAAHIRQVLQAYRHN CTLA4 F3 26 MGSWNYFKDHLAWIKNSLEALGGSEAELAHFETAIASFERQLQEYKGKGNP EVEALRKEAAAIRDELQAYRHN DR5 Fl 27 MGSWLYFKEHLAHIKAWLEALGGSEAELAHFELAIADFEYHLQEYKGKGNP EVEALRKEAAAIRDELQAYRHN DR5 Fl 28 MGSWVYFKEHLAWIKTELEALGGSEAELAHFEHSIADFEMSLQFYKGKGNP EVEALRKEAAAIRDELQAYRHN DR5 Fl 29 MGSWFYFKQHLAWIKSYLEALGGSEAELAHFERAIAAFEQHLQMYKGKGNP EVEALRKEAAAIRDELQAYRHN DR5 Fl thirty MGSWHYFKDHLAEIKGLLEALGGSEAELAHFEMAIADFEHNLQYYKGKGNP EVEALRKEAAAIRDELQAYRHN DR5 Fl 31 MGSWHYFKGHLAEIKNHLEALGGSEAELAHFERAIAAFERSLQWYKGKGNP EVEALRKEAAAIRDELQAYRHN DR5 Fl 32 MGSWIYFKEHLAYIKKELEALGGSEAELAHFESAIAVFESTLQYYKGKGNP EVEALRKEAAAIRDELQAYRHN DR5 Fl 33 MGSWTYFKEHLAEIKYMLEALGGSEAELAHFEVAIADFEKMLQYYKGKGNP EVEALRKEAAAIRDELQAYRHN DR5 Fl 34 MGSWWLFKDHLAEIKTALEALGGSEAELAHFEMAIAAFEKQLQYYKGKGNP EVEALRKEAAAIRDELQAYRHN DR5 Fl 35 MGSWSEFYNRLDAIESRLLALGGSEAELALFEIQIARFEKVLQAYKGKGNP EVEALRGEARAIFAELYAYRHN KIR C2 36 MGSWYEFYNRLYAIEIRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVERLRVRAAKIRVILQAYRHN KIR F3 37 MGSWLWFKIFLAEIKYFLEALGGSEAELAAFDFEIHAFHVELFAYKGKGNP EVEVLREVAAEIRWDLQAYRHN KIR Cl 38 MGSWTEFQSRLDAIHSRLRALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVELLRDDAAFIRHFLQAYRHN PD-L1 F3 39 MGSWQEFDDRLNAIKARLQALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRDDAAFIRRFLQAYRHN PD-L1 F3 40 MGSWYEFQNRLHAIHERLNALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVELLRDDAAFIRHFLQAYRHN PD-L1 F3 41 MGSWFEFQDRLTAINERLSALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVETLRSDAAFIRRFLQAYRHN PD-L1 F3 42 MGSWYEFESRLDAIHERLHALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVENLRGDAAFIRHFLQAYRHN PD-L1 F3 43 MGSWYEFNHRLDAISKRLNALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEELRGDAAFIRHFLQAYRHN PD-L1 F3

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44 MGSWFEFENRLHAIVHRLGALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVETLRADAAFIRHYLQAYRHN PD-L1 F3 45 MGSWWFKVDLATIKYILEALGGSEAELAEFEGEIAGFEYSLQFYKGKGNP EVEALRKEAAAIRDELQAYRHN TIM3 Fl 46 MGSWTIFKEWLAFIKTDLEALGGSEAELAFFEGWIASFEMELQKYKGKGNP EVEALRKEAAAIRDELQAYRHN PD1 Fl 47 MGSWVMFKWLLADIKSHLEALGGSEAELAFFEGFIAAFETHLQVYKGKGNP EVEALRKEAAAIRDELQAYRHN PD1 Fl 48 MGSWYAFKDYLADIKGWLEALGGSEAELAFFEIFIARFELELQAYKGKGNP EVEALRKEAAAIRDELQAYRHN PD1 Fl 49 MGSWAEFKQRLAAIKTRLQALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEALRKEAAAIRDELQAYRHN None 51 MGSWVEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEKLRQRAAFIRRFRLQAYRHNGGGGSHHHHHH CD137 F3 52 MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEHLRDQAAFIRHKLQAYRHNGGGGSHHHHHH CD137 F3 53 MGSWYEFRHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEGLREAAAFIRAKLQAYRHNGGGGSHHHHHH CD137 F3 54 MGSWYEFSMRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEALRAKAAYIRWKLQAYRHNGGGGSHHHHHH CD137 F3 55 MGSWFEFNHRLWAINERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVERLRSMAAFIRYKLQAYRHNGGGGSHHHHHH CD137 F3 56 MGSWYEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRETAAHIRTRLQAYRHNGGGGSHHHHHH CD137 F3 57 MGSWYEFHYRLHAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEELRIKAAFIRRDRLQAYRHNGGGGSHHHHHH CD137 F3 58 MGSWAEFKQRLAAIKTRLEALGGSEAELAAFLGEIWAFEMELAAYKGKGNP EVEALGREAAAIRMELQAYRHNGGGGSHHHHHH CD137 F2 60 MGSWIEFEDRLDAITDRLWALGGSEAELAEFEHQIAFFEEDLQWYKGKGNP EVEALHMEAEAIMEELGAYRHN CD123 C2 61 MGSWVEFEYRLDAISDRLWALGGSEAELAFFENEIASFESDLQFYKGKGNP EVEALMFEAEAIDDELHAYRHN CD123 C2 62 MGSWYEFEDRLAAIEARLWALGGSEAELADFEEEIAYFEHGLQWYKGKGNP EVEALESEAMAIIDELHAYRHN CD123 C2 63 MGSWYEFEERLDAIEDRLIALGGSEAELAIFEDIIAFFEQDLQYYKGKGNP EVEALEMEEAISIELDAYRHN CD123 C2 64 MGSWWEFEDRLWAIDRRLMALGGSEAELAVFEQMIAHFEQILQVYKGKGNP EVEALHFEAHAIGMELAAYRHN CD123 C2 65 MGSWEEFHERLDAIDERLEALGGSEAELAFFEDDIASFEDWLQWYKGKGNP EVEALSREADAINFELEAYRHN CD123 C2

-80043883

66 MGSWEEFDKRLDAITRRLMALGGSEAELAEFESTIAWFEWDLQEYKGKGNP EVEALDWEAYAIDYELGAYRHN CD123 C2 67 MGSWSEFVDRLDAIFDRLWALGGSEAELAWFEDTIAHFEWNLQEYKGKGNP EVEALNGEADAITDELHAYRHN CD123 C2 68 MGSWWEFTDRLDAIFDRLWALGGSEAELAAFEESIAIFEQDLQYYKGKGNP EVEALEYEANAIQYELEAYRHN CD123 C2 69 MGSWWEFTDRLEAIEDRLWALGGSEAELAHFEDSIAQFEQELQWYKGKGNP EVEALADEADAIES ELHAYRHN CD123 C2 70 MGSWEWFKSDLASIKWELEALGGSEAELAWFEHDIAEFEEDLQWYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 71 MGSWDHFKNDLAWIKKHLEALGGSEAELAEFEAVIAYFELYLQGYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 72 MGSWEFFKEVLAEIKYDLEALGGSEAELAWFETDIAGFEIDLQVYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 73 MGSWYDFKEDLADIKWMLEALGGSEAELAEFENVIAYFENDLQEYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 74 MGSWSFFKDDLAEIKYFLEALGGSEAELAMFEQTIAEFEYDLQDYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 75 MGSWVTFKDELADIKDFLEALGGSEAELAFFEVDIAEFEAELQFYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 76 MGSWSWFKEDLADIKFELEALGGSEAELAWFELDIADFEQALQQYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 77 MGSWWEFKEDLAEIKWFLEALGGSEAELAWFEHDIAKFEFELQYYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 78 MGSWDEFKEDLAHIKTDLEALGGSEAELALFEDEIADFEMYLQHYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 79 MGSWFMFKEELADIKDWLEALGGSEAELASFESYIAWFEQDLQWYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 80 MGSWQIFKGELAYIKQYLEALGGSEAELAFFEFDIAEFEEDLQYYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 81 MGSWYIFKEDLAEIKEELEALGGSEAELAYFEEEIALFEMELQWYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 82 MGSWYYFKDELADIKWDLEALGGSEAELAWFEMLIAQFELDLQWYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 83 MGSWFNFKEELAVIKFQLEALGGSEAELAFFEWVIADFEDDLQEYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 84 MGSWYMFKEELADIKWYLEALGGSEAELAWFEDDIAGFEWDLQAYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 85 MGSWHVFKTELADIKFYLEALGGSEAELAMFELWIAEFEHELQDYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl

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86 MGSWYVFKDELAEIKQFLEALGGSEAELAWFEDDIAEFETQLQHYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 87 MGSWTEFKGELAEIKWILEALGGSEAELAFFEDEIAAFEWDLQKYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 88 MGSWFWFKEDLAFIKEDLEALGGSEAELAWFEDGIAFFEWDLQDYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 89 MGSWSWFKEDLASIKAVLEALGGSEAELAFFESDIAEFEQELQYYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 90 MGSWILFKDDLAWIKETLEALGGSEAELAFFEDNIADFEEQLQGYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 91 MGSWQWFKDDLAYIKETLEALGGSEAELALFEDMIADFEFELQWYKGKGNP EVEALRKEAAAIRDELQAYRHN CD123 Fl 92 MGSWEEFHSRLDAIDDRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRWEAATIRETLQAYRHN CD123 F3 93 MGSWSEFWQRLEAIEDRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRENAAMIRDELQAYRHN CD123 F3 94 MGSWTEFAWRLDAIYDRLLALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRHVAANIRRELQAYRHN CD123 F3 95 MGSWDEFYYRLEAIEMRLGALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEELRHYAAQIRHMLQAYRHN CD123 F3 96 MGSWIEFNMRLDAIYERLVALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRKVAANIRLELQAYRHN CD123 F3 97 MGSWSEFNMRLDAIYERLTALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRHSAARIRLELQAYRHN CD123 F3 98 MGSWVEFNIRLDAIYERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEKLRHWAASIRRELQAYRHN CD123 F3 99 MGSWDEFGRRLYAIEWRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEKLREIAAVIRSNLQAYRHN CD123 F3 100 MGSWIEFYDRLEAIYDRLDALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLREDAAFIRSWLQAYRHN CD123 F3 101 MGSWTEFDRRLDAIWDRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLREEAADIRDYLQAYRHN CD123 F3 102 MGSWTEFDRRLDAIWDRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLREEAADIRDYLQAYRHN CD123 F3 103 MGSWIEFEVRLDAIYNRLAALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVERLRRYAAANIRHELQAYRHN CD123 F3 104 MGSWTEFHDRLEAIDDRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLREEAAQIRWELQAYRHN CD123 F3 105 MGSWYEFHHRLDAIYERLLALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRS SAANIRKELQAYRHN CD123 F3

- 82 043883

106 MGSWHEFDQRLWAIEERLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVETLRLYAALIRHDLQAYRHN CD123 F3 107 MGSWIEFESRLWAIEDRLLALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEFLRLEAADIREDLQAYRHN CD123 F3 108 MGSWYEFENRLGAIGDRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRDEAAYIRAVLQAYRHN CD123 F3 109 MGSWNEFYDRLSAIYFRLQALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEHLRWYAADIRMILQAYRHN CD123 F3 110 MGSWYEFEYRLEAIEDRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLREEAAWIRVWLQAYRHN CD123 F3 111 MGSWVEFENRLEAIENRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLREDAAQIRMMLQAYRHN CD123 F3 112 MGSWYEFWDRLEAIDDRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEALRQEAAWIREELQAYRHN CD123 F3 113 MGSWFEFWDRLDAIEDRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEELRDEAAWIRGTLQAYRHN CD123 F3 114 MGSWTEFDRRLDAIWDRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLREEAADIRDYLQAYRHN CD123 F3 115 MGSWWEFEMRLEAIEDRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVESLRWEAAFIRDILQAYRHN CD123 F3 116 MGSWVEFYDRLHAIYFRLLALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRWYAADIRLVLQAYRHN CD123 F3 117 MGSWYEFYNRLSAIYARLQALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRWYAADIRYMLQAYRHN CD123 F3 118 MGSWFEFWGRLEAIESRLKALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEELREHAAWIRAYLQAYRHN CD123 F3 119 MGSWTEFSIRLEAIYDRLVALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEVLRTYAAANIRHELQAYRHN CD123 F3 120 MGSWYEFENRLEAIEERLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEMLREEAAFIRDWLQAYRHN CD123 F3 121 MGSWYEFVIRLEAIEDRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEVLRWYAADIRHELQAYRHN CD123 F3 122 MGSWIEFEDRLEAIEDRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRQEAAEIRLMLQAYRHN CD123 F3 123 MGSWTEFNLRLDAIYDRLMALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRASAAAIRVELQAYRHN CD123 F3 124 MGSWSEFYLRLDAIYDRLDALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRKTAANIREELQAYRHN CD123 F3 125 MGSWSEFHVRLDAIYARLDALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVERLREWAANIRRELQAYRHN CD123 F3

- 83 043883

126 MGSWHEFGVRLDAIYDRLMALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEFLRQAAANIRSELQAYRHN CD123 F3 127 MGSWYEFSMRLDAIYDRLMALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEQLRGYAANIRNELQAYRHN CD123 F3 128 MGSWDEFGRRLYAIEWQLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEKLREIAAVIRSNLQAYRHN CD123 F3 129 MGSWDEFGRRLYAIEWRLYALGGEEAELAAFEKEIAAFESELQAYKGKGNP EVEKLREIAAVIRSNLQAYRHN CD123 F3 130 MGSWDEFGRRLYAIEWRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEKLREIAAVIRENLQAYRHN CD123 F3 131 MGSWDEFGRRLYAIEWQLYALGGEEAELAAFEKEIAAFESELQAYKGKGNP EVEKLREIAAVIRSNLQAYRHN CD123 F3 132 MGSWDEFGRRLYAIEWQLYALGGTEAELAAFEKEIAAFESELQAYKGKGNP EVEKLREIAAVIRSNLQAYRHN CD123 F3 133 MGSWDEFGRRLYAIEWQLYALGGGEEAELAAFEKEIAAFESELQAYKGKGNP EVEKLREIAAVIRSNLQAYRHN CD123 F3 134 MGSWDEFGRRLYAIEWQLYALGGEEAELAAFEKEIAAFESELQAYKGKGNP EVEKLREIAAVIRENLQAYRHN CD123 F3 135 MGSWDEFGRRLYAIEWQLYALGGTEAELAAFEKEIAAFESELQAYKGKGNP EVEKLREIAAVIRENLQAYRHN CD123 F3 136 MGSWDEFGRRLYAIEWQLYALGGGEEAELAAFEKEIAAFESELQAYKGKGNP EVEKLREIAAVIRENLQAYRHN CD123 F3 137 MGSWEEFELRLNAIEERLYALGGSEAELAYFEYVIADFEGNLQRYKGKGNP EVEALYFEADAIFEELVAYRHN CD19 C2 138 MGSWFEFNHRLWAIFERLMALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRAMAAVIRYHLQAYRHN CD19 F3 139 MGSWEEFDGRLFAIEQRLQALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEVLRWFAAGIRDFLQAYRHN CD19 F3 140 MGSWAEFYHRLYAIETRLSALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRHWAAWIRTYLQAYRHN CD19 F3 141 MGSWVEFSDRLYAIEERLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEELRELAAIIRHSLQAYRHN CD19 F3 142 MGSWWEFEGRLYAIEERLTALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLREWAAWIRQMLQAYRHN CD19 F3 143 MGSWWEFEHRLYAIEERLVALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRNWAAYIRMALQAYRHN CD19 F3 144 MGSWWEFEARLYAIEFRLSALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRSWAAYIRTSLQAYRHN CD19 F3 145 MGSWWEFEARLWAIESRLKALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRHWAAYIRVILQAYRHN CD19 F3

- 84 043883

146 MGSWWEFEARLYAIEFRLSALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRSWAAYIRTSLQAYRHN CD19 F3 147 MGSWEEFYHRLDAIELRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRWYAAEIREILQAYRHN CD19 F3 148 MGSWYEFYERLDAIDTRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEFLREYAAEIRHFLQAYRHN CD19 F3 149 MGSWNEFFDRLDAILYRLDALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRFVAADIRSWLQAYRHN CD19 F3 150 MGSWIEFDDRLLAIMDRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRDVAADIRHYLQAYRHN CD19 F3 151 MGSWYEFWERLDAITFRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRTWAADIRAILQAYRHN CD19 F3 152 MGSWEEFYIRLDAIMERLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRYAAADIRHFLQAYRHN CD19 F3 153 MGSWIEFEERLYAIETRLLALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEFLRWAADIREWLQAYRHN CD19 F3 154 MGSWIEFEHRLSAINDRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLREWAADIRSLLQAYRHN CD19 F3 155 MGSWFEFEMRLDAIMARLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRYAAADIRDYLQAYRHN CD19 F3 156 MGSWYEFVYRLDAIYDRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRYAAADIRDFLQAYRHN CD19 F3 157 MGSWVEFEDRLDAILERLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRELAADIRDFLQAYRHN CD19 F3 158 MGSWFEFEERLIAIEERLFALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEYLRWIAADIRDVLQAYRHN CD19 F3 159 MGSWIEFADRLDAILDRLDALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLREIAADIRAYLQAYRHN CD19 F3 160 MGSWLEFEYRLDAILDRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLREVAADIRMLLQAYRHN CD19 F3 161 MGSWYEFHDRLDAITNRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRDWAADIRVWLQAYRHN CD19 F3 162 MGSWQEFEQRLDAINWRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEELREWAADIRIFLQAYRHN CD19 F3 163 MGSWYEFYSRLDAIDSRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEFLRDYAAEIRRYLQAYRHN CD19 F3 164 MGSWEEFHDRLEAISDRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEDLRDWAADIRFYLQAYRHN CD19 F3 165 MGSWWEFDERLYAIEDRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNP EVEWLRIVAADIREILQAYRHN CD19 F3

- 85 043883

MGSWEEFEYRLMAIEVRLWALGGSEAELAAFEKEIAAFESELQAYKGKGNP 166 EVEVLREIAADIRQILQAYRHN CD19 F3 MGSWWFKQRLAYIKDLLEALGGSEAELAYFEMSIAFFEEDLQVYKGKGNP 167 EVEALRKEAAAIRDELQAYRHN CD22 Fl MGSWYEFKNDLAWIKVHLEALGGSEAELAYFEFRIAHFENALQYYKGKGNP 168 EVEALRKEAAAIRDELQAYRHN CD22 Fl MGSWVEFYNRLWAIDHRLHALGGSEAELAAFEKEIAAFESELQAYKGKGNP 169 EVEVLRYHAASIRVTLQAYRHN CD22 F3 MGSWSEFYDRLHAIHHRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP 170 EVEALRDTAAFIRTRLQAYRHN CD22 F3 MGSWKEFHFRLHAIEHRLIALGGSEAELAAFEKEIAAFESELQAYKGKGNP 171 EVEFLRAKAANIRTHLQAYRHN CD22 F3 MGSWFEFHGRLHAIYGRLSALGGSEAELAAFEKEIAAFESELQAYKGKGNP 172 EVEHLRAHAAHIRDHLQAYRHN CD22 F3 MGSWYEFADRLHAIHQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP 173 EVEALRMTAAFIRSRLQAYRHN CD22 F3 MGSWNEFYNRLHAIHQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP 174 EVESLRQTAAYIDRRLQAYRHN CD22 F3 MGSWNEFADRLHAIHQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP 175 EVESLRMTAAFIRSRLQAYRHN CD22 F3 MGSWTEFSYRLGAIQSRLHALGGSEAELAAFEKEIAAFESELQAYKGKGNP 176 EVEHLRYNAAKIRHFLQAYRHN CD22 F3 MGSWQEFTTRLEAIYHRLRALGGSEAELANFEGFIAEFEGNLQMYKGKGNP 177 EVEALVH EAYAIMEELHAYRHN DR5 C2 MGSWVEFFDRLKAIHDRLEALGGSEAELAHFEKLIAHFEHRLQNYKGKGNP 178 EVEALEKEADAILYELAAYRHN DR5 C2 MGSWYYFKHHLAWIKMELEALGGS EAELAHFES SIAS FERDLQQYKGKGNP 179 EVEALRKEAAAIRDELQAYRHN DR5 Fl MGSWVEFHIRLHAIQYRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNP 180 EVEELRHWAAFIRLQLQAYRHN DR5 F3 MGSWNEFHDRLNAIHARLHALGGSEAELAAFEKEIAAFESELQAYKGKGNP 181 EVENLRDDAAFIRRFLQAYRHN PD-L1 F3 MGSWYEFTVRLEAIHERLKALGGSEAELAAFEKEIAAFESELQAYKGKGNP 182 EVEILRDDAAFIRRFLQAYRHN PD-L1 F3 MGSWKEFDDRLNAIKARLQALGGSEAELAAFEKEIAAFESELQAYKGKGNP 183 EVEDLRDDAAFIRRFLQAYRHN PD-L1 F3 MGSWYEFDDRLNAIHDRLQALGGSEAELAAFEKEIAAFESELQAYKGKGNP 184 EVEDLRDDAAFIRRFLQAYRHN PD-L1 F3 MGSWNEFKNRLDAIHKRLNALGGSEAELAAFEKEIAAFESELQAYKGKGNP 185 EVENLRDAAFIRHFLQAYRHN PD-L1 F3 MGSWTEFEQRLEAIHNRLQALGGSEAELAAFEKEIAAFESELQAYKGKGNP 186 EVEELRNDAAFIRHFLQAYRHN PD-L1 F3

In some embodiments, the invention includes one or more sequences defined in table. 1. In other embodiments, the invention includes one or more sequences with 60-70%, 70-75%, 75-80%, 80-85%, 85-90%, 95-99% homology (and overlapping ranges therein) with the sequences defined in table. 1. In several embodiments, the sequences having such homology are functionally similar or identical to the corresponding sequence defined in table. 1. In several embodiments, the invention includes one or more polypeptides that compete with (in whole or in part) one or more sequences in table. 1 for their respective target. In several embodiments, competition may be assessed using standard competitive analysis. In some embodiments, competition does not require that the competing polypeptide compete for the same specific target as the polypeptides in the table. 1, rather they may compete to bind a sterically inhibitory epitope, an overlapping epitope, etc.

Affinity maturation of DBDpp.

Affinity maturation strategies can be used to create high-affinity DBDpp, which can be used in the DBDpp fusion proteins described in the present invention.

Mutagenesis libraries can be generated and screened to further optimize the sequence of the best binders. Lowman, Ann. Rev. Biophys. Biomol. Struct. 26: 401-24 (1997).

An improved DBDpp that specifically binds the desired target can also be derived from the known DBDpp sequence. For example, at least one, two, three, four, five or more amino acid mutations (eg, conservative or non-conservative substitutions), deletions or insertions can be introduced into a known DBDpp sequence, and the resulting DBDpp can be screened for binding to the desired target and biological activity, such as the ability to antagonize the biological activity of the target or agonistically affect the biological activity of the target.

Products.

The present invention provides products, including kits. The article may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, vials, ampoules or syringes. Containers can be made from a variety of materials, such as glass or plastic. The container contains one or more DBDpp, nucleic acids encoding DBDpp, and/or vectors or host cells as described herein. The label or package insert may include instructions for performing affinity-based screening, detection and/or purification.

Kits containing DBDpp are also provided. Such kits have applications including, but not limited to, detection or isolation of a target of interest to which DBDpp specifically binds. Such an assay kit can be used in screening for the presence of a target of interest and/or quantifying concentrations of a target of interest in a fluid, such as a biological fluid (eg, blood, serum, or synovial fluid).

In one embodiment, a DBDpp assay kit is contemplated that includes one or more containers of a DBDpp that specifically binds a target of interest, and optionally detection means for determining the presence or absence of a target/DBDpp interaction or absence thereof. The kit also optionally contains a protein target of interest, which can be used, for example, as a control or standard. DBDpp can be free or expressed on the surface of a host cell or on the surface of a phage. In a certain embodiment, the DBDpp or target of interest provided in the kit is labeled. Any label known in the art may be used. In some embodiments, the label is selected from the group consisting of biotin, fluorogen, enzyme, epitope, chromogen, or radionuclide. In some embodiments, DBDpp is immobilized on a solid support. The detection means used to detect the tag will depend on the nature of the tag and may be anything known in the art, for example, radionuclide detection film; an enzyme substrate that produces or enhances a detectable signal to detect the presence of a target of interest.

Preferably, the kit also includes a solid support for DBDpp, which can be provided as a separate element or immobilized on it with DBDpp that specifically binds the target of interest. Therefore, the DBDpp that specifically binds the target of interest in the kit can be immobilized on a solid support, or they can be immobilized on such a support that is included in the kit or provided separately from the kit. Preferably, the microtiter plate is coated with the DBDpp polypeptide. In some embodiments, the detection includes a signal-enhancing molecule. In cases where the signal enhancing molecule is an enzyme, the kit optionally further includes substrates and cofactors required for the enzyme, and where the signal enhancing molecule is a fluorophore. The kit optionally further includes a dye precursor that produces a detectable chromophore.

The kit may also contain instructions for performing the assay, as well as other additives such as stabilizers, wash and incubation buffers, etc. The components of the kit will be provided in predetermined ratios, with the relative amounts of the various reagents preferably varying to produce concentrations in the reagent solution that significantly enhance the sensitivity of the assay and/or the ability to purify the target of interest. In particular,

- 87 043883 reagents can be provided in the form of dry powders, usually lyophilized, containing excipients which, when dissolved, provide a solution of reagents having a suitable concentration for combining with the test sample.

Various formats and methods of binding assays that can be used are known in the art and include, but are not limited to, immobilization on filters such as nylon or nitrocellulose; two-dimensional arrays, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays, fluorometric microvolume assay technology (FMATTM), Luminex™ assays, fluorescence resonance energy transfer ( FRET), bioluminescence resonance energy transfer (BRET), electroimmunoassays, AlphaScreen™, nanoparticle techniques and surface plasmon resonance (SPR).

Binding assays can be homogeneous or semi-homogeneous. A homogeneous assay is an assay in which all components are mixed together, incubated and then analyzed. A semi-homogeneous assay is an assay in which most of the reaction occurs in a complex mixture, but a wash step is required before the final reagent and assay are added, as opposed to a typical sequential sandwich assay where each component is added, then washed off before the next component is added. In some embodiments, the assay is an immunoassay. In some embodiments, the assay is a semi-homogeneous enzyme-linked immunosorbent assay (EIA).

Applications.

DBDpp, alone, as fusion proteins, as chemical conjugates, or as other embodiments described in the present invention, has many uses. In some embodiments, DBDpp are used as detection reagents, capture reagents, separation reagents, diagnostic reagents, or analytical reagents. Some embodiments have in vivo, in vitro and/or ex vivo applications. Methods in which DBDpp is used in vitro can be performed in a variety of formats, for example, in microtiter plates, on protein matrices, on biosensor surfaces, on tissue sections, as well as in additional formats that would be obvious to one skilled in the art. Likewise, methods that employ DBDpp in vivo can be used in a variety of formats, which include, but are not limited to, DBDpp-Fc fusion proteins, CAR cells, and DBDpp multispecific antibodies. In certain embodiments, DBDpp, such as DBDpp fusion proteins, are used as a therapeutic agent.

Analytical and diagnostic applications.

DBDpp, whether alone, as fusion proteins, as chemical conjugates, or as other embodiments described in the present invention, have many uses. In some embodiments, DBDpp are used as reagents to detect targets of interest in a variety of different types of samples.

In one embodiment, DBDpp are used to detect a target of interest in solutions used in manufacturing processes such as protein expression and purification. Samples may contain, but are not limited to, water, buffers, processing samples for purification, bulk drug substance, and finished pharmaceutical product. In other additional embodiments, DBDpp may be used to detect and/or remove impurities or contaminants from a sample, such as a water supply or water (or other liquid) used in production.

In another embodiment, DBDpp are used to detect a target of interest in diagnostic samples. Samples may include, but are not limited to, tissue homogenates, cell extracts, biopsy specimens, serum, plasma, lymph, blood, blood fractions, urine, synovial fluid, cerebrospinal fluid, saliva, mucous secretions, sputum, pleural fluid, nipple aspirates, Fluid from the respiratory tract, intestinal and genitourinary tracts, tear fluid, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, fluid from visceral systems, ascitic fluid, tumor cyst fluid, amniotic fluid, and cultured cell media or lysate.

In one embodiment, DBDpp can be used to detect the presence of a factor or multiple factors (eg, antigens or organisms) in a biological sample. The term detection as used herein covers quantitative or qualitative detection. In some embodiments, the biological sample includes a cell, tissue, or fluid. In some embodiments, such tissues include normal and/or cancerous tissues.

Various detection formats and techniques are known in the art and include, but are not limited to, Western blotting, immunohistochemistry, ELISA, FACS analysis, enzymatic assays, autoradiography, and any of the binding assays specified herein.

- 88 043883

In one embodiment, a method is provided for detecting a target of interest in a solution containing the target, comprising: (a) contacting the solution with a DBDpp that specifically binds the target of interest, under conditions suitable for the DBDpp to specifically bind to the target, and (b) detecting the binding DBDpp and targets. DBDpp can be free or immobilized. Sufficient time is allowed for the target of interest to bind to DBDpp, and optional components in the solution or mixture are removed or washed away. Formation of a binding complex between DBDpp and a target of interest can then be detected, for example, by detecting a signal from a label on DBDpp that is one of the components of the binding complex. The label can be any label that generates a signal that can be detected by standard methods, such as a fluorescent label, a radioactive compound, or an enzyme that reacts with a substrate to generate a detectable signal. Examples of suitable labels for such purposes are described in the present invention and/or are otherwise known in the art.

DBDpp that bind to something of interest can be labeled for detection using radioisotopes, affinity tags (such as biotin, avidin, etc.), enzyme tags (such as horseradish peroxidase, alkaline phosphatase, etc.) etc.) using methods known in the art, such as those described in WO 00/70023 and (Harlow and Lane (1989) Antibodies, Cold Spring Harbor Laboratory, p. 1-726).

A detectable marker or label can be any that is capable of generating, directly or indirectly, a measurable signal, such as a radioactive, chromogenic, luminescent or fluorescent signal that can be used to quantify the amount of bound detectable molecule or label in a sample. Detectable labels known in the art include radioisotopes such as 3H, 14C, 32P, 35 or 125I, electrochemiluminescent labels (such as a ruthenium (Ru) catalyst in combination with substrates, etc.), luminescent or bioluminescent tags (e.g. europium, vanadium), fluorescent or chemiluminescent compounds such as fluorescein isothiocyanate, rhodamine or luciferin, enzymes (e.g. enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase), colorimetric tags such as colloidal gold, colored glass or plastic beads (e.g. polystyrene, polypropylene, latex, etc.), paramagnetic atoms or magnetic substances, electron-dense reagents, nano- or microsphere-containing fluorescent dye, nanocrystals, quantum dot, quantum sphere, nanotag, fluorescently labeled dendrimers , a microtransponder, an electron-donating molecule or molecular structure, or a light-reflecting particle, wherein the microparticles may be quantum dots or nanocrystals. Nanocrystals are substances that absorb photons of light, then re-emit photons of a different wavelength (fluorophores). In addition, additional fluorescent tags or secondary antibodies can be conjugated to the nanocrystals. Nanocrystals are commercially available from sources such as Invitrogen and Evident Technologies (TROY, NY). Other labels include (E)-5-[2-(methoxycarbonyl)ethenyl]cytidine, which is a non-fluorescent molecule that upon exposure to ultraviolet (UV) radiation forms the product, 3-beta-Oribofuranosyl-2,7-dioxopyrido[2,3 -d]pyrimidine, which gives a strong fluorescent signal.

Competitive inhibition can be determined using any method known in the art, for example, competitive ELISA assays. A DBDpp, such as a DBDpp fusion protein (e.g., DBDpp-Fc, DBDpp-CAR, DBDpp-scFv) or other molecule, is said to competitively inhibit the binding of a reference molecule to a given epitope if it binds to such an epitope to such an extent that it blocks, to a certain level, the binding of the reference molecule to the epitope. As used herein, DBDpp (e.g., a DBDpp fusion protein) or other molecule can be said to competitively inhibit the binding of a reference molecule to a given epitope, e.g., by at least 90%, by at least 80%, by at least 70% %, by at least 60%, by at least 50%, by at least 40%, by at least 30%, or by at least 20%. The terms compete, compete, and compete with are relative terms that are used to describe a DBDpp, such as a DBDpp fusion protein that produces at least 20%, at least 30%, at least 40%, or at least 50% inhibition binding of a reference molecule to a DBDpp target, such as a DBDpp fusion protein (e.g., DBDpp-Fc, CAR DBDpp, DBDpp-scFv, and DBDpp antibody), when determined in a standard competition assay as described herein or otherwise known in the art, including, but not limited to, competitive assay systems using techniques such as radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), preferably enzyme-linked immunosorbent assays (ELISAs), sandwich immunoassays, immunoradiometric assays, fluorescent immunoassays, fluorescent, electrochemical fluorescent and electrophoresis immunoassays . Methods for determining the binding and affinity of candidate binding molecules are known in the art and include, but are not limited to, affinity chromatography, size exclusion chromatography, equilibrium

- 89 043883 dialysis, fluorescent probe displacement and plasma resonance.

Affinity purification.

In affinity chromatography-based purification, target proteins are selectively isolated according to their ability to specifically and reversibly bind to a ligand, which is typically covalently linked to the chromatography matrix. In one embodiment, DBDpp can be used as reagents for affinity purification of targets of interest from recombinant sources or from natural sources, such as biological samples (eg, serum).

In another embodiment, a method is provided for isolating a target of interest from a solution that contains the target of interest. Such a method involves: (a) contacting the solution with DBDpp under conditions that allow DBDpp to bind to the target of interest; and (b) isolating the target of interest. In another embodiment, a method is provided for isolating a target of interest from a solution that contains the target of interest, comprising: (a) contacting the solution with DBDpp under conditions suitable for specific binding of DBDpp to the target; and (b), separating the complex(es) formed by the target of interest and/or DBDpp from other components of the solution. In another embodiment, the method further includes the steps of: (c) separating DBDpp from the target of interest and (d) isolating the dissociated target of interest.

In some embodiments, DBDpp, which specifically binds a target of interest, is immobilized on beads and then used to purify the target protein.

Methods for covalently linking proteins to a surface are known in the art, and peptide tags that can be used to attach DBDpp to a solid surface are known in the art. In addition, DBDpp can be attached (ie, cross-linked, bonded or bonded) to a solid surface using any reagents or methods known in the art. In some embodiments, the solid support is selected from: sphere, glass, glass slides, chips, and gelatin. Thus, the DBDpp series can be used to form a matrix on a solid surface using methods known in the art. For example, in patent publ. US 2004/0009530 discloses methods for producing matrices. Contents of the patent publication. US 2004/0009530 is hereby incorporated by reference in its entirety.

In another embodiment, DBDpp is used to isolate a target of interest using affinity chromatography. Any standard chromatographic method can be used. In some embodiments, DBDpp is immobilized on a solid support. DBDpp can be immobilized on a solid support using methods and reagents described in the present invention or otherwise known in the art. Suitable solid supports are described in the present invention or otherwise known in the art and, in some embodiments, are suitable for packing a chromatography column. The immobilized DBDpp can then be applied or contacted with a solution under conditions favorable for the formation of a complex between the DBDpp and the target of interest. Unbound materials can be removed by washing. Suitable washing conditions can be readily determined by one skilled in the art. Examples of suitable wash conditions include, but are not limited to, PBS/0.01% Tween 20, pH 7.2, and 1M NaCl/10 mM Tris, pH 7.5. Tris-containing wash buffers may be preferred since phosphates may precipitate in 50% ethylene glycol. Typically, under non-limiting conditions, wash buffers have a pH of 7.0, optionally containing 0.0-1.5 M NaCl, more preferably 1 M NaCl. In addition, wash buffers may optionally contain a mild detergent such as Tween 20, Tween 80 or NP-80. The target of interest can be eluted from the DBDpp binding complex by creating solution conditions that promote dissociation of the binding complex. Suitable elution solutions can be readily determined by one skilled in the art and include, but are not limited to, 50% ethylene glycol/10 mM NaOAc. As a non-limiting example, suitable elution buffers contain 40-60% ethylene glycol, preferably 50% ethylene glycol; and 50-100 mM NaOAc, at a pH ranging from pH 4-7, more preferably pH 4-6 and most preferably pH 4.5-5.5. Preferably, Fast Flow affinity chromatography technique is used to bind DBDpp to the target of interest and from which the purified target of interest is eluted.

Alternatively, chromatography can be performed by mixing a solution containing the target of interest and DBDpp and then isolating complexes of the target of interest and DBDpp. For this type of separation, many methods are known and can be applied in a standard manner. For example, DBDpp can be immobilized on a solid support such as spheres, then separated from solution along with the target of interest through filtration. In another example, DBDpp may be a fusion protein that contains a peptide tag, such as a poly-HIS tail or streptavidin-binding region, which can be used to isolate DBDpp after complexation using an immobilized metal affinity chromatography resin or streptavidin-coated substrate. Once separated, the target of interest can be released from DBDpp under elution conditions and isolated in purified form.

Therapeutic agents.

The DBDs described herein can be used in a variety of fields, including, but not limited to, therapeutic treatments, which may be in vitro, ex vivo or in vivo methods.

Use as a therapeutic entity is an indication of the target-binding specificity of DBDpp. Incorporation of DBDpp into various molecular compositions (eg, DBD-antibody fusions, DBD-drug conjugates, and DBD-chimeric receptors) allows for use in a range of therapeutic indications and modalities, which include, but are not limited to, soluble and cell-associated compositions.

In one embodiment, DBDpp is a soluble fusion protein (which is schematically shown in Fig. 5C and consists of an optional epitope tag 10 and a targeting domain 20) that binds to a target that is associated with a metabolic, cardiovascular, musculoskeletal disease or disorder , neurological or skeletal system. In other embodiments, DBDpp is a soluble fusion protein that binds to a target that is associated with a yeast, fungal, viral or bacterial infection or disease. In some embodiments, DBDpp is a soluble fusion protein that binds to a target that is associated with a disease or disorder of the immune system.

Also provided are therapeutic compositions that can be used to carry out the therapeutic methods described in the present invention. In one embodiment, the therapeutic compositions provided herein comprise a physiologically tolerated carrier along with at least one DBDpp fusion construct as described herein dissolved or dispersed therein as an active ingredient. In another embodiment, the therapeutic compositions provided herein comprise a physiologically tolerable carrier together with at least one species of DBDpp, as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a human patient for therapeutic purposes.

The preparation of a pharmacological composition which contains the active ingredients dissolved or dispersed therein is well known in the art. Typically, such compositions are prepared as sterile injectable solutions, either as liquid solutions or suspensions, aqueous or non-aqueous. However, solid forms suitable for preparing a solution or suspension in liquid before use can also be prepared. The drug can also be emulsified. Thus, the DBDpp-containing composition may take the form of solutions, suspensions, tablets, capsules, sustained release dosage forms or powders or other compositional forms. In some embodiments, DBDpp compositions (eg, DBDpp fusion proteins) are formulated to ensure or optimize in vivo distribution. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds and, if necessary, formulations are formulated to increase BBB passage, such as liposome formulations. For methods of producing liposomes, see, for example, US Pat. US 4,522,811, 5,374,548 and 5,399,331. Liposomes may include one or more molecules that are selectively transported to specific cells or organs, thereby improving targeted drug delivery (see, for example, Ranade, Clin. Pharmacol. 29:685 (1989)).

DBDpp (eg, DBDpp fusion protein) can be mixed with other active ingredients and/or excipients that are pharmaceutically acceptable and compatible with the active ingredient, and in amounts suitable for use in the therapeutic methods described in the present invention. Suitable excipients are, for example, water, saline, dextrose, glycerin, ethanol and the like, and combinations thereof. In addition, if necessary, the composition may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, etc., which increase the effectiveness of the active substance.

The DBDpp therapeutic agent may include pharmaceutically acceptable salts of the components. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be obtained from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. .P.

Physiologically tolerated carriers are known in the art. Examples of liquid carriers are sterile aqueous solutions that do not contain other materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH, saline sodium chloride, or both, such as phosphate buffered saline solution. In addition, aqueous vehicles may contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other solutes.

Liquid compositions may also contain liquid phases in addition to and other than water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate and water-in-oil emulsions.

In one embodiment, the therapeutic composition contains a DBDpp fusion protein, typically in an amount of at least 0.1 percent by weight of the DBDpp fusion protein based on the total weight of the therapeutic composition. The percentage by weight is the weight ratio of the fused DBDpp structure to the complete composition. Thus, for example, 0.1 percent by weight is 0.1 grams of DBDpp per 100 grams of the total composition.

A therapeutic composition containing a DBDpp fusion protein typically contains from about 10 micrograms (μg) per milliliter (ml) to about 100 milligrams (mg) per ml of DBDpp fusion protein active ingredient per volume of composition, and more preferably contains from about 1 mg/ml to approximately 10 mg/ml (ie, approximately 0.1-1 percent by weight).

Dosage ranges for administration of DBDpp (eg, DBDpp fusion protein) are large enough to produce the desired effect in which the symptoms of the disease mediated by the target molecule are alleviated. The dose should not be so large as to cause undesirable side effects such as hyperviscosity syndrome, pulmonary edema, congestive heart failure, etc. Typically, the dosage varies depending on the age, condition, gender and extent of the disease in the patient and can be determined by a person skilled in the art. The dose may be adjusted by the individual physician in case of any complication.

DBDpp (eg, DBDpp fusion protein) can be administered parenterally by injection or gradual infusion over time. Although the target molecule can typically be accessed in the body through systemic administration, and therefore treatment is most often accomplished by intravenous administration of therapeutic compositions, other tissues and delivery vehicles are contemplated where there is a likelihood that the target tissue contains the molecule -target. Thus, DBDpp can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitarily, transdermally, and can be delivered using peristaltic devices. DBDpp fusion proteins can also be delivered as an aerosol to the airways and lungs.

Therapeutic compositions containing DBDpp can usually be administered intravenously, for example, by injection of a unit dose. The term unit dose when used in relation to a therapeutic composition of the present invention refers to physically discrete units suitable as a unit dose for a subject, each unit containing a specified amount of active material calculated to produce the desired therapeutic effect, in combination with necessary diluent; for example, a carrier or solvent. In a certain embodiment, therapeutic compositions containing DBDpp are administered subcutaneously.

In some embodiments, DBDpp (eg, a DBDpp fusion protein) is administered in a route compatible with the dosage form and in a therapeutically effective amount. The amount administered depends on the subject to be treated, the subject's body's ability to utilize the active substance, and the degree of therapeutic effect desired. The exact amounts of active substance that will need to be administered depend on the judgment of the healthcare practitioner and are individual for each person. However, suitable dosage ranges for systemic administration are disclosed herein and depend on the route of administration. Suitable dosing regimens also vary, but are characterized by an initial administration followed by repeated doses at intervals of one or more hours upon subsequent injection or other administration. An alternative is a continuous intravenous infusion sufficient to maintain blood concentrations within the ranges established for in vivo therapy.

DBDpp compositions are prepared, dosed and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the route of administration, the schedule of administration and other factors known to health care professionals. The dosage ranges for administration of DBDpp are large enough to produce the desired effect in which the symptoms of the disease mediated by the target molecule are alleviated. The dose should not be so large as to cause undesirable side effects such as hyperviscosity syndrome, pulmonary edema, congestive heart failure, etc. Typically, the dosage varies depending on the age, condition, gender and extent of the disease in the patient and can be determined by a person skilled in the art. The dose may be adjusted by the individual physician in case of any complication.

- 92 043883

Dosage schedule and amounts administered effective for therapeutic and prophylactic use, i.e. dosage regimen will depend on a number of factors, including the cause, stage and severity of the disease or disorder, health status, physical condition, age of the mammal being treated, and the site and mode of delivery of the DBD. The therapeutic efficacy and toxicity of the complex and formation can be determined using standard pharmaceutical, pharmacological and toxicological procedures in cell cultures or experimental animals. Data obtained from these procedures can similarly be used to develop dose ranges for human use. In addition, the therapeutic index (ie, the dose therapeutically effective for 50 percent of the population divided by the dose lethal for 50 percent of the population (ED50/LD50)) can be easily determined using known techniques. The dose is preferably within a concentration range that includes the ED50 with minimal or no toxicity and may vary within this range depending on the dosage form used, patient sensitivity and route of administration.

The administration schedule also takes into account pharmacokinetic parameters known in the art, such as drug absorption rate, bioavailability, metabolism and clearance (see, for example, Hidalgo-Aragones, J. Steroid Biochem. Mol. Biol. 58:611-617 (1996 ); Groning et al., Pharmazie 51:337-341 (1996); Fotherby, Contraception 54:59-69 (1996); and Johnson et al., J. Pharm. Sci. 84:1144-1146 (1995)) . The healthcare provider is knowledgeable about how to tailor the dosing schedule for each subject being treated. In addition, single or multiple administrations of DBDpp compositions can be administered at a dosage and frequency level according to the need and tolerance of the subject. The duration of prophylactic and therapeutic treatment varies depending on the specific disease or condition being treated. Some diseases can be treated immediately, while others require long-term, ongoing therapy. DBDpp can be administered intermittently or concomitantly with an additional therapeutic agent.

In some embodiments, DBDpp is administered in an amount of from about 1 mg/kg to about 50 mg/kg, from about 1 mg/kg to about 25 mg/kg, from about 1 mg/kg to about 20 mg/kg, from about 1 mg/kg to about 15 mg/kg, about 1 mg/kg to about 10 mg/kg, or about 1 mg/kg to about 5 mg/kg.

In another embodiment, DBDpp is administered in combination with one or more additional therapeutic agents.

A therapeutically effective amount of DBDpp, such as a DBDpp fusion protein, may be an amount that, when administered in a physiologically tolerable composition, is sufficient to produce a plasma concentration of from about 0.1 micrograms (μg) per milliliter (mL) to about 100 μg /ml, preferably from about 1 μg/ml to about 5 μg/ml and usually about 5 μg/ml. In other words, the dose may range from about 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or more dosages per day, for one or more days.

In one embodiment, the disease or disorder is a disease or disorder of the immune system, such as inflammation or an autoimmune disease.

In some embodiments, DBDpp is a soluble protein that specifically binds to a target that is associated with a metabolic, cardiovascular, musculoskeletal, neurological, or skeletal disease or disorder.

In other embodiments, DBDpp is a soluble protein that specifically binds to a target that is associated with a yeast, fungal, viral or bacterial infection or disease. In some embodiments, DBDpp is a soluble protein that specifically binds to a target that is associated with a disease or disorder of the immune system.

In embodiments, DBDpp fusion proteins can be used to inhibit tumor growth, reduce neovascularization, reduce angiogenesis, induce differentiation, reduce tumor volume, and/or reduce tumor tumorigenicity.

In some embodiments, the DBDpp described herein can be used to treat cancer. Thus, some embodiments of the invention provide methods for treating cancer comprising administering a therapeutically effective amount of DBDpp (eg, a DBDpp fusion construct) to a patient.

Cancers that can be treated include tumors that are not vascularized or not yet significantly vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematologic tumors, such as leukemias and lymphomas) or may include solid tumors. The types of cancers that will be treated with DBDpp include, but are not limited to, carcinoma, blastoma and sarcoma, and some leukemias or lymphoid neoplasias, benign and malignant tumors, and malignancies such as sarcomas, carcinomas and melanomas. We. Also included are adult tumors/cancers and pediatric tumors/cancers.

Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid neoplasia, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas, papillary sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma cinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma and central nervous system tumors (such as glioma (such as brainstem and mixed gliomas), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and head metastases nom brain).

In another embodiment, the DBDpp described in the present invention can be used to treat a patient having hematological cancers. Examples of hematologic (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic leukemia ny ( granulocytic leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and poorly differentiated forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodys splazy.

In additional embodiments, the DBDpp fusion protein binds: (1) a target on a cell or tissue of interest (eg, a tumor antigen on a tumor cell), and (2) a target on an effector cell, such as a T cell receptor molecule. In one embodiment, binding of one or more targets by a DBDpp fusion protein is used to direct an immune response against an infectious agent, cell, tissue, or other site of interest in a patient. For example, in some embodiments, DBDpp specifically binds a target on the surface of an effector cell. Thus, in some embodiments, DBDpp specifically binds a target on the surface of a T cell. In certain embodiments, DBDpp specifically binds CD3. In other embodiments, DBDpp specifically binds CD2. In another embodiment, DBDpp specifically binds to the T cell receptor (TCR). In additional embodiments, DBDpp specifically binds a target on the surface of an NK cell. Thus, in some embodiments, DBDpp specifically binds the NKG2D (natural killer group 2D) receptor. In additional embodiments, DBDpp specifically binds CD16 (ie, Fc gamma RIII), CD64 (ie, Fc gamma RI), or CD32 (ie, Fc gamma RII).

In one embodiment, the DBDpp fusion protein binds a target on a leukocyte and a tumor antigen on a tumor cell. In some embodiments, the DBDpp fusion protein binds NKG2D. In another embodiment, the DBDpp fusion protein binds NKG2D and a target selected from ErbB2, EGFR, IGF1R, CD19, CD20, CD80 and EPCAM. In one embodiment, the DBDpp fusion protein binds CD3. In certain embodiments, DBDpp specifically binds CD3 epsilon. In one embodiment, the DBDpp fusion protein binds CD4.

In one embodiment, the DBDpp fusion construct is bispecific and specifically binds to two different targets expressed on the surface of two different cell types. In one embodiment, the bispecific DBDpp fusion protein specifically binds to a target on a cancer cell and a target on an effector immune cell. In one embodiment, the DBDpp bispecific fusion protein specifically binds a target expressed on a cancer cell (eg, CD19) and a target expressed on the surface of a T cell (eg, CD3).

In some embodiments, DBDpp can mimic ligand binding. In some embodiments, DBDpp may mimic the biological activity of a ligand (DBDpp agonist) or inhibit the bioactivity of a ligand (DBDpp antagonist), for example, through competitive binding. DBDpp in DBDpp fusion proteins can also affect targets in other ways, such as by neutralizing, blocking, stabilizing, aggregating, or cross-linking the DBDpp target.

DBDpp-drug conjugates.

In another embodiment, the DBDpp fusion protein may be linked to other organic or inorganic molecules or substrates through chemical conjugation. In one embodiment, DBDpp-drug conjugates are designed to facilitate local delivery of cytotoxic agents using the targeting specificity of DBDpp. This combination of targeting specificity and cytotoxicity allows for drug targeting and intracellular accumulation within tumors, whereas systemic administration of such unconjugated drugs may result in unacceptable levels of toxicity to normal cells along with the tumor cells that must be eliminated (Baldwin et al. , Lancet pages 603-05 (1986); Thorpe, Antibody Carriers Of Cytotoxic agents In Cancer Therapy: A Review, Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al., (ed.s), pp. 475 -506) (1985)).

Cytotoxic agents include chemotherapeutic agents, growth inhibitory agents, toxins (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), radioactive isotopes (ie, radioconjugate), etc. Chemotherapeutic agents useful in creating such immunoconjugates include, for example, methotrexate, adriamycin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents. Chemotherapeutic agents useful in creating such immunoconjugates also include antitubulin agents such as auristatins, including monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). Enzymatically active toxins and fragments thereof that can be used according to the invention include diphtheria toxin A chain, non-binding active diphtheria toxin fragments, exotoxin A chain, ricin A chain, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, diantina proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes.

In one embodiment, DBDpp (eg, a DBDpp fusion protein) is conjugated to a radioisotope. In another embodiment, DBDpp is conjugated to an isotope selected from 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re, using any of many known chelating agents or direct labeling. In other embodiments, DBDpp is associated with drugs, prodrugs, or lymphokines, such as interferon. DBDpp-cytotoxin conjugates can be prepared in a standard manner using a variety of bifunctional protein cross-linkers such as N-succinimidyl-3-(2pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyladipimidate-HCl), 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 1,5-difluoro2,4-dinitrobenzene). In a certain embodiment, the toxin is conjugated to a DBDpp fusion protein through an enzyme-cleavable linker system (eg, such as is present in SGN35). Conjugates of DBDpp and one or more low molecular weight toxins, such as calicheamicin, maytansinoids, trichothene and CC1065, and derivatives of such toxins that have toxin activity may also be used.

In some embodiments, the cytotoxic agent is covalently attached to DBDpp via a linker. In some embodiments, the linker through which DBDpp and the cytotoxic agent are connected may be cleaved by a protease.

Therapeutic use as a cell-associated receptor.

In one embodiment of the invention, DBDpp-CARs are used to redirect transduced T cells against a tumor target using the binding specificity of DBDpp-CARs. In one example, primary T cells are transduced with a lentiviral vector encoding a CAR that combines the target binding domain of the DBD with the transmembrane domain and intracellular domain of CD3-zeta, CD28, 4-1BB. The resulting population of transduced T cells can then induce a DBDpp-CAR-mediated T cell response. In one embodiment, the T cells are genetically modified to express DBDpp-CAR, and the DBDpp-CAR T cell is administered to a recipient in need thereof. The injected cell is capable of killing tumor cells in the recipient. Several embodiments of the invention are particularly advantageous because they include one, more, or all of the following advantages: (i) target-binding specificity, (ii) increased therapeutic efficacy, (iii) reduced off-target side effects, (iv) ability to tailor to specific markers. patient or group of patients, (v) increased stability during manufacturing and processing, and (vi) the ability to target one, two or more specific targets to improve target-directed therapy.

Genetically modified cells, redirected cells, recombinant cells or modified cells as used herein refer to cells that express the DBDpp of the present invention. In a specific embodiment, the genetically modified cells express a DBDpp fusion protein, such as DBDppCAR. In another embodiment, the genetically modified cells express and display DBDpp-CAR on the surface of the cells.

Disease targeted by genetically modified cells, as used herein, includes targeting any cell involved in any way in any disease by genetically modified cells, regardless of whether the genetically modified cells target diseased cells or on healthy cells to obtain a therapeutically beneficial result. Genetically modified cells include, but are not limited to, genetically modified T cells, NK cells, hematopoietic stem cells, pluripotent embryonic stem cells, or embryonic stem cells. Genetically modified cells express DBDpp-CAR, which can specifically interact with any of the antigens expressed on the surface of target cells.

In one embodiment, the DBDpp portion of the DBDpp-CAR is designed to treat a specific cancer. Cancers that can be treated include tumors that are not vascularized or not yet significantly vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematologic tumors, such as leukemias and lymphomas) or may include solid tumors. The types of cancers that will be treated with DBDpp include, but are not limited to, carcinoma, blastoma and sarcoma, and certain leukemias or lymphoid neoplasias, benign and malignant tumors, and malignancies such as sarcomas, carcinomas and melanomas. Also included are adult tumors/cancers and pediatric tumors/cancers.

Examples of hematologic (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic leukemia ny ( granulocytic leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and poorly differentiated forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodys splazy.

Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid neoplasia, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas, papillary sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma cinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma and central nervous system tumors (such as glioma (such as brainstem and mixed gliomas), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoy craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and brain metastases brain).

In one embodiment, cancers and disorders can be treated using a cell expressing a DBDpp-CAR that targets CD19, CD20, CD22 and ROR1. In one specific embodiment, a DBD-CAR that targets CD22 can be created to treat B-cell lymphoma. In another embodiment, a cell expressing a DBDpp-CAR that contains an engineered DBDpp that targets CD19 can be used to treat cancers and disorders including, but not limited to, pre-B-ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, rescue after allogeneic bone marrow transplantation, etc.

B-cell-associated diseases as used herein include B-cell immunodeficiencies, autoimmune diseases, and/or excessive/uncontrolled B-cell-associated cell proliferation (including lymphomas and/or leukemias). Examples of such diseases for which DBDpp-CAR may be used therapeutically include, but are not limited to, systemic lupus erythematosus (SLE), diabetes, rheumatoid arthritis (RA), reactive arthritis, multiple sclerosis (MS), pemphigus vulgaris, celiac disease, Crohn's disease, inflammatory bowel disease, ulcerative colitis, autoimmune thyroid disease, X-linked agammaglobulinemia, non-specific B-cell acute lymphoblastic

- 96 043883 leukemia, systemic lupus erythematosus, variable unclassified immunodeficiency, chronic lymphocytic leukemia, diseases associated with selective IgA deficiency and/or deficiency of the IgG subclass, B-cell line lymphoma (Hodgkin lymphoma and/or non-Hodgkin lymphoma), immunodeficiency with thymoma, transient hypogammaglobulinemia and/or hyper-IgM syndrome, as well as virally mediated B-cell diseases such as EBV-mediated lymphoproliferative disease, and chronic infections in which B-cells are involved in the pathophysiology.

In one embodiment, a DBDpp-CAR that targets mesothelin can be engineered to treat mesothelioma, pancreatic cancer, ovarian cancer, and the like. In one embodiment, a CD33/IL3Ra-targeted DBDpp-CAR may be engineered to treat acute myelogenous leukemia or the like. In one embodiment, a c-Met-targeted DBDpp-CAR may be engineered to treat triple-negative breast cancer, non-small cell lung cancer, or the like. In one embodiment, a PSMA-targeted DBDpp-CAR may be engineered to treat prostate cancer and the like. In one embodiment, a DBDpp-CAR may be engineered to target glycolipid F77 for the treatment of prostate cancer and the like. In one embodiment, a DBDpp-CAR that targets EGFRvIlI may be engineered to treat glioblastoma and the like. In one embodiment, a DBDpp-CAR that targets GD-2 can be engineered to treat neuroblastoma, melanoma, and the like. In one embodiment, a DBDppCAR that targets NY-ESO-1 can be engineered to treat myeloma, sarcoma, melanoma, and the like. In one embodiment, a DBDpp-CAR that targets MAGE A3 can be engineered to treat myeloma, sarcoma, melanoma, and the like. However, it should be understood that the invention should not be limited only to the target antigens and diseases disclosed in the present invention. On the contrary, it should be understood that the invention includes any antigenic target that is associated with a disease where DBDpp-CAR can be used to treat such disease.

In a preferred embodiment, DBDpp-CAR is expressed in a T cell, and a method of treating or preventing cancer is provided, comprising administering host cells expressing DBDpp-CAR to a cancer patient in which the cancer cell expresses a tumor antigen on its surface, and wherein DBDpp specifically binds the target antigen. Examples of target antigens that DBDpp and DBDpp-CAR bind include, but are not limited to, CD19, CD123, TSLPR, and CD267.

DBDpp-CAR-modified T cells can also serve as a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. Preferably the mammal is human.

The DBDpp-CAR modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or other components such as chemotherapeutic agents, antibodies, cytokines or cell populations. The compositions of the present invention are preferably formulated for intravenous administration and may be administered one or more times.

Loss-of-antigen escape variants, as used herein, refer to cells that exhibit reduced expression or loss of expression of a target antigen, where such antigens are the target of the CAR receptor of the present invention.

Various embodiments of the invention are illustrated below by describing experiments carried out in accordance therewith. The examples that follow are presented to facilitate the practical application of the disclosed embodiments and should not be construed as limiting the remainder of the description in any way. In the examples, reference is made to the accompanying figures.

Examples

Example 1. Assessment of immunogenicity of DBDpp.

DBDpp sequences, particularly those administered to a subject and/or used in the purification of a composition administered to a subject, are preferably not antigenic to the subject (eg, human). In some embodiments, the DBDpp sequence does not contain a human HLA-DR binding motif or cleavage sites for proteasomes and immune proteasomes. In certain embodiments, the DBDpp sequence does not contain an antigenic sequence as determined by a version of a computer predictive model existing as of the filing date of this disclosure. In certain embodiments, the DBDpp sequence does not contain an MHC binding region sequence (class I or class II) as predicted using an algorithm selected from ProPred (see, e.g., Singh, Bioinformatics 17(12):1236-1237 (2001)) , ProPred1 (Singh, Bioinformatics 19(8):1009-14 (2003)), SYFPEITHI (see, for example, Schuler, Immunoinf. Meth. in Mol. Biol. 409(1):75-93 (2007)), SMM-align (see, for example, Nielsen, BMC Bioinformatics 8:238 (2007)), RANKPEP (see, for example, Reche, Hum Immunol 63: 701-709. (2004)) or

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TEPITOPE (see, Stumiolo, Nat Biotechnol 17:555-561 (1999)), where the version of the algorithm and the applied database exists as of the filing date of this application.

In silico analysis of the alpha3D amino acid sequence (MGSWAEFKQRLAAIKTRLQALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 49) revealed a 9 amino acid sequence (i.e. LAAIKTRLQ (SEQ ID NO: 50)) that shares properties with the high affinity sequence (binding threshold less than 6%) and mixed (existing in more than 50% of the corresponding alleles) T-cell epitopes (Singh, Bioinformatics 17:1236-1237, 2001). This epitope is present in the invariant region of some DBDpp libraries. Thus, in order to reduce potential immunogenicity, the Q19E substitution was introduced in SEQ ID NO: 49. This conservative and superficial substitution was expected to be unlikely to result in significant disruption of the hydrophobic core (see, e.g., Fig. IB).In silico analysis of the resulting sequence (SEQ ID NO: 1) showed lower immunogenicity scores.

Example 2: Development, construction and screening of the DBDpp library.

Unlike natural ligands and binding proteins, the synthetic DBD framework sequence (ie, SEQ ID NO: 1) has no known binding partner. When designing DBDpps that bind to targets, residues were selected for mutation (i.e., randomization in the library) if they were considered surface-exhibiting significant solvent accessibility. There are many methods for assessing the solvent accessibility of certain molecular structures. For example, PyMOL is a publicly available software package developed for molecular imaging and analysis and can be used to calculate solvent accessible surface area using the method of Lee and Richards (Lee et al., J. Mol. Biol. 55:379-400 (1971) )). In particular, when using PyMOL (version 1.4.1) with the parameters set to dot solvent 1, solvent radius 1.4 A and dot density 4, the solvent accessible surface area can be calculated for each amino acid in the homology model SEQ ID NO: 1 per matrix-based, PDB 2A3D. In table Table 2 lists the calculated area (in square angstroms) for each residue, when measured relative to a domain (area D) and for an individual amino acid, regardless of steric hindrance created by neighboring residues (area I). The relative accessibility of a residue in a domain (area D) compared to the isolated state (area I) is expressed as a percentage (%A).

table 2

Solvent accessible reference framework sequence (SEQ ID NO: 1)

provisions e ak Square I Square D %A position ak Square I Square D %A 1 M 295.0 156, 9 53.2 38 F 316, 3 12.6 4.0 2 G 187.1 56, 6 30.2 39 E 289.6 112.0 38.7 3 S 227.0 23.6 10.4 40 S 227.0 93.7 41.3 4 W 345.8 107.4 31.1 41 E 285.0 57.9 20.2 5 A 211.9 53.7 25.4 42 L 281.8 21.5 7.6 6 E 286, 8 104.7 36.5 43 Q 294.3 120.5 41.0 7 F 3181 7.8 2.5 44 A 209.6 79.0 37.7 8 TO 303.7 142.0 46, 8 45 Y 323.7 33.1 10.2 9 Q 293.7 123.0 41.9 46 TO 303.8 70.1 23.1 10 R 341.2 102.9 30.2 47 G 187.2 64.4 34.4 eleven L 274.1 19.0 6, 9 48 TO 298.8 149.1 49.9 12 A 211.5 54.8 25.9 49 G 185.6 6, 9 3.1 13 A 211.3 43.4 20.6 50 N 267.4 80.5 30.1 14 I 279.0 180 6, 5 51 R 238.9 92.8 38.2 15 TO 300.5 134.3 44.7 52 E 293.1 110.3 37.6 16 T 246.5 89.7 36, 4 53 V 249.2 5.7 2.1 17 R 339.6 1318 38.8 54 E 290.7 91.0 31.3 18 L 279.6 27.8 9.9 55 A 211.2 67.2 31.8 19 E 269.3 135.1 50.2 56 L 274.5 10.5 3.2 20 A 211.6 61.6 29.1 57 R 336, 4 146, 8 43.6 21 L 278.0 89 3.2 58 TO 306, 7 165.1 53.8 22 G 186, 9 67.8 36, 3 59 E 290.1 114.0 39.3

-98043883

23 G 186, 6 47.9 25.7 60 A 211.1 13.2 6, 3 24 S 225.9 12.2 5.4 61 A 211.7 47.6 22.5 25 E 286, 9 130.2 45.4 62 A 211.0 56, 7 26, 9 26 A 210.5 92.4 43.9 63 I 275.2 26, 3 9.5 27 E 281.6 57.7 20.5 64 R 337.5 119.5 35.4 28 L 269.8 44 1.6 65 D 261.1 106.5 40.8 29 A 211.4 53.0 25.1 66 E 284.7 102.5 36, 0 thirty A 210.7 54.2 25.7 67 L 272.8 7.3 2.7 31 F 317.0 14.8 4.7 68 Q 277.9 131.3 47.2 32 E 284.7 96, 0 33.7 69 A 211.4 40.4 19.1 33 TO 306, 8 1588 51.8 70 Y 329.3 50.8 15.4 34 E 281.1 83.1 29.5 71 R 341.0 149.1 43.7 35 I 276, 3 22.9 8.3 72 N 279.1 135.5 48.5 36 A 211.4 585 27.7 73 N 275.1 130.5 47.4 37 A 209.8 51.4 24.5

Residues in the domain with %A values that are less than 10-11% (e.g. F7, L11, I14, L18, L21, S24, L28, F31, I35, F38, L42, Y45, G49, V53, L56, A60, I63 and L67 in SEQ ID NO: 1; Table 2A) were considered to be relatively inaccessible to external solvent and were therefore considered to be internal core DBD residues. On the other hand, residues with %A values that exceeded 10-11% (for example, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69 and Y70 SEQ ID NO: 1) are predicted to be located in regions of the polypeptide associated with alpha-helical secondary structure and to occupy positions that have greater potential for interaction with macromolecular targets of interest. These solvent-accessible alpha-helical residues were considered candidates for the greatest degree of substitutional variability (including conservative and non-conservative substitutions) in the library.

Non-alpha helix residues of the reference framework sequence correspond, in several embodiments, to positions M1, L21, G22, G23, S24, E25, A26, E27, Y45, K46, G47, K48, G49, N50, P51, R71, H72 and N73 SEQ ID NO: 1. In additional embodiments, the reference framework sequence residues included in the alpha helices correspond to positions M1, G22, G23, S24, E25, A26, E27, K46, G47, K48, G49, N50, P51, R71, H72 and N73 SEQ ID NO: 1. These residues were also considered candidates for conservative and non-conservative substitutions in the library.

Some DBDpp libraries have been generated by selectively or randomly mutating positions of certain solvent-accessible positions of the DBDpp amino acid sequence. In one series of experiments, libraries, referred to herein as plane libraries or F libraries, were designed such that the replaced residues of the reference polypeptide framework SEQ ID NO: 1 were grouped on the same plane of the domain and formed one continuous binding surface. Plane libraries were constructed for all three planes (F1, F2 and F3) of the polypeptide structure SEQ ID NO: 1. Due to the asymmetry of the domain structure, each pair of alpha helices, and therefore each plane, forms a unique geometric topology (Figs. 1 and 2). As modeled in the reference scaffold, the large number of target residues correspond to a continuous surface area greater than 1400 square angstroms, significantly larger than the binding surfaces measured in a study of non-immunoglobulin binding scaffolds (Gilbreth et al., Curr. Opin. Struct. Biol. 22:413- 420 (2012)).

In another set of experiments, libraries, referred to herein as combination libraries or C-libraries, were constructed to identify DBDpps that potentially exhibit multiplanar binding to the target of interest (Table 3 and FIGS. 1 and 2). Combination libraries (C1 and C2) were constructed by combining residues from each of the three helices used in the F-series libraries.

In these experiments, a total of 32 residue positions were mutagenized. Each mutagenesis position is present in at least 2 libraries. In addition, each mutagenesis position is represented in each of the two library architectures; F and C.

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Table 3

DBDpp library sequence profiles

Library Sequence profile Fl MGSWX5X ₆ FKX ₉ X ₁ oLAX ₁ 3lKX ₁₆ X ₁₇ LEALGGSEAELAX3oFEX33X34lAX ₃₇ FEX4oX4i LQX ₄₄ YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 2) F2 MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX ₃ 2X33EIX3 ₆ AFX3 ₉ X ₄ oELX ₄₃ A YKGKGNPEVEALX ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO: 3) F3 MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALGGSEAELAAFEKEIAAFESELQAYKGK GNPEVEX ₅₅ LRX5 ₈ X ₅₉ AAX ₆ 2lRX65X66LQAYRHN (SEQ ID NO: 4) Cl MGSWX ₅ X ₆ FKX ₉ X ₁ oLAX ₁ 3lKX ₁₆ X ₁₇ LEALGGSEAELAAFX3 ₂ X33EIX3 ₆ AFX3 ₉ X ₄ o ELX ₄₃ AYKGKGNPEVEX55LRX5 ₈ X5 ₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO: 5) C2 MGSWX5EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALGGSEAELAX3oFEX33X3 ₄ IAX3 ₇ FEX ₄ oX ₄ iL QX ₄₄ YKGKGNPEVEALX ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO: 6) FlLp _x F2Lp _x F3Lp _x CILpx MGSWX ₅ X ₆ FKX ₉ X ₁ oLAX ₁ 3lKX ₁₆ X ₁₇ LEALZ ₁ EAELAX ₂₈ FEX3 ₁ X3 ₂ IAX35FEX3 ₈ X3 ₉ LQ X ₄₂ YZ ₂ NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 7) MGSWAEFKQRLAAIKTRLEALZ4EAELAA FX30X31EIX34AFX37X38ELX4 4AYZ2NPEV EALX ₅₂ X53EAX5 ₆ AIX5 ₉ X ₆ oELX ₆₃ AYRHN (SEQ ID NO: 8) MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALZ ₁ EAELAAFEKEIAAFESELQAY Z ₂ NPEVEX ₅ oLRX53X5 ₄ AAX ₅₇ IRX ₆ oX ₆ iLQAYRHN (SEQ ID NO: 9) MGSWX ₅ X ₆ FKX ₉ X ₁ oLAX ₁ 3lKX ₁₆ X ₁₇ LEALZ ₁ EAELAAFX3oX3iEIX3 ₄ AFX3 ₇ X3 ₈ ELX ₄₁ AYZ2NPEVEX ₅ oLRX ₅₃ X54AAX ₅₇ IRX ₆ oX _6:l LQAYRHN (SEQ ID NO: 10) C2Lp _x MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁ 5X ₁₆ RLX ₁₉ ALZ ₁ EAELAX ₂₈ FEX3 ₁ X3 ₂ IAX35FEX3 ₈ X ₃₉ L QX ₄₂ YZ ₂ X ₅₂ X53EAX5 ₆ AIX5 ₉ X ₆ oELX ₆₃ AYRHN (SEQ ID NO: 11) X=all amino acid residues 2=amino acid sequence corresponding to loop1 ( _Z4 ) or loop2 ( _Z2 ) as described herein

Construction of the DBDpp F2 _nnk library.

An F2 library was constructed that targeted 12 surface residues on plane 2 (helices 2 and 3) (Table 3 and Figures 2C and 2D). This library, designated F2NNK, was generated by Kunkel mutagenesis using oliages containing NNK codons. Libraries were constructed using a modified pComb phagemid vector in which DBDpp is fused C-terminally to the N-terminus of M13 pIII. These DBDpp are also fused at their N-terminus to the C-terminus of the FLAG epitope tag. The entire DBDpp fusion protein is under the secretory control of the DsbA signal peptide (Fig. 3B).

Construction of the trinucleotide phosphoramidite library DBDpp.

The following libraries were constructed by Kunkel mutagenesis in the same modified pComb phagemid vector as the F2NNK library. In several embodiments, the FLAG tag is optional (see, for example, FIG. 3C), or may be replaced by another tag. These libraries were constructed using a mixture of trinucleotide phosphoramidites (codons). These mixtures were designed to exclude the stop codon and the cysteine and proline codons and ensure equal representation of the remaining amino acids. Libraries were constructed using all five sequence profiles (F1, F2, F3, C1 and C2), as shown in Table. 3.

Selection using the F2 _nnk library.

The F2 _NNK DBD library was used in five rounds of selection against recombinant human biotinylated 4-1BB/TNFRSF9/CD137-FC. ELISA screening of the rescued phage showed that 89 of 95 clones bound 4-1BB/CD137 with an average OD of 5.3 times greater than the control (IgG Fc). Binding distribution (ELISA optical density values) for 89 clones: CD137 0.353 (0.134-0.617), control 0.067 (0.056-0.125). Sequencing of the individual phage showed that all 89 clones were identical at the nucleotide level. Specifically, this clone, named bb10, contained substitutions at only 8 (shown in bold in the sequence below) of the 12 randomized positions in SEQ ID NO: 1, which are underlined in the sequence:

MGSWAEFKQRLAAIKTRLEALGGSEAELAAFLGEIWAFEMELAAYKGKGNPEVEALGREAAAIRMEL^AYRHN (bb10: SEQ ID NO: 19).

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Selection using trinucleotide phosphoramidite libraries F1, F2, F3, C1 and C2.

Screens were also made using trinucleotide phosphoramidite libraries F1, F2, F3, C1 and C2. In most cases, libraries were pooled before use in screening. When equal volumes of individual libraries were combined, Group F (libraries F1, F2 and F3) and Group C (libraries C1 and C2) were obtained. These libraries were used in screens for DBDpp binding 41BB/CD137, as well as a larger group of purified recombinant target Fc proteins including CD47, CTLA4, DR5, KIR, LAG3, OX40, PD1, PD-L1, and TIM3. (Many of these targets are considered immunoregulatory factors (Pardoll et al., Nat. Rev. Cancer 12:252-264 (2012)). After incubating the target with pools of DBDpp phage libraries, bound target phage complexes were captured and separated from unbound phage at using protein A beads (the target proteins were Fc fusion proteins).After three rounds of selection, the rescued phage clones were screened by ELISA for binding to the selected target.

For each target, approximately 90 DBDpp phage clones were used in ELISA screening for binding to the target protein as well as a nonspecific control (eg, IgG1-Fc). Individual DBDpp clones were sequenced and exhibited a target-specific binding signal that was 3-fold higher than the nonspecific control. In some cases, if the majority of DBDpp clones were positive in an ELISA screening plate, the entire plate was sequenced. The results of the sequence analysis showed that a total of approximately 70% of the DBDpp clones were in the correct reading frame and matched one of the five expected (Table 3) library sequence profiles. Sequences that did not fit the expected profile typically consisted of either misread sequences, frameshift mutations, truncations, concatemerizations, or other cloning artifacts.

DBDpp bind to multiple targets.

In table Figure 4 shows the distribution of clones for each library depending on the target and binding data. The three subtables correspond to the distribution for all sequences (top) and sequences with target-specific binding ratios greater than or equal to 2 (middle) or 3 (bottom). (In cases where sequences are represented by more than one clone, the average binding value is used). Of a total of 794 sequences, 330 represented unique clones, of which 278 showed an ELISA signal of 3 above background.

Table 4

AND 1 162 NIN! 64 short circuit ί 1 TO seams 74 26 5 3 m 2 AND 2 IYAYI ID 43 24 EATING And P 36 7 F2 7 7 5· 5: 2 2 F3 402 209 114 49 5.2 31 74 2 2 1 15 14 3 3 52 40 75 62 15 7 :ι 3 3 1 1 2 2 □ 92 14 3- 2 70 9 14 2 5 1 hell 1 50 1

Total I 756 I 238 I 208 | 53 | 123 | 41 | 74 2 « 29 | 26 | 1S | 10 | 10 | 4 | 4 | 95 | M | 75 | 62 | I'm M~

Fl 159 61 1 1 74 26 5 3 2 2 42 23 35 6 F2 5 5 4 4 1 1 F3 310 195 114 49 51 thirty 2 1 14- 13 3· 3· 49 WE 66 55 eleven 6 Cl 2 2 1 1 1 1 C2 92 14 3 2 70 9 14 2 5 1 F2NNK 90 1 90 1

[Total I 658 | 278 | 207 I 52 | 122 | 40 | | | 90 | 29 | 25 | 18 | 9 | 9 | 2 | 2 | 91 | 61 | 66 | 55 | 46 | 1G~

In table 5 lists exemplary sequences obtained from the experiments described above. For each sequence, the Target, library of origin (Bib.), number of screens (Qty.) and target signal to background ratio (ELISA ratio) are indicated. In several embodiments, a DBDpp with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% homology to those described above (and elsewhere herein) description) retains significant functional equivalence. In several

- 101 043883 embodiments, this is preferred since deviation in homology may provide some advantages, such as reduced immunogenicity, increased cross-reactivity, increased specificity, etc.

Table 5

Sequences of clones of DBDpp libraries isolated from screened libraries

SEQ ID NO: Subsequence Target Bib. Qty Attitude ELISA 12 MGSWVEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVEKLRQRAAFIRRLQAYRHN CD137 F3 3 20,606 13 MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVEHLRDQAAFIRHKLQAYRHN CD137 F3 7 16,055 14 MGSWYEFRHRLWAIDQRLYALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVEGLREAAAFIRAKLQAYRHN CD137 F3 4 12,974 15 MGSWYEFSMRLWAIDQRLYALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVEALRAKAAYIRWKLQAYRHN CD137 F3 2 12,040 16 MGSWFEFNHRLWAINERLYALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVERLRSMAAFIRYKLQAYRHN CD137 F3 4 11,925 17 MGSWYEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVEYLRETAAHIRTRLQAYRHN CD137 F3 3 7,707 18 MGSWYEFHYRLHAIDQRLYALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVEELRIKAAFIRRLQAYRHN CD137 F3 3 7,262 19 MGSWAEFKQRLAAIKTRLEALGGSEAELAAFLGEIWAFEM ELAAYKGKGNPEVEALGREAAAIRMELQAYRHN CD137 F2 90 5,269 20 MGSWYEFDLRLHAIYDRLVALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVEILRDNAAYIRQMLQAYRHN CD47 F3 2 14,087 21 MGSWTEFTYRLSAIEWRLWALGGSEAELAWFEQKIAFFED FLQYYKGKGNPEVEALKHEAGAILNELMAYRHN CD47 C2 24 12,517 22 MGSWAEFDHRLHAIRERLHALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVEILRGNAAYIRALLQAYRHN CD47 F3 3 11,651 23 MGSWTEFVGRLAAIEFRLWALGGSEAELAWFEAHIAFFED YLQWYKGKGNPEVEALREEAGAIMEELKAYRHN CD47 C2 3 8,230 24 MGSWTEFYSRLEAIWVRLQALGGSEAELAMFEDRIAHFEW FLQQYKGKGNPEVEALHEEAIAIRKELAAYRHN CD47 C2 37 4,578 25 MGSWHEFHDRLQAIHERLYALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVESLRIAAAHIRQVLQAYRHN CTLA4 F3 73 2,950 26 MGSWNYFKDHLAWIKNSLEALGGSEAELAHFETAIASFER QLQEYKGKGNPEVEALRKEAAAIRDELQAYRHN DR5 Fl 12 12,993 27 MGSWLYFKEHLAHIKAWLEALGGSEAELAHFELAIADFEY HLQEYKGKGNPEVEALRKEAAAIRDELQAYRHN DR5 Fl 5 12,309 28 MGSWVYFKEHLAWIKTELEALGGSEAELAHFEHSIADFEM SLQFYKGKGNPEVEALRKEAAAIRDELQAYRHN DR5 Fl 4 12,117 29 MGSWFYFKQHLAWIKSYLEALGGSEAELAHFERAIAAFEQ HLQMYKGKGNPEVEALRKEAAAIRDELQAYRHN DR5 Fl 5 11,836 thirty MGSWHYFKDHLAEIKGLLEALGGSEAELAHFEMAIADFEH NLQYYKGKGNPEVEALRKEAAAIRDELQAYRHN DR5 Fl 5 11,436

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31 MGSWHYFKGHLAEIKNHLEALGGSEAELAHFERAIAAFER SLQWYKGKGNPEVEALRKEAAAIRDELQAYRHN DR5 F1 7 10,822 32 MGSWIYFKEHLAYIKKELEALGGSEAELAHFESAIAVFES TLQYYKGKGNPEVEALRKEAAAIRDELQAYRHN DR5 F1 4 10,677 33 MGSWTYFKEHLAEIKYMLEALGGSEAELAHFEVAIADFEK MLQYYKGKGNPEVEALRKEAAAIRDELQAYRHN DR5 F1 8 10,256 34 MGSWWLFKDHLAEIKTALEALGGSEAELAHFEMAIAAFEK QLQYYKGKGNPEVEALRKEAAAIRDELQAYRHN DR5 F1 3 9,748 35 MGSWSEFYNRLDAIESRLLALGGSEAELALFEIQIARFEK VLQAYKGKGNPEVEALRGEARAIFAELYAYRHN KIR C2 5 8,399 36 MGSWYEFYNRLYAIEIRLYALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVERLRVRAAKIRVILQAYRHN KIR F3 2 4,244 37 MGSWLWFKIFLAEIKYFLEALGGSEAELAAFDFEIHAFHV ELFAYKGKGNPEVEVLREVAAEIRWDLQAYRHN KIR C1 1 4,170 38 MGSWTEFQSRLDAIHSRLRALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVELLRDDAAFIRHFLQAYRHN PD-L1 F3 2 8,682 39 MGSWQEFDDRLNAIKARLQALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVEDLRDDAAFIRRFLQAYRHN PD-L1 F3 2 7,413 40 MGSWYEFQNRLHAIHERLNALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVELLRDDAAFIRHFLQAYRHN PD-L1 F3 2 6, 345 41 MGSWFEFQDRLTAINERLSALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVETLRSDAAFIRRFLQAYRHN PD-L1 F3 2 6,015 42 MGSWYEFESRLDAIHERLHALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVENLRGDAAFIRHFLQAYRHN PD-L1 F3 6 4,882 43 MGSWYEFNHRLDAISKRLNALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVEELRGDAAFIRHFLQAYRHN PD-L1 F3 2 2,982 44 MGSWFEFENRLHAIVHRLGALGGSEAELAAFEKEIAAFES ELQAYKGKGNPEVETLRADAAFIRHYLQAYRHN PD-L1 F3 2 2,764 45 MGSWWFKVDLATIKYILEALGGSEAELAEFEGEIAGFEY SLQFYKGKGNPEVEALRKEAAAIRDELQAYRHN TIM3 F1 2 5,788 46 MGSWTIFKEWLAFIKTDLEALGGSEAELAFFEGWIASFEM ELQKYKGKGNPEVEALRKEAAAIRDELQAYRHN PD1 F1 14 17,145 47 MGSWVMFKWLLADIKSHLEALGGSEAELAFFEGFIAAFET HLQVYKGKGNPEVEALRKEAAAIRDELQAYRHN PD1 F1 4 8,132 48 MGSWYAFKDYLADIKGWLEALGGSEAELAFFEIFIARFEL ELQAYKGKGNPEVEALRKEAAAIRDELQAYRHN PD1 F1 2 3.295

N-terminal FLAG-tagged constructs pb04 (SEQ ID NO: 182) and oc3D (SEQ ID NO: 49) were expressed and purified from E. soy cultures. Purified FLAG-pbO4 was assessed by ELISA to dose-dependently bind to PD-Ll-Fc-coated wells in a microtiter plate. In contrast, FLAGa3D did not show detectable binding to the target protein PD-Ll-Fc (Fig. 3D). The results demonstrate that modification of the reference framework sequence (SEQ ID NO: 1) is effective in providing a reliable source of DBDpp that are capable of binding, with novel specificity, to a diverse set of targets of interest.

Example 3 DBDpp fusion proteins.

To evaluate the modular nature of DBDpp as a binding element, the CO137-binding DBDpp, bbl0 (SEQ ID NO: 19), was reformatted as a construct fused to the N- or C-terminus of the heavy chain of an antibody derived from the sequence of the RSV-specific monoclonal antibody palivizumab ( Synagis®) (schematically shown in Fig. 4A and 4B, respectively). As a comparison construct, similar fusion constructs were generated using the parent sequence DBDpp (SEQ ID NO: 1), which is not known to exhibit any binding specificity. Proteins were produced in a suspension of HEK293F cells that were transiently transfected with equimolar ratios of independent heavy chain-bblO fusion constructs and light chain cDNA expression constructs, and purified using standard protein A affinity methods. Separation of purified samples by SDS-PAGE revealed that migration of fusion proteins

- 103 043883 heavy chain-bb10 (DBDpp) was consistent with molecular weight-based predictions (data not shown).

SEC analysis showed that the bb10 DBDpp antibody fusion constructs did not aggregate and migrated as predicted relative to size standards (data not shown). The SYN-bb10 and bb10-SYN fusion constructs exhibited similar migration relative to each other, with both moving faster than the parent Synagis® antibody.

The bispecific antibodies, SYN-bb10 and bb10-SYN, demonstrate binding to both CD137 and RSV (Fig. 4C-D; filled squares indicate bb10-SYN, filled circles indicate SYN-bb10)), indicating that the new binding activity was conferred on the original DBD sequence, with DBDpp functionality retained in both N-terminal and C-terminal fusions. In contrast, fusions between an untargeted alpha-helical protein scaffold and SYN (DBD-SYN for N-terminal fusion, open circles; SYN-DBD for C-terminal fusion, open squares) showed binding only to RSV, but binding to CD137 was not reported.

The binding of DBDpp, bb10 to CD137 was demonstrated using two different experimental methods: ELISA (Fig. 4C-D) and FACS (Fig. 6A-C). In these assays, the target antigen is presented and ultimately recognized in three different formats: either directly bound to plastic (Figure 4C-D) or in situ as part of the cell membrane (Figure 6A-C).

Treatment of PBMCs with SYN-bb10 and bb10-SYN demonstrates that, in addition to binding, both fusion proteins are capable of inducing a subsequent biological response in target cells (Figure 7AD).

In vivo stability is critical to the clinical efficacy of most biotherapeutics. Pharmacokinetic measurements of the bb10 fusion constructs (SYN-bb10 and bb10-SYN) were performed to assess the relative stability of DBDpp compared to the mAb fusion partner (Fig. 8). In vivo stability was determined by analyzing the binding of RSV and CD137 bispecific antibodies present in the serum of CD1 mice that received a single intravenous injection (1 mg/kg) of the fusion proteins. Serum samples were collected after 15 min and 48 h and tested by ELISA. Both N-terminal and C-terminal DBDpp fusion proteins exhibit long-term stability in vivo.

Example 4: Application of DBDpp in Affinity Purification.

Eight CD137-binding DBDpp ligands (SEQ ID NO: 12-19) were reformatted as N-terminal hexahistidine fusion proteins. Their tagged sequences and corresponding parent sequences are shown in Table. 5.

Table 5

N-terminal hexahistidine fusion proteins derived from SEQ ID NO: 12-19.

Original SEQ ID NO: New Seq ID His fusion protein sequence 12 51 MGSWVEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEKLRQRAA FIRFRLQAYRHNGGGGSHHHHHH 13 52 MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEHLRDQAA FIRHKLQAYRHNGGGGSHHHHHH 14 53 MGSWYEFRHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEGLREAAA FIRAKLQAYRHNGGGGSHHHHHH 15 54 MGSWYEFSMRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEALRAKAA YIRWKLQAYRHNGGGGSHHHHHH 16 55 MGSWFEFNHRLWAINERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVERLRSMAA FIRYKLQAYRHNGGGGSHHHHHH 17 56 MGSWYEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEYLRETAA HIRTRLQAYRHNGGGGSHHHHHH 18 57 MGSWYEFHYRLHAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEELRIKAA FIRDRLQAYRHNGGGGSHHHHHH 19 58 MGSWAEFKQRLAAIKTRLEALGGSEAELAAFLGEIWAFEMELAAYKGKGNPEVEALGREAA AIRMELQAYRHNGGGGSHHHHHH

Each fusion protein was expressed separately in E. Coli BL21 (DE3) cells. After cell lysis and purification by metal ion chromatography, each CD137-104 043883 DBDpp binding ligand was purified again by reverse phase HPLC. HPLC columns (20x100 mm C-4, Western Analytical) were eluted with 0.1% TFA:acetonitrile (88:12 over 2 min, followed by a linear gradient to 58:42 over 15 min). The main peak at approximately 12 min corresponded to the target ligand (see arrow in Fig. 9). Purified ligands were lyophilized until further use.

The identity and purity of each ligand was confirmed by electrospray mass spectrometry (Table 6) and SDS-PAGE (Fig. 10). In table Figure 6 shows the calculated molecular mass for each of the CO137-targeted DBDpps based on their sequence. In table Figure 6 also shows the expected molecular weight for each DBDpp after replacing the N-terminal methionine with a hexahistidine tag. In table 6 also shows the observed molecular weights. For each CD 137directed DBDpp, excluding SEQ ID NO. 54, the observed molecular weight was in good agreement with the expected molecular weight, indicating that the dominant component in the purified sample included the hexahistidine tag. In the case of SEQ ID NO: 54, the methionine containing component was the main component.

Table 6

Comparison of expected molecular mass and observed molecular mass ________________ for 8 proteins SEQ ID 51-58________________

Seq ID Calc. they say weight Expected pier mass (without Met) Observed pier weight 51 9596 9465 9464 52 9501 9370 9369 53 9500 9369 9368 54 9569 9438 9568 55 9651 9520 9518 56 9585 9454 9453 57 9643 9512 9511 58 9207 9076 9075

In fig. 10 shows SDS-PAGE analysis with Coomassie Blue staining of each hexahistidine-tagged CD 137-targeted DBDpp. Lane 1 is a molecular weight marker (kilodaltons), lane 2 is SEQ ID NO: 58, and lanes 3-9 correspond to SEQ ID NO: 51-57, respectively. Each of the tracks has a clear and precise banding with no shmear or presence of smaller bands that could indicate DBDpp splitting.

In fig. 11 shows an expanded electrospray mass spectrum for DBDpp according to SEQ ID NO: 54, from which it is clear that the dominant component in the sample remained the N-terminal methionine protein rather than the His6 tag.

In accordance with several embodiments disclosed in the present invention, the production methods disclosed in the present invention for producing labeled DBDpp result in a dominant component (e.g., greater than 50%, greater than 60%, greater than 70%, greater than 80%, etc. .) in a given batch of product that is a labeled component. As discussed above, tags other than His6 may be used, depending on the embodiment.

The target protein, CD137, was constructed as a CD137-Fc-His6 fusion protein. A hexahistidine-tagged chimeric construct (Leu24-Glnl86)/rHuman Fc (Lysl00-Lys329) (SEQ ID: 59) was generated by transient transfection of HEK293 cells and purified from clarified cell supernatant using immobilized metal ion chromatography (IMAC) and then replaced buffer in PBS. This material had identical properties to authentic protein purchased from commercial sources (RnD systems) and was used as a positive control for CD 137.

SEQ ID: 59 is shown below:

LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTR

KECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNC

SLDGKSVLVNGTKERDWCGPSPADLSPGASSVTPPAPAREPGHSPQDIEGRMDKSCDKTHTCP

PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

Binding of eight his-tagged DBDpp ligands (SEQ ID NO: 51-58) to the target protein CD137

- 105 043883 was assessed using biolayer interferometry (ForteBio, Menlo Park, CA) according to established methods. The binding assay was performed by immobilizing CD137-Fc-His6 via protein A and then incubating in solutions of each DBDpp ligand for 5 min to measure the association phase. The sensors were then placed in buffer to monitor the dissociation phase, and the complete sensorgrams (time in seconds on the x-axis and nm on the y-axis) are shown in FIG. 12A, 12C, 12E, 12G, 12I, 12K, 12M and 120 for DBDpp SEQ ID NO: 51-58, respectively. The data were approximated using steady state analysis, which is shown in each Fig. 12D, 12D, 12F, 12H, 12J, 12L, 12H and 12P for the corresponding DBDpp. The data showed affinity constants for CD137 ranging from 140 nM to 1.7 μM (Table 7).

Table 7

Calculated affinity constants for 8 ligands SEQ ID 51-59

SEQ ID K _D (Μ) 51 6, 4xl0~ ⁷ ± 1.8xl0- ⁷ 52 4, 6xl0~ ⁷ + 8.5xl0- ⁸ 53 1, 4xl0~ ⁷ ± 3, OxiO- ⁸ 54 4, 0xl0~ ⁷ ± 9.4xl0- ⁸ 55 1, 7xl0~ ⁷ ± 4, OxiO- ⁸ 56 7, 7xl0~ ⁷ ± 1.2xl0- ⁷ 57 1, OxlCT ⁶ ± 1.7xl0- ⁷ 58 1, 7xl0- ⁶ ± 2.6xl0- ⁷

Four of the eight His-fusion proteins were coupled to NHS-activated Sepharose 4 Fast Flow (GE Healthcare Life Sciences) using the manufacturer's instructions for 4 h at room temperature and then washed before ligand density analysis (Table 8).

Table 8

Measured ligand density of affinity resins

Ligand concentration Ligand Density Seq ID upon binding, mg/ml resin, mg/ml 51 4.9 10 52 1.7 3.3 53 3.9 7 56 3.3 6.5 58 3.9 5.4

For each resin, a portion of the washed resin was packed into a 3x25 mm glass column (Omnifit) and mounted on a BioLogic chromatograph (Bio-Rad). The resins were equilibrated with 1 mL of phosphate-buffered saline (PBS) at a flow rate of 0.5 mL/min. A supernatant sample of Chinese hamster ovary (CHO) cells containing CD137 (which was added at a concentration of 0.25 mg/ml) was applied to a column (550 μg CD137-Fc-His at 0.2 ml/min) followed by washing (7, 5 ml PBS at 0.5 ml/min) and elution (2 ml 50 mM Na acetate, pH 3, at 0.2 ml/min). Before assessing the binding of another sample, the resins were regenerated with 1 ml of 6 M guanidine-HCl at 0.5 ml/min and re-equilibrated with 25 column volumes of PBS at 0.5 ml/min.

In fig. 13 shows the chromatogram after purification of the CD137-Fc-His6 protein from the CHO supernatant. The peak eluted at 8-9 ml corresponds to the eluted CD137-Fc-His6 protein. These data demonstrate that the DBDpp disclosed in the present invention can be successfully used to capture a target protein from a sample with a high degree of specificity, even when the sample loaded onto the column includes a complex mixture of biological proteins that are capable of disrupting target-binding interactions.

To further evaluate binding specificity, fractions from the elution phase were examined by SDS-PAGE to confirm that the affinity resin was suitable for CD137 purification, ensuring removal of a significant portion of the host cell protein. In fig. 14A shows a Coomassie blue stained gel with a molecular weight marker in Lane 1 (kilodaltons). Lane 2 shows the positive control, IMAC-purified CD137 protein. Lane 3 shows the negative control, CD137-Fc-His6, spiked into the CHO supernatant at a concentration of 0.25 mg/ml, which represents no purification of CD137 from the supernatant. Lane 4 - eluate after purification using DBDpp SEQ ID NO. 58, lane 5 - eluate after purification using DBDpp SEQ ID NO. 51, lane 6 - eluate after purification using DBDpp SEQ ID NO. 52, lane 7 - eluate after purification using DBDpp SEQ ID NO. 57, and lane 8 - eluate after purification using DBDpp SEQ ID NO. 57 These data indicate that DBDpp specifically purify target proteins, a non-limiting example of which is CD137. Lane 3 clearly shows additional protein material in the lane that

- 106 043883 is the result of protein components of the supernatant of CHO cells. In comparison, lanes 4–8 show clear bands with minimal or no other proteins, similar to the positive control, IMAC-purified CD137.

In addition, Western blot analysis shown in FIG. 14B also confirms the ability of the DBDpp described in the present invention to provide the isolation of a specific target protein from a complex protein-containing sample. The arrangement of the tracks is the same as described above for FIG. 14A. HRP-conjugated anti-penta-His antibody (Qiagen) was used for detection. As initially hypothesized after Coomassie staining, Western blot analysis confirmed that DBDpp described in the present invention can successfully bind a target protein with specificity, and then be used to specifically isolate such a protein from a complex starting sample. Each of the CD137-targeted DBDpps in Lanes 4–8 showed the ability to purify CD137 as well as, if not better than, purification by immobilized metal ion chromatography (compare Lanes 4–8 with Lane 2). Since CD137 is just one non-limiting embodiment, the DBDpp disclosed in the present invention, according to additional embodiments, can be used to purify a variety of target proteins from a variety of starting samples. In some embodiments, the starting material is a biological fluid, while in other embodiments, the starting material is other types of liquid or gas samples from which the target protein is to be captured and purified.

Example 5: DBDpp stability study.

The present example was performed to evaluate the stability of DBDpp according to the embodiments disclosed in the present invention.

DBDpp-6xHIS fusion constructs (pb04 and pb06) and scFv-6xHIS fusion constructs were expressed through in vitro transcription and translation reactions (NEB, PureExpress). Samples were diluted 30-fold in ELISA blocking buffer (Thermo Fisher). Individual samples were then incubated at 25°C, 40°C, 55°C, 70°C, or 100°C for 2 min and then rapidly cooled to room temperature. An increase in temperature accompanied by rapid cooling can cause protein denaturation or other disruption of the three-dimensional structure of proteins, which reduces interaction and/or binding to the target. The ability of proteins to bind to a ligand, such as a target tumor antigen, after exposure to denaturing conditions indicates either increased stability when exposed to elevated temperature or the ability to refold and maintain function after exposure. In any case, increased thermal stability makes such proteins attractive candidates for maintaining function during and after exposure to the harsh conditions of production, storage, thawing, and clinical use.

Heat-exposed samples were serially diluted in ELISA buffer and binding to PD-L1-Fc was measured in ELISA. Bound proteins were detected with an HRP-conjugated rabbit polyclonal antibody against 6xHIS (Abcam). In fig. 15A shows the binding of DR5 scFv to cells expressing PD-L1. Since this particular scFv is not designed to bind PD-L1, this acts as a negative control and, as expected, no binding was detected for any of the DR5 scFvs, regardless of the temperatures to which they were exposed. In fig. 15B shows that scFv directed against PD-L1 successfully binds to its target after exposure to elevated temperatures of approximately 55°C. However, exposure to temperatures around 70°C showed a decrease in the ability of scFv to successfully bind to PD-L1, indicating the thermolabile nature of scFv. After heating the scFv to 100°C, PD-L1 target binding was completely abolished.

In contrast, DBDpp exhibit increased thermal stability. In fig. 15C presents data demonstrating the ability of DBDpp (pb04 in this non-limiting embodiment) to bind to the PD-L1 target, even after exposure to elevated temperatures on the order of 100°C. In fig. 15D shows similar data for another, non-limiting embodiment of DBDpp (pb06). Again, DBDpp retain the ability to bind their target after exposure to temperatures up to 100°C.

These data suggest that DBDpp, according to several embodiments disclosed in the present invention, has the ability to resist denaturation and/or undergo refolding after exposure to elevated temperatures, while retaining the ability to bind the desired target. As discussed above, this makes DBDpp an attractive targeting molecule because their increased thermal stability suggests that they are robust enough to withstand manufacturing processes (or other manufacturing/processing techniques) that may involve elevated temperatures better than other types of targeting molecules. , such as scFv.

Example 6. Interspecies reactivity of DBDpp.

The present example was performed to determine the species specificity of DBDpp as disclosed in the present invention.

In fig. 16A shows that soluble DBDpp selected to bind human PD-L1 (pb04 in this non-limiting embodiment) can also bind cynomolgus PD-L1 (Macaca fascicularis) as assessed by ELISA. Human PD-L1 or cynomolgus PD-L1 was immobilized in separate wells of an ELISA plate. Soluble DBDpp (FLAG-tagged) directed against PD-L1 was incubated in the corresponding plates to assess the extent of binding to PD-L1 of each species. pb04 DBDpp has been shown to bind to human and cynomolgus PD-L1. In fig. 16B further demonstrates that human and cynomolgus PD-L1 is bound by a CAR receptor including DBDpp (pb04 in this non-limiting embodiment) expressed on the surface of human T cells. As demonstrated by flow cytometry data, PD-L1-targeted DBDpp CAR T cells can bind soluble human PD-L1 and cynomolgus PD-L1 (97.7% for human and 97.1% for cynomolgus).

Example 7: CAR T cells expressing DBDpp polypeptides bind target molecules.

The present example was performed to determine the ability of transiently expressed CAR receptors, including DBDpp in this non-limiting embodiment, to bind a tumor target comprising an amino acid sequence that is at least 95% identical to residues 19-305 in SEQ ID NO: 187 (CD123).

293T cells were transiently transfected with pcDNA3 expression vectors encoding DBDpp containing CAR receptors using (eg, Fig. 5B) Lipofectamine 3000 (Life Technologies). After 24 hours, cells were harvested using CellStripper™. Cells were examined for CAR expression using a fluorescently labeled anti-FLAG antibody. DBDpp-containing CAR receptors binding to CD123 were measured by incubating cells with an Fc fusion protein comprising the extracellular domain of CD123 fused to the human IgG1 Fc in cell culture medium at 37°C for 30 min, washing, and then detecting with a PE antibody anti-human IgG. CD123 binding and CAR (FLAG tag) expression were analyzed by flow cytometry. The data is shown in Fig. 17, with each data point indicating the average FLAG expression and CD123 binding for each DBDpp CAR receptor from multiple experiments.

In fig. 17 summarizes flow cytometry data demonstrating that CAR receptors including CD123-binding scFv (32716) or DBDpp expressed in human T cells bind soluble CD123, as a non-limiting target of interest. The X-axis corresponds to the level of CAR expression on human T cells. The Y-axis represents the binding of FC-CD123 by the CAR receptor.

Example 8 T cells expressing CAR receptors including DBDpp (DBDpp-CAR) induce intracellular signaling.

To evaluate the ability of DBDpp-incorporating CAR receptors (DBDpp-CARs) to initiate signal transduction, a reporter Jurkat cell line containing a nuclear factor of activated T cell (NFAT) enhancer coupled to a luciferase reporter gene was stably expressed in Jurkat cells. Various CAR constructs were introduced by electroporation into the reporter Jurkat cell line. 24 h after electroporation, CAR expression was assessed by detection with a fluorescently labeled anti-FLAG monoclonal antibody. DBDpp-CAR-expressing Jurkat cells were then cocultured with CD123+ (BDCM, acute myelogenous leukemia, as a non-limiting embodiment) tumor cells for 6 hours, after which NFAT-mediated signaling was measured by adding luciferase assay reagent (Promega) to the cells and quantitating relative luminescence units (RLU) as shown in FIG. 18.

These results demonstrate that, in this non-limiting embodiment, DBDppCAR receptors are expressed on cells (e.g., human T cells), and when expressed can initiate an intracellular signaling cascade upon contact with a target (FC-CD123 in this non-limiting embodiment) bound DBDpp.

Example 9 DBDpp-CAR receptors expressed in human T cells produce cytokines upon target binding.

Capture of a target by a CAR-expressing T cell can lead to the secretion of cytokines.

Thus, 293T cells were transiently transfected with 3rd generation lentiviral packaging vectors (PRSV-REV, pMDLg/pRRE and pMD2.G) with pELNS vectors encoding DBDppCAR receptors using Lipofectamine 3000. 6 h after transfection, the media was replaced, then Lentivirus-containing media were collected 30 and 54 h after transfection, pooled, and then centrifuged to remove cellular debris. The lentivirus was then aliquoted and stored at −80°C until used for viral transduction. Transduction of human T cells with CARlentivirus was performed using total human PBMCs activated with aCD3/CD28 T cell activation beads in culture media supplemented with 40 U/ml IL-2. After 24 hours, 2x106 PBMCs were seeded into the wells of a 6-well tissue culture plate with 1 ml of culture media and 3 ml of lentivirus-containing media, supplemented with 40 U/ml IL-2 and protamine sulfate. The plates were then centrifuged for 2 h at 1000xg and 32°C, and then incubated overnight at 37°C. The next day, the lentiviral transduction procedure was repeated with new media for

- 108 043883 cultivation and media containing lentivirus. 72 hours after initial cell activation, T cell activation beads were removed, after which T cells were cultured for expansion at ~0.25-0.5x10 ⁶ T cells/ml in new media supplemented with 100 U/ml IL- 2. Additional T cell media and IL-2 were added to the T cells every 2–3 days until they were used for cytokine assays (described below), 7–10 days after primary activation.

Cytokine production in response to expression of a target antigen (CD123 in this non-limiting embodiment) was assessed by culturing 25,000 transduced T cells (7 days after activation) with 25,000 non-target (K562, CD123-) or targeted (BDCM, CD123+) tumor cells per well in 96-well plates. After 24 h, culture supernatants were collected and cytokine production was assessed by ELISA. Culture supernatants were diluted 1:5 before ELISA. Similarly, cytokine production in response to expression of the target antigen, PD-L1, was assessed by culturing 25,000 transduced T cells (7 days after activation) with 25,000 nontarget (K562, PD-1-) or target (SUDHL-1, PD-L1+) tumor cells per well in 96-well plates. After 24 h, culture supernatants were collected and cytokine production was assessed by ELISA. Culture supernatants were diluted 1:5 before ELISA.

In fig. 19A demonstrates that T cells expressing CD123-binding DBDppCAR receptors produce interferon (IFN) after stimulation with CD123+ BDCM cells, but not the CD123- cell line K562. In fig. 19B demonstrates that T cells expressing CD123-binding DBDpp-CAR receptors produce interleukin 2 (IL2) after stimulation with CD123+ BDCM cells, but not the CD123-cell line K562. In fig. 20A demonstrates that T cells expressing PD-L1-binding DBDpp-CAR receptors produce interferon (IFN) after stimulation with PD-L1+ SUDHL-1 cells, but not the PD-1− cell line K562. In fig. 20B demonstrates that T cells expressing PD-L1-binding DBDpp-CAR receptors produce interleukin 2 (IL2) after stimulation with PD-L1+ SUDHL-1 cells, but not the PD-1 cell line K562.

Example 10 T cells expressing DBDpp-CAR receptors proliferate when co-cultured with target-expressing tumor cells.

T cells expressing target-binding CAR receptors can proliferate after capturing soluble target or target-expressing tumor cells.

Proliferation of DBDpp-CAR transduced human T cells in response to tumor cells expressing a target antigen (of which CD123 is a non-limiting example) was assessed by culturing transduced T cells (1x105, day 10 post-activation) with 1x105 mitomycin-C pretreated tumor cells cells in 24-well plates. Tumor cells included non-target-expressing K562 (CD123-), intermediate target-expressing lines KG1a and MOLM-13 (intermediate CD123), and BDCM (high CD123). Transduced T cells were collected and enumerated after 96 h of coculture with tumor cells. This approach has also been used to assess T cell proliferation in response to tumor cells expressing the target antigen PD-L1. Tumor cells included non-target-expressing K562 cells (PD-1-), intermediate target-expressing lines BDCM and H460 (PD-L1 intermediate), and SUDHL-1 (PD-L1 high). Cells were collected and counted after culture for 96 hours. Data from proliferation experiments are shown in FIG. 21 and fig. 22, respectively.

The histogram bars in Fig. 21 correspond to the following (from left to right): culture of DBDpp-CAR T cells alone, coculture with CD123-negative K562 cells, coculture with low-level CD123-expressing KG1a cells, coculture with low-level CD123-expressing MOLM-13 cells, and coculture with high-level CD123-expressing BDCM cells. As indicated by the height of the histogram bars, when incubated with cells that express CD123 (at intermediate or high levels), there is a corresponding increase in T cell proliferation. These data indicate that CD123-specific DBDpp-CAR T cells proliferate in response to CD123 binding to a comparable or, in some embodiments, greater extent than CD123-specific scFvs. Likewise, in FIG. 22, the histogram bars correspond to the following (from left to right): PD-L1-DBDpp-CAR T cell culture only, coculture with PD-L1-negative K562 cells, coculture with PD-L1 intermediate-expressing BDCM cells, coculture with PDL2 -high-level expressing SUDHL-1 cells and coculture with intermediate-level PD-L1-expressing H460 cells. These data demonstrate the specificity of the T cell response against the DBDpp target (eg, in the absence of the target, limited or no response is observed). Thus, as above, these data indicate that DBDpp-CAR T cells directed against PD-L1 specifically proliferate in response to PD-L1 binding. As discussed above, CD137, CD123, and PD-L1 are just non-limiting examples of targets that DBDpp can specifically bind, and thus can (shared with CARs in a T cell, NK cell, etc.) induce target function -specific immunocytes.

- 109 043883

Example 11 DBDpp-CAR-transduced T cells do not exhibit phenotypes associated with T cell exhaustion.

Chronic exposure of T cells to antigen and/or inflammatory signals can lead to T cell exhaustion, which is characterized by loss of effector function and expression of numerous inhibitory receptors such as LAG-3, PD-1, and TIM-3. Such depletion may also occur as a result of spontaneous stimulation of T cells through antigen-independent mechanisms that cause aggregation of T cell receptors. A consequence of T cell depletion may be reduced tumor control, and thus avoidance of excessive depletion is a preferred feature in T cell-based antitumor immunotherapy.

To assess potential antigen-independent exhaustion of T cells expressing DBDppCAR receptors, transduced T cells (day 10 after activation) were stained with antibodies against CD3 and markers of T-cell exhaustion (LAG3, PD1 and TIM3). In fig. Figure 23A summarizes data from separate experiments with multiple T cell donors. Data demonstrate that expression of exhaustion markers was not increased in various CD123-binding DBDpp-CAR T cells. In fig. 23B shows representative flow cytometry data for the expression of LAG-3, PD1 and TIM-3 in T cells transduced with either scFv-containing CAR (top row) or DBDpp-CAR (in this particular experiment CD123-targeted cg06), 10 days after primary activation of T cells. The similarity of these data, again, demonstrates that DBDpp-CAR T cells do not increase the expression of exhaustion markers, providing further support for their effectiveness in antitumor immunotherapy.

Example 12 DBDpp-CAR-expressing T cells exhibit target-specific degranulation and antitumor cytotoxicity.

Degranulation of T cells, NK cells, and many monocytic lineage cells (all of which may be used depending on the embodiment). Degranulation may result in the release, depending on the cell type, of antimicrobial, cytotoxic, or other molecules from secretory granules in the immunocyte. Molecules such as perforin (a pore-forming cytotoxin) or granzymes (serine proteases that cause apoptosis in the target cell) help T cells and NK cells destroy tumor cells (or other types of cells).

To assess degranulation of T cells expressing DBDpp-CAR receptors, 1x105 transduced T cells (day 9 post-activation) were cultured in T cell medium for 4 h in the presence of monensin and PE-conjugated CD107a/LAMP1. T cells were cultured alone or in the presence of 2x105 non-target tumor cells (K562, which are CD123-) or target-expressing tumor cells (BDCM, CD123+), then washed and stained for CD3 expression. T cell degranulation was then assessed by flow cytometry, first sorting CD3+SSC-low cells (non-tumor) and then CD3+CD107a+ cells. Symbols indicate samples from individual experiments using multiple donors.

In fig. 24A-24D summarize this data. In fig. 24A shows CD107a production (as a marker of DBDpp-CAR T cell degranulation) equivalent to negative controls when CD123-targeted DBDpp-CAR T cells were cultured separately. In fig. 24B shows limited expression of CD107a when DBDpp-CAR T cells are cocultured with CD123 negative K562 tumor cells. In fig. 24C shows significant CD107a expression when CD123-targeted DBDpp-CAR T cells are co-cultured with CD123-positive BDCM cells, indicating that T cells are activated, signaled, and degranulated, leading to tumor eradication. In fig. 24D shows data from replicate co-culture experiments of CD123-targeted DBDpp-CAR Ts with CD123-positive BDCM cells. In fig. 25A-25D show similar findings associated with degranulation of T cells expressing PD-L1-DBDpp-CAR receptors. These data not only demonstrate that DBDpp-CAR T cells effectively degranulate, but these data also provide further support for target-dependent activation of DBDpp-CAR-expressing T cells.

Example 13 DBDpp-CAR-mediated antitumor cytotoxicity is target specific.

The target-specific function of DBDpp-CAR-expressing human T cells was expanded to include in vitro cytotoxicity against tumor cells, as described in FIG. 26 and 27. These experiments used CD123+ (BDCM, acute myelogenous leukemia) and CD123− (K562, chronic myelogenous leukemia) tumor cells that were preloaded with fluorescence enhancing ligand (BATDA). CD123-targeted DBDpp-CAR T cells were cultured with tumor cells for 2 h at different effector to target (E:T) ratios. Mock coculture and CD123-targeted SCFV-CAR coculture were used as controls. In an additional set of experiments, PD-L1+ (SU-DHL-1, large cell lymphoma) and PD-1- (K562, chronic myelogenous leukemia) tumor cells were preloaded with BATDA. PD-Ll-targeted DBDpp-CAR T cells were cultured with tumor cells for 2 h at different E:T ratios. A mock coculture was used as a control. The BATDA ligand is released by cytolysis of target cells and upon addition of europium (Eu) solution forms a fluorescent and stable chelate, which was measured using a Synergy 2 time-resolved fluorimeter (Biotek).

In fig. 26A-26D and 27A-27F summarize data from these experiments. In fig. 26A shows that coculture of CD123-targeted DBDpp-CAR T cells with K562 cells (without CD123 expression) produces a kill rate less than mock control cocultures. At the same time, in FIG. 26B demonstrates that each of the groups of T cells expressing the CD123-targeted DBDppCAR kills CD123-positive tumor cells more efficiently than mock control co-cultures and, in the case of some DBDpp, compared to T cells directed against CD123 via scFv. In fig. 26C and 26D show similar data with cells from a single donor.

As another example, FIG. 27A-27F show the percentage of killing for PD-L1-directed DBDpp-CAR T cells. As discussed above, limited or no cytotoxicity is observed when cocultured with cells not expressing PD-L1 (27A, 27C, 27E). However, when cocultured with cells that do express the target marker PD-L1, there is cytotoxicity that is measured and substantially exceeds the cytotoxicity found in mock control cocultures (27B, 27D, 27F). These data expand the functional signature of DBDpp-expressing T cells to include target-specific killing of tumor cells.

Example 14 DBDpp domains can be deimmunized while retaining function.

Many therapeutic agents have the potential to cause unwanted side effects while providing effective therapy. In some cases, patients may have an immune response to a therapeutic agent (either a drug or a cell-based agent). Since the DBDpps disclosed herein are non-human proteins, in silico studies were performed to identify potentially immunogenic epitopes and eliminate them without compromising the functional properties of the DBDpps.

In silico analysis of the cg06 amino acid sequence (SEQ ID NO: 99) identified three 9 amino acid sequences that have high affinity (binding threshold less than 6%) and mixed (present in more than 50% of the corresponding alleles) T cell sequence properties epitopes (Singh, Bioinformatics 17:1236-1237, 2012). Certain amino acid substitutions in cg06 were identified as reducing the number of predicted T cell epitopes. Appropriate point mutations were introduced into cg06, alone or in combination, to produce a series of 'deimmunized' DBDpp-CAR receptors.

The 3D model of DBDpp (cg06) is shown in Fig. 28A. In fig. 28B represents cg06 with one of (three) potentially immunogenic epitopes modified to reduce potential immunogenicity. In fig. 28C is represented by cg06 with modification of two (of three) potentially immunogenic epitopes. In fig. 28D is represented by cg06 with all three potentially immunogenic epitopes modified.

Jurkat cells with the reporter were electroporated with the indicated deimmunized DBDppCAR receptors. After 24 h of culture, CAR expression on Jurkat cells was assessed by staining with an anti-FLAG monoclonal antibody. Cells were cocultured for 6 h with CD123+ target tumor cells (KG1a with low CD123 expression and BDCM with high CD123 expression). NFAT-mediated signaling was measured by adding luciferase assay reagent (Promega) to cells and quantifying relative luminescence units (RLU).

Data from these experiments are shown in FIG. 29A-29B. In fig. 29A demonstrates that deimmunized CD123-binding DBDpp-CAR receptors (cg06-1 - cg06-6) induce NFAT signaling when co-cultured with CD123-positive BDCM cells. In fig. 29B also demonstrates that CD123-binding DBDpp-CAR receptors also induce NFAT signaling when co-cultured with cells having lower expression of CD123 (KG1a)—notably reduced levels of RLU compared to FIG. 29A. Taken together, these data demonstrate that the immunogenic potential of target-binding DBDpp can be reduced by substitutions of specific amino acid residues in DBDpp that do not alter DBDpp function or target density dependence of CAR-mediated intracellular signaling—the greater the degree of target presence, the greater the degree of response. These data also indicate that DBDpp according to several embodiments disclosed in the present invention can be used as therapeutic agents and, if necessary, can be modified to reduce the likelihood of immune responses against DBDpp-CAR containing cells (e.g., T cells, NK cells, etc.).

- 111 043883

Example 15. Bispecific DBDpp.

In some cases, target cells (eg, tumor cells) may express more than one marker. In some embodiments, DBD-CAR receptors are specific for a single marker (eg, a unique cancer cell marker) on a target cell. In some embodiments, however, certain markers are not unique to cancer cells but are also expressed on normal cells (albeit likely at different levels). Thus, in several embodiments, targeting two markers represents an opportunity to increase the specificity of an immunotherapeutic agent by engineering a CAR to express bispecific DBDpp. In such embodiments, the target cells that are most effectively killed may be cells that express both markers targeted by DBDpp (although cells expressing one or the other marker may be killed). To test this approach, bispecific DBDpp-CAR receptors were expressed on Jurkat cells and intracellular signaling in response to tumor cell expression of one or both targets was measured.

In fig. 30A-30B determined the surface expression of CD123 and PD-L1, respectively, on K562 cell lines (CML); KG-1a (AML); BDCM (AML); SU-DHL-1 (LCL) and H460 (lung carcinoma). K562 does not express either target, whereas KG1a and BDCM express CD123 and low levels of PDL1 compared to the relatively high PD-L1 expression observed on CD123-negative cells, SU-DHL and H460. These cell lines provide an opportunity to determine whether intracellular signaling of the CD123-binding DBDpp-CAR (cg06) can be enhanced by a bispecific CAR including cg06 (anti-CD123) fused to a second PD-L1-specific DBDpp (pb04). In fig. 31A demonstrates that DBDpp-CAR receptors comprising cg06 only (Fig. 31A), pb04 only (Fig. 31B), cg06 fused to the N terminus of pb04 (cg06-pb04, Fig. 31C), and pb04 fused to N-terminus cg06 9pb-04cg06, Fig. 31D) can be transduced and expressed in the Jurkat NFAT cell line with a reporter, as assessed using an anti-FLAG mAb that binds to CAR receptors. The ability of monospecific and bispecific CAR receptors to activate the NFAT pathway was assessed by co-cultivating various CAR receptors with tumor cells having different levels of PD-L1 and/or CD123 expression. Cells were cocultured with target cells for 6 h. NFAT-mediated signaling was measured by adding luciferase assay reagent (Promega) to cells and quantifying relative luminescence units (RLU) as an indicator of induced intracellular signaling.

In fig. Figure 31E shows the results of this experiment. The group of bars and histogram on the left shows the relative killing effect for cg06 DBDpp against different cell types. The signaling response after coculture with highly CD123+ BDCM was greatest with this DBDpp-CAR. The next panel to the right shows data showing intracellular signaling after co-culture of pb04 DBDpp against the same cell types. Signaling was highest in BDCM, followed by SUHDL1 and H460 (referring to Figures 30A-30B, these are the cell lines with the highest expression of CD123 and PD-L1). The next panel to the right shows data showing intracellular signaling of the bispecific cg06-pb04 DBDpp (cg06 is more distant from the T cell membrane compared to pb04). Finally, the panel on the right shows intracellular signaling from a second bispecific DBPpp (pb04-cg06 DBDpp, where pb04 is more distant from the T cell membrane compared to cg06). These two groups show that bispecific DBDpp-CAR receptors do function to induce intracellular signaling. In accordance with several embodiments, the bispecific DBDpp-CAR receptors exhibit increased activity (the amount of intracellular signaling in the pb04-cg06 group with BDCM cells is greater than that observed for pb04 DBDpp alone). Thus, in several embodiments, DBDppCAR receptors comprising two DBDpps can act together to enhance T cell function. In several embodiments, there is synergy between the different DBDpps used in bispecific (or other multimeric) DBDpp-CARs.

Example 16. DBDpp-mediated antitumor immunotherapy in vivo.

In several embodiments, DBDpp-CAR expressing cells effectively generate antitumor cytotoxicity in vivo. Experiments will be conducted in which tumor marker-specific DBDpp-CARs are expressed on the surface of T cells (or, in other experiments, NK cells). A mouse model will be used in which mice are genetically modified to express a solid tumor or a suspension tumor, where the tumor cells express a tumor marker that is targeted by specific DBDpp-CAR receptors. Control mice with a tumor that does not express the target marker will be used as controls, as will mice receiving a therapeutic T-cell placebo.

DBDpp-CAR cells will be administered to mice and tumor burden will be assessed over time for mice receiving different DBDpp-CARs. Tumor burden will be assessed using established methods (eg, in vivo imaging) and mortality will be assessed over time.

As a result of obtaining target-specific DBD-pp-CAR cells, the tumor burden will be

- 112 043883 decrease in mice expressing a marker with which DBDpp specifically interacts. The reduction will be significant compared to placebo-treated mice. Likewise, the reduction would be significant compared to mice bearing tumor cells that do not express the marker that DBDpp specifically interacts with. In addition, mortality was reduced in mice receiving target-specific DBDpp-CAR receptors compared to a placebo group and a group of mice with tumors that do not express a specific target marker. The results of this experiment will demonstrate that immunocytes expressing DBDpp-CAR receptors are effective therapeutic agents and generate target-specific cytotoxicity against tumor cells in vivo.

Example 17. DBDpp-mediated antitumor immunotherapy in vivo.

As discussed above, DBDpp-CAR expressing cells efficiently generate antitumor cytotoxicity in vivo. Experiments will be conducted in which tumor marker-specific DBDpp-CARs are expressed on the surface of T cells (or, in other experiments, NK cells). Clinical studies will demonstrate the safety of DBDpp-CAR cells. Additional studies will be conducted to administer DBDpp-CAR cells to people who have tumors that express a marker with which DBDpp specifically interacts.

DBDpp-CAR cells will be administered according to a schedule to be determined in accordance with standard skill in the art. Tumor development, tumor burden and mortality will be assessed over time.

As a result of the introduction of target-specific DBDpp-CAR cells, tumor development will slow down and the overall tumor burden will decrease. The reduction will be significant compared to previously obtained data using standard antitumor methods such as radiation therapy or chemotherapy. The mortality rate will also be reduced compared to such treatments. Limited off-target cytotoxic effects will be present. The results of this study will demonstrate that immunocytes expressing DBDpp-CAR receptors are effective therapeutic agents and generate target-specific cytotoxicity against tumor cells in vivo.

Example 18 Competitive inhibition assay to determine target epitope specificity of DBDpp comprising different amino acid sequences.

DBDpp can be defined by various structural and functional properties including, but not limited to, primary amino acid sequence, pI, melting point, target specificity, binding affinity, and target epitope specificity. The target epitope specificity of the first DBDpp can be compared with that of the second DBDpp using a competition assay format in which binding of a fixed concentration of the first DBDpp to the target is performed in the presence of increasing concentrations of the second DBDpp. If the first and second DBDpp bind to the same epitope or a partially overlapping epitope, as determined by amino acid sequence or spatially, then the second DBDpp inhibits (eg, competes for) the binding of the first DBDpp to the target. If, however, the second DBDpp does not inhibit the binding of the first DBDpp, then the DBDpps bind to different epitopes. This assay format was used to evaluate the ability of a PD-L1-binding DBDpp, pb04, to inhibit the binding of a fixed concentration (11.1 pmol/well) of a second PD-L1-binding DBDpp, pb06. In fig. 32 demonstrated concentration-dependent inhibition of binding of soluble FLAG-tagged pb06 to FC-PD-L1 soluble pb04 at a pb04 IC50 concentration of 19 pmol/well. Thus, pb04 and pb06 show binding to a common epitope even though their primary amino acid sequences are different (SEQ ID NOs: 182 and 184). This assay format can be easily adapted to test the ability of DBDpp to inhibit ligand binding to a DBDpp target.

Example 19: Generation and selection of CD123-targeted DBDpp.

In accordance with several embodiments of the methods disclosed above, the reference sequence of SEQ ID NO: 1 has been modified at multiple positions. In one experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 6. Non-limiting examples of DBDpp demonstrating specificity for CD123 are represented by SEQ ID NO: 60-69.

In an additional experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 2. Non-limiting examples of DBDpp demonstrating specificity for CD123 are represented by SEQ ID NO: 70-91.

In an additional experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 4. Non-limiting examples of DBDpp demonstrating specificity for CD123 are represented by SEQ ID NO: 92-127.

In any of the libraries created according to the methods disclosed in the present invention, any of the Xn positions in the library sequences may be replaced by a natural or unnatural amino acid, depending on the embodiment. In some embodiments, cis

- 113 043883 theine and/or proline are not used for such substitutions.

Example 20: Generation and selection of deimmunized CD123-targeted DBDpp.

In accordance with several embodiments of the methods disclosed above, the reference sequence of SEQ ID NO: 1 has been modified at multiple positions. In one experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 4. According to the methods disclosed in the present invention, selected library elements were identified and deimmunized by identifying and modifying potentially immunogenic residues. In this experiment, DBDpp SEQ ID NO: 99 was modified by substituting S65E to produce SEQ ID NO: 130, demonstrating reduced immunogenicity.

Example 21 Deimmunization of CD123-targeted DBDpp.

DBDpp SEQ ID NO: 99 was created and identified according to the methods disclosed in the present invention. Other modifications have been made to reduce the potential immunogenicity of DBDpp. In this experiment, the substitution R17Q was made to produce DBDpp SEQ ID NO: 128. In addition, the substitution S24E was made to produce DBDpp SEQ ID NO: 129. In addition, in accordance with several embodiments, multiple deimmunizing substitutions can be made. For example, SEQ ID NO: 99 was modified by: (i) substitutions R17Q, S24E, resulting in DBDpp SEQ ID NO: 131, (ii) substitutions R17Q, S24T, resulting in DBDpp SeQ ID NO: 132, (iii) substitutions R17Q , S24G, obtaining DBDpp SEQ ID NO: 133, (iv) replacements R17Q, S24E, S65E, obtaining DBDpp SEQ ID NO: 134, (v) replacements R17Q, S24T, S65E, obtaining DBDpp SEQ ID NO: 135, and replacements R17Q, s24g, S65E, resulting in DBDpp SEQ ID NO: 136.

Example 22: Generation and selection of CD19-targeted DBDpp.

In accordance with several embodiments of the methods disclosed above, the reference sequence of SEQ ID NO: 1 has been modified at multiple positions. In one experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 6. Non-limiting examples of DBDpp demonstrating specificity for CD19 are represented by SEQ ID NO: 137.

In an additional experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 4. Non-limiting examples of DBDpp demonstrating specificity for CD19 are represented by SEQ ID NO: 138-166.

In any of the libraries created according to the methods disclosed in the present invention, any of the X _n positions in the library sequences can be replaced by a natural or unnatural amino acid, depending on the embodiment. In some embodiments, cysteine and/or proline are not used for such substitutions.

Example 23: Generation and selection of CD22-targeted DBDpp.

In accordance with several embodiments of the methods disclosed above, the reference sequence of SEQ ID NO: 1 has been modified at multiple positions. In one experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 2. Non-limiting examples of DBDpp demonstrating specificity for CD22 are represented by SEQ ID NO: 167-168.

In an additional experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 4. Non-limiting examples of DBDpp demonstrating specificity for CD22 are represented by SEQ ID NO: 169-176.

In any of the libraries created according to the methods disclosed in the present invention, any of the Xn positions in the library sequences can be replaced by a natural or unnatural amino acid, depending on the embodiment. In some embodiments, cysteine and/or proline are not used for such substitutions.

Example 24. Generation and selection of DR5-targeted DBDpp.

In accordance with several embodiments of the methods disclosed above, the reference sequence of SEQ ID NO: 1 has been modified at multiple positions. In one experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 6. Non-limiting examples of DBDpp demonstrating specificity for DR5 are represented by SEQ ID NO: 177-178.

In an additional experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 2. Non-limiting examples of DBDpp demonstrating specificity for DR5 are represented by SEQ ID NO: 179.

In an additional experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 4. Non-limiting examples of DBDpp demonstrating specificity for DR5 are represented by the sequence SEQ ID NO: 180.

In any of the libraries created according to the methods disclosed in the present invention, any of the Xn positions in the library sequences can be replaced by a natural or unnatural amino acid, depending on the embodiment. In some embodiments, cysteine and/or proline are not used for such substitutions.

- 114 043883

Example 25: Generation and selection of PD-L1-targeted DBDpp.

In accordance with several embodiments of the methods disclosed above, the reference sequence of SEQ ID NO: 1 has been modified at multiple positions. In one experiment, modifications resulted in a DBDpp library corresponding to SEQ ID NO: 4. Non-limiting examples of DBDpp demonstrating specificity for PD-L1 are represented by SEQ ID NO: 181-186.

In any of the libraries created according to the methods disclosed in the present invention, any of the X _n positions in the library sequences can be replaced by a natural or unnatural amino acid, depending on the embodiment. In some embodiments, cysteine and/or proline are not used for such substitutions.

All publications, patents, patent applications, Internet sites and database accession numbers/sequences (including polynucleotide and polypeptide sequences) cited herein are incorporated by reference in their entirety in all respects to the same extent as if it has been expressly and individually stated that each individual publication, patent, patent application, internet site or database registration number/sequence is hereby incorporated by reference.

It is contemplated that various combinations or subcombinations of specific features and aspects of the embodiments disclosed above can be made and will still be included in one or more inventions. In addition, the description herein of any particular feature, aspect, method, property, indicator, quality, attribute, element, or the like, as applied to an embodiment may be used in all other embodiments presented herein. Thus, it should be understood that various features and aspects of the disclosed embodiments may be combined or interchanged with each other to produce different embodiments of the disclosed inventions. Thus, it is intended that the scope of the inventions disclosed herein should not be limited to the specific disclosed embodiments described above. Moreover, although the invention is susceptible to various modifications and alternative forms, specific examples thereof have been shown in the drawings and described in detail herein. It is to be understood, however, that the invention is not intended to be limited to the specific forms or methods disclosed, but rather, the invention covers all modifications, equivalents and alternatives included within the spirit and scope of the various embodiments described and the appended claims. Any methods described herein are not necessarily performed in the order listed. The methods disclosed herein include certain actions taken by a practitioner; however, they may also include any third party instruction to do so, directly or indirectly. For example, acts such as administering a T cell including a DBDpp-CAR include instructions for administering a T cell including a DBDpp-CAR. In addition, where features or aspects of the present specification are described in relation to Markush groups, those skilled in the art will recognize that the present specification is also described in relation to any individual element or subset of elements of a Markush group.

The ranges disclosed herein also cover any possible overlapping ranges, subranges, and combinations thereof. Expressions such as up to, at least more than, less than, between, etc. include the specified number. Numbers preceded by the term approximately or approximately include the numbers indicated. For example, approximately 10 nanometers includes 10 nanometers.

- 115 043883

Sequence List <110> Subdomain, LLC Arcellx, Inc. LaFleur, David W Hilbert, David M < 120> POLYPEPTIDES CONTAINING DE NOVO BINDING DOMAIN AND THEIR APPLICATIONS < 130> ENC.002WO < 140> Unknown < 141> attached < 150> 62/143,772 < 151> 2015-04-0 6 < 160>187 < 170> PatentIn version 3.5 < 210>1 < 211>73 < 212> Protein < 213> Homo sapiens < 400>1

Met Gly Ser Trp Ala Glu Phe Lys Gln Arg Leu Ala Ala Ile Lys Thr

5 1015

Arg Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Glu

2530

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln Ala Tyr Lys Gly Lys

4045

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu Ala Ala Ala Ile Arg

5560

Asp Glu Leu Gln Ala Tyr Arg His Asn 6570 < 210>2 < 211>73 < 212> Protein < 213> Homo sapiens <220>

<221> OPTION <222> (5) ..(6) <223> Xaa is any natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (9)..(10) < 223> Xaa is any natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (13)..(13) < 223> Xaa is any natural or unnatural amino acid residue <220>

<221> OPTION <222> (16)..(17) <223> Xaa is any natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (30)..(30) < 223> Xaa is any natural or unnatural amino acid residue

- 116 043883 remainder

<220> <221> <222> <223> OPTION any natural or unnatural amino acid (33) Xaa . . (34) is <220> <221> <222> <223> OPTION (37)..(37) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (40) . . (41) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (44)..(44) Xaa is any natural or unnatural amino acid <400> 2 Met Gly 1 Ser Trp Xaa 5 Xaa Phe Lys Xaa Xaa 10 Leu Ala Xaa Ile Lys 15 Xaa Xaa Leu Glu Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Xaa 30 Phe Glu Xaa Xaa Ile 35 Ala Xaa Phe Glu Xaa 40 Xaa Leu Gln Xaa Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Ala 55 Leu Arg Lys Glu Ala 60 Ala Ala Ile Arg Asp Glu 65 Leu Gln Ala Tyr 70 Arg His Asn

remainder remainder remainder < 210 > 3 < 211 > 73 < 212 > Protein < 213 > Homo sapiens

<220> <221> <222> <223> OPTION (32)..(33) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (36)..(36) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (39) .. (40) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (43)..(43) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (57).. (58) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (61)..(61) Xaa is any natural or unnatural amino acid

the rest the rest the rest the rest the rest the rest

- 117 043883 <220>

<221> OPTION <222> (64)..(65) <223> Xaa is any natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (68)..(68) < 223> Xaa is any natural or unnatural amino acid residue < 400>3

Met Gly Ser Trp Ala Glu Phe Lys Gln Arg Leu Ala Ala Ile Lys Thr

5 1015

Arg Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Xaa 20 2530

Xaa Glu Ile Xaa Ala Phe Xaa Xaa Glu Leu Xaa Ala Tyr Lys Gly Lys 35 4045

Gly Asn Pro Glu Val Glu Ala Leu Xaa Xaa Glu Ala Xaa Ala Ile Xaa 50 5560

Xaa Glu Leu Xaa Ala Tyr Arg His Asn 6570 < 210>4 < 211>73 < 212> Protein < 213> Homo sapiens <220>

< 221> OPTION < 222> (5)..(5) < 223> Xaa is any natural or unnatural amino acid residue <220>

<221> OPTION <222> (8)..(9) <223> Xaa is any natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (12)..(12) < 223> Xaa is any natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (15)..(16) < 223> Xaa is any natural or unnatural amino acid residue <220>

<221> OPTION <222> (19)..(19) <223> Xaa is any natural or unnatural amino acid residue <220>

<221> OPTION <222> (55)..(55) <223> Xaa is any natural or unnatural amino acid residue <220>

<221> OPTION <222> (58)..(59) <223> Xaa is any natural or unnatural amino acid residue <220>

<221> OPTION <222> (62)..(62) <223> Xaa is any natural or unnatural amino acid residue

- 118 043883 <220>

<221>

<222>

<223>

OPTION (65)..(66)

Xaa is any natural or unnatural amino acid residue <400> 4

Met Gly Ser Trp Xaa Glu Phe Xaa 1 5

Xaa Arg Leu Xaa Ala Ile Xaa Xaa

15

Arg Leu Xaa Ala Leu Gly Gly Ser 20

Glu Ala Glu Leu Ala Ala Phe Glu

thirty

Lys Glu Ile Ala Ala Phe Glu Ser

40

Glu Leu Gln Ala Tyr Lys Gly Lys 45

Gly Asn Pro Glu Val Glu Xaa Leu

55

Arg Xaa Xaa Ala Ala Xaa Ile Arg 60

Xaa Xaa Leu Gln Ala Tyr Arg His Asn 6570 <210>5 <211>73 <212> Protein <213> Homo sapiens

<220> <221> <222> <223> OPTION (5)..(6) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (9) . . (10) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (13)..(13) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (16)..(17) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (32)..(33) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (36)..(36) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (39)..(40) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (43)..(43) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (55)..(55) Xaa is any natural or unnatural amino acid the remainder

- 119 043883 <220>

<221> OPTION <222> (58)..(59) <223> Xaa is any natural or unnatural amino acid <220>

<221> OPTION <222> (62).. (62) <223> Xaa is any natural or unnatural amino acid <220>

< 221> OPTION < 222> (65)..(66) < 223> Xaa is any natural or unnatural amino acid < 400> 5

Met Gly Ser Trp Xaa Xaa Phe Lys Xaa Xaa Leu Ala Xaa Ile Lys Xaa

5 10 15 the rest the rest the rest

Xaa Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Xaa 20 2530

Xaa Glu Ile Xaa Ala Phe Xaa Xaa Glu Leu Xaa Ala Tyr Lys Gly Lys 35 4045

Gly Asn Pro Glu Val Glu Xaa Leu Arg Xaa Xaa Ala Ala Xaa Ile Arg

5560

Xaa Xaa Leu Gln Ala Tyr Arg His Asn 6570 <210>6 <211>73 <212> Protein <213> Homo sapiens

<220> <221> <222> <223> OPTION (5)..(5) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (8) . . (9) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (12)..(12) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (15)..(16) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (19)..(19) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (30)..(30) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (33)..(34) Xaa is any natural or unnatural amino acid

the rest the rest the rest the rest the rest the rest the rest

- 120 043883 balance

<220> <221> <222> <223> OPTION (37)..(37) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (40)..(41) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (44)..(44) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (57)..(58) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (61)..(61) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (64)..(65) Xaa is any natural or unnatural amino acid <220> <221> <222> <223> OPTION (68)..(68) Xaa is any natural or unnatural amino acid <400> 6 Met Gly Ser Trp Xaa Glu Phe Xaa Xaa Arg Leu Xaa Ala Ile Xaa Xaa 1 5 10 15

the rest the rest the rest the rest the rest the rest

Arg Leu Xaa Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Xaa 30 Phe Glu Xaa Xaa Ile 35 Ala Xaa Phe Glu Xaa 40 Xaa Leu Gln Xaa Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Ala 55 Leu Xaa Xaa Glu Ala 60 Xaa Ala Ile Xaa Xaa 65 Glu Leu Xaa Ala Tyr 70 Arg His Asn

<210> 7 <211> 68 <212> Protein <213> Homo sapiens <220>

<221> OPTION <222> (5)..(6) <223> Xaa is any natural or unnatural amino acid <220>

< 221> OPTION < 222> (9)..(10) < 223> Xaa is any natural or unnatural amino acid <220>

< 221> OPTION < 222> (13)..(13) < 223> Xaa is any natural or unnatural amino acid

- 121 043883 <220>

< 221> OPTION < 222> (16)..(17) < 223> Xaa is any natural or unnatural amino acid < 220>

< 221> OPTION < 222> (22)..(22) < 223> Xaa is 20-30 natural or unnatural amino acid residues < 220>

< 221> OPTION <222> (28).. (28)

<223> Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (31)..(32) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (35)..(35) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (38)..(39) Xaa is any natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (42)..(42) Xaa is any natural or unnatural amino acid the remainder

<220>

< 221> OPTION < 222> (44)..(44) < 223> Xaa is 20-30 natural or unnatural amino acid residues < 400>7

Met Gly Ser Trp Xaa Xaa Phe 15

Lys Xaa Xaa Leu Ala Xaa 10

Ile Lys Xaa 15

Xaa Leu

Ile Ala

Glu Ala

Glu Ala Leu Xaa Glu Ala Glu Leu Ala Xaa Phe Glu Xaa Xaa

2530

Xaa Phe Glu Xaa Xaa Leu Gln Xaa Tyr Xaa Asn Pro Glu Val 35 4045

Leu Arg Lys Glu Ala Ala Ala Ile Arg Asp Glu Leu Gln Ala 5560

Tyr Arg His Asn < 210>8 < 211>68 < 212> Protein < 213> Homo sapiens <220>

<221> OPTION <222> (22)..(22) <223> Xaa is 2-30 natural or unnatural amino acid residues <220>

<221> OPTION <222> (30)..(31) <223> Xaa is a natural or unnatural amino acid residue

- 122 043883 <220>

<221> OPTION <222> (34) .. (34) <223> Xaa is a natural or unnatural amino acid residue <220>

<221> OPTION <222> (37).. (38) <223> Xaa is a natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (41)..(41) < 223> Xaa is a natural or unnatural amino acid residue <220>

<221> OPTION <222> (44)..(44) <223> Xaa is 2-30 natural or unnatural amino acid residues <220>

< 221> OPTION < 222> (52)..(53) < 223> Xaa is a natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (56) . . (56) <223> Xaa is a natural or unnatural amino acid residue <220>

<221> OPTION <222> (59) ..(60) <223> Xaa is a natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (63)..(63) < 223> Xaa is a natural or unnatural amino acid residue <400>

Met Gly Ser Trp Ala Glu Phe Lys Gln Arg Leu Ala Ala Ile Lys Thr

Arg Leu Glu Ala Leu Xaa Glu Ala Glu Leu Ala Ala Phe Xaa Xaa Glu

Ile Xaa Ala Phe Xaa Xaa Glu Leu Xaa Ala Tyr Xaa Asn Pro Glu Val

Glu Ala Leu Xaa Xaa Glu Ala Xaa Ala Ile Xaa Xaa Glu Leu Xaa Ala

Tyr Arg His Asn 65 <210> 9 <211> 68 <212> Protein <213> Homo sapiens <220>

< 221> OPTION < 222> (5)..(5) < 223> Xaa is a natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (8)..(9) < 223> Xaa is a natural or unnatural amino acid residue

- 123 043883 <220>

< 221> OPTION < 222> (12)..(12) < 223> Xaa is a natural or unnatural amino acid residue < 220>

< 221> OPTION < 222> (15)..(16) < 223> Xaa is a natural or unnatural amino acid residue < 220>

< 221> OPTION < 222> (19)..(19) < 223> Xaa is a natural or unnatural amino acid residue < 220>

<221> OPTION <222> (22)..(22) <223> Xaa is 2-30 natural or unnatural amino acid residues <220>

< 221> OPTION < 222> (44)..(44) < 223> Xaa is 2-30 natural or unnatural amino acid residues

<220> <221> <222> <223> OPTION (50)..(50) Xaa is natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (53)..(54) Xaa is natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (57) . . (57) Xaa is natural or unnatural amino acid the remainder <220> <221> <222> <223> OPTION (60)..(61) Xaa is natural or unnatural amino acid the remainder <400> 9

Met Gly Ser Trp Xaa Glu Phe Xaa Xaa Arg Leu Xaa Ala 1 5 10

Ile Xaa Xaa

Arg Leu Xaa Ala Leu Xaa Glu Ala 20

Ile Ala Ala Phe Glu Ser Glu Leu 3540

Glu Xaa Leu Arg Xaa Xaa Ala Ala 5055

Glu Leu Ala Ala Phe Glu Lys Glu 2530

Gln Ala Tyr Xaa Asn Pro Glu Val 45

Xaa Ile Arg Xaa Xaa Leu Gln Ala 60

Tyr Arg His Asn <210> 10 <211> 68 <212> Protein <213> Homo sapiens <220>

< 221> OPTION < 222> (5)..(6) < 223> Xaa is a natural or unnatural amino acid residue

- 124 043883 <220>

<221> OPTION <222> (9)..(10) <223> Xaa is a natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (13)..(13) < 223> Xaa is a natural or unnatural amino acid residue <220>

<221> OPTION <222> (16)..(17) <223> Xaa is a natural or unnatural amino acid residue <220>

<221> OPTION <222> (22)..(22) <223> Xaa is 2-30 natural or unnatural amino acid residues <220>

< 221> OPTION < 222> (30)..(31) < 223> Xaa is a natural or unnatural amino acid residue <220>

<221> OPTION <222> (34) .. (34) <223> Xaa is a natural or unnatural amino acid residue <220>

<221> OPTION <222> (37).. (38) <223> Xaa is a natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (41)..(41) < 223> Xaa is a natural or unnatural amino acid residue <220>

<221> OPTION <222> (44)..(44) <223> Xaa is 2-30 natural or unnatural amino acid residues <220>

<221> OPTION <222> (50) .. (50) <223> Xaa - natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (53)..(54) < 223> Xaa - natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (57)..(57) < 223> Xaa - natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (60)..(61) < 223> Xaa - natural or unnatural amino acid residue < 400> 10

Met Gly Ser Trp Xaa Xaa Phe Lys Xaa Xaa Leu Ala Xaa Ile Lys Xaa

5 10 15

Xaa Leu Glu Ala Leu Xaa Glu Ala Glu Leu Ala Ala Phe Xaa Xaa Glu

Ile Xaa Ala Phe Xaa Xaa Glu Leu Xaa Ala Tyr Xaa Asn Pro Glu Val

- 125 043883

Glu Xaa Leu Arg Xaa Xaa Ala Ala Xaa Ile Arg Xaa Xaa Leu Gln Ala

Tyr Arg His Asn 65 <210> 11 <211> 68 <212> Protein <213> Homo sapiens <220>

< 221> OPTION < 222> (5) . . (5) <223> Xaa is a natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (8)..(9) < 223> Xaa is a natural or unnatural amino acid residue < 220>

< 221> OPTION < 222> (12)..(12) < 223> Xaa is a natural or unnatural amino acid residue < 220>

< 221> OPTION < 222> (15)..(16) < 223> Xaa is a natural or unnatural amino acid residue < 220>

< 221> OPTION < 222> (19)..(19) < 223> Xaa is a natural or unnatural amino acid residue < 220>

<221> OPTION <222> (22)..(22) <223> Xaa is 2-30 natural or unnatural amino acid residues <220>

< 221> OPTION < 222> (28)..(28) < 223> Xaa is a natural or unnatural amino acid residue < 220>

< 221> OPTION < 222> (31)..(32) < 223> Xaa is a natural or unnatural amino acid residue < 220>

< 221> OPTION < 222> (35)..(35) < 223> Xaa is a natural or unnatural amino acid residue < 220>

< 221> OPTION < 222> (38)..(39) < 223> Xaa is a natural or unnatural amino acid residue < 220>

< 221> OPTION < 222> (42)..(42) < 223> Xaa is a natural or unnatural amino acid residue < 220>

<221> OPTION <222> (44)..(44) <223> Xaa is 2-30 natural or unnatural amino acid residues <220>

<221>OPTION

- 126 043883 <222> (52)..(53) <223> Xaa is a natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (56) . . (56) <223> Xaa is a natural or unnatural amino acid residue <220>

<221> OPTION <222> (59) ..(60) <223> Xaa is a natural or unnatural amino acid residue <220>

< 221> OPTION < 222> (63)..(63) < 223> Xaa is a natural or unnatural amino acid residue < 400> 11

Met Gly Ser Trp Xaa Glu Phe Xaa Xaa Arg Leu Xaa Ala Ile Xaa Xaa 1 5 10 15

Arg Leu Xaa Ala Leu Xaa Glu Ala Glu Leu Ala Xaa Phe Glu Xaa Xaa 20 25 30

Ile Ala Xaa Phe Glu Xaa Xaa Leu Gln Xaa Tyr Xaa Asn Pro Glu Val 35 40 45

Glu Ala Leu Xaa Xaa Glu Ala Xaa Ala Ile Xaa Xaa Glu Leu Xaa Ala

55 60

Tyr Arg His Asn < 210>12 < 211>73 < 212> Protein < 213> Homo sapiens < 400>12

Met Gly Ser Trp Val Glu Phe Gly His Arg Leu Trp Ala Ile Asp Gln

5 1015

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Glu 20 25 30

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln Ala Tyr Lys Gly Lys 35 40 45

Gly Asn Pro 50

Glu Val Glu Lys Leu Arg Gln Arg Ala Ala Phe

60

Ile Arg

Phe Arg Leu

Gln Ala Tyr Arg His Asn 70 <210>13 <211>73 <212> Protein <213> Homo sapiens <400>13

Met Gly Ser Trp Val Glu Phe Ala Asn Arg Leu Trp Ala Ile Asp Gln 1 5 10 15

- 127 043883

Arg Leu Phe Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Glu 20 2530

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln Ala Tyr Lys Gly Lys

4045

Gly Asn Pro Glu Val Glu His Leu Arg Asp Gln Ala Ala Phe Ile Arg 50 5560

His Lys Leu Gln Ala Tyr Arg His Asn 6570 <210>14 <211>73 <212> Protein <213> Homo sapiens <400>14

Met Gly Ser 1

Trp Tyr Glu Phe Arg His Arg Leu Trp Ala Ile Asp Gln

1015

Arg Leu Tyr Ala Leu Gly 20

Lys Glu Ile Ala Ala Phe 35

Gly Asn Pro Glu Val Glu 50

Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Gly 55 Leu Arg Glu Ala Ala 60 Ala Phe Ile Arg

Ala Lys Leu Gln Ala Tyr Arg His Asn 6570

<210> <211> <212> <213> <400> 15 73 Protein Phe Ser Met Arg 10 Leu Trp Ala Ile Asp 15 Gln Homo 15 Ser sapiens Trp Tyr 5 Glu Met 1 Gly Arg Leu Tyr Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Lys Glu Ile 35 Ala Ala Phe Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Ala 55 Leu Arg Ala Lys Ala 60 Ala Tyr Ile Arg Trp 65 Lys Leu Gln Ala Tyr 70 Arg His Asn

<210>16 <211>73 <212> Protein <213> Homo sapiens <400>16

Met Gly Ser Trp Phe Glu Phe Asn

His Arg Leu

Trp Ala Ile Asn Glu

- 128 043883

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Glu 20 2530

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln Ala Tyr Lys Gly Lys 35 4045

Gly Asn Pro Glu Val Glu Arg Leu Arg Ser Met Ala Ala Phe Ile Arg 50 5560

Tyr Lys Leu Gln Ala Tyr Arg His Asn 6570 <210>17 <211>73 <212> Protein <213> Homo sapiens <400>17

Met 1 Gly Ser Trp Tyr 5 Glu Phe Gly His Arg 10 Leu Trp Ala Ile Asp 15 Gln Arg Leu Tyr Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Lys Glu Ile 35 Ala Ala Phe Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Tyr 55 Leu Arg Glu Thr Ala 60 Ala His Ile Arg Thr 65 Arg Leu Gln Ala Tyr 70 Arg His Asn

<210>18 <211>73 <212> Protein <213> Homo sapiens <400>18

Met Gly Ser Trp Tyr Glu Phe His Tyr Arg Leu His Ala 1 510

Ile Asp Gln 15

Arg Leu Tyr Ala Leu Gly 20

Lys Glu Ile Ala Ala Phe 35

Gly Asn Pro Glu Val Glu 50

Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Glu 55 Leu Arg Ile Lys Ala 60 Ala Phe Ile Arg

Asp Arg Leu Gln Ala Tyr Arg His Asn 6570 <210>19 <211>73 <212> Protein <213> Homo sapiens

- 129 043883 <400> 19

Met Gly Ser Trp Ala Glu Phe Lys Gln Arg Leu

5 10

Arg Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Gly Glu Ile Trp Ala Phe Glu Met Glu Leu Ala

40

Gly Asn Pro Glu Val Glu Ala Leu Gly Arg Glu 50 55

Met Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>20 <211>73 <212> Protein <213> Homo sapiens <400>20

Met Gly Ser Trp Tyr Glu Phe Asp Leu Arg Leu

510

Arg Leu Val Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ile Leu Arg Asp Asn 50 55

Gln Met Leu Gln Ala Tyr Arg His Asn 65 70 <210>21 <211>73 <212> Protein <213> Homo sapiens <400>21

Met Gly Ser Trp Thr Glu Phe Thr Tyr Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Gln Lys Ile Ala Phe Phe Glu Asp Phe Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Lys His Glu 50 55

Ala Ile Lys Thr 15

Ala Ala Phe Leu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Ala Ile Tyr Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Tyr Ile Arg

Ala Ile Glu Trp 15

Ala Trp Phe Glu 30

Tyr Lys Gly Lys 45

Gly Ala Ile Leu

Asn Glu Leu Met Ala Tyr Arg His Asn 65 70 <210> 22 <211> 73

- 130 043883

Ala Ile Arg Glu <212> Protein <213> Homo sapiens <400> 22

Met Gly Ser Trp Ala Glu Phe Asp His Arg Leu

5 10

Arg Leu His Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ile Leu Arg Gly Asn

55

Ala Leu Leu Gln Ala Tyr Arg His Asn 65 70 <210>23 <211>73 <212> Protein <213> Homo sapiens <400>23

Met Gly Ser Trp Thr Glu Phe Val Gly Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Ala His Ile Ala Phe Phe Glu Asp Tyr Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Glu Glu

55

Glu Glu Leu Lys Ala Tyr Arg His Asn 65 70 <210>24 <211>73 <212> Protein <213> Homo sapiens <400>24

Met Gly Ser Trp Thr Glu Phe Tyr Ser Arg Leu

510

Arg Leu Gln Ala Leu Gly Gly Ser Glu Ala Glu

25

Asp Arg Ile Ala His Phe Glu Trp Phe Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu His Glu Glu

55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Tyr Ile Arg

Ala Ile Glu Phe

Ala Trp Phe Glu 30

Tyr Lys Gly Lys 45

Gly Ala Ile Met

Ala Ile Trp Val 15

Ala Met Phe Glu

Tyr Lys Gly Lys 45

Ile Ala Ile Arg

Lys Glu Leu Ala Ala Tyr Arg His Asn 65 70

- 131 043883

Ala Ile His Glu <210>25 <211>73 <212> Protein <213> Homo sapiens <400>25

Met Gly Ser Trp His Glu Phe His Asp Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ser Leu Arg Ile Ala 50 55

Gln Val Leu Gln Ala Tyr Arg His Asn 65 70 <210>26 <211>73 <212> Protein <213> Homo sapiens <400>26

Met Gly Ser Trp Asn Tyr Phe Lys Asp His Leu

510

Ser Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Thr Ala Ile Ala Ser Phe Glu Arg Gln Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu 50 55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>27 <211>73 <212> Protein <213> Homo sapiens <400>27

Met Gly Ser Trp Leu Tyr Phe Lys Glu His Leu

510

Trp Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Leu Ala Ile Ala Asp Phe Glu Tyr His Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu 50 55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala His Ile Arg

Trp Ile Lys Asn 15

Ala His Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

His Ile Lys Ala 15

Ala His Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

- 132 043883

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>28 <211>73 <212> Protein <213> Homo sapiens <400>28

Met Gly Ser Trp Val Tyr Phe Lys Glu His Leu

510

Glu Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

His Ser Ile Ala Asp Phe Glu Met Ser Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu 50 55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>29 <211>73 <212> Protein <213> Homo sapiens <400>29

Met Gly Ser Trp Phe Tyr Phe Lys Gln His Leu

510

Tyr Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Arg Ala Ile Ala Ala Phe Glu Gln His Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>30 <211>73 <212> Protein <213> Homo sapiens <400>30

Met Gly Ser Trp His Tyr Phe Lys Asp His Leu

510

Leu Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Met Ala Ile Ala Asp Phe Glu His Asn Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

Trp Ile Lys Thr 15

Ala His Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Trp Ile Lys Ser 15

Ala His Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Glu Ile Lys Gly 15

Ala His Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

- 133 043883

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>31 <211>73 <212> Protein <213> Homo sapiens <400>31

Met Gly Ser Trp His Tyr Phe Lys Gly His Leu

510

His Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Arg Ala Ile Ala Ala Phe Glu Arg Ser Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>32 <211>73 <212> Protein <213> Homo sapiens <400>32

Met Gly Ser Trp Ile Tyr Phe Lys Glu His Leu

510

Glu Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Ser Ala Ile Ala Val Phe Glu Ser Thr Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>33 <211>73 <212> Protein <213> Homo sapiens <400>33

Met Gly Ser Trp Thr Tyr Phe Lys Glu His Leu

510

Met Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Val Ala Ile Ala Asp Phe Glu Lys Met Leu Gln

40

Glu Ile Lys Asn 15

Ala His Phe Glu 30

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Tyr Ile Lys Lys 15

Ala His Phe Glu 30

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Glu Ile Lys Tyr 15

Ala His Phe Glu 30

Tyr Lys Gly Lys 45

- 134 043883

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>34 <211>73 <212> Protein <213> Homo sapiens <400>34

Met Gly Ser Trp Trp Leu Phe Lys Asp His Leu

510

Ala Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Met Ala Ile Ala Ala Phe Glu Lys Gln Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>35 <211>73 <212> Protein <213> Homo sapiens <400>35

Met Gly Ser Trp Ser Glu Phe Tyr Asn Arg Leu

510

Arg Leu Leu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Ile Gln Ile Ala Arg Phe Glu Lys Val Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Gly Glu 50 55

Ala Glu Leu Tyr Ala Tyr Arg His Asn 65 70 <210>36 <211>73 <212> Protein <213> Homo sapiens <400>36

Met Gly Ser Trp Tyr Glu Phe Tyr Asn Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu

25

Ala Ala Ile Arg

Glu Ile Lys Thr 15

Ala His Phe Glu 30

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Ala Ile Glu Ser

Ala Leu Phe Glu

Tyr Lys Gly Lys 45

Arg Ala Ile Phe

Ala Ile Glu Ile

Ala Ala Phe Glu

- 135 043883

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Arg Leu Arg Val Arg 50 55

Val Ile Leu Gln Ala Tyr Arg His Asn 65 70 <210>37 <211>73 <212> Protein <213> Homo sapiens <400>37

Met Gly Ser Trp Leu Trp Phe Lys Ile Phe Leu

510

Phe Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Phe Glu Ile His Ala Phe His Val Glu Leu Phe

40

Gly Asn Pro Glu Val Glu Val Leu Arg Glu Val 50 55

Trp Asp Leu Gln Ala Tyr Arg His Asn 65 70 <210>38 <211>73 <212> Protein <213> Homo sapiens <400>38

Met Gly Ser Trp Thr Glu Phe Gln Ser Arg Leu

510

Arg Leu Arg Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Leu Leu Arg Asp Asp

55

His Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>39 <211>73 <212> Protein <213> Homo sapiens <400>39

Met Gly Ser Trp Gln Glu Phe Asp Asp Arg Leu

510

Tyr Lys Gly Lys 45

Ala Lys Ile Arg

Glu Ile Lys Tyr 15

Ala Ala Phe Asp 30

Tyr Lys Gly Lys 45

Ala Glu Ile Arg

Ala Ile His Ser

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile Lys Ala 15

- 136 043883

Arg Leu Gln Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Asp Asp

55

Arg Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>40 <211>73 <212> Protein <213> Homo sapiens <400>40

Met Gly Ser Trp Tyr Glu Phe Gln Asn Arg Leu

510

Arg Leu Asn Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Leu Leu Arg Asp Asp 50 55

His Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>41 <211>73 <212> Protein <213> Homo sapiens <400>41

Met Gly Ser Trp Phe Glu Phe Gln Asp Arg Leu

510

Arg Leu Ser Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Thr Leu Arg Ser Asp 50 55

Arg Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>42 <211>73 <212> Protein <213> Homo sapiens <400>42

Met Gly Ser Trp Tyr Glu Phe Glu Ser Arg Leu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile His Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile Asn Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile His Glu

- 137 043883

Arg Leu His Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Glu 20 2530

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln Ala Tyr Lys Gly Lys 35 4045

Gly Asn Pro Glu Val Glu Asn Leu Arg Gly Asp Ala Ala Phe Ile Arg 50 5560

His Phe Leu Gln Ala Tyr Arg His Asn 6570 < 210>43 < 211>73 < 212> Protein < 213> Homo sapiens < 400>43

Met Gly Ser Trp Tyr Glu Phe Asn His Arg Leu Asp Ala Ile Ser Lys

5 1015

Arg Leu Asn Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Glu 20 2530

Lys Glu Ile Ala Ala Phe 35

Gly Asn Pro Glu Val Glu 50

Glu Ser Glu Leu Gln Ala Tyr Lys

45

Glu Leu Arg Gly Asp Ala Ala Phe 55 60

Gly Lys

Ile Arg

His Phe Leu Gln Ala Tyr Arg His Asn < 210>44 < 211>73 < 212> Protein < 213> Homo sapiens < 400>44

Met 1 Gly Ser Trp Phe 5 Glu Phe Glu Asn Arg 10 Leu His Ala Ile Val 15 His Arg Leu Gly Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Lys Glu Ile 35 Ala Ala Phe Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Thr 55 Leu Arg Ala Asp Ala 60 Ala Phe Ile Arg His 65 Tyr Leu Gln Ala Tyr 70 Arg His Asn

< 210 > 45 < 211 > 73 < 212 > Protein < 213 > Homo sapiens

- 138 043883 <400> 45

Met Gly Ser Trp Val Val Phe Lys Val Asp Leu

5 10

Ile Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Gly Glu Ile Ala Gly Phe Glu Tyr Ser Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>46 <211>73 <212> Protein <213> Homo sapiens <400>46

Met Gly Ser Trp Thr Ile Phe Lys Glu Trp Leu

510

Asp Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Gly Trp Ile Ala Ser Phe Glu Met Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>47 <211>73 <212> Protein <213> Homo sapiens <400>47

Met Gly Ser Trp Val Met Phe Lys Trp Leu Leu

510

His Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Gly Phe Ile Ala Ala Phe Glu Thr His Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Thr Ile Lys Tyr 15

Ala Glu Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Phe Ile Lys Thr 15

Ala Phe Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Asp Ile Lys Ser 15

Ala Phe Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210> 48 <211> 73

- 139 043883

Asp Ile Lys Gly <212> Protein <213> Homo sapiens <400> 48

Met Gly Ser Trp Tyr Ala Phe Lys Asp Tyr Leu

5 10

Trp Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Ile Phe Ile Ala Arg Phe Glu Leu Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>49 <211>73 <212> Protein <213> Homo sapiens <400>49

Met Gly Ser Trp Ala Glu Phe Lys Gln Arg Leu

510

Arg Leu Gln Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>50 <211>9 <212> Protein <213> Homo sapiens <400>50

Leu Ala Ala Ile Lys Thr Arg Leu Gln <210>51 <211>84 <212> Protein <213> Homo sapiens <400>51

Met Gly Ser Trp Val Glu Phe Gly His Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu

25

Ala Phe Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Ala Ile Lys Thr 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Ala Ile Asp Gln 15

Ala Ala Phe Glu

- 140 043883

Lys Glu Ile Ala Ala 35

Phe Glu Ser Glu Leu Gln Ala Tyr

45

Lys Gly Lys

Gly Asn Pro Glu Val 50

Glu Lys Leu Arg Gln Arg Ala Ala

60

Phe Ile Arg

Phe Arg Leu Gln Ala 65

Tyr Arg His Asn Gly Gly Gly Gly 70 75

Ser His His

His His His His <210>52 <211>84 <212> Protein <213> Homo sapiens <400>52

Met Gly Ser Trp Val Glu Phe Ala

Asn Arg Leu Trp Ala Ile Asp Gln

15

Arg Leu Phe Ala Leu Gly Gly Ser 20

Glu Ala Glu Leu Ala Ala Phe Glu

thirty

Lys Glu Ile Ala Ala Phe Glu Ser

40

Glu Leu Gln Ala Tyr Lys Gly Lys 45

Gly Asn Pro Glu Val Glu His Leu

55

Arg Asp Gln Ala Ala Phe Ile Arg

His Lys Leu Gln Ala Tyr Arg His 65 70

Asn Gly Gly Gly Gly Ser His His

80

His His His His <210>53 <211>84 <212> Protein <213> Homo sapiens <400>53

Met Gly Ser Trp Tyr Glu Phe Arg

His Arg Leu Trp Ala Ile Asp Gln

15

Arg Leu Tyr Ala Leu Gly Gly Ser 20

Glu Ala Glu Leu Ala Ala Phe Glu

thirty

Lys Glu Ile Ala Ala Phe Glu Ser

40

Glu Leu Gln Ala Tyr Lys Gly Lys 45

Gly Asn Pro Glu Val Glu Gly Leu

55

Arg Glu Ala Ala Ala Phe Ile Arg

Ala Lys Leu Gln Ala Tyr Arg His 65 70

Asn Gly Gly Gly Gly Ser His His

80

His His His

- 141 043883

Ala Ile Asp Gln <210>54 <211>84 <212> Protein <213> Homo sapiens <400>54

Met 1 Gly Ser Trp Tyr 5 Glu Phe Ser Met Arg 10 Leu Arg Leu Tyr Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Lys Glu Ile 35 Ala Ala Phe Glu Ser 40 Glu Leu Gln Gly Asn 50 Pro Glu Val Glu Ala 55 Leu Arg Ala Lys Trp 65 Lys Leu Gln Ala Tyr 70 Arg His Asn Gly Gly 75 His His His His

<210>55 <211>84 <212> Protein <213> Homo sapiens <400>55

Met Gly Ser Trp Phe Glu Phe Asn His Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Arg Leu Arg Ser Met

55

Tyr Lys Leu Gln Ala Tyr Arg His Asn Gly Gly 65 70 75

His His His His <210>56 <211>84 <212> Protein <213> Homo sapiens <400>56

Met Gly Ser Trp Tyr Glu Phe Gly His Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Tyr Ile Arg

Gly Ser His His

Ala Ile Asn Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Gly Ser His His

Ala Ile Asp Gln 15

Ala Ala Phe Glu

Tyr Lys Gly Lys

- 142 043883

Ala His Ile Arg

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg Glu Thr

55

Thr Arg Leu Gln Ala Tyr Arg His Asn Gly Gly 65 70 75

His His His His <210>57 <211>84 <212> Protein <213> Homo sapiens <400>57

Met Gly Ser Trp Tyr Glu Phe His Tyr Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Glu Leu Arg Ile Lys

55

Asp Arg Leu Gln Ala Tyr Arg His Asn Gly Gly

70 75

His His His His <210>58 <211>84 <212> Protein <213> Homo sapiens <400>58

Met Gly Ser Trp Ala Glu Phe Lys Gln Arg Leu

510

Arg Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Gly Glu Ile Trp Ala Phe Glu Met Glu Leu Ala

40

Gly Asn Pro Glu Val Glu Ala Leu Gly Arg Glu

55

Met Glu Leu Gln Ala Tyr Arg His Asn Gly Gly 65 70 75

Gly Ser His His

Ala Ile Asp Gln 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Gly Ser His His

Ala Ile Lys Thr 15

Ala Ala Phe Leu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Gly Ser His His

His His His His <210> 59

- 143 043883

Phe Cys Asp Asn <211>406 <212> Protein <213> Homo sapiens <400>59

Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly

510

Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro

25

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg

40

Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser

55

Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly 65 70 75

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys

90

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg

100 105

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val

115 120

Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro

130 135

Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala

145 150 155

Ser Pro Gln Asp Ile Glu Gly Arg Met Asp Lys

165 170

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

180 185

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

195 200

Thr Pro Glu Val Thr Cys Val Val Val Asp Val

210 215

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

225 230 235

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

245 250

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

260 265

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

275 280

Ser Phe Ser Ser 30

Cys Lys Gly Val 45

Ala Glu Cys Asp

Ser Met Cys Glu 80

Gly Cys Lys Asp

Ile Cys Arg Pro

110

Val Asn Gly Thr 125

Asp Leu Ser Pro

Glu Pro Gly His

160

Cys Asp Lys Thr 175

Gly Gly Pro Ser

190

Met Ile Ser Arg 205

His Glu Asp Pro

Val His Asn Ala

240

Tyr Arg Val Val

255

Gly Lys Glu Tyr

270

Ile Glu Lys Thr 285

- 144 043883

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

290 295

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

305 310 315

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

325 330

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

340 345

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

355 360

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

370 375

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

385 390 395

His His His His His

405 <210>60 <211>73 <212> Protein <213> Homo sapiens <400>60

Met Gly Ser Trp Ile Glu Phe Glu Asp Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

His Gln Ile Ala Phe Phe Glu Glu Asp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu His Met Glu 50 55

Glu Glu Leu Gly Ala Tyr Arg His Asn 65 70 <210>61 <211>73 <212> Protein <213> Homo sapiens <400>61

Met Gly Ser Trp Val Glu Phe Glu Tyr Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Asn Glu Ile Ala Ser Phe Glu Ser Asp Leu Gln

40

Val Tyr Thr Leu

Ser Leu Thr Cys

320

Glu Trp Glu Ser

335

Pro Val Leu Asp 350

Val Asp Lys Ser 365

Met His Glu Ala

Ser Pro Gly Lys

400

Ala Ile Thr Asp 15

Ala Glu Phe Glu

Tyr Lys Gly Lys 45

Glu Ala Ile Met

Ala Ile Ser Asp 15

Ala Phe Phe Glu

Tyr Lys Gly Lys 45

- 145 043883

Gly Asn Pro Glu Val Glu Ala Leu Met Phe Glu Ala

55 60

Asp Glu Leu His Ala Tyr Arg His Asn 65 70 <210>62 <211>73 <212> Protein <213> Homo sapiens <400>62

Met Gly Ser Trp Tyr Glu Phe Glu Asp Arg Leu Ala

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu Leu 20 25

Glu Glu Ile Ala Tyr Phe Glu His Gly Leu Gln Trp

40

Gly Asn Pro Glu Val Glu Ala Leu Glu Ser Glu Ala

55 60

Asp Glu Leu His Ala Tyr Arg His Asn 65 70 <210>63 <211>73 <212> Protein <213> Homo sapiens <400>63

Met Gly Ser Trp Tyr Glu Phe Glu Glu Arg Leu Asp

510

Arg Leu Ile Ala Leu Gly Gly Ser Glu Ala Glu Leu 20 25

Asp Ile Ile Ala Phe Phe Glu Gln Asp Leu Gln Tyr

40

Gly Asn Pro Glu Val Glu Ala Leu Glu Met Glu Ala

55 60

Ile Glu Leu Asp Ala Tyr Arg His Asn 65 70 <210>64 <211>73 <212> Protein <213> Homo sapiens <400>64

Met Gly Ser Trp Trp Glu Phe Glu Asp Arg Leu Trp

510

Arg Leu Met Ala Leu Gly Gly Ser Glu Ala Glu Leu 20 25

Gln Met Ile Ala His Phe Glu Gln Ile Leu Gln Val

Ala Ile Asp

Ile Glu Ala

Asp Phe Glu 30

Lys Gly Lys

Ala Ile Ile

Ile Glu Asp 15

Ile Phe Glu 30

Lys Gly Lys

Ala Ile Ser

Ile Asp Arg 15

Val Phe Glu 30

Lys Gly Lys

- 146 043883

His Ala Ile Gly

40

Gly Asn Pro Glu Val Glu Ala Leu His Phe Glu

55

Met Glu Leu Ala Ala Tyr Arg His Asn 65 70 <210>65 <211>73 <212> Protein <213> Homo sapiens <400>65

Met Gly Ser Trp Glu Glu Phe His Glu Arg Leu

510

Arg Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Asp Asp Ile Ala Ser Phe Glu Asp Trp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Ser Arg Glu

55

Phe Glu Leu Glu Ala Tyr Arg His Asn 65 70 <210>66 <211>73 <212> Protein <213> Homo sapiens <400>66

Met Gly Ser Trp Glu Glu Phe Asp Lys Arg Leu

510

Arg Leu Met Ala Leu Gly Gly Ser Glu Ala Glu

25

Ser Thr Ile Ala Trp Phe Glu Trp Asp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Asp Trp Glu

55

Tyr Glu Leu Gly Ala Tyr Arg His Asn 65 70 <210>67 <211>73 <212> Protein <213> Homo sapiens <400>67

Met Gly Ser Trp Ser Glu Phe Val Asp Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Ala Ile Asp Glu 15

Ala Phe Phe Glu

Tyr Lys Gly Lys 45

Asp Ala Ile Asn

Ala Ile Thr Arg 15

Ala Glu Phe Glu

Tyr Lys Gly Lys 45

Tyr Ala Ile Asp

Ala Ile Phe Asp 15

Ala Trp Phe Glu 30

- 147 043883

Asp Thr Ile Ala His Phe Glu Trp Asn Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Asn Gly Glu

55

Asp Glu Leu His Ala Tyr Arg His Asn 65 70 <210>68 <211>73 <212> Protein <213> Homo sapiens <400>68

Met Gly Ser Trp Trp Glu Phe Thr Asp Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Glu Ser Ile Ala Ile Phe Glu Gln Asp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Glu Tyr Glu 50 55

Tyr Glu Leu Glu Ala Tyr Arg His Asn 65 70 <210>69 <211>73 <212> Protein <213> Homo sapiens <400>69

Met Gly Ser Trp Trp Glu Phe Thr Asp Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Asp Ser Ile Ala Gln Phe Glu Gln Glu Leu Gln 35 40

Gly Asn Pro Glu Val Glu Ala Leu Ala Asp Glu 50 55

Ser Glu Leu His Ala Tyr Arg His Asn 65 70 <210>70 <211>73 <212> Protein <213> Homo sapiens <400>70

Met Gly Ser Trp Glu Trp Phe Lys Ser Asp Leu

510

Tyr Lys Gly Lys 45

Asp Ala Ile Thr

Ala Ile Phe Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Asn Ala Ile Gln

Ala Ile Glu Asp 15

Ala His Phe Glu 30

Tyr Lys Gly Lys 45

Asp Ala Ile Glu

Ser Ile Lys Trp 15

- 148 043883

Glu Leu Glu Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Trp 30 Phe Glu His Asp Ile 35 Ala Glu Phe Glu Glu 40 Asp Leu Gln Trp Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Ala 55 Leu Arg Lys Glu Ala 60 Ala Ala Ile Arg Asp 65 Glu Leu Gln Ala Tyr 70 Arg His Asn

<210>71 <211>73 <212> Protein <213> Homo sapiens <400>71

Met

His

Gly

Leu

Ala Val

Gly Asn 50

Ser

Glu

Trp

Ala 20

Asp 5

Leu

His

Gly

Phe

Gly

Lys

Ser

Asn

Glu 25

Asp 10

Ala

Ile Ala Tyr Phe Glu Leu Tyr Leu 35 40

Pro Glu Val Glu Ala Leu Arg Lys 55

Leu

Glu

Ala

Leu

Trp

Ala

Ile

Glu

Lys 15

Lys

Phe

Glu

Gln Gly Tyr Lys Gly Lys 45

Glu Ala Ala Ala Ile Arg 60

Asp Glu 65

Leu Gln Ala Tyr Arg His Asn <210>72 <211>73 <212> Protein <213> Homo sapiens <400>72

Met Gly Ser Trp Glu Phe Phe Lys Glu Val Leu Ala Glu Ile Lys Tyr

5 1015

Asp Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Trp Phe Glu 20 2530

Thr Asp Ile Ala Gly Phe Glu Ile Asp Leu Gln Val Tyr Lys Gly Lys

4045

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu Ala Ala Ala Ile Arg

5560

Asp Glu Leu Gln Ala Tyr Arg His Asn 6570 <210>73 <211>73 <212> Protein <213> Homo sapiens <400>73

- 149 043883

Met Gly Ser Trp Tyr Asp Phe Lys Glu Asp Leu 1 5 10

Met Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Asn Val Ile Ala Tyr Phe Glu Asn Asp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu 50 55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>74 <211>73 <212> Protein <213> Homo sapiens <400>74

Met Gly Ser Trp Ser Phe Phe Lys Asp Asp Leu

510

Phe Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Gln Thr Ile Ala Glu Phe Glu Tyr Asp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>75 <211>73 <212> Protein <213> Homo sapiens <400>75

Met Gly Ser Trp Val Thr Phe Lys Asp Glu Leu

510

Phe Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Val Asp Ile Ala Glu Phe Glu Ala Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu 50 55

Asp Ile Lys Trp 15

Ala Glu Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Glu Ile Lys Tyr 15

Ala Met Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Asp Ile Lys Asp 15

Ala Phe Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210> 76 <211> 73 <212> Protein <213> Homo sapiens

- 150 043883 <400> 76

Met Gly Ser Trp Ser Trp Phe Lys Glu Asp Leu

5 10

Glu Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Leu Asp Ile Ala Asp Phe Glu Gln Ala Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>77 <211>73 <212> Protein <213> Homo sapiens <400>77

Met Gly Ser Trp Trp Glu Phe Lys Glu Asp Leu

510

Phe Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

His Asp Ile Ala Lys Phe Glu Phe Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>78 <211>73 <212> Protein <213> Homo sapiens <400>78

Met Gly Ser Trp Asp Glu Phe Lys Glu Asp Leu

510

Asp Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Asp Glu Ile Ala Asp Phe Glu Met Tyr Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Ile Lys Phe 15

Ala Trp Phe Glu 30

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Glu Ile Lys Trp 15

Ala Trp Phe Glu 30

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

His Ile Lys Thr 15

Ala Leu Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210> 79

- 151 043883 <211>73 <212> Protein <213> Homo sapiens <400>79

Met Gly Ser Trp Phe Met Phe Lys Glu Glu Leu Ala Asp Ile Lys Asp

5 1015

Trp Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ser Phe Glu 20 2530

Ser Tyr Ile Ala Trp Phe Glu Gln Asp Leu Gln Trp Tyr Lys Gly Lys 35 4045

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu Ala Ala Ala Ile Arg 50 5560

Asp Glu Leu Gln Ala Tyr Arg His Asn 6570 <210>80 <211>73 <212> Protein <213> Homo sapiens <400>80

Met Gly Ser Trp Gln Ile Phe Lys Gly Glu Leu Ala Tyr Ile Lys Gln

5 1015

Tyr Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Phe Phe Glu 20 2530

Phe Asp Ile Ala Glu Phe Glu Glu Asp Leu Gln Tyr Tyr Lys Gly Lys

4045

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu Ala Ala Ala Ile Arg 50 5560

Asp Glu Leu Gln Ala Tyr Arg His Asn 6570 <210>81 <211>73 <212> Protein <213> Homo sapiens <400>81

Met Gly Ser Trp Tyr Ile Phe Lys Glu Asp Leu Ala Glu Ile Lys Glu

5 1015

Glu Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Tyr Phe Glu 20 2530

Glu Glu Ile Ala Leu Phe Glu Met Glu Leu Gln Trp Tyr Lys Gly Lys

4045

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu Ala Ala Ala Ile Arg

5560

Asp Glu Leu Gln Ala Tyr Arg His Asn 6570

- 152 043883

Asp Ile Lys Trp <210>82 <211>73 <212> Protein <213> Homo sapiens <400>82

Met Gly Ser Trp Tyr Tyr Phe Lys Asp Glu Leu

510

Asp Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Met Leu Ile Ala Gln Phe Glu Leu Asp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>83 <211>73 <212> Protein <213> Homo sapiens <400>83

Met Gly Ser Trp Phe Asn Phe Lys Glu Glu Leu

510

Gln Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Trp Val Ile Ala Asp Phe Glu Asp Asp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>84 <211>73 <212> Protein <213> Homo sapiens <400>84

Met Gly Ser Trp Tyr Met Phe Lys Glu Glu Leu

510

Tyr Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Asp Asp Ile Ala Gly Phe Glu Trp Asp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Ala Trp Phe Glu 30

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Val Ile Lys Phe 15

Ala Phe Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Asp Ile Lys Trp 15

Ala Trp Phe Glu 30

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

- 153 043883

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>85 <211>73 <212> Protein <213> Homo sapiens <400>85

Met Gly Ser Trp His Val Phe Lys Thr Glu Leu

510

Tyr Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Leu Trp Ile Ala Glu Phe Glu His Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu 50 55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>86 <211>73 <212> Protein <213> Homo sapiens <400>86

Met Gly Ser Trp Tyr Val Phe Lys Asp Glu Leu

510

Phe Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Asp Asp Ile Ala Glu Phe Glu Thr Gln Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu 50 55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>87 <211>73 <212> Protein <213> Homo sapiens <400>87

Met Gly Ser Trp Thr Glu Phe Lys Gly Glu Leu

510

Ile Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Asp Glu Ile Ala Ala Phe Glu Trp Asp Leu Gln

40

Asp Ile Lys Phe 15

Ala Met Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Glu Ile Lys Gln 15

Ala Trp Phe Glu 30

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Glu Ile Lys Trp 15

Ala Phe Phe Glu

Tyr Lys Gly Lys 45

- 154 043883

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>88 <211>73 <212> Protein <213> Homo sapiens <400>88

Met Gly Ser Trp Phe Trp Phe Lys Glu Asp Leu

510

Asp Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Asp Gly Ile Ala Phe Phe Glu Trp Asp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu 50 55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>89 <211>73 <212> Protein <213> Homo sapiens <400>89

Met Gly Ser Trp Ser Trp Phe Lys Glu Asp Leu

510

Val Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Ser Asp Ile Ala Glu Phe Glu Gln Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>90 <211>73 <212> Protein <213> Homo sapiens <400>90

Met Gly Ser Trp Ile Leu Phe Lys Asp Asp Leu

510

Thr Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Asp Asn Ile Ala Asp Phe Glu Glu Gln Leu Gln

Ala Ala Ile Arg

Phe Ile Lys Glu 15

Ala Trp Phe Glu 30

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Ser Ile Lys Ala 15

Ala Phe Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Trp Ile Lys Glu 15

Ala Phe Phe Glu

Tyr Lys Gly Lys

- 155 043883

Ala Ala Ile Arg

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>91 <211>73 <212> Protein <213> Homo sapiens <400>91

Met Gly Ser Trp Gln Trp Phe Lys Asp Asp Leu

510

Thr Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Asp Met Ile Ala Asp Phe Glu Phe Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>92 <211>73 <212> Protein <213> Homo sapiens <400>92

Met Gly Ser Trp Glu Glu Phe His Ser Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Trp Leu Arg Trp Glu

55

Glu Thr Leu Gln Ala Tyr Arg His Asn 65 70 <210>93 <211>73 <212> Protein <213> Homo sapiens <400>93

Met Gly Ser Trp Ser Glu Phe Trp Gln Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Tyr Ile Lys Glu 15

Ala Leu Phe Glu

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Ala Ile Asp Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Thr Ile Arg

Ala Ile Glu Asp 15

Ala Ala Phe Glu

- 156 043883

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu

3540

Gly Asn Pro Glu Val Glu Trp Leu Arg Glu 5055

Gln Ala Tyr Lys 45

Gly Lys

Asn Ala Ala Met

Ile Arg

Asp Glu Leu Gln Ala Tyr Arg His Asn 6570

<210> <211> <212> <213> <400> 94 73 Protein Homo 94 Ser sapiens Phe Ala Trp Arg 10 Leu Asp Ala Ile Tyr 15 Asp Met 1 Gly Trp Thr 5 Glu Arg Leu Leu Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Lys Glu Ile 35 Ala Ala Phe Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Trp 55 Leu Arg His Val Ala 60 Ala Asn Ile Arg Arg 65 Glu Leu Gln Ala Tyr 70 Arg His Asn

<210>95 <211>73 <212> Protein <213> Homo sapiens <400>95

Met 1 Gly Ser Trp Asp 5 Glu Phe Tyr Tyr Arg 10 Leu Glu Ala Ile Glu 15 Met Arg Leu Gly Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Lys Glu Ile 35 Ala Ala Phe Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Glu 55 Leu Arg His Tyr Ala 60 Ala Gln Ile Arg His 65 Met Leu Gln Ala Tyr 70 Arg His Asn

<210>96 <211>73 <212> Protein <213> Homo sapiens <400>96

Met Gly Ser Trp Ile

Glu Phe Asn Met Arg

5 10

Leu Asp Ala

Ile Tyr Glu 15

- 157 043883

Arg Leu Val Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Trp Leu Arg Lys Val

55

Leu Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>97 <211>73 <212> Protein <213> Homo sapiens <400>97

Met Gly Ser Trp Ser Glu Phe Asn Met Arg Leu

510

Arg Leu Thr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Trp Leu Arg His Ser 50 55

Leu Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>98 <211>73 <212> Protein <213> Homo sapiens <400>98

Met Gly Ser Trp Val Glu Phe Asn Ile Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Lys Leu Arg His Trp

55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asn Ile Arg

Ala Ile Tyr Glu 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Arg Ile Arg

Ala Ile Tyr Glu 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Ser Ile Arg

Arg Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>99 <211>73 <212> Protein <213> Homo sapiens <400>99

- 158 043883

Met Gly Ser Trp Asp Glu Phe Gly Arg Arg Leu 1 5 10

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Lys Leu Arg Glu Ile 50 55

Ser Asn Leu Gln Ala Tyr Arg His Asn 65 70 <210>100 <211>73 <212> Protein <213> Homo sapiens <400>100

Met Gly Ser Trp Ile Glu Phe Tyr Asp Arg Leu

510

Arg Leu Asp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln 35 40

Gly Asn Pro Glu Val Glu Trp Leu Arg Glu Asp 50 55

Ser Trp Leu Gln Ala Tyr Arg His Asn 65 70 <210>101 <211>73 <212> Protein <213> Homo sapiens <400>101

Met Gly Ser Trp Thr Glu Phe Asp Arg Arg Leu

510

Arg Leu Phe Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln 35 40

Gly Asn Pro Glu Val Glu Trp Leu Arg Glu Glu 50 55

Ala Ile Glu Trp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

Ala Ile Tyr Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile Trp Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Asp Tyr Leu Gln Ala Tyr Arg His Asn 65 70 <210> 102 <211> 73 <212> Protein <213> Homo sapiens

- 159 043883 <400> 102

Met Gly Ser Trp Thr Glu Phe Asp Arg Arg Leu

5 10

Arg Leu Phe Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Trp Leu Arg Glu Glu 50 55

Asp Tyr Leu Gln Ala Tyr Arg His Asn 65 70 <210>103 <211>73 <212> Protein <213> Homo sapiens <400>103

Met Gly Ser Trp Ile Glu Phe Glu Val Arg Leu

510

Arg Leu Ala Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Arg Leu Arg Arg Tyr

55

His Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>104 <211>73 <212> Protein <213> Homo sapiens <400>104

Met Gly Ser Trp Thr Glu Phe His Asp Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg Glu Glu 50 55

Ala Ile Trp Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Tyr Asn 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asn Ile Arg

Ala Ile Asp Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Gln Ile Arg

Trp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210> 105

- 160 043883

Ala Ile Tyr Glu <211>73 <212> Protein <213> Homo sapiens <400>105

Met Gly Ser Trp Tyr Glu Phe His His Arg Leu

510

Arg Leu Leu Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Trp Leu Arg Ser Ser

55

Lys Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>106 <211>73 <212> Protein <213> Homo sapiens <400>106

Met Gly Ser Trp His Glu Phe Asp Gln Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Thr Leu Arg Leu Tyr

55

His Asp Leu Gln Ala Tyr Arg His Asn 65 70 <210>107 <211>73 <212> Protein <213> Homo sapiens <400>107

Met Gly Ser Trp Ile Glu Phe Glu Ser Arg Leu

510

Arg Leu Leu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Phe Leu Arg Leu Glu

55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asn Ile Arg

Ala Ile Glu Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Leu Ile Arg

Ala Ile Glu Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Glu Asp Leu Gln Ala Tyr Arg His Asn 65 70

- 161 043883 <210>108 <211>73 <212> Protein <213> Homo sapiens <400>108

Met 1 Gly Ser Trp Tyr 5 Glu Phe Glu Asn Arg 10 Leu Gly Ala Ile Gly 15 Asp Arg Leu Trp Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Lys Glu Ile 35 Ala Ala Phe Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Trp 55 Leu Arg Asp Glu Ala 60 Ala Tyr Ile Arg Ala 65 Val Leu Gln Ala Tyr 70 Arg His Asn

< 210>109 < 211>73 < 212> Protein < 213> Homo sapiens < 400>109

Met Gly Ser Trp Asn Glu Phe Tyr Asp Arg Leu Ser Ala Ile Tyr Phe

5 1015

Arg Leu Gln Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Glu 20 2530

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln Ala Tyr Lys Gly Lys 35 4045

Gly Asn Pro Glu Val Glu His Leu Arg Trp Tyr Ala Ala Asp Ile Arg 50 5560

Met Ile Leu Gln Ala Tyr Arg His Asn 6570 < 210>110 < 211>73 < 212> Protein <213> Homo sapiens <400>110

Met Gly Ser Trp Tyr Glu Phe Glu Tyr 15

Arg Leu Glu Ala Ile Glu Asp

15

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Glu 20 2530

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln Ala Tyr Lys Gly Lys 35 4045

Gly Asn Pro Glu Val Glu Tyr Leu Arg Glu Glu Ala Ala Trp Ile Arg 50 5560

- 162 043883

Val Trp Leu Gln Ala Tyr Arg His Asn 65 70 <210>111 <211>73 <212> Protein <213> Homo sapiens <400>111

Met Gly Ser Trp Val Glu Phe Glu Asn Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Trp Leu Arg Glu Asp 50 55

Met Met Leu Gln Ala Tyr Arg His Asn 65 70 <210>112 <211>73 <212> Protein <213> Homo sapiens <400>112

Met Gly Ser Trp Tyr Glu Phe Trp Asp Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Gln Glu 50 55

Glu Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>113 <211>73 <212> Protein <213> Homo sapiens <400>113

Met Gly Ser Trp Phe Glu Phe Trp Asp Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Ala Ile Glu Asn

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Gln Ile Arg

Ala Ile Asp Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Trp Ile Arg

Ala Ile Glu Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

- 163 043883

Gly Asn Pro Glu Val Glu Glu Leu Arg Asp

5055

Glu Ala Ala

Trp Ile Arg

Gly Thr Leu Gln Ala Tyr Arg His Asn 6570 <210>114 <211>73 <212> Protein <213> Homo sapiens <400>114

Met Gly Ser Trp 1

Thr Glu Phe Asp Arg 5

Arg Leu Asp Ala Ile 10

Trp Asp 15

Arg Leu

Phe Ala Leu Gly Gly Ser Glu Ala

25

Lys Glu

Ile Ala Ala Phe Glu Ser Glu Leu

40

Gly Asn 50

Pro Glu Val Glu Trp Leu Arg Glu 55

Glu Leu Ala Ala Phe Glu 30

Gln Ala Tyr Lys Gly Lys 45

Glu Ala Ala Asp Ile Arg 60

Asp Tyr 65

Leu Gln Ala Tyr Arg His Asn <210>115 <211>73 <212> Protein <213> Homo sapiens <400>115

Met Gly Ser Trp Trp Glu Phe Glu Met Arg Leu Glu Ala 1 5 10

Ile Glu Asp 15

Arg Leu Phe Ala Leu Gly 20

Lys Glu Ile Ala Ala Phe 35

Gly Asn Pro Glu Val Glu 50

Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Ser 55 Leu Arg Trp Glu Ala 60 Ala Phe Ile Arg

Asp Ile Leu Gln Ala Tyr Arg His Asn 65 70 <210>116 <211>73 <212> Protein <213> Homo sapiens <400>116

Met Gly Ser Trp Val Glu Phe Tyr Asp Arg Leu His Ala 1 5 10

Ile Tyr Phe 15

Arg Leu Leu Ala Leu Gly Gly Ser Glu Ala 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu

Glu Leu Ala Ala Phe Glu

Gln Ala Tyr Lys Gly Lys

- 164 043883

Ala Asp Ile Arg

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Trp Tyr

55

Leu Val Leu Gln Ala Tyr Arg His Asn 65 70 <210>117 <211>73 <212> Protein <213> Homo sapiens <400>117

Met Gly Ser Trp Tyr Glu Phe Tyr Asn Arg Leu

510

Arg Leu Gln Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Trp Tyr

55

Tyr Met Leu Gln Ala Tyr Arg His Asn 65 70 <210>118 <211>73 <212> Protein <213> Homo sapiens <400>118

Met Gly Ser Trp Phe Glu Phe Trp Gly Arg Leu

510

Arg Leu Lys Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Glu Leu Arg Glu His

55

Ala Tyr Leu Gln Ala Tyr Arg His Asn 65 70 <210>119 <211>73 <212> Protein <213> Homo sapiens <400>119

Met Gly Ser Trp Thr Glu Phe Ser Ile Arg Leu

510

Arg Leu Val Ala Leu Gly Gly Ser Glu Ala Glu

25

Ala Ile Tyr Ala 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Glu Ser

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Trp Ile Arg

Ala Ile Tyr Asp 15

Ala Ala Phe Glu

- 165 043883

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Val Leu Arg Thr Tyr 50 55

His Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>120 <211>73 <212> Protein <213> Homo sapiens <400>120

Met Gly Ser Trp Tyr Glu Phe Glu Asn Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Met Leu Arg Glu Glu 50 55

Asp Trp Leu Gln Ala Tyr Arg His Asn 65 70 <210>121 <211>73 <212> Protein <213> Homo sapiens <400>121

Met Gly Ser Trp Tyr Glu Phe Val Ile Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Val Leu Arg Trp Tyr

55

His Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>122 <211>73 <212> Protein <213> Homo sapiens <400>122

Met Gly Ser Trp Ile Glu Phe Glu Asp Arg Leu

510

Tyr Lys Gly Lys 45

Ala Asn Ile Arg

Ala Ile Glu Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile Glu Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Glu Asp 15

- 166 043883

Arg Leu Phe Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Lys Glu Ile 35 Ala Ala Phe Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Trp 55 Leu Arg Gln Glu Ala 60 Ala Glu Ile Arg Leu 65 Met Leu Gln Ala Tyr 70 Arg His Asn

<210>123 <211>73 <212> Protein <213> Homo sapiens <400>123

Met Gly Ser Trp Thr Glu Phe Asn Leu Arg Leu Asp Ala Ile Tyr Asp

5 1015

Arg Leu Met Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Glu 20 2530

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln Ala Tyr Lys Gly Lys 35 4045

Gly Asn Pro Glu Val Glu Trp Leu Arg Ala Ser Ala Ala Ala Ile Arg 50 5560

Val Glu Leu Gln Ala Tyr Arg His Asn 6570 <210>124 <211>73 <212> Protein <213> Homo sapiens <400>124

Met Gly Ser Trp Ser Glu Phe Tyr Leu Arg Leu Asp Ala Ile Tyr Asp

5 1015

Arg Leu Asp Ala Leu Gly Gly Ser Glu Ala Glu Leu Ala Ala Phe Glu 20 2530

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln Ala Tyr Lys Gly Lys 35 4045

Gly Asn Pro Glu Val Glu Trp Leu Arg Lys Thr Ala Ala Asn Ile Arg 50 5560

Glu Glu Leu Gln Ala Tyr Arg His Asn 6570 <210>125 <211>73 <212> Protein <213> Homo sapiens <400>125

- 167 043883

Met Gly Ser Trp Ser Glu Phe His Val Arg Leu 1 5 10

Arg Leu Asp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Arg Leu Arg Glu Trp 50 55

Arg Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>126 <211>73 <212> Protein <213> Homo sapiens <400>126

Met Gly Ser Trp His Glu Phe Gly Val Arg Leu

510

Arg Leu Met Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Phe Leu Arg Gln Ala 50 55

Ser Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>127 <211>73 <212> Protein <213> Homo sapiens <400>127

Met Gly Ser Trp Tyr Glu Phe Ser Met Arg Leu

510

Arg Leu Met Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Gln Leu Arg Gly Tyr 50 55

Ala Ile Tyr Ala 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asn Ile Arg

Ala Ile Tyr Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asn Ile Arg

Ala Ile Tyr Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asn Ile Arg

Asn Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210> 128 <211> 73 <212> Protein <213> Homo sapiens

- 168 043883 <400> 128

Met Gly Ser Trp Asp Glu Phe Gly Arg Arg Leu

5 10

Gln Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Lys Leu Arg Glu Ile 50 55

Ser Asn Leu Gln Ala Tyr Arg His Asn 65 70 <210>129 <211>73 <212> Protein <213> Homo sapiens <400>129

Met Gly Ser Trp Asp Glu Phe Gly Arg Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Glu Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Lys Leu Arg Glu Ile

55

Ser Asn Leu Gln Ala Tyr Arg His Asn 65 70 <210>130 <211>73 <212> Protein <213> Homo sapiens <400>130

Met Gly Ser Trp Asp Glu Phe Gly Arg Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Lys Leu Arg Glu Ile 50 55

Ala Ile Glu Trp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

Ala Ile Glu Trp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

Ala Ile Glu Trp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

Glu Asn Leu Gln Ala Tyr Arg His Asn 65 70 <210> 131

- 169 043883

Ala Ile Glu Trp <211>73 <212> Protein <213> Homo sapiens <400>131

Met Gly Ser Trp Asp Glu Phe Gly Arg Arg Leu

510

Gln Leu Tyr Ala Leu Gly Gly Glu Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln 35 40

Gly Asn Pro Glu Val Glu Lys Leu Arg Glu Ile

55

Ser Asn Leu Gln Ala Tyr Arg His Asn 65 70 <210>132 <211>73 <212> Protein <213> Homo sapiens <400>132

Met Gly Ser Trp Asp Glu Phe Gly Arg Arg Leu

510

Gln Leu Tyr Ala Leu Gly Gly Thr Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Lys Leu Arg Glu Ile

55

Ser Asn Leu Gln Ala Tyr Arg His Asn 65 70 <210>133 <211>73 <212> Protein <213> Homo sapiens <400>133

Met Gly Ser Trp Asp Glu Phe Gly Arg Arg Leu

510

Gln Leu Tyr Ala Leu Gly Gly Gly Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Lys Leu Arg Glu Ile

55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

Ala Ile Glu Trp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

Ala Ile Glu Trp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

Ser Asn Leu Gln Ala Tyr Arg His Asn 65 70

- 170 043883

Ala Ile Glu Trp <210>134 <211>73 <212> Protein <213> Homo sapiens <400>134

Met Gly Ser Trp Asp Glu Phe Gly Arg Arg Leu

510

Gln Leu Tyr Ala Leu Gly Gly Glu Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Lys Leu Arg Glu Ile

55

Glu Asn Leu Gln Ala Tyr Arg His Asn 65 70 <210>135 <211>73 <212> Protein <213> Homo sapiens <400>135

Met Gly Ser Trp Asp Glu Phe Gly Arg Arg Leu

510

Gln Leu Tyr Ala Leu Gly Gly Thr Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln 35 40

Gly Asn Pro Glu Val Glu Lys Leu Arg Glu Ile 50 55

Glu Asn Leu Gln Ala Tyr Arg His Asn 65 70 <210>136 <211>73 <212> Protein <213> Homo sapiens <400>136

Met Gly Ser Trp Asp Glu Phe Gly Arg Arg Leu

510

Gln Leu Tyr Ala Leu Gly Gly Gly Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Lys Leu Arg Glu Ile

55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

Ala Ile Glu Trp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

Ala Ile Glu Trp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

- 171 043883

Glu Asn Leu Gln Ala Tyr Arg His Asn 65 70 <210>137 <211>73 <212> Protein <213> Homo sapiens <400>137

Met Gly Ser Trp Glu Glu Phe Glu Leu Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Tyr Val Ile Ala Asp Phe Glu Gly Asn Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Tyr Phe Glu 50 55

Glu Glu Leu Val Ala Tyr Arg His Asn 65 70 <210>138 <211>73 <212> Protein <213> Homo sapiens <400>138

Met Gly Ser Trp Phe Glu Phe Asn His Arg Leu

510

Arg Leu Met Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg Ala Met 50 55

Tyr His Leu Gln Ala Tyr Arg His Asn 65 70 <210>139 <211>73 <212> Protein <213> Homo sapiens <400>139

Met Gly Ser Trp Glu Glu Phe Asp Gly Arg Leu

510

Arg Leu Gln Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Ala Ile Glu Glu

Ala Tyr Phe Glu 30

Tyr Lys Gly Lys 45

Asp Ala Ile Phe

Ala Ile Phe Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Val Ile Arg

Ala Ile Glu Gln

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

- 172 043883

Gly Asn Pro Glu Val Glu Val Leu Arg Trp Phe

55

Asp Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>140 <211>73 <212> Protein <213> Homo sapiens <400>140

Met Gly Ser Trp Ala Glu Phe Tyr His Arg Leu

510

Arg Leu Ser Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg His Trp 50 55

Thr Tyr Leu Gln Ala Tyr Arg His Asn 65 70 <210>141 <211>73 <212> Protein <213> Homo sapiens <400>141

Met Gly Ser Trp Val Glu Phe Ser Asp Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Glu Leu Arg Glu Leu

55

His Ser Leu Gln Ala Tyr Arg His Asn 65 70 <210>142 <211>73 <212> Protein <213> Homo sapiens <400>142

Met Gly Ser Trp Trp Glu Phe Glu Gly Arg Leu

510

Arg Leu Thr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

Ala Gly Ile Arg

Ala Ile Glu Thr

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Trp Ile Arg

Ala Ile Glu Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Ile Ile Arg

Ala Ile Glu Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys

- 173 043883

Ala Trp Ile Arg

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg Glu Trp

55

Gln Met Leu Gln Ala Tyr Arg His Asn 65 70 <210>143 <211>73 <212> Protein <213> Homo sapiens <400>143

Met Gly Ser Trp Trp Glu Phe Glu His Arg Leu

510

Arg Leu Val Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg Asn Trp

55

Met Ala Leu Gln Ala Tyr Arg His Asn 65 70 <210>144 <211>73 <212> Protein <213> Homo sapiens <400>144

Met Gly Ser Trp Trp Glu Phe Glu Ala Arg Leu

510

Arg Leu Ser Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg Ser Trp

55

Thr Ser Leu Gln Ala Tyr Arg His Asn 65 70 <210>145 <211>73 <212> Protein <213> Homo sapiens <400>145

Met Gly Ser Trp Trp Glu Phe Glu Ala Arg Leu

510

Arg Leu Lys Ala Leu Gly Gly Ser Glu Ala Glu

25

Ala Ile Glu Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Tyr Ile Arg

Ala Ile Glu Phe

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Tyr Ile Arg

Ala Ile Glu Ser

Ala Ala Phe Glu

- 174 043883

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg His Trp 50 55

Val Ile Leu Gln Ala Tyr Arg His Asn 65 70 <210>146 <211>73 <212> Protein <213> Homo sapiens <400>146

Met Gly Ser Trp Trp Glu Phe Glu Ala Arg Leu

510

Arg Leu Ser Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg Ser Trp 50 55

Thr Ser Leu Gln Ala Tyr Arg His Asn 65 70 <210>147 <211>73 <212> Protein <213> Homo sapiens <400>147

Met Gly Ser Trp Glu Glu Phe Tyr His Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg Trp Tyr 50 55

Glu Ile Leu Gln Ala Tyr Arg His Asn 65 70 <210>148 <211>73 <212> Protein <213> Homo sapiens <400>148

Met Gly Ser Trp Tyr Glu Phe Tyr Glu Arg Leu

510

Tyr Lys Gly Lys 45

Ala Tyr Ile Arg

Ala Ile Glu Phe

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Tyr Ile Arg

Ala Ile Glu Leu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Glu Ile Arg

Ala Ile Asp Thr 15

- 175 043883

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Phe Leu Arg Glu Tyr

55

His Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>149 <211>73 <212> Protein <213> Homo sapiens <400>149

Met Gly Ser Trp Asn Glu Phe Phe Asp Arg Leu

510

Arg Leu Asp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg Phe Val 50 55

Ser Trp Leu Gln Ala Tyr Arg His Asn 65 70 <210>150 <211>73 <212> Protein <213> Homo sapiens <400>150

Met Gly Ser Trp Ile Glu Phe Asp Asp Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Asp Val

55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Glu Ile Arg

Ala Ile Leu Tyr 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Met Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

His Tyr Leu Gln Ala Tyr Arg His Asn 65 70 <210> 151 <211> 73 <212> Protein <213> Homo sapiens <400> 151

- 176 043883

Met Gly Ser Trp Tyr Glu Phe Trp Glu Arg Leu 1 5 10

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Thr Trp 50 55

Ala Ile Leu Gln Ala Tyr Arg His Asn 65 70 <210>152 <211>73 <212> Protein <213> Homo sapiens <400>152

Met Gly Ser Trp Glu Glu Phe Tyr Ile Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Tyr Ala 50 55

His Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>153 <211>73 <212> Protein <213> Homo sapiens <400>153

Met Gly Ser Trp Ile Glu Phe Glu Glu Arg Leu

510

Arg Leu Leu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Phe Leu Arg Val Val 50 55

Ala Ile Thr Phe

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Met Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Glu Thr

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Glu Trp Leu Gln Ala Tyr Arg His Asn 65 70 <210> 154 <211> 73 <212> Protein <213> Homo sapiens

- 177 043883 <400> 154

Met Gly Ser Trp Ile Glu Phe Glu His Arg Leu

5 10

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Glu Trp

55

Ser Leu Leu Gln Ala Tyr Arg His Asn 65 70 <210>155 <211>73 <212> Protein <213> Homo sapiens <400>155

Met Gly Ser Trp Phe Glu Phe Glu Met Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Tyr Ala

55

Asp Tyr Leu Gln Ala Tyr Arg His Asn 65 70 <210>156 <211>73 <212> Protein <213> Homo sapiens <400>156

Met Gly Ser Trp Tyr Glu Phe Val Tyr Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Tyr Ala

55

Ala Ile Asn Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Met Ala

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Tyr Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Asp Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210> 157

- 178 043883

Ala Ile Leu Glu <211>73 <212> Protein <213> Homo sapiens <400>157

Met Gly Ser Trp Val Glu Phe Glu Asp Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Glu Leu

55

Asp Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>158 <211>73 <212> Protein <213> Homo sapiens <400>158

Met Gly Ser Trp Phe Glu Phe Glu Glu Arg Leu

510

Arg Leu Phe Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Tyr Leu Arg Trp Ile

55

Asp Val Leu Gln Ala Tyr Arg His Asn 65 70 <210>159 <211>73 <212> Protein <213> Homo sapiens <400>159

Met Gly Ser Trp Ile Glu Phe Ala Asp Arg Leu

510

Arg Leu Asp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Trp Leu Arg Glu Ile

55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Glu Glu

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Leu Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Tyr Leu Gln Ala Tyr Arg His Asn 65 70

- 179 043883

Ala Ile Leu Asp <210>160 <211>73 <212> Protein <213> Homo sapiens <400>160

Met Gly Ser Trp Leu Glu Phe Glu Tyr Arg Leu

510

Arg Leu Phe Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Glu Val

55

Met Leu Leu Gln Ala Tyr Arg His Asn 65 70 <210>161 <211>73 <212> Protein <213> Homo sapiens <400>161

Met Gly Ser Trp Tyr Glu Phe His Asp Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Asp Trp 50 55

Val Trp Leu Gln Ala Tyr Arg His Asn 65 70 <210>162 <211>73 <212> Protein <213> Homo sapiens <400>162

Met Gly Ser Trp Gln Glu Phe Glu Gln Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Glu Leu Arg Glu Trp

55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Thr Asn

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Asn Trp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

- 180 043883

Ile Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>163 <211>73 <212> Protein <213> Homo sapiens <400>163

Met Gly Ser Trp Tyr Glu Phe Tyr Ser Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Phe Leu Arg Asp Tyr 50 55

Arg Tyr Leu Gln Ala Tyr Arg His Asn 65 70 <210>164 <211>73 <212> Protein <213> Homo sapiens <400>164

Met Gly Ser Trp Glu Glu Phe His Asp Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Asp Trp 50 55

Phe Tyr Leu Gln Ala Tyr Arg His Asn 65 70 <210>165 <211>73 <212> Protein <213> Homo sapiens <400>165

Met Gly Ser Trp Trp Glu Phe Asp Glu Arg Leu

510

Arg Leu Phe Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Ala Ile Asp Ser 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Glu Ile Arg

Ala Ile Ser Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Ala Ile Glu Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

- 181 043883

Gly Asn Pro Glu Val Glu Trp Leu Arg Ile Val

55

Glu Ile Leu Gln Ala Tyr Arg His Asn 65 70 <210>166 <211>73 <212> Protein <213> Homo sapiens <400>166

Met Gly Ser Trp Glu Glu Phe Glu Tyr Arg Leu

510

Arg Leu Trp Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Val Leu Arg Glu Ile 50 55

Gln Ile Leu Gln Ala Tyr Arg His Asn 65 70 <210>167 <211>73 <212> Protein <213> Homo sapiens <400>167

Met Gly Ser Trp Val Val Phe Lys Gln Arg Leu

510

Leu Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu

25

Met Ser Ile Ala Phe Phe Glu Glu Asp Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>168 <211>73 <212> Protein <213> Homo sapiens <400>168

Met Gly Ser Trp Tyr Glu Phe Lys Asn Asp Leu

510

His Leu Glu Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Phe Arg Ile Ala His Phe Glu Asn Ala Leu Gln

Ala Asp Ile Arg

Ala Ile Glu Val

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Asp Ile Arg

Tyr Ile Lys Asp 15

Ala Tyr Phe Glu 30

Tyr Lys Gly Lys 45

Ala Ala Ile Arg

Trp Ile Lys Val 15

Ala Tyr Phe Glu 30

Tyr Lys Gly Lys

- 182 043883

Ala Ala Ile Arg

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Lys Glu

55

Asp Glu Leu Gln Ala Tyr Arg His Asn 65 70 <210>169 <211>73 <212> Protein <213> Homo sapiens <400>169

Met Gly Ser Trp Val Glu Phe Tyr Asn Arg Leu

510

Arg Leu His Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Val Leu Arg Tyr His

55

Val Thr Leu Gln Ala Tyr Arg His Asn 65 70 <210>170 <211>73 <212> Protein <213> Homo sapiens <400>170

Met Gly Ser Trp Ser Glu Phe Tyr Asp Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Asp Thr

55

Thr Arg Leu Gln Ala Tyr Arg His Asn 65 70 <210>171 <211>73 <212> Protein <213> Homo sapiens <400>171

Met Gly Ser Trp Lys Glu Phe His Phe Arg Leu

510

Arg Leu Ile Ala Leu Gly Gly Ser Glu Ala Glu

25

Ala Ile Asp His 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Ser Ile Arg

Ala Ile His His

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile Glu His

Ala Ala Phe Glu

- 183 043883

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Phe Leu Arg Ala Lys

55

Thr His Leu Gln Ala Tyr Arg His Asn 65 70 <210>172 <211>73 <212> Protein <213> Homo sapiens <400>172

Met Gly Ser Trp Phe Glu Phe His Gly Arg Leu

510

Arg Leu Ser Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu His Leu Arg Ala His 50 55

Asp His Leu Gln Ala Tyr Arg His Asn 65 70 <210>173 <211>73 <212> Protein <213> Homo sapiens <400>173

Met Gly Ser Trp Tyr Glu Phe Ala Asp Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ala Leu Arg Met Thr 50 55

Ser Arg Leu Gln Ala Tyr Arg His Asn 65 70 <210>174 <211>73 <212> Protein <213> Homo sapiens <400>174

Met Gly Ser Trp Asn Glu Phe Tyr Asn Arg Leu

510

Tyr Lys Gly Lys 45

Ala Asn Ile Arg

Ala Ile Tyr Gly 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala His Ile Arg

Ala Ile His Gln

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile His Gln

- 184 043883

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ser Leu Arg Gln Thr

55

Asp Arg Leu Gln Ala Tyr Arg His Asn 65 70 <210>175 <211>73 <212> Protein <213> Homo sapiens <400>175

Met Gly Ser Trp Asn Glu Phe Ala Asp Arg Leu

510

Arg Leu Tyr Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Ser Leu Arg Met Thr 50 55

Ser Arg Leu Gln Ala Tyr Arg His Asn 65 70 <210>176 <211>73 <212> Protein <213> Homo sapiens <400>176

Met Gly Ser Trp Thr Glu Phe Ser Tyr Arg Leu

510

Arg Leu His Ala Leu Gly Gly Ser Glu Ala Glu 20 25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu His Leu Arg Tyr Asn

55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Tyr Ile Arg

Ala Ile His Gln

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile Gln Ser

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Lys Ile Arg

His Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210> 177 <211> 73 <212> Protein <213> Homo sapiens <400> 177

- 185 043883

Met Gly Ser Trp Gln Glu Phe 1 5

Thr Thr Arg Leu Glu Ala Ile Tyr His

15

Arg Leu Arg Ala Leu Gly Gly 20

Gly Phe Ile Ala Glu Phe Glu 35

Gly Asn Pro Glu Val Glu Ala 50 55

Ser Glu Ala Glu Leu Ala Asn Phe Glu

thirty

Gly Asn Leu Gln Met Tyr Lys Gly Lys 40 45

Leu Val His Glu Ala Tyr Ala Ile Met 60

Glu Glu Leu His Ala Tyr Arg His Asn 6570

<210> <211> <212> <213> <400> 178 73 Protein Asp Arg 10 Leu Lys Ala Ile His 15 Asp Homo 178 Ser sapiens Phe Met 1 Gly Trp Val 5 Glu Phe Arg Leu Glu Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala His 30 Phe Glu Lys Leu Ile 35 Ala His Phe Glu His 40 Arg Leu Gln Asn Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Ala 55 Leu Glu Lys Glu Ala 60 Asp Ala Ile Leu Tyr 65 Glu Leu Ala Ala Tyr 70 Arg His Asn

<210>179 <211>73 <212> Protein <213> Homo sapiens <400>179

Met Gly Ser Trp Tyr Tyr Phe Lys

His His Leu Ala Trp Ile Lys Met

15

Glu Leu Glu Ala Leu Gly Gly Ser

Glu Ala Glu Leu Ala His Phe Glu

thirty

Ser Ser Ile Ala Ser Phe Glu Arg

40

Asp Leu Gln Gln Tyr Lys Gly Lys 45

Gly Asn Pro Glu Val Glu Ala Leu

55

Arg Lys Glu Ala Ala Ala Ile Arg

Asp Glu Leu Gln Ala Tyr Arg His Asn 6570 <210>180 <211>73 <212> Protein <213> Homo sapiens

- 186 043883 <400>180

Met Gly Ser Trp Val Glu Phe His

Ile Arg Leu His Ala Ile Gln Tyr

1015

Arg Leu Tyr Ala Leu Gly Gly Ser 20

Glu Ala Glu Leu Ala Ala Phe Glu

thirty

Lys Glu Ile Ala Ala Phe Glu Ser

40

Glu Leu Gln Ala Tyr Lys Gly Lys 45

Gly Asn Pro Glu Val Glu Glu Leu

55

Arg His Trp Ala Ala Phe Ile Arg

Leu Gln Leu Gln Ala Tyr Arg His 6570

Asn <210>181 <211>73 <212> Protein <213> Homo sapiens <400>181

Met 1 Gly Ser Trp Asn 5 Glu Phe His Asp Arg 10 Leu Asn Ala Ile His 15 Ala Arg Leu His Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Lys Glu Ile 35 Ala Ala Phe Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Asn 55 Leu Arg Asp Asp Ala 60 Ala Phe Ile Arg Arg 65 Phe Leu Gln Ala Tyr 70 Arg His Asn

< 210>182 < 211>73 < 212> Protein < 213> Homo sapiens < 400>182

Met 1 Gly Ser Trp Tyr 5 Glu Phe Thr Val Arg 10 Leu Glu Ala Ile His 15 Glu Arg Leu Lys Ala 20 Leu Gly Gly Ser Glu 25 Ala Glu Leu Ala Ala 30 Phe Glu Lys Glu Ile 35 Ala Ala Phe Glu Ser 40 Glu Leu Gln Ala Tyr 45 Lys Gly Lys Gly Asn 50 Pro Glu Val Glu Ile 55 Leu Arg Asp Asp Ala 60 Ala Phe Ile Arg Arg 65 Phe Leu Gln Ala Tyr 70 Arg His Asn

<210> 183

- 187 043883

Ala Ile Lys Ala <211>73 <212> Protein <213> Homo sapiens <400>183

Met Gly Ser Trp Lys Glu Phe Asp Asp Arg Leu

510

Arg Leu Gln Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Asp Asp

55

Arg Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>184 <211>73 <212> Protein <213> Homo sapiens <400>184

Met Gly Ser Trp Tyr Glu Phe Asp Asp Arg Leu

510

Arg Leu Gln Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asp Leu Arg Asp Asp

55

Arg Phe Leu Gln Ala Tyr Arg His Asn 65 70 <210>185 <211>73 <212> Protein <213> Homo sapiens <400>185

Met Gly Ser Trp Asn Glu Phe Lys Asn Arg Leu

510

Arg Leu Asn Ala Leu Gly Gly Ser Glu Ala Glu

25

Lys Glu Ile Ala Ala Phe Glu Ser Glu Leu Gln

40

Gly Asn Pro Glu Val Glu Asn Leu Arg Asp Asp

55

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile His Asp 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

Ala Ile His Lys 15

Ala Ala Phe Glu

Tyr Lys Gly Lys 45

Ala Phe Ile Arg

His Phe Leu Gln Ala Tyr Arg His Asn 65 70

- 188 043883

Glu Ala Ile His Asn <210>186 <211>73 <212> Protein <213> Homo sapiens <400>186

Met Gly Ser Trp Thr 1 5

Arg Leu Gln Ala Leu

Lys Glu Ile Ala Ala

Gly Asn Pro Glu Val 50

Glu Phe Glu Gln Arg Leu 10

Gly Gly Ser Glu Ala Glu 25

Phe Glu Ser Glu Leu Gln 40

Glu Glu Leu Arg Asn Asp 55

Leu Ala Ala Phe Glu

Ala Tyr Lys Gly Lys 45

Ala Ala Phe Ile Arg 60

His Phe Leu Gln Ala Tyr Arg His Asn 6570 < 210>187 < 211>378 < 212> Protein < 213> Homo sapiens <220>

<221> other features <223> CD123 (interleukin-3 receptor alpha subunit) <400>187

Met Val Leu Leu Trp Leu Thr Leu Leu Leu Ile Ala Leu Pro Cys Leu 1 5 1015

Leu Gln Thr Lys Glu Asp Pro Asn Pro Pro Ile Thr Asn Leu Arg Met 20 2530

Lys Ala Lys Ala Gln Gln Leu Thr Trp Asp Leu Asn Arg Asn Val Thr 35 4045

Asp Ile Glu Cys Val Lys Asp Ala Asp Tyr Ser Met Pro Ala Val Asn 50 5560

Asn Ser Tyr Cys Gln Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn 65 70 7580

Tyr Thr Val Arg Val Ala Asn Pro Pro Phe Ser Thr Trp Ile Leu Phe 85 9095

Pro Glu Asn Ser Gly Lys Pro Trp Ala Gly Ala Glu Asn Leu Thr Cys

100 105110

Trp Ile His Asp Val Asp Phe Leu Ser Cys Ser Trp Ala Val Gly Pro

115 120125

Gly Ala Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Val Ala Asn

130 135140

Arg Arg Gln Gln Tyr Glu Cys Leu His Tyr Lys Thr Asp Ala Gln Gly

- 189 043883

145

150

155

160

Thr

Arg

Ile

Gly

Cys

165

Arg

Phe

Asp

Asp

Ile

170

Ser

Arg

Leu

Ser

Ser

175

Gly

Ser

Gln

Ser

Ser

180

His

Ile

Leu

Val

Arg 185

Gly

Arg

Ser

Ala

Ala

190

Phe

Gly

Ile

Pro

Cys

195

Thr

Asp

Lys

Phe

Val

200

Val

Phe

Ser

Gln

Ile

205

Glu

Ile

Leu

Thr

Pro

210

Pro

Asn

Met

Thr

Ala

215

Lys

Cys

Asn

Lys

Thr

220

His

Ser

Phe

Met

His

225

Trp

Lys

Met

Arg

Ser

230

His

Phe

Asn

Arg

Lys 235

Phe

Arg

Tyr

Glu

Leu

240

Gln

Ile

Gln

Lys

Arg 245

Met

Gln

Pro

Val

Ile

250

Thr

Glu

Gln

Val

Arg 255

Asp

Arg

Thr

Ser

Phe

260

Gln

Leu

Leu

Asn

Pro

265

Gly

Thr

Tyr

Thr

Val

270

Gln

Ile

Arg

Ala

Arg 275

Glu

Arg

Val

Tyr

Glu

280

Phe

Leu

Ser

Ala

Trp

285

Ser

Thr

Pro

Gln

Arg 290

Phe

Glu

Cys

Asp

Gln

295

Glu

Glu

Gly

Ala

Asn

300

Thr

Arg

Ala

Trp

Arg 305

Thr

Ser

Leu

Leu

Ile

310

Ala

Leu

Gly

Thr

Leu

315

Leu

Ala

Leu

Val

Cys 320

Val

Phe

Val

Ile

Cys 325

Arg

Arg

Tyr

Leu

Val

330

Met

Gln

Arg

Leu

Phe

335

Pro

Arg

Ile

Pro

His

340

Met

Lys

Asp

Pro

Ile

345

Gly

Asp

Ser

Phe

Gln

350

Asn

Asp

Lys

Leu

Val

355

Val

Trp

Glu

Ala

Gly 360

Lys

Ala

Gly

Leu

Glu

365

Glu

Cys

Leu

Val

Thr

370

Glu

Val

Gln

Val

Val

375

Gln

Lys

Thr

Claims (29)

1. A target-binding polypeptide comprising three antiparallel alpha helices, wherein (a) the target-binding polypeptide is a synthetic peptide resulting from modifications in the amino acid sequence of SEQ ID NO: 1, wherein the modifications include one or more conservative or non-conservative substitutions amino acid residues accessible to the solvent or inaccessible to the solvent; (b) the target-binding polypeptide specifically binds a target of interest, where the specific binding of the target-binding polypeptide to the target of interest is greater than the binding of a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to the target of interest, where the target of interest is tumor antigen; (c) the target-binding polypeptide includes an amino acid sequence MGSWX ₅ X ₆ FKX ₉ X ₁₀ LAX ₁₃ IKX ₁₍ X ₁₇ LEALGGSEAELAX ₃₍₎ FEX ₃₃ X ₃₄ IAX ₃₇ FEX ₄ ()X ₄₁ LQX ₄₄ YKGKGNPE VEALRKEAAAIRDELQAYRHN (SEQ ID NO: 2), MGSWAEFKQRLAAIKTRLEAGGSEAELAAFX ₃₂ X ₃₃ EIX ₃₆ AFX ₃₉ X ₄₀ ELX ₄₃ AYKGKGNPEVEALX ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO: 3), MGSWX ₅ EFX ₈ X ₉ RLX ₁₂ AIX ₁₅ X ₁₆ RLX ₁₉ ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX ₅₅ LR X ₅₈ X ₅ 9AAX62IRX6 ₅ X66LQAYRHN (SEQ ID NO: 4), MGSWX ₅ x ₆ FKX ₉ X _{10 LAX 13} IKX ₁₆ x ₁₇ Leaggseeelafx ₃₂ x ₃₃ EIX ₃₆ AFX ₃₉ X4 ₀ ELX4 ₃ Aykgknpe VEX ₅₈ X ₅₉ AAX _{62 IRX 66 LQARHN} (Seq ID No: 5), MGSWX5EFX8X9RLX12AIX15X16RLX19ALGGSEAELAX30FEX33X34IAX37FEX40X41LQX44YKGKGNPE VEAL X ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO: 6), mgswx ₅ x ₆ fkx ₉ x ₁₀ lax ₁₃ ikx ₁₆ x ₁₇ lealz ₁ eaelax ₂₈ fex ₃₁ x ₃₂ iax ₃₅ fex ₃₈ x ₃₉ lqx ₄₂ yz ₂ npeveal RKEAAAIRDELQAYRHN (SEQ ID NO: 7), MGSWAEFKQRLAAIKTRLEALZ1EAELAAFX30X31EIX34AFX37X38ELX41AYZ2NPEVEALX52X53EA X ₅₆ AIX ₅₉ X ₆₀ ELX ₆₃ AYRHN (SEQ ID NO: 8), MGSWX5EFX8X9RLX12AIX15X16RLX19ALZ1EAELAAFEKEIAAFESELQAYZ2NPEVEX50LRX53X54A AX ₅₇ IRX ₆₀ X ₆₁ LQAYRHN (SEQ ID NO: 9), MGSWX5X6FKX9X10LAX13IKX16X17LEALZ1EAELAAFX30X31EIX34AFX37X38ELX41AYZ2NPEVEX50L RX ₅₃ X ₅₄ AAX ₅₇ IRX ₆₀ X ₆₁ LQAYRHN (SEQ ID NO: 10) or MGSWX5EFX8X9RLX12AIX15X16RLX19ALZ1EAELAX28FEX31X32IAX35FEX38X39LQX42YZ2NPEVEAL X ₅₂ X ₅₃ EAX ₅₆ AIX ₅₉ X ₆₀ ELX ₆₃ AYRHN (SEQ ID NO: 11), where X _n is a natural or unnatural amino acid, and Z1 and Z ₂ contain from 2 to 30 natural or unnatural amino acids; and (d) the target binding polypeptide does not contain the amino acid sequence of SEQ ID NO: 50. 2. The target-binding polypeptide according to claim 1, comprising the amino acid sequence of SEQ ID NO: 4. 3. The target-binding polypeptide according to claim 1 or 2, wherein _Xn is not cysteine or proline. 4. The target-binding polypeptide according to any one of claims 1 to 3, wherein the target of interest is PD-L1, CD137 or CD123. 5. A de novo binding domain polypeptide (DBDpp) comprising three antiparallel alpha helices that are connected by linker peptides, wherein (a) DBDpp is a synthetic peptide resulting from modifications in the amino acid sequence of SEQ ID NO: 1, wherein the modifications consist of 1-30 conservative or non-conservative amino acid substitutions selected from positions 1-6, 8-10, 12, 13, 15-17, 19, 2027, 29, 30, 32-34, 36, 37, 39-41, 43 -52, 54, 55, 57-59, 61, 62, 64-66 and 68-73 SEQ ID NO: 1, wherein the substitutions do not include cysteine or proline substitutions; (b) DBDpp specifically binds to a target of interest, wherein the specific binding of DBDpp to the target of interest is greater than the binding of a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to the target of interest, wherein the target of interest is a tumor antigen; and (c) DBDpp does not contain the amino acid sequence of SEQ ID NO: 50. 6. DBDpp according to claim 5, including the amino acid sequence MGSWX5X6FKX9X10LAX13IKX16X17LEALGGSEAELAX30FEX33X34IAX37FEX40X41LQX44YKGKGNPE VEALRKEAAAIRDELQAYRHN (SEQ ID NO: 2), MGSWAEFKQRLAAIKTRLEAGGSEAELAAFX32X33EIX36AFX39X40ELX43AYKGKGNPEVEALX57 X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO: 3), MGSWX5EFX8X9RLX12AIX15X16RLX19ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX55LR X ₅₈ X ₅₉ AAX ₆₂ IRX ₆₅ X ₆₆ LQAYRHN (SEQ ID NO: 4) or - 191 043883 MGSWX ₅ X ₆ FKX ₉ X ₁₀ LAX ₁₃ IKX ₁₆ X ₁₇ LEALGGSEAELAAFX ₃₂ X ₃₃ EIX ₃₆ AFX ₃₉ X ₄₀ ELX ₄₃ AYKGKGNPE VEX55LRX58X59AAX62IRX65X66LQAYRHN (SEQ ID NO: 5), where X _n is a natural or unnatural amino acid. 7. DBDpp according to claim 5 or 6, which specifically binds to a protein containing amino acids 19305 CD123. 8. A fusion protein comprising a first and a second DBDpp according to any one of claims 5 to 7, wherein the first and second DBDpp exhibit binding specificity for a tumor target. 9. The target binding polypeptide of any one of claims 1 to 4, the DBDpp of any one of claims 5 to 7, or the fusion protein of claim 8, wherein the target binding protein, DBDpp, or fusion protein is labeled. 10. The target binding polypeptide of any one of claims 1 to 4 and 9, the DBDpp of any of claims 5 to 7 and 9, or the fusion protein of claim 8 or 9, wherein the target binding protein, DBDpp or fusion protein is conjugated to therapeutic or cytotoxic agent. 11. A pharmaceutical composition comprising a target-binding polypeptide according to any one of claims 1-4, 9 and 10, a DBDpp according to any one of claims 5-7, 9 and 10 or a fusion protein according to any one of claims 8-10, further comprising pharmaceutically acceptable carrier. 12. A kit comprising a target binding polypeptide according to any one of claims 1-4, 9 and 10, a DBDpp according to any one of claims 5-7, 9 and 10 or a fusion protein according to any one of claims 8-10, wherein the kit is intended for the treatment of a tumor. 13. An isolated nucleic acid molecule encoding a target-binding polypeptide according to any one of claims 1-4, a DBDpp according to any one of claims 5-7, or a fusion protein according to claim 8. 14. A vector comprising an isolated nucleic acid molecule encoding a target binding polypeptide according to any one of claims 1 to 4, a DBDpp according to any one of claims 5 to 7, or a fusion protein according to claim 8. 15. A host cell, comprising a vector encoding a target binding polypeptide according to any one of claims 1 to 4, a DBDpp according to any one of claims 5 to 7, or a fusion protein according to claim 8. 16. A cell line genetically modified to express a target-binding polypeptide according to any one of claims 1-4, a DBDpp according to any one of claims 5-7, or a fusion protein according to claim 8. 17. A chimeric antigen receptor (CAR), wherein the CAR includes (a) a targeting domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the targeting domain includes a target binding polypeptide according to any one of claims 1 to 4, DBDpp according to any one of claims 1 to 4, DBDpp according to any of claims 5-7 or a fusion protein according to claim 8. 18. The CAR of claim 17, wherein the intracellular signaling domain is selected from the group consisting of a human CD3 zeta domain, a 41BB domain, a CD28 domain, and any combination thereof. 19. The CAR of claim 17, wherein the target binding polypeptide, DBDpp, or fusion protein binds to a tumor antigen associated with hematologic malignancy or a solid tumor. 20. CAR according to claim 19, where the tumor antigen is selected from the group consisting of CD137, PD-L1, CD123, CTLA4, CD47, KIR, DR5, TIM3, PD1, EGFR, TCR, CD19, CD20, CD22, ROR 1, mesothelin, CD33/lL3Ra, cMet, PSMA, glycolipid F77, EGFRvIII, GD2, NY-SO-1, MAGE A3 and combinations thereof. 21. The CAR of claim 17, wherein the intracellular signaling domain includes a co-stimulatory signaling region. 22. The CAR of claim 21, wherein the costimulatory signaling region comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, lymphocyte function-associated antigen 1 (LFA -1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and any combination thereof. 23. An isolated nucleic acid molecule comprising a sequence encoding a CAR according to claim 17 or 18. 24. A cell obtained using genetic engineering methods to express CAR according to claim 17 or 18. 25. A method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of an immune cell containing a CAR coding sequence according to claim 17 or 18. 26. The method of claim 25, wherein the immune cell is a T cell or a natural killer (NK) cell. 27. A method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a T cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a target-binding polypeptide according to claim 1, (b) a transmembrane domain selected from 41BB and CD28, and (c) an intracellular domain, wherein the intracellular domain includes a signaling domain selected from T cell receptor alpha, beta or zeta chain . 28. A method of converting a reference polypeptide into a target-binding polypeptide having specific binding to a target of interest, where the method includes: - 192 043883 (a) modifying a plurality of amino acid residues from a reference polypeptide to produce a plurality of candidate target binding polypeptides, wherein the candidate target binding polypeptides comprise a variant amino acid sequence of SEQ ID NO: 1, wherein the candidate target binding polypeptides comprise three antiparallel alpha helices which are linked by linker peptides, wherein the amino acid residues being modified are solvent accessible or solvent inaccessible, and where the modification involves one or more conservative or non-conservative amino acid substitutions, and does not include a cysteine or proline substitution; (b) packaging a plurality of candidate target-binding polypeptides into a plurality of vectors to produce a candidate library; and (c) screening the candidate library for candidate target-binding polypeptides that exhibit specific binding to a target of interest, wherein the candidate target-binding polypeptides comprise an amino acid sequence selected from the group consisting of mgswx ₅ x ₆ fkx ₉ x ₁₀ lax ₁₃ ikx ₁₆ x ₁₇ lealggseaelax ₃₀ fex ₃₃ x ₃₄ iax ₃₇ fex ₄₀ x ₄₁ lqx ₄₄ ykgkgnpe VEALRKEAAAIRDELQAYRHN (SEQ ID NO: 2), mgswaefkqrlaaiktrlealggseaelaafx ₃₂ x ₃₃ eix ₃₆ afx ₃₉ x ₄₀ elx ₄₃ aykgkgnpevealx ₅₇ X58EAX61AIX64X65ELX68AYRHN (SEQ ID NO: 3), mgswx ₅ efx ₈ x ₉ rlx ₁₂ aix ₁₅ x ₁₆ rlx ₁₉ alggseaelaafekeiaafeselqaykgkgnpevex ₅₅ lr X58X59AA X62IRX65X66LQAYRHN (SEQ ID NO: 4), MGSWX5X6FKX9X10LAX13IKX16X17LEALGGSEAELAAFX32X33EIX36AFX39X40ELX43AYKGKGNPE VEX55LR X58X59AAX62IRX65X66LQAYRHN (SEQ ID NO: 5), MGSWX5EFX8X9RLX12AIX15X16RLX19ALGGSEAELAX30FEX33X34IAX37FEX40X41LQX44YKGKGNPE VEAL X ₅₇ X ₅₈ EAX ₆₁ AIX ₆₄ X ₆₅ ELX ₆₈ AYRHN (SEQ ID NO: 6), MGSWX5X6FKX9X10LAX13IKX16X17LEALZ1EAELAX28FEX31X32IAX35FEX38X39LQX42YZ2NPEVEAL RKEAAAIRDELQAYRHN (SEQ ID NO: 7), MGSWAEFKQRLAAIKTRLEALZ1EAELAAFX30X31EIX34AFX37X38ELX41AYZ2NPEVEALX52X53EA X56AIX59 X60ELX63AYRHN (SEQ ID NO: 8), MGSWX5EFX8X9RLX12AIX15X16RLX19ALZ1EAELAAFEKEIAAFESELQAYZ2NPEVEX50LRX53X54A AX ₅₇ IRX ₆₀ X ₆₁ LQAYRHN (SEQ ID NO: 9), MGSWX5X6FKX9X10LAX13IKX16X17LEALZ1EAELAAFX30X31EIX34AFX37X38ELX41AYZ2NPEVEX50L RX ₅₃ X ₅₄ AAX ₅₇ IRX ₆₀ X ₆₁ LQAYRHN (SEQ ID NO: 10) or MGSWX5EFX8X9RLX12AIX15X16RLX19ALZ1EAELAX28FEX31X32IAX35FEX38X39LQX42YZ2NPEVEAL X ₅₂ X ₅₃ EAX ₅₆ AIX ₅₉ X ₆₀ ELX ₆₃ AYRHN (SEQ ID NO: 11), where Xn is a natural or unnatural amino acid, and Z1 and Z ₂ contain from 2 to 30 natural or unnatural amino acids. 29. A virus-like particle (VLP) comprising a fusion protein that includes DBDpp according to any one of claims 5 to 7, wherein the VLP is suitable for use as an immunogen for generating antibodies, wherein said antibodies are directed against DBDpp.