EA045226B1 - RADIO-LABELED ANTI-PD-L1 ANTI-BODIES FOR IMMUNO-PET IMAGING - Google Patents

Description

Field of technology to which the present invention relates

The invention relates to radiolabeled anti-PD-L1 antibodies and their use in immuno-PET imaging.

List of sequences

An official copy of the sequence listing is provided simultaneously with the description electronically via EFS-Web as a sequence listing in ASCII format with a file name of 10305WO01_SEQ_LIST_ST25.txt, a creation date of December 01, 2017, and a size of approximately 117 KB. The sequence listing contained in this formatted ASCII document is part of the specification and is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Programmed cell death ligand 1 (PD-L1) (also called B7-H1 or CD274) is a 290 amino acid receptor protein ligand widely expressed in both lymphoid and non-lymphoid tissues such as CD4 and CD8 T cells, macrophages cell lines, in peripheral tissues, and in tumor cells and virus-infected cells (Dong et al. 1999, Nature Med.). PD-L1 binds to the PD-1 and B7-1 receptors, which belong to the CD28/CTLA-4 (cytotoxic T-lymphocyte antigen)/ICOS (inducible co-stimulator) T cell coinhibitory receptor family (Chen et al. 2013, Nature Rev. Immunol 13: 227-242), and weakens the immune response by inhibiting T cell activation. PD-L1 binding to PD-1 or B7-1 leads to decreased T cell proliferation and cytokine secretion, and impaired humoral and cellular immune responses in diseases such as cancer and viral infection. PD-L1 expression on tumor cells and virus-infected cells is used by tumors and chronic viral infections to evade the immune response. PD-L1 is expressed in a wide variety of tumors, and studies in animal models have shown that PD-L1 in tumors inhibits T cell activation and tumor cell lysis and may lead to increased death of tumor-specific T cells. During chronic viral infections, PD-L1 expressed on virus-infected cells binds to PD-1 on virus-specific T cells, and these T cells become exhausted with loss of effector functions and proliferative capacity (Freeman 2008, PNAS 105: 10275- 10276). The PD-1:PD-L1 system also plays an important role in inducing the development of T regulatory (Treg) cells and in maintaining Treg function (Francisco et al. 2010, Immunol. Rev. 236: 219-242). Blocking PD-L1 by antagonists, including monoclonal antibodies, has been studied in the treatment of cancer and chronic viral infections (Ribas 2012, NEJM 366: 2517-2519; Freeman 2008, PNAS 105: 10275-10276; Sheridan 2012, Nature Biotechnology 30: 729-730 ). Immunopositron emission tomography (PET) is a diagnostic imaging tool that uses monoclonal antibodies labeled with positron emitters, combining the targeting properties of the antibody with the sensitivity of positron emission tomography cameras. See, for example, The Oncologist, 12: 1379 (2007); Journal of Nuclear Medicine, 52(8): 1171 (2011). Immuno-PET provides visualization and quantification of antigen and antibody accumulation in vivo and, as such, can serve as an important tool for diagnosis and complementary therapy. For example, immuno-PET can help in selecting potential patient candidates for a particular therapy, as well as in monitoring treatment. As both PD1 and PD-L1 have emerged as targets for immunotherapy, there is a need for diagnostic tools for anti-PD1 and/or anti-PD-L1 antibody-based therapies, including, but not limited to, diagnostic tools that can detect suitable patient candidates for the indicated therapy.

Brief Disclosure of the Present Invention

The present disclosure includes radiolabeled anti-PD-L1 antibody conjugates for use in immuno-PET imaging. In one aspect, the conjugate comprises an anti-PD-L1 antibody or an antigen-binding fragment thereof, a chelating moiety, and a positron emitter. The present document also provides processes for the synthesis of these conjugates and synthetic intermediates useful therefor.

Also provided herein are methods for imaging tissue that expresses PD-L1, wherein the methods comprise introducing a radiolabeled anti-PD-L1 antibody conjugate described herein into the tissue and visualizing PD-L1 expression using positron emission tomography imaging ( PAT).

Also provided herein are methods for detecting PD-L1 in tissue, the methods comprising introducing a radiolabeled anti-PD-L1 antibody conjugate described herein into the tissue and visualizing PD-L1 expression using PET imaging. In one embodiment, the tissue is located in a human subject. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject has a disease or disorder, such as a cancer, inflammatory disease, or infection.

In some aspects, a dose of 5 mg, or 10 mg, or 20 mg of a radiolabeled anti-PD-L1 antibody conjugate is administered to the subject.

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Also provided herein are methods for identifying a patient suitable for anticancer therapy comprising an inhibitor of the PD-1/PD-L1 signaling axis, the methods comprising selecting a patient with a solid tumor, administering a radiolabeled antibody conjugate described herein, and visualizing the injected radiolabeled antibody conjugate into the tumor using PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor identifies the patient as eligible for anticancer therapy including an inhibitor of the PD-1/PD-L1 signaling axis.

Also provided herein are methods of treating a tumor, the methods comprising selecting a subject with a solid tumor; determining that the solid tumor is PDLI positive; and administering anticancer therapy to a subject in need thereof. In some embodiments, the anticancer therapy includes an inhibitor of the PD1/PD-L1 signaling axis (eg, an anti-PD-1 antibody or an anti-PD-L1 antibody). In some embodiments, a subject is administered a radiolabeled antibody conjugate described herein and the localization of the radiolabeled antibody conjugate is visualized by positron emission tomography (PET) imaging to determine whether the tumor is PD-LI positive.

Also provided herein are methods for monitoring the effectiveness of anticancer therapy in a subject, the methods comprising selecting a subject with a solid tumor and the subject receiving treatment with anticancer therapy; administering to a subject a radiolabeled conjugate as described herein; visualization of the localization of the injected radiolabeled conjugate into the tumor using PET imaging and determination of tumor growth, while a decrease from the initial level of conjugate uptake or radiolabeled signal indicates regression and effectiveness of anticancer therapy. In some embodiments, the anticancer therapy includes an inhibitor of the PD-1/PDL1 signaling axis (eg, an anti-PD-1 antibody).

Also provided herein are methods for predicting the response of a patient to an anticancer therapy that includes an inhibitor of the PD-1/PD-L1 signaling axis, the methods comprising selecting a patient with a solid tumor and determining whether the tumor is PD-L1 positive, wherein if the tumor is PD-LI positive, which indicates a positive response of the patient to anticancer therapy, including an inhibitor of the PD-1/PD-L1 signaling axis. In some embodiments, a tumor is determined to be positive by administering a radiolabeled antibody conjugate in accordance with the present disclosure and the localization of the radiolabeled antibody conjugate in the tumor is visualized using PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is PD-LI positive.

Brief description of graphic materials

In fig. 1 depicts SDS-PAGE and SEC of unmodified anti-PD-L1 antibody and DFO-modified anti-PD-L1 antibody.

In fig. 2A and 2B depict radio-SEC-HPLC after ⁸⁹ Zr radiolabeling for Study 1.

In fig. Figure 3 depicts radio-SEC-HPLC DFO-conjugate (anti-PD-L1 antibody) after radiolabeling with ⁸⁹ Zr for Study 2.

In fig. Figure 4 depicts radio-SEC-HPLC SEC after radiolabeling with ⁸⁹ Zr for Study 3.

In fig. Figure 5 shows a UV280-SEC-HPLC and radio-iTLC chromatogram after ⁸⁹ Zr radiolabeling for Study 1.

In fig. 6A, 6B, 6C and 6D show the expression of hPD-L1 by tumor cell lines MC38cOVA/eGFP-mPD-L1-/-hPD-L1 ^Tg (Fig. 6A), LOX-IMVI (Fig. 6B), MDA-MB-231 ( Fig. 6C) and SK-Br-3 (Fig. 6D) in vitro, as described in Example 5 herein.

In fig. 7 shows hPD-L1 expression by MC38-cOVA/eGFP-mPD-L1-/-hPDL1 ^Tg and LOX-IMVI tumor cells with or without interferon-gamma treatment in vitro in a second experiment as described in Example 5 herein.

In fig. 8 shows chromatograms obtained by SEC-HPLC analysis using samples from the ⁸⁹ Zr-DFO-anti-PD-L1 conjugate radioimmunoconjugates for the assays shown in A, B, D, and E, and the isotype control radioimmunoconjugate ⁸⁹ Zr-DFOIgG4 ^P for studies shown in C and F. Chromatograms for absorbance at 280 nm are shown in AC and radiochromatograms for γ-emission intensity are shown in DF. Elution of buffer components was also detected on AC. These sample buffer salt peaks (retention time >25 min, asterisk *) were excluded from peak area integration. Peaks were labeled to indicate HMW (high molecular weight) immunoconjugate (1), monomeric immunoconjugate (2), unincorporated ⁸⁹ Zr (3), and salts in sample buffer (*). Abbreviations: mAU = milliabsorption units; cps = number of pulses per second.

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In fig. 9 shows ex vivo biodistribution data for the ⁸⁹ Zr-DFO-anti-PD-L1 antibody conjugate in PD-1hu/hu-PD-L1hu/hu mice. Sixteen mice (2 groups of 8 animals each) were administered a single IV dose of 50 μCi (1 mg/kg) of ⁸⁹ Zr-DFO-anti-PD-L1 conjugate on day 0 and sacrificed on day 6 (black bars) or day 10 (gray bars) after dosing. Blood collected by cardiac puncture and these collected tissues were weighed and radioactivity determined. The percentage of injected dose per gram (% ID/g) for individual samples collected on day 6 or 10 was calculated relative to the radioactivity of the dose standard from the injected material ( ⁸⁹ ZrDFO-anti-PD-L1 antibody conjugate) and the weight of the individual samples. Data are presented as mean ±SD.

Detailed Disclosure of the Present Invention

I. Definitions.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly known to one of ordinary skill in the art to which the subject matter disclosed relates. The term PD-L1 refers to programmed death ligand 1, also known as CD274 and B7H1. The amino acid sequence of full-length PD-L1 is submitted to GenBank under accession number NP_054862.1. The term PDL1 also includes variants of the PD-L1 protein. The term PD-L1 includes recombinant PDL1 or a fragment thereof. The term also includes PD-L1 or a fragment thereof linked to, for example, a histidine tag, a mouse or human Fc, or a signal sequence such as ROR1. For example, the term includes sequences containing mouse Fc (mIgG2a) or human Fc (hlgGl) at the C terminus fused to amino acid residues 19-239 of full-length PD-L1 (NP_054862.1). The protein variants contain a histidine tag at the C-terminus connected to amino acid residues 19-239 of NP_054862.1. Unless the origin of a non-human species is indicated, the term PD-L1 means human PD-L1. PD-L1 is a 290 amino acid protein with extracellular IgV-like and IgC-like domains (amino acids 19–239 of full-length PD-L1), a transmembrane domain, and an intracellular domain of approximately 30 amino acids. PD-L1 is constitutively expressed on many cells, such as antigen-presenting cells (eg, dendritic cells, macrophages, and B cells) and on hematopoietic and non-hematopoietic cells (eg, vascular endothelial cells, pancreatic islets, and sites of immune privilege). PD-L1 is also expressed in a wide range of tumors and virus-infected cells and is a component of the immunosuppressive environment (Ribas 2012, NEJM 366: 2517-2519). PD-L1 binds to one of two T cell co-inhibitors PD-1 and B7-1.

The term PD-1 refers to programmed death protein 1, T cell coinhibitor, also known as CD279. The amino acid sequence of full-length PD-1 is submitted to GenBank under accession number NP_005009.2. The term also includes PD-1 or a fragment thereof coupled, for example, to a histidine tag, a mouse or human Fc, or a signal sequence such as ROR1. For example, the term includes sequences containing mouse Fc (mIgG2a) or human Fc (hlgGl) at the C terminus fused to amino acid residues 25-170 of NPJ305009.2 with a C93S substitution. PD-1 is a member of the CD28/CTLA4/ICOS family of T cell co-inhibitors. PD-1 is a 288 amino acid protein with an extracellular N-terminal domain that is IgV-like, a transmembrane domain, and an intracellular domain containing a tyrosine-containing immunoreceptor inhibitory motif (ITIM) and a tyrosine-containing immunoreceptor switch motif (ITSM) (Chattopadhyay et al. 2009, Immunol. Rev.). The PD-1 receptor is characterized by the presence of two ligands PD-L1 and PD-L2.

The term B7-1 refers to T-lymphocyte activation antigen, also known as co-stimulatory factor CD80. B7-1 is a 288 amino acid membrane receptor with an extracellular N-terminal domain that contains IgV-like (aa 37-138) and IgC-like (aa 154-232) regions, a transmembrane domain (aa 243-263) and C -terminal intracellular region (aa 263-288). The amino acid sequence of full-length B7-1 is submitted to GenBank under accession number NP_005182.1.

As used herein, the term T cell co-inhibitor refers to a ligand and/or receptor that modulates the immune response through T cell activation or suppression. The term T cell co-inhibitor, also known as T cell co-signaling molecule, includes, but is not limited to, PD-1, lymphocyte activation gene protein 3 (LAG-3, also known as CD223), cytotoxic T lymphocyte antigen-4 (CTLA- 4), B and T lymphocyte attenuator (BTLA), CD-28, 2B4, LY108, T cell immunoglobulin and mucin-3 (TIM3), T cell immunoreceptor with immunoglobulin and ITIM (TIGIT; also known as VSIG9) , leukocyte-associated immunoglobulin-like receptor 1 (LAIR1; also known as CD305), inducible T-cell co-stimulator (ICOS; also known as CD278), B7-1 (CD80), and CD160.

Each heavy chain consists of a heavy chain variable region (HCVR or VH) and a heavy chain constant region (consisting of CH1, CH2 and _CH3 domains). Each light chain consists of a light chain variable region (LCVR or VL) and a light chain constant region (CL). Regions

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VH and V _L can be further subdivided into regions of hypervariability, which are called complementarity determining regions (CDR), interspersed with regions that are more conserved, which are called framework regions (FR). Each of VH and V _L consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FR of an antibody (or an antigen binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on comparative analysis of two or more CDRs.

It is also possible to replace one or more CDR residues or omit one or more CDRs. Antibodies have been described in the scientific literature that can dispense with one or two CDRs to bind. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only approximately one-fifth to one-third of the CDR residues actually contact the antigen. Padlan has also discovered many antibodies in which one or two CDRs are not characterized by the presence of amino acids in contact with the antigen (see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

Non-antigen contact CDR residues can be identified based on previous studies (eg, residues H60-H65 in CDRH2 are often dispensable) from Kabat CDR regions outside the Chothia CDR, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, they are typically replaced by an amino acid occupying a corresponding position in another human antibody sequence or consensus of such sequences. The positions to be substituted within the CDR and the amino acid to be substituted may also be selected empirically. Empirical substitutions may be conservative or non-conservative substitutions.

The fully human anti-PDL1 monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains relative to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present disclosure provides antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated at the corresponding residue(s). a) the germline sequence from which the antibody is derived, or in relation to the corresponding residue(s) of another human germline sequence, or in relation to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes collectively referred to herein as germline mutations). One of skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, will readily be able to produce various antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In some embodiments, all framework and/or CDR residues within the VH and/or VL domains are mutated back to residues found in the original germline sequence from which the antibody originated. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues occurring within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only mutated residues occurring within CDR1, CDR2, or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated relative to the corresponding residue(s) of another germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived ). Moreover, antibodies in accordance with the present disclosure may contain any combination of two or more germline mutations within framework and/or CDR regions, for example, where certain individual residues are mutated relative to the corresponding residue of a particular germline sequence, while certain others residues that differ from the original germline sequence are conserved or mutated relative to the corresponding residue of another germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (if any) ), reduced immunogenicity, etc. Antibodies and antigen-binding fragments prepared in this general manner are provided

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The present invention also provides fully human anti-PD-L1 monoclonal antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein, characterized by one or more conservative substitutions. For example, the present disclosure provides anti-PD-L1 antibodies characterized by HCVR, LCVR and/or CDR amino acid sequences, e.g., with 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein. The term human antibody, as used herein, is intended to include antibodies characterized by variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs according to the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDR and in particular CDR3. However, the term human antibody as used herein is not intended to include mAbs in which CDR sequences derived from the germ line of another mammalian species (eg, mouse) have been grafted onto human FR sequences. As used herein, the term multispecific antigen binding molecules refers to bispecific, trispecific or multispecific antigen binding molecules and antigen binding fragments thereof. Multispecific antigen binding molecules may be specific for different epitopes of a single target polypeptide or may contain antigen binding domains specific for epitopes of more than one target polypeptide. The multispecific antigen binding molecule may be a single multifunctional polypeptide, or it may be a multimeric complex of two or more polypeptides that are covalently or non-covalently linked to each other. The term multispecific antigen-binding molecules includes antibodies in accordance with the present disclosure that can be associated with or can be co-expressed with another functional molecule, for example, with another peptide or protein. For example, an antibody or fragment thereof may be operably linked (e.g., by chemical coupling, genetic fusion, non-covalent linkage, or otherwise) to one or more other molecular entities, such as a protein or fragment thereof, to form a bispecific or multispecific antigen-binding molecule with a second binding specificity. According to the present disclosure, the term multispecific antigen binding molecules also includes bispecific, trispecific or multispecific antibodies or antigen binding fragments thereof. In certain embodiments, an antibody according to the present disclosure is operably linked to another antibody or an antigen-binding fragment thereof to produce a bispecific antibody with a second binding specificity. Bispecific and multispecific antibodies in accordance with the present disclosure are described elsewhere herein.

The term specifically binds or specifically binds to or the like means that the antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x 10' ⁸ M or less (eg, a lower KD means stronger binding). Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies that specifically bind to PD-L1 have been identified using surface plasmon resonance, such as BIACORE™. Moreover, multispecific antibodies that bind to one domain in PD-L1 and one or more additional antigens, or bispecifics that bind to two different regions of PD-L1, are nevertheless considered to be antibodies that specifically bind, as used in this document.

As used herein, the terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. As used herein, the terms antigen binding antibody fragment or antibody fragment refer to one or more antibody fragments that retain the ability to bind PD-L1.

The term isolated antibody, as used herein, is intended to refer to an antibody that does not substantially include other antibodies (Abs) having different characteristics.

- 5 045226 antigen specificities (for example, an isolated antibody that specifically binds to

PD-L1, or a fragment thereof, does not essentially include Abs that specifically bind to antigens other than PD-L1.

As used herein, the term surface plasmon resonance refers to an optical phenomenon that allows the analysis of biomolecular interactions in real time by detecting changes in protein concentrations within a biosensor array, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway , N.J.).

The term KD as used herein is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

The term epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and may have different biological effects. The term epitope also refers to a site on an antigen with which B and/or T cells react. It also refers to the region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, that is, consisting of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface moieties of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may be characterized by particular three-dimensional structural characteristics and/or particular characteristics charge.

The term substantial identity or essentially identical, referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with corresponding nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90 % and more preferably at least about 95, 96, 97, 98 or 99% of the nucleotide bases as measured by any well known sequence identity algorithm such as FASTA, BLAST or GAP.

When applied to polypeptides, the term substantially similar or substantially similar means that two peptide sequences, when optimally aligned, for example by the GAP or BESTFIT programs using default gap opening penalties, have at least 90% sequence identity, even more preferably in at least 95, 98 or 99% sequence identity. Preferably, the positions of residues that are not identical differ by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue characterized by a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not essentially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of groups of amino acids that contain side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are as follows: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative substitution is any change characterized by a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, incorporated herein by reference. A moderately conservative substitution is any change characterized by a non-negative value in the PAM250 log-likelihood matrix. Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software checks for similar sequence matches using similarity scores assigned to various substitutions, deletions and other modifications, including conserved amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; programs in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and percentage sequence identities for regions of best overlap between query and search sequences (Pearson (2000), supra). Another preferred algorithm when comparing a sequence according to the present disclosure to a database containing a large number of sequences from various organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410, and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

By the phrase a therapeutically effective amount is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of treatment and can be determined by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

As used herein, the term subject refers to an animal, preferably a mammal, in need of alleviation, prevention and/or treatment of a disease or disorder, such as a chronic viral infection, cancer or autoimmune disease.

II. Radiolabeled anti-PD-L1 antibody immunoconjugates for immuno-PET imaging.

Presented herein are radiolabeled antigen-binding proteins that bind programmed cell death ligand 1 (PD-L1). In some embodiments, the radiolabeled antigen binding proteins include an antigen binding protein covalently linked to one or more chelating moieties, which are chemical moieties capable of chelating a positron emitter.

In some embodiments, provided herein are antigen binding proteins that bind PD-L1, such as antibodies, wherein said antigen binding proteins that bind PD-L1 are covalently linked to one or more moieties having the following structure:

-L-Mz, wherein L is a chelating moiety; M represents a positron emitter; a z is independently equal to 0 or 1 in each case; and at least one of z is equal to 1.

In some embodiments, the radiolabeled antigen binding protein is a compound of formula (I):

M-L-A-[L-Mz]k (I), wherein A is a protein that binds PD-L1; L represents a chelating moiety; M represents a positron emitter; z is 0 or 1; and k is an integer from 0 to 30. In some embodiments, k is 1.

In some embodiments, the radiolabeled antigen binding protein is a compound of formula (II):

A-[LM] _k (P), wherein A is a protein that binds PD-L1; L represents a chelating moiety; M represents a positron emitter; and k is equal to an integer from 1 to 30.

In some embodiments, provided herein are compositions comprising a conjugate having the following structure:

AL _k , wherein A is a protein that binds PD-L1; L represents a chelating moiety; and k is equal to an integer from 1 to 30; wherein the conjugate is chelated with a positron emitter in an amount sufficient to provide specific activity suitable for clinical PET imaging. Suitable binding proteins, chelating moieties and positron emitters are presented below.

A. PD-L1 binding proteins.

Suitable PD-L1 binding proteins are those that specifically bind to PD-L1, including those described in US Patent Publication No. US 2015-0203580 A1.

- 7 045226 incorporated herein by reference in its entirety. Exemplary anti-PD-L1 antibodies in accordance with the present disclosure are shown in table. 1 of US Patent Publication No. US 2015-0203580 A1, also shown below.

Table 1 _____Aino acid sequence identifiers_____

SEQ GO NO: Antibody designation HCVR HCDR 1 HCDR 2 HCDR3 LCVR LCDR1 LCDR2 LCDR 3 H2M8306N 2 4 6 8 10 12 14 16 H2M8307N 18 20 22 24 26 28 thirty 32 H2M8309N 34 36 38 40 42 44 46 48 H2M8310N 50 52 54 56 58 60 62 64 H2M8312N 66 68 70 72 74 76 78 80 H2M8314N 82 84 86 88 90 92 94 96 H2M8316N 98 100 102 104 106 108 110 112 H2M8317N 114 116 118 120 122 124 126 128 H2M8321N 130 132 134 136 138 140 142 144 H2M8323N 146 148 150 152 154 156 158 160 H2M8718N 162 164 166 168 170 172 174 176 H2M8718N2 178 180 182 184 170 172 174 176 H2M8719N 186 188 190 192 194 196 198 200 Н1Н9323Р 202 204 206 208 210 212 214 216 Н1Н9327Р 218 220 222 224 226 228 230 232 Н1Н9329Р 234 236 238 240 242 244 246 248 Н1Н9336Р 250 252 254 256 258 260 262 264 N1N9344R2 266 268 270 272 274 276 278 280 N1N9345R2 282 284 286 288 274 276 278 280 N1N9351R2 290 292 294 296 274 276 278 280 N1N9354R2 298 300 302 304 274 276 278 280 N1N9364R2 306 308 310 312 274 276 278 280 N1N9373R2 314 316 318 320 274 276 278 280 Н1Н9382Р2 322 324 326 328 274 276 278 280 N1N9387R2 330 332 334 336 274 276 278 280 N1N9396R2 338 340 342 344 274 276 278 280

In table 1 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HCVR), light chain variable regions (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-PD antibodies -L1.

In some embodiments, the binding protein is an antibody or antigen binding fragment comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the binding protein is an antibody or antigen binding fragment comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the binding protein is an antibody or antigen binding fragment comprising a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), including any of the amino acid sequences HCVR listed in Table. 1, paired with any of the LCVR amino acid sequences given in table. 1. In some embodiments, the present disclosure relates to antibodies or antigen binding fragments thereof comprising the amino acid sequence pair HCVR/LCVR contained in any of the exemplary anti-PD-L1 antibodies set forth in Table. 1. In some embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/170, 186/194, 202/210, 218/226, 234/242, 250/258, 266/274, 282/274, 290/ 274, 298/274, 306/274,

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314/274, 322/274, 330/274 and 338/274. In some embodiments, the HCVR/LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 82/90 (e.g., H2M8314N), 162/170 (e.g., H2M8718N), 306/274 (e.g., H1H9364P2), and 314/274 (e.g. , H1H9373P2). In some other embodiments, the HCVR/LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 98/106 (e.g., H2M8316N), 146/154 (e.g., H2M8323N), 290/274 (e.g., H1H9351P2), and 330/274 ( for example H1H9387P2).

In some embodiments, the binding protein is an antibody or antigen binding fragment comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In some embodiments, the binding protein is an antibody or antigen binding fragment comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In some embodiments, the binding protein is an antibody or antigen binding fragment comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In some embodiments, the binding protein is an antibody or antigen binding fragment comprising a heavy chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In some embodiments, the binding protein is an antibody or antigen binding fragment comprising a heavy chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In some embodiments, the binding protein is an antibody or antigen binding fragment comprising a heavy chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In some embodiments, the binding protein is an antibody or antigen binding fragment comprising a pair of amino acid sequences HCVR3 and LCVR3 (HCVR3/LCVR3), including any of the amino acid sequences of HCVR3 listed in Table. 1, paired with any of the amino acid sequences of LCVR3 given in table. 1. In some embodiments, the present disclosure relates to antibodies or antigen binding fragments thereof comprising the HCVR3/LCVR3 amino acid sequence pair contained in any of the exemplary anti-PD-L1 antibodies set forth in Table. 1. In some embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 88/96 (e.g., H2M8314N), 168/176 (e.g., H2M8718N), 312/280 (e.g., H1H9364P2), and 320/280 (for example, H1H9373P2). In some other embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 104/112 (e.g., H2M8316N), 152/160 (e.g., H2M8323N), 296/280 (e.g., H1H9351P2), and 336 /280 (for example, H1H9387P2).

In some embodiments, the binding protein is an antibody or antigen binding fragment comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-PD-L1 antibodies listed in Table. 1. In some embodiments, the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is selected from the group consisting of SEQ ID NO: 8486-88-92-94-96 (eg, H2M8314N); 164-166-168-172-174-176 (for example, H2M8718N); 308-310-312276-278-280 (for example, H1H9364P2) and 316-318-320-276-278-280 (for example, H1H9373P2). According to some

- 9 045226 In other embodiments, the set of amino acid sequences HCDR1-HCDR2HCDR3 -LCDR1-LCDR2-LCDR3 is selected from the group consisting of SEQ ID NO: 100-102-104-108-110-112 (for example, H2M8316N); 148-150-152-156-158-160 (for example, H2M8323N); 292-294-296-276-278-280 (for example, H1H9351P2) and 332-334-336-276-278-280 (for example, H1H9387P2). In some embodiments, the binding protein is an antibody or antigen binding fragment comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in the amino acid sequence pair HCVR/LCVR defined in any of illustrative antibodies against PD-L1 shown in table. 1. For example, in some embodiments, the binding protein is an antibody or antigen binding fragment comprising the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 contained in the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NO: 82/90 (eg H2M8314N), 98/106 (eg H2M8316N), 146/154 (eg H2M8323N), 162/170 (eg H2M8718N), 290/274 (eg H1H9351P2), 306/ 274 (eg H1H9364P2), 314/274 (eg H1H9373P2) and 330/274 (eg H1H9387P2). Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in certain HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary rules that may be used to identify the boundaries of a CDR include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al, J. Mol. Biol. 273:927-948 (1997); and Martin et al, Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). There are also publicly available databases for identifying CDR sequences in an antibody.

In some embodiments, the binding proteins are antibodies and antigen binding fragments thereof that compete for specific binding to PD-L1 with an antibody or antigen binding fragment thereof comprising a CDR from HCVR and a CDR from LCVR, wherein each of HCVR and LCVR has an amino acid sequence , selected from the HCVR and LCVR sequences given in table. 1.

In some embodiments, the binding proteins are isolated antibodies and antigen binding fragments thereof that block the binding of PD-L1 to PD-1 or B7-1. In some embodiments, an antibody or antigen-binding fragment thereof that blocks PD-L1 binding to PD-1 or to B7-1 may bind to the same epitope on PD-L1 as PD-1/B7-1, or may bind with a different epitope on PD-L1 than PD-1/B7-1. In some embodiments, antibodies of the present disclosure that block PD-L1 binding to PD-1 or B7-1 include a CDR from HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table . 1; and a CDR from LCVR having an amino acid sequence selected from the group consisting of LCVR sequences given in table. 1.

In alternative embodiments, the binding proteins are isolated antibodies and antigen binding fragments thereof that do not block PD-L1 binding to PD-1 or B7-1. In some embodiments, the present disclosure provides isolated antibodies or antigen binding fragments thereof that bind PD-L1, wherein the antibodies or antigen binding fragments thereof enhance the binding of PD-L1 to PD-1 or to B7-1. In some embodiments, isolated antibodies or antigen binding fragments thereof that enhance the binding of PD-L1 to PD-1/B7-1 include CDRs from HCVR, wherein HCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 66, 114, 130, 202, 218, 266, 282, 298, 322 and 338; and a CDR from LCVR, wherein LCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 74, 122, 138, 210, 226, and 274. In some embodiments, the isolated antibodies or antigen binding fragments thereof include a pair HCVR/LCVR amino acid sequences selected from the group consisting of SEQ ID NO: 18/26 (eg, H2M8307N), 66/74 (eg, h2m8312N), 114/122 (eg, H2M8317N), 130/138 (eg, H2M8321N ), 202/210 (eg H1H9323P), 218/226 (eg H1H9327P), 266/274 (eg H1H9344P2), 282/274 (eg H1H9345P2), 298/274 (eg H1H9354P2), 322/274 (eg H1H9382P2) and 338/274 (eg H1H9396P2).

In some embodiments, the binding proteins are antibodies and antigen binding fragments thereof that specifically bind to PD-L1 from humans or other species. In some embodiments, the antibodies may bind to human PD-L1 and/or cynomolgus PD-L1. In some embodiments, the binding proteins are antibodies and antigen-binding fragments thereof that cross-compete for binding to PD-L1 with a reference antibody or antigen-binding fragment thereof including a CDR from

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HCVR and CDR from LCVR, wherein each of HCVR and LCVR has an amino acid sequence selected from the sequences of HCVR and LCVR given in table. 1.

In one embodiment, the binding protein is an isolated antibody or antigen binding fragment that has one or more of the following characteristics: (a) blocks the binding of PD-L1 to PD-1 or to B7-1; (b) specifically binds to human PD-L1 and/or cynomolgus PD-L1; (c) inhibit T cell proliferation in a mixed lymphocyte reaction (MLR) assay; and (d) increase the secretion of IL-2 and/or interferon gamma in an MLR assay.

In some embodiments, the binding protein is an antibody or antigen binding fragment thereof that can bind specifically to PD-L1 in an agonistic manner, i.e. they enhance or stimulate PD-L1 binding and/or activity; in other embodiments, the antibody may bind specifically to PD-L1 in an antagonistic manner, i.e. it blocks PD-L1 from binding to its receptor.

In some embodiments, the antibodies or antigen binding fragments are bispecific, comprising a first binding specificity for PD-L1 and a second binding specificity for a second target epitope. The second target epitope may be another epitope on PD-L1 or on another protein, such as a T cell coinhibitor. In certain embodiments, the target epitope may be on another cell, including, for example, another T cell, a B cell, a tumor cell, an autoimmune tissue cell, or a virus-infected cell.

In some embodiments, antibodies and antigen-binding antibody fragments bind monomeric PD-L1 (e.g., at 25°C or 37°C) with a KD of less than about 318 pM, as measured by surface plasmon resonance, e.g., using an assay format defined in Example 3 of US Patent Publication No. US 20150203580 A1, or a substantially similar analysis. In some embodiments, antibodies or antigen-binding fragments thereof bind monomeric PD-L1 with a KD of less than about 300 pM, less than about 250 pM, less than about 150 pM, less than about 100 pM, or less than about 50 pM, as measured by surface plasmon resonance, for example, using the assay format defined in Example 3 of US Patent Publication No. US 2015-0203580 A1, or a substantially similar assay. In some embodiments, antibodies and antigen-binding antibody fragments bind dimeric PD-L1 (e.g., at 25°C or 37°C) with a KD of less than about 15 pM, as measured by surface plasmon resonance, e.g., using an assay format defined in Example 3 of US Patent Publication No. US 2015-0203580 A1, or a substantially similar analysis. In some embodiments, antibodies or antigen binding fragments thereof bind dimeric PD-L1 with a KD of less than about 12 pM, less than about 10 pM, less than about 8 pM, or less than about 5 pM, as measured by surface plasmon resonance, e.g. using the analysis format defined in Example 3 of US Patent Publication No. US 2015-0203580 A1, or a substantially similar analysis.

In some embodiments, the antibodies or antigen-binding fragments thereof bind cynomolgus monkey (Macaca fascicularis) PD-L1 (e.g., at 25°C or 37°C) with a KD of less than about 28 nM, as measured by surface plasmon resonance, e.g. using the analysis format defined in Example 3 of US Patent Publication No. US 2015-0203580 A1. In some embodiments, the antibodies or antigen-binding fragments thereof bind cynomolgus PD-L1 with a KD of less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, or less than about 5 nM, as measured with using surface plasmon resonance, for example, using the assay format defined in Example 3 of US Patent Publication No. US 2015-0203580 A1, or a substantially similar assay.

In some embodiments, antibodies and antigen-binding antibody fragments bind PD-L1 with a dissociation half-life (t ¹ / ₂ ) of greater than about 1 minute, as measured by surface plasmon resonance at 25°C or 37°C, for example, using the format the analysis defined in Example 3 of US Patent Publication No. US 2015-0203580 A1, or a substantially similar analysis. In some embodiments, the antibodies or antigen binding fragments bind PD-L1 to t1/ ₂ for more than about 5 minutes, more than about 10 minutes, more than about 30 minutes, more than about 50 minutes, more than about 60 minutes, more than about 70 minutes, more than about 80 minutes, more than about 90 minutes, more than about 100 minutes, more than about 200 minutes, more than about 300 minutes, more than about 400 minutes, more than about 500 minutes, more than about 600 minutes, more than about 700 minutes or more than about 800 minutes, as measured by surface plasmon resonance at 25°C or 37°C,

- 11 045226 for example, using the assay format defined in Example 3 of US Patent Publication No. US 2015-0203580 A1 (eg, mAb capture or antigen capture format), or a substantially similar assay.

In some embodiments, the antibodies or antigen-binding fragments thereof block the binding of PD-L1 to PD-1 with an IC ₅₀ of less than about 770 pM, as determined using an ELISA-based immunoassay, for example, as shown in Example 4 of U.S. Patent Publication No. 2015 -0203580 A1, or essentially a similar analysis. In some embodiments, the antibodies or antigen-binding fragments thereof block the binding of PD-L1 to B71 with an IC ₅₀ of less than about 10 nM as determined using an ELISA-based immunoassay, for example, as shown in Example 4 of US Patent Publication No. US 20150203580 A1, or essentially a similar analysis. In some embodiments, the antibodies and antigen-binding fragments thereof bind to PD-L1 and enhance the binding of PD-L1 to PD-1 or to B7-1. In some embodiments, the antibodies bind to the extracellular domain of PD-L1 or a fragment of the domain. In some embodiments, the antibodies bind to more than one domain (cross-reactive antibodies). In some embodiments, the antibodies bind to an epitope located in the extracellular domain comprising amino acid residues 19-239 of NP_054862.1.

In some embodiments, the antibodies function by blocking or inhibiting PD-1 binding or B7-1 binding activity associated with PD-L1 by binding to any other region or fragment of the full-length protein. In some embodiments, the antibodies attenuate or modulate the interaction between PD-L1 and PD-1/B7-1. In some embodiments, the antibodies are bispecific antibodies. Bispecific antibodies can bind one epitope in one domain and can also bind a second epitope in another domain of PD-L1. In some embodiments, bispecific antibodies bind two different epitopes in the same domain. In one embodiment, the multispecific antigen binding molecule comprises a first antigen binding specificity, wherein the first binding specificity comprises a PD1 extracellular domain or fragment thereof; and a second antigen binding specificity to a different PD-L1 epitope. In another embodiment, the multispecific antigen binding molecule comprises a first antigen binding specificity, wherein the first binding specificity comprises a B7-1 extracellular domain or fragment thereof; and a second antigen binding specificity to a different PD-L1 epitope. In one embodiment, the antibody or fragment thereof is a fully human monoclonal antibody or antigen binding fragment thereof that binds PD-L1, wherein the antibody or fragment thereof has one or more of the following characteristics: (i) contains an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 186, 202, 218, 234, 250, 258, 266, 274, 282 , 290, 298, 306, 314, 322, 330 and 338, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) contains an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 194, 210, 226, 242, 258 and 274, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) contains an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 192, 208, 224 , 240, 256, 272, 280, 288, 296, 304, 312, 320, 328, 336 and 344, or a substantially similar sequence thereof, characterized by at least 90%, at least 95%, at least 98 % or at least 99% sequence identity; and an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 200, 216, 232, 248, 264, and 280, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iv) contains an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 188, 204, 220 , 236, 252, 268, 284, 292, 300, 308, 316, 324, 332 and 340, or a substantially similar sequence thereof, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 190, 206, 222, 238, 254 , 270, 286, 294, 302, 310, 318, 326, 334, and 342, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99 % sequence identity; an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 196, 212, 228, 244, 260, and 276 , or a sequence substantially similar thereto, characterized by at least 90%, at least 95%, at least

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98% or at least 99% sequence identity; and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 198, 214, 230, 246, 262 and 278, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (v) is a multispecific antigen-binding molecule comprising a first binding specificity for PD-L1 and a second binding specificity for an antigen selected from the group consisting of PD-L1, a tumor-specific antigen, a virus-infected cell antigen, and a T-cell coinhibitor; (vi) binds to human PD-L1 with a K _D from about 4 pM to about 645 nM; (vii) binds to cynomolgus PD-L1 with a K _D of about 70 pM to about 400 nM; (viii) blocks or enhances the binding of PD-L1 to PD-1 with an IC ₅₀ <770 pM; (ix) blocks or enhances the binding of PD-L1 to B7-1 with an IC ₅₀ <10 nM; (x) blocks PD-1-induced T cell down-regulation and/or restores T cell signaling in a T cell/APC luciferase reporter gene assay; (xi) stimulates T cell proliferation and activity in a mixed lymphocyte reaction (MLR) assay; (xii) induces the production of IL-2 and/or IFNy in an MLR assay; and (xiii) inhibits tumor growth and increases survival in subjects with cancer. In one embodiment, the antibody or fragment thereof is a fully human monoclonal antibody or antigen binding fragment thereof that blocks PD-L1 binding to PD-1 or to B7-1, wherein the antibody or fragment thereof has one or more of the following characteristics: (i ) contains HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 98, 146, 162, 290, 306, 314 and 330, or a substantially similar sequence thereof, characterized by at least 90% at least 95%, at least 98%, or at least 99% sequence identity; (ii) contains an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 90, 106, 154, 170 and 274, or a substantially similar sequence thereof, characterized by at least 90%, at least 95% , at least 98% or at least 99% sequence identity; (iii) contains an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 88, 104, 152, 168, 296, 312, 320 and 336, or a substantially similar sequence thereof having at least 90 %, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 96, 112, 160, 176, and 280, or a substantially similar sequence thereof, characterized by at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) contains an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 100, 148, 164, 292, 308, 316 and 332, or a substantially similar sequence thereof having at least 90 %, at least 95%, at least 98%, or at least 99% sequence identity; an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 102, 150, 166, 294, 310, 318 and 334, or a substantially similar sequence thereof, characterized by at least 90% of at least 95%, at least 98%, or at least 99% sequence identity; an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 92, 108, 156, 172 and 276, or a substantially similar sequence thereof, characterized by at least 90%, at least 95%, at least at least 98% or at least 99% sequence identity; and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 94, 110, 158, 174 and 278, or a substantially similar sequence thereof, characterized by at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (v) is a multispecific antigen-binding molecule comprising a first binding specificity for PD-L1 and a second binding specificity for an antigen selected from the group consisting of another PD-L1 epitope, a tumor-specific antigen, a virus-infected cell antigen, and a T-cell coinhibitor; (vi) binds to human PD-L1 with a K _D <10 ^-10 M; (vii) binds to cynomolgus PD-L1 with a K _D <10 ^-7 M; (viii) blocks PD-1-induced T cell down-regulation and/or restores T cell signaling in a T cell/APC luciferase reporter gene assay; (xi) stimulates T cell proliferation and activity in a mixed lymphocyte reaction (MLR) assay; (xii) induces the production of IL-2 and/or IFNy in an MLR assay; and (xiii) inhibits tumor growth and increases survival in subjects with cancer.

In some embodiments, anti-PD-L1 antibodies or antigen-binding fragments thereof bind an epitope within any one or more of the regions exemplified by PD-L1, either in natural or recombinantly produced form, or a fragment thereof. In some embodiments, antibodies of the present disclosure bind to an extracellular region comprising one or more amino acids selected from the group consisting of amino acid residues 19-239 of PD-L1. In certain embodiments, antibodies of the present disclosure bind to a region including one or more amino acids selected from the group consisting of amino acid residues 1-221 of cynomolgus PD-L1.

In some embodiments, the antibodies of the present disclosure shown in Table. 1, interact with at least one amino acid sequence selected from the group consisting of amino acid residues ranging from approximately position 19 to approximately position 130 in PD-L1; or amino acid residues ranging from about position 130 to about position 153 in PD-L1; or amino acid residues ranging from about position 153 to about position 210 in PD-L1; or amino acid residues ranging from about position 210 to about position 239 in PD-L1. In some embodiments, anti-PD-L1 antibodies bind to the same epitope or portion of an epitope as any of the specific exemplary antibodies described herein in Table. 1, or an antibody having the CDR sequences of any of the exemplary antibodies described in table. 1. Likewise, suitable antibodies also include anti-PD-L1 antibodies that compete for binding to PD-L1 or a fragment of PD-L1 with any of the specific exemplary antibodies described herein in Table. 1, or an antibody having the CDR sequences of any of the exemplary antibodies described in table. 1. For example, suitable antibodies include anti-PD-L1 antibodies that cross-compete for binding to PD-L1 with one or more of the antibodies defined in Example 6 herein (e.g., H2aM8309N, H1H9329P, H1H9336P, H2aM8314N, H2aM8316N, H2AM8718N , H1H9387P2, H1H9351P2, H1H9364P2, H1H9373P2 and H2aM8306N). The present disclosure also relates to anti-PD-L1 antibodies that cross-compete for binding to PD-L1 with one or more of the antibodies defined in Example 6 of US Patent Publication No. US 2015-0203580 A1 (e.g., H1H9396P2, H2aM8317N, H2aM8321N, H1H9323P , H1H9382P2, H1H9344P2, H1H9345P2 and H1H9354P2).

The antibodies and antigen binding fragments described herein specifically bind to PD-L1 and modulate the interaction of PD-L1 with PD-1 or with B7-1. Anti-PD-L1 antibodies can bind to PD-L1 with high affinity or low affinity. In some embodiments, the antibodies are blocking antibodies, wherein the antibodies bind to PD-L1 and block the interaction of PD-L1 with PD-1 or with B7-1. In some embodiments, blocking antibodies according to the present disclosure block the binding of PDL1 to PD-1 or to B7-1 and/or stimulate or enhance T cell activation. In some embodiments, blocking antibodies are useful for stimulating or enhancing an immune response and/or for treating a subject suffering from a cancer or chronic viral infection. Antibodies, when administered to a subject in need thereof, can reduce chronic infection caused by a virus, such as HIV, LCMV, or HBV, in the subject. They can be used to inhibit the growth of tumor cells in a subject. They may be used alone or as adjuvant therapy with other therapeutic moieties or methods known in the art for the treatment of cancer or viral infection. In some embodiments, anti-PD-L1 antibodies that bind to PD-L1 with low affinity are used as multispecific antigen-binding molecules, wherein the first binding specificity binds to PD-L1 with low affinity and the second binding specificity binds to an antigen selected from the group consisting of another epitope in PD-L1, a T cell co-inhibitor such as PD-1, a tumor-specific antigen, and an infected cell-specific antigen.

In some embodiments, the antibodies of the present disclosure are agonistic antibodies, wherein the antibodies bind to PD-L1 and enhance the interaction of PD-L1 and PD-1/B7-1. In some embodiments, activating antibodies enhance the binding of PD-L1 to PD-1 or B7-1 and/or inhibit or suppress T cell activation. Activating antibodies according to the present disclosure may be useful for inhibiting an immune response in a subject and/or treating an autoimmune disease.

In some embodiments, anti-PD-L1 antibodies are multispecific antigen-binding molecules, wherein they include a first binding specificity for PD-L1 and a second binding specificity for an antigen selected from the group consisting of another PD-L1 epitope, a T cell coinhibitor , such as PD-1, tumor-specific antigen and infected cell-specific antigen. In some embodiments, the first binding specificity binds to PD-L1 with low affinity, for example, a KD of 10-8M, ^107M , or greater.

Certain anti-PD-L1 antibodies of the present disclosure are capable of binding to PD-L1 and neutralizing its activity, as determined by in vitro or in vivo assays. The ability of antibodies according to the present disclosure to bind to and neutralize PD-L1

- 14 045226 activity can be measured using any standard method known to those skilled in the art, including binding assays or activity assays described herein.

Non-limiting exemplary in vitro assays for measuring binding activity are illustrated in Example 3 of US Patent Publication No. US 2015-0203580 A1. In Example 3, the binding affinities and kinetic constants of human anti-PD-L1 antibody to human PD-L1 and cynomolgus PD-L1 were determined by surface plasmon resonance and measurements were performed on a T200 Biacore apparatus. In Examples 4 and 5 of US Patent Publication No. US 2015-0203580 A1, blocking assays were used to determine the ability of an anti-PD-L1 antibody to block PD-L1 binding to PD-1 or B7-1 in vitro. In Example 6 of US Patent Publication No. US 2015-0203580 A1, blocking assays were used to determine cross-competition between different anti-PD-L1 antibodies. Example 7 of US Patent Publication No. US 2015-0203580 A1 describes the binding of antibodies to cells overexpressing PD-L1. Example 8 of US Pat. No. US 2015-0203580 A1 used a luciferase assay to determine the ability of anti-PD-L1 antibodies to antagonize PD-1/PD-L1 signaling in T cells.

Unless specifically stated otherwise, the term antibody as used herein should be understood to include antibody molecules containing two immunoglobulin heavy chains and two immunoglobulin light chains (ie, complete antibody molecules), as well as antigen-binding fragments thereof. As used herein, the terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms antigen binding antibody fragment or antibody fragment refer to one or more antibody fragments that retain the ability to specifically bind PD-L1. The antibody fragment may include a Fab fragment, an F(ab')2 fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In certain embodiments, the term antigen binding fragment refers to a polypeptide or fragment thereof of a multispecific antigen binding molecule. In such embodiments, the term antigen binding fragment includes, for example, the extracellular domain of PD-1 that specifically binds PD-L1. Antigen-binding antibody fragments can be prepared, for example, from complete antibody molecules using any suitable standard techniques, such as treatment with proteolytic enzymes or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and/or readily available, for example from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and chemically manipulated or molecular biology techniques used, for example, to arrange one or more variable and/or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc. d.

Non-limiting examples of antigen binding fragments include the following: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR), such as the CDR3 peptide), or the FR3-CDR3-FR4 restricted peptide. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.) , a small modular protein (SMIP) immunopharmaceutical and shark IgNAR variable domains are also provided within the term antigen binding fragment as used herein.

An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR that is adjacent or in frame to one or more framework sequences. In antigen binding fragments characterized by a VH domain linked to a VL domain, the VH and _VL domains may be located relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain _VH - _VH , VH-VL or VL-VL dimers. Alternatively, the antigen binding fragment of the antibody may comprise a monomeric VH or VL domain. In certain embodiments, an antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen binding fragment of an antibody according to the present disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) V _H Ch1-Ch2; (v) Vh-Ch1-Ch2-Ch3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) VlCH1-CH2; (xii) V _L -C _H 1-C _H 2-C _H 3; (xiii) VL-CH2-CH3 and (xiv) V _L -C _L . In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can either be directly connected to each other or can be connected by a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen binding fragment of an antibody according to the present disclosure may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric VH or V domains _L (eg via disulfide bond(s)). Similar to full antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding antibody fragment will typically contain at least two different variable domains, with each variable domain being capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen binding fragment of an antibody according to the present disclosure using routine techniques available in the art.

Anti-PD-L1 antibodies and antibody fragments according to the present disclosure include proteins having amino acid sequences that differ from those of the described antibodies, but which retain the ability to bind PD-L1. Such variant antibodies and antibody fragments contain one or more insertions, deletions or amino acid substitutions from the original sequence, but exhibit biological activity that is substantially equivalent to the biological activity of the described antibodies. Likewise, antibody-encoding DNA sequences according to the present disclosure include sequences that contain one or more insertions, deletions, or nucleotide substitutions relative to the disclosed sequence, but that encode an antibody or antibody fragment that is substantially bioequivalent to the antibody or antibody fragment in accordance with this disclosure.

Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not demonstrate significant differences when administered at the same molar dose under similar experimental conditions, or in a single dose, or in multiple doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption but not in their rate of absorption, and may still be considered bioequivalent because such differences in rate of absorption are intentional and reflected on the label and are not significant to achieve effective concentrations of the drug in the body, for example, during chronic use, and are considered not medically significant for the specific drug product being studied.

In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, or potency.

In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch one or more times from the reference product to the biologic product without a predicted increase in the risk of adverse effects, including a clinically significant change in immunogenicity or reduced efficacy, compared to continuous therapy without such switching .

In one embodiment, two antigen binding proteins are bioequivalent if they both act through a common mechanism or mechanisms of action for conditions or conditions of use, to the extent such mechanisms are known.

Bioequivalence can be demonstrated using in vivo and/or in vitro methods. Bioequivalence parameters include, for example, (a) in vivo testing in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum or other biological fluid as a function of time; (b) in vitro testing that has been correlated with in vivo bioavailability data in humans and that is reasonably predictive of these data; (c) in vivo testing in humans or other mammals, in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of the antibody. Bioek

- 16 045226 vivalent variants of antibodies in accordance with the present disclosure can be constructed, for example, by creating various substitutions of residues or sequences or deletion of terminal or internal residues or sequences that are not necessary for biological activity. For example, cysteine residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or irregular intermolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants containing amino acid changes that modify the glycosylation characteristics of the antibodies, such as mutations that eliminate or remove glycosylation.

In certain embodiments of the present disclosure, anti-PD-L1 antibodies include an Fc domain containing one or more mutations that increase or decrease the binding of the antibody to the FcRn receptor, for example, at an acidic pH compared to a neutral pH. For example, the present disclosure relates to anti-PD-L1 antibodies containing a mutation in the C _H 2 or C _H 3 region of the Fc domain, wherein the mutation(s) increase(s) the affinity of the Fc domain for FcRn in an acidic environment (eg, endosome, where the pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (eg L/Y/F/W or T), 254 (eg S or T) and 256 (eg S/R/Q/E/D or T); or modification at position 428 and/or 433 (for example, H/L/R/S/P/Q or K) and/or 434 (for example, A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modification at position 250 and/or 428; or modification at position 307 or 308 (eg, 308F, V308F) and 434. According to one embodiment, the modification includes modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259 I (for example, v259I) and 308F (for example, V308F); modification 433K (for example, H433K) and 434 (for example, 434Y); modification of 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L); as well as a modification of 307 and/or 308 (for example, 308F or 308P). According to yet another embodiment, the modification includes modification 265A (eg, D265A) and/or 297A (eg, N297A).

For example, the present disclosure relates to anti-PD-L1 antibodies comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (eg, T250Q and M248L); 252Y, 254T and 256E (for example, M252Y, S254T and T256E); 428L and 434S (for example, M428L and N434S); 257I and 311I (for example, P257I and Q311I); 257I and 434H (for example, P257I and N434H); 376V and 434H (for example, D376V and N434H); 307A, 380A and 434A (eg T307A, E380A and N434A); and 433K and 434F (for example, H433K and N434F). In one embodiment, the present disclosure provides anti-PD-L1 antibodies comprising an Fc domain containing an S108P mutation in the IgG4 hinge region to promote dimer stabilization. All possible combinations of the above Fc domain mutations and other mutations within the variable domains of the antibodies disclosed herein are contemplated within the scope of this disclosure.

The present disclosure also relates to anti-PD-L1 antibodies comprising a chimeric heavy chain constant ( _CH ) region, wherein the chimeric _CH region contains segments derived from the _CH regions of more than one immunoglobulin isotype. For example, antibodies in accordance with the present disclosure may contain a chimeric _CH region containing part or all of a _CH 2 domain derived from a human IgG1, human IgG2, or human IgG4 molecule combined with part or all of a CH3 domain derived from a human IgG1 molecule, Human IgG2 or human IgG4. In certain embodiments, antibodies of the present disclosure comprise a chimeric CH region characterized by a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge amino acid sequence (amino acid residues from position 216 to position 227 according to EU numbering) derived from the hinge region of human IgG1, human IgG2 or human IgG4 combined with a lower hinge sequence (amino acid residues from position 228 to position 236 according to EU numbering) originating from the hinge region of human IgG1, human IgG2 or human IgG4. In certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. An antibody containing a chimeric CH region described herein may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, USSN. 14/170166, filed January 31, 2014, the entire disclosure of which is hereby incorporated by reference).

B. Positron emitters and chelating moieties.

Suitable positron emitters include, but are not limited to, those that form stable complexes with the chelating moiety and have physical half-lives suitable for immuno-PET imaging purposes. Exemplary positron emitters include, but are not limited to, ⁸⁹ Zr, ⁶⁸ Ga, ⁶⁴ Cu, ⁴⁴ Sc, and ⁸⁶ Y. Suitable positron emitters also include those that directly bind to the PD-L1 binding protein, including but not limited to ⁷⁶ Br and ¹²⁴ I, and those introduced through a prosthetic group, for example, ¹⁸ F.

Chelating moieties described herein are chemical moieties that are covalently linked to a PD-L1 binding protein, such as an anti-PD-L1 antibody, and include a portion capable of chelating a positron emitter, i.e. capable of reacting with a positron emitter to form a coordinated chelate complex. Suitable moieties include those that provide efficient loading of a particular metal and form metal-chelator complexes that are sufficiently stable in vivo for diagnostic applications, for example, immuno-PET imaging. Exemplary chelating moieties include those that minimize positron emitter dissociation and accumulation in mineral deposits in bone, plasma proteins, and/or bone marrow to an extent suitable for diagnostic applications.

Examples of chelating moieties include, but are not limited to, those that form stable complexes with the positron emitters ⁸⁹ Zr, ⁶⁸ Ga, ⁶⁴ Cu, ⁴⁴ Sc, and/or ⁸⁶ Y. Exemplary chelating moieties include, but are not limited to, those described in Nature Protocols. 5(4): 739, 2010; Bioconjugate Chem., 26(12): 2579 (2015); Chem Commun (Camb), 51(12): 2301 (2015); Mol. Pharmaceutics, 12: 2142 (2015); Mol. Imaging Biol., 18: 344 (2015); Eur. J. Nucl. Med. Mol. Imaging, 37:250 (2010); Eur.. J. Nucl. Med. Mol. Imaging (2016). doi:10.1007/s00259-016-3499-x; Bioconjugate Chem., 26(12): 2579 (2015); in International Application No. WO 2015/140212A1 and US Patent No. US 5,639,879, incorporated by reference in their entirety.

Exemplary chelating moieties also include, but are not limited to, those that include desferrioxamine (DFO), 1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), (1,4 ,7,10tetraazacyclododecane-1,4,7,10-tetra(methylenephosphinic) acid (DOTP), 1R,4R,7R,10R)-a'aa'tetramethyl-1,4,7,10-tetraazacyclododecane-1,4 ,7,10-tetraacetic acid (DOTMA), 1,4,8,11tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), H ₄ octapa, H ₆ phospa, H ₂ dedpa, H ₅ decapa, H ₂ azapa, HOPO, DO2A, 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7-triazacyclononane-N,N',N-triacetic acid (NOTA), 1,4,7,10-tetrakis(carbamoylmethyl)1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazabicyclo[6,6,2]hescadecane-4,11 -acetic acid (CB-TE2A), 1,4,7,10-tetraazacyclododecane (cyclen), 1,4,8,11-tetraazacyclotetradecane (cyclam), octadentate chelators, hexadentate chelators, phosphonate-based chelators, macrocyclic chelators, chelators , including macrocyclic terephthalamide ligands, bifunctional chelators, chelators based on fusarin C and fusarin C derivatives, triacetyl fusarin C (TAFC), ferrioxamine E (FOXE), ferrioxamine B (FOXB), ferrichrome A (FCHA), etc.

In some embodiments, the chelating moieties are covalently linked to a PD-L1 binding protein, such as an antibody or an antigen binding fragment thereof, through a linker moiety that covalently attaches the chelating portion of the chelating moiety to the binding protein. In some embodiments, these linker moieties are formed by a reaction between a reactive moiety of the PD-L1 binding protein, such as a cysteine or lysine of the antibody, and a reactive moiety that is attached to a chelator including, for example, a p-isothiocyanatobenzyl group and the reactive moieties represented by in the conjugation methods below. In addition, such linker moieties optionally include chemical groups used for the purpose of controlling polarity, solubility, steric interactions, rigidity and/or length between the chelating moiety and the PD-L1 binding protein.

C. Preparation of radiolabeled anti-PD-L1 antibody conjugates.

Radiolabeled anti-PD-L1 antibody conjugates can be prepared by (1) reacting a PD-L1 binding protein, such as an antibody, with a molecule comprising a positron emitter chelator and a moiety reactive at a desired PD-L1 binding site of conjugation. L1 protein, and (2) loading the desired positron emitter.

Suitable conjugation sites include, but are not limited to, lysine and cysteine, both of which may be, for example, native or engineered, and may, for example, be present on the heavy or light chain of the antibody. Cysteine conjugation sites include, but are not limited to, those obtained by mutation, insertion, or reduction of disulfide bonds in antibodies. Methods for producing cysteine engineered antibodies include, but are not limited to, those described in International Application No. WO 2011/056983. Site-specific conjugation methods can also be used to direct the conjugation reaction to specific sites on an antibody, achieving desired stoichiometry and/or achieving desired drug-to-antibody ratios (DARs). such conjugation methods are known to those skilled in the art and include, without limitation, the method

- 18 045226 cysteine construction, enzymatic and chemoenzymatic methods, including but not limited to glutamine conjugation, Q295 conjugation and transglutaminase-mediated conjugation, as well as those described in J. Clin. Immunol., 36: 100 (2016), incorporated herein by reference in its entirety. Suitable moieties reactive at the desired conjugation site will generally provide efficient and facile binding of the PD-L1 binding protein, such as an antibody and a positron emitter chelator. Moieties reactive at lysine and cysteine sites include electrophilic groups that are known to those skilled in the art. In some aspects, when the desired conjugation site is lysine, the reactive moiety is an isothiocyanate, such as a p-isothiocyanatobenzyl group or a reactive ester. In some aspects, when the desired conjugation site is a cysteine, the reactive moiety is a maleimide.

When the chelator is desferrioxamine (DFO), suitable reactive moieties include, but are not limited to, isothiocyanatobenzyl groups, nhydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, n-succinimidyl S-acetylthioacetate and those described in BioMed Research International , Vol 2014, Article ID 203601, incorporated herein by reference in its entirety. In some embodiments, the PD-L1 binding protein is an antibody and the molecule including a positron emitter chelator and a conjugation site reactive moiety is p-isothiocyanatobenzyl-desferrioxamine (p-SCN-Bn-DFO):

Loading of a positron emitter is accomplished by incubating the PDL1 binding protein/chelator conjugate with the positron emitter for a time sufficient to ensure coordination of said positron emitter with the chelator, for example, by the methods described in the examples presented herein or a substantially similar method.

D. Exemplary Embodiments of Conjugates.

The present disclosure provides radiolabeled antibody conjugates comprising an antibody or an antigen-binding moiety thereof that binds human programmed cell death ligand 1 (PD-L1), a chelating moiety, and a positron emitter.

In some embodiments, the chelating moiety comprises a chelator capable of forming a complex with ⁸⁹ Zr. In some embodiments, the chelating moiety comprises desferrioxamine. In some embodiments, the chelating moiety is p-isothiocyanatobenzyl-desferrioxamine.

In some embodiments, the positron emitter is ⁸⁹ Zr.

In some embodiments, the ratio of chelating moiety to antibody in the conjugate is between 1 and 2.

In a specific embodiment, the chelating moiety is pisothiocyanatobenzyl-desferrioxamine and the positron emitter is ⁸⁹ Zr. In another specific embodiment, the chelating moiety is pisothiocyanatobenzyl-desferrioxamine, the positron emitter is ⁸⁹ Zr, and the ratio of chelating moiety to antibody in the conjugate is 1 to 2.

In some embodiments, provided herein are antigen binding proteins that bind PD-L1, wherein said antigen binding proteins that bind PD-L1 are covalently linked to one or more moieties having the following structure:

wherein L represents a chelating moiety; M represents a positron emitter; a z is independently equal to 0 or 1 in each case; and wherein at least one of z is equal to 1. In some embodiments, the radiolabeled antigen binding protein is a compound of formula (I):

MLA-[LM _z ]k (I),

- 19 045226 wherein A is a protein that binds PD-L1; L represents a chelating moiety; M represents a positron emitter; z is 0 or 1; and k is an integer from 0 to 30. In some embodiments, k is 1.

In some embodiments, L is

In some embodiments, M is ⁸⁹ Zr.

In some embodiments, k is an integer between 1 and 2. In some embodiments, k is 1.

In some embodiments, -L-M is

The present disclosure also provides methods for synthesizing radiolabeled antibody conjugates comprising contacting a compound of formula (III):

with ⁸⁹ Zr, wherein A is an antibody or antigen binding fragment thereof that binds PD-L1. In some embodiments, a compound of formula (III) is synthesized by contacting a PD-L1 binding antibody or antigen binding fragment thereof with p-SCN-Bn-DFO.

Also provided herein is the reaction product between a compound of formula (III) and ⁸⁹ Zr.

Presented herein are compounds of formula (III):

wherein A is an antibody or antigen binding fragment thereof that binds PD-L1, and k is an integer from 0 to 30. In some embodiments, k is 1 or 2.

In some embodiments, provided herein are compositions comprising a conjugate having the following structure:

A-Lk, wherein A is a protein that binds PD-L1; L represents a chelating moiety; and k is equal to an integer from 0 to 30; wherein the conjugate is chelated with a positron emitter in an amount sufficient to provide specific activity suitable for clinical PET imaging. In some embodiments, the amount of the chelated positron emitter is an amount sufficient to provide a specific activity of 1-3 mCi per 1-50 mg of protein that binds PD-L1.

In some embodiments, the antibody or antigen binding fragment thereof binds monomeric human programmed cell death ligand 1 (PD-L1) with an equilibrium binding dissociation constant (KD) of less than about 310 pM, as measured in a surface plasmon resonance assay at 37°C.

In some embodiments, the antibody or antigen binding fragment thereof binds monomeric human PD-L1 with a KD of less than about 180 pM in a surface plasmon resonance assay at 25°C.

In some embodiments, the antibody or antigen binding fragment thereof binds dimeric human PD-L1 with a KD of less than about 15 pM, as measured in a surface plasmon resonance assay at 37°C.

In some embodiments, the antibody or antigen binding fragment thereof binds dimeric human PD-L1 with a KD of less than about 8 pM in a surface plasmon resonance assay at 25°C.

In some embodiments, the antibody or antigen binding fragment thereof competes for binding to human PD-L1 with a reference antibody including HCVR complementarity determining regions (CDRs), wherein HCVR has an amino acid sequence selected from the group consisting of the HCVR sequences set forth in table 1; and CDR LCVR, wherein LCVR has an amino acid sequence selected from the group consisting of LCVR sequences given in table. 1. In some embodiments, the reference antibody or antigen binding fragment thereof comprises the amino acid sequence pair HCVR/LCVR set forth in Table. 1. In some embodiments, the reference antibody comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 82/90, 98/106, 146/154, 162/170, 290/274, 306/274, 314/274 and 330/274.

In some embodiments, the antibody or antigen binding fragment thereof enhances the binding of PD-L1 to one of PD-1 or B7-1. In some embodiments, the antibody or antigen binding fragment thereof blocks the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, the antibody or antigen binding fragment thereof does not increase or decrease the binding of PD-L1 to its ligands.

In some embodiments, the antibody or antigen binding fragment thereof comprises HCVR complementarity determining regions (CDRs), wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 66, 114, 130, 202, 218, 266, 282 , 298, 322 and 338; and a CDR LCVR, wherein the LCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 74, 122, 138, 210, 226, and 274. In some embodiments, the isolated antibody comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 18/26, 66/74, 114/122, 130/138, 202/210, 218/226, 266/274, 282/274, 298/274, 322/274 and 338 /274.

In some embodiments, the antibody is a human monoclonal antibody or antigen binding fragment thereof that specifically binds to human PDL1, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of HCVR sequences given in table. 1.

In some embodiments, the antibody is a human monoclonal antibody or antigen binding fragment thereof that specifically binds to human PDL1, wherein the antibody or antigen binding fragment thereof comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of LCVR sequences given in table. 1.

In some embodiments, the antibody is a human monoclonal antibody or antigen binding fragment thereof that specifically binds to human PDL1, wherein the antibody or antigen binding fragment thereof comprises (a) an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences set forth in table 1; and (b) LCVR having an amino acid sequence selected from

- 21 045226 group consisting of LCVR sequences given in table. 1.

In some embodiments, the antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in any of the heavy chain variable region (HCVR) sequences set forth in Table. 1; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in any of the light chain variable region (LCVR) sequences shown in table. 1.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 188, 204, 220, 236, 252, 268, 284, 292, 300, 308, 316, 324, 332 and 340;

(b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 190, 206, 222, 238, 254, 270, 286, 294, 302, 310, 318, 326, 334 and 342;

(c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 192, 208, 224, 240, 256, 272, 288, 296, 304, 312, 320, 328, 336 and 344;

(d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 196, 212, 228, 244, 260 and 276;

(e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 198, 214, 230, 246, 262 and 278; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 200, 216, 232, 248 , 264 and 280.

In some embodiments, the antibody or antigen binding fragment comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 82/90, 98/106, 146/154, 162/170, 290/274, 306/274, 314/274 and 330/274.

In some embodiments, the antibody or antigen binding fragment thereof comprises a CDR of HCVR, wherein HCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 34, 50, 82, 98, 146, 162, 178, 186, 234, 250, 290, 306, 314 and 330; and the CDR LCVR, wherein LCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 42, 58, 90, 106, 154, 170, 194, 242, 258 and 274.

E. Large-scale production to produce anti-PD-L1 antibody-chelator conjugates.

The present disclosure relates to large-scale manufacturing processes for the production of chelator-conjugated anti-PD-L1 antibodies. Anti-PD-L1 antibody-chelator conjugates are in a form suitable for radiolabeling.

Good manufacturing processes are followed in all aspects of receipt, including maintaining a sterile environment, using aseptic procedures, maintaining records of all processes, and documenting the quality, purity, strength and identity of the product, as well as any deviations therefrom. The large scale production process in some embodiments is much faster than the research and development production process. In some embodiments, the large-scale production process may take less than 12 hours, or less than 10 hours, or less than 8 hours, or less than 6 hours, or less than 4 hours, or less than or about 2 hours.

In some embodiments, the first step involves ultrafiltration and diafiltration (UFDF) using a 30-50 kDa anti-PD-L1 antibody membrane to remove anticonjugation agents and salts that inhibit the conjugation process. Exemplary membrane polymers include polyethersulfone (PES), cellulose acetate (CA), and regenerated cellulose (RC). At this stage, the antibody solution is buffered with a non-reacting buffer solution of low ionic strength. The pH of the buffer may be from about 4.5 to about 6, or from about 5 to about 6, or from about 5.3 to about 5.7, or about 5.5. Buffer systems contemplated as applicable herein include any buffer system that does not contain a primary amine. Exemplary buffers include acetate, phosphate, or citrate buffers. The buffer ensures protein stability during processing prior to conjugation. The treatment volume can be further reduced to concentrate the antibody, then sterile filtered.

After preconjugation with UFDF, the concentrated and filtered antibody can be transferred to an amine-free carbonate buffer system. The carbonate buffer system may have a pH ranging from about 8.5 to about 9.6, or from about 9.0 to about 9.6, or from about 9.2 to about 9.4, or from about 9.4 to approximately 9.6, or pH approximately 9.4.

Add a chelator, such as DFO, to the solvent to the target concentration in the buffer system,

- 22 045226 containing the antibody, and additional solvent can be added to the solution to the desired percentage. The chelator can be added in a molar excess relative to the antibody, for example, 3.5-5:1 chelator to antibody. The total reaction volume can be up to 5 liters.

Reaction temperature and reaction time are inversely related. For example, if the reaction temperature is higher, the reaction time is shorter. If the reaction temperature is lower, the reaction time is higher. By way of illustration, at temperatures above about 18°C the reaction may take less than 2 hours; at temperatures below 18°C the reaction may take more than 2 hours.

The conjugation reaction can be terminated by quenching, for example by adding acetic acid.

In some embodiments, the antibody is conjugated with deferoxamine to produce DFO-mAb conjugates. In some embodiments, the antibody is conjugated with p-SCN-Bn-deferoxamine to produce DFO-mAb conjugates.

Exemplary chelator solvents include DMSO and DMA. Subsequent UFDF steps use membranes, and the membrane is selected based on the solvent system used in the conjugation step. For example, DMA dissolves PES membranes, so they cannot be used in the same system.

Carbonate buffers are not preferred for conjugate stability during long-term storage. Thus, once antibody-chelator conjugates are formed, they can be replaced with a buffer selected specifically for long-term storage and stability. Exemplary buffers include citrate, acetate, phosphate, arginine and histidine buffers. An additional UFDF step may be performed to remove residual salts and to ensure a suitable concentration, excipient level, and pH of the conjugated monoclonal antibody. The resulting antibody-chelator conjugates can be sterile filtered and stored for later formulation.

III. Methods for using radiolabeled immunoconjugates.

In some aspects, the present disclosure relates to diagnostic and therapeutic methods of using radiolabeled antibody conjugates in accordance with the present disclosure.

In one aspect, the present disclosure provides methods for detecting PD-L1 in tissue, the methods comprising administering a radiolabeled antibody conjugate provided herein to the tissue and visualizing PD-L1 expression using positron emission tomography (PET) imaging. In some embodiments, the tissue comprises cells or cell lines. In some embodiments, the tissue is present in a subject, wherein the subject is a mammal. In some embodiments, the subject is a human subject. In some embodiments, the subject has a disease or disorder selected from the group consisting of a cancer, an infectious disease, and an inflammatory disease. In one embodiment, the subject has a malignant tumor. In some embodiments, the infectious disease is a bacterial or viral infection caused by, for example, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Mycobacterium tuberculosis.

In one aspect, the present disclosure provides methods for imaging tissue that expresses PD-L1, comprising administering a radiolabeled antibody conjugate according to the present disclosure to the tissue and visualizing PD-L1 expression using positron emission tomography (PET) imaging. In one embodiment, the tissue is contained within a tumor. In one embodiment, the tissue is contained in a culture of tumor cells or a tumor cell line. In one embodiment, the tissue is contained in a tumor lesion in a subject.

In one aspect, the present disclosure relates to methods for measuring response to therapy, wherein response to therapy is measured by measuring inflammation. The methods of this aspect involve administering a radiolabeled antibody conjugate provided herein to a subject in need thereof and visualizing PD-L1 expression using positron emission tomography (PET) imaging. In some embodiments, inflammation is present in a tumor in a subject. In some embodiments, increased PD-L1 expression correlates with increased inflammation in the tumor.

In one aspect, the present disclosure relates to methods for determining whether a patient is suitable for an anticancer therapy comprising an inhibitor of the PD-1/PD-L1 signaling axis, the methods comprising selecting a patient with a solid tumor, administering a radiolabeled antibody conjugate according to with the present disclosure and visualization of the localization of the administered radiolabeled antibody conjugate in the tumor using PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor identifies the patient as suitable for anticancer therapy including a PD-1 signaling axis inhibitor /PD-L1.

In one aspect, the present disclosure relates to methods for identifying a candidate for anticancer therapy comprising an inhibitor of the PD-1/PD-L1 signaling axis, the methods comprising selecting a patient with a solid tumor, administering a radiolabeled antibody conjugate in accordance with the present disclosure, and visualization of the localization of the administered radiolabeled antibody conjugate in the tumor using PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor identifies the patient as suitable for anticancer therapy, including an inhibitor of the PD-1/PDL1 signaling axis.

In one aspect, the present disclosure relates to methods for predicting the response of a patient to an anticancer therapy comprising an inhibitor of the PD-1/PD-L1 signaling axis, the methods comprising selecting a patient with a solid tumor, determining whether the tumor is PD-LI positive , and a positive response of the patient is predicted if the tumor is PD-LI positive. In some embodiments, a tumor is determined to be positive by administering a radiolabeled antibody conjugate in accordance with the present disclosure and visualizing the localization of the radiolabeled antibody conjugate in the tumor using PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is PD-LI positive.

In one aspect, the present disclosure relates to methods for detecting a PD-LI positive tumor in a subject. The methods of this aspect include selecting a subject with a solid tumor; administering a radiolabeled antibody conjugate according to the present disclosure to a subject and localizing the radiolabeled antibody conjugate by PET imaging, wherein the presence of the radiolabeled antibody conjugate in a tumor indicates that the tumor is PD-LI positive.

As used herein, subject in need means a human or non-human mammal that exhibits one or more symptoms or signs of a cancer, and/or has been diagnosed with a cancer, including a solid tumor, and is in need of treatment. her. In many embodiments, the term subject may be used interchangeably with the term patient. For example, a human subject may be diagnosed with a primary or metastatic tumor and/or one or more symptoms or indications, including, but not limited to, unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, night sweats, bone pain, shortness of breath, bloating, chest pain/pressure, enlarged spleen, and increased levels of a cancer-related biomarker (eg, CA125). This expression includes subjects with primary or established tumors. In specific embodiments, the expression includes human subjects who have a solid tumor and/or are in need of treatment for a solid tumor, for example, colon cancer, breast cancer, lung cancer, prostate cancer, skin cancer, liver cancer, bone cancer , ovarian cancer, cervical cancer, pancreatic cancer, head and neck cancer and brain cancer. The term includes subjects with primary or metastatic tumors (advanced malignancies). In some embodiments, the term "subject in need" includes patients with a solid tumor that is resistant or refractory to treatment or inadequately controlled by prior therapy (eg, treatment with an anticancer agent). For example, the expression includes subjects who have been treated with one or more lines of prior therapy, such as treatment with chemotherapy (eg, carboplatin or docetaxel). In some embodiments, the term subject in need includes patients with a solid tumor who have been treated with one or more lines of prior therapy but have subsequently relapsed or metastasized. In some embodiments, the term includes subjects having an inflammatory disease or disorder, including, but not limited to, cancer, rheumatoid arthritis, atherosclerosis, periodontitis, hay fever, heart disease, coronary artery disease, infectious disease, bronchitis, dermatitis, meningitis, asthma, tuberculosis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, hepatitis, sinusitis, psoriasis, allergies, fibrosis, lupus, vesiculitis, ankylosing spondylitis, Graves' disease, celiac disease, fibromyalgia and transplant rejection.

In some embodiments, the methods of the present disclosure are used in a subject with a solid tumor. The terms tumor, cancer and malignant tumor are used interchangeably herein. As used herein, the term solid tumor refers to an abnormal mass of tissue that typically does not contain cysts or areas of fluid. Solid tumors can be benign (non-cancerous) or malignant (cancerous). For purposes of the present disclosure, the term solid tumor means malignant solid tumors. The term includes various types of solid tumors, named for the type of cells that form them, namely sarcomas, carcinomas and lymphomas. According to some options

- 24 045226 implementation of the term solid tumor includes malignant tumors, including without limitation colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer , colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma.

In one aspect, the present disclosure relates to methods of treating a tumor in a subject. The methods of this aspect include selecting a subject with a solid tumor; determining that the tumor is PD-LI positive; and administering one or more doses of a PD-1/PD-L1 signaling axis inhibitor.

In some embodiments, a tumor is determined to be PD-LI positive by administering a radiolabeled antibody conjugate according to the present disclosure to a subject and visualizing the radiolabeled antibody conjugate in the tumor using PET imaging, wherein the presence of a radiolabeled antibody conjugate in the tumor indicates that the tumor is PD-LI positive.

As used herein, the terms treat, treat or the like. means relieving symptoms, eliminating the cause of symptoms either temporarily or permanently, delaying or inhibiting tumor growth, reducing tumor cell load or tumor burden, causing tumor regression, causing tumor shrinkage, necrosis and/or disappearance, preventing tumor recurrence, preventing or inhibiting metastasis, inhibiting the growth of a metastatic tumor and/or increasing the survival time of the subject.

In one aspect, the present disclosure relates to methods for monitoring the effectiveness of an anticancer therapy in a subject, the methods comprising selecting a subject with a solid tumor, wherein the subject is receiving treatment with an anticancer therapy; administering a radiolabeled antibody conjugate according to the present disclosure to a subject; visualization of the localization of the injected radiolabeled conjugate in the tumor using PET imaging; and determining tumor growth, whereby a decrease from baseline in the radiolabeled signal indicates tumor regression and effectiveness of anticancer therapy. In some embodiments, the anticancer therapy includes an inhibitor of the PD-1/PD-L1 signaling axis (eg, an anti-PD-1 antibody).

In some embodiments, the present disclosure provides methods for assessing changes in the inflammatory state of a tumor, the methods comprising selecting a subject with a solid tumor, wherein the subject is receiving treatment with anticancer therapy; administering a radiolabeled antibody conjugate provided herein to a subject and visualizing the localization of the administered radiolabeled conjugate in the tumor using PET imaging, wherein an increase from baseline in the radiolabeled signal indicates increased inflammation and the effectiveness of anticancer therapy. In some embodiments, the anticancer therapy includes an inhibitor of the PD-1/PD-L1 signaling axis (eg, an anti-PD-1 antibody).

As used herein, the term baseline in relation to tumor PD-L1 expression means the numerical value of uptake of the radiolabeled conjugate in a subject before or during dosing of anticancer therapy. The uptake of the radiolabeled conjugate is determined using methods known in the art (see, for example, Oosting et al. 2015, J. Nucl. Med. 56: 63-69). In some embodiments, the anticancer therapy includes an inhibitor of the PD-1/PD-L1 signaling axis.

To determine the presence of tumor regression, uptake of the radiolabeled conjugate is quantified at the baseline visit and at one or more time points after administration of a PD-1/PD-L1 signaling axis inhibitor (eg, anti-PD-1 antibody). For example, the uptake of an administered radiolabeled antibody conjugate (eg, a radiolabeled anti-PD-L1 antibody conjugate) can be measured on days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14 , 15, 22, 25, 29, 36, 43, 50, 57, 64, 71, 85 or at the end of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or later after initiation of treatment with a PD-1/PD-L1 signaling axis inhibitor (eg, anti-PD-1 antibody). The difference between the uptake value at a specific time point after the start of treatment and the uptake value at the baseline visit is used to determine whether there was a difference in the amount of tumor tissue (tumor regression or progression). For example, a decrease from baseline in uptake upon treatment with at least one dose of a PD-1/PD-L1 signaling axis inhibitor signifies tumor regression and indicates the effectiveness of anticancer therapy.

In some embodiments, the radiolabeled antibody conjugate is administered to a subject intravenously or subcutaneously. In some embodiments, the radiolabeled antibody conjugate is administered intratumorally. After administration, the radiolabeled antibody conjugate is localized to the tumor. The localized radiolabeled antibody conjugate is visualized using PET imaging, and tumor uptake of the radiolabeled antibody conjugate is measured by methods known in the art.

In some embodiments, imaging is performed 1, 2, 3, 4, 5, 6, or 7 days after administration of the radiolabeled conjugate. In some embodiments, imaging is performed on the same day after administration of the radiolabeled antibody conjugate.

In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PD-L1. In some embodiments, the anti-PD-L1 antibody comprises a CDR of HCVR, wherein HCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 34, 50, 82, 98, 146, 162, 178, 186, 234, 250, 290, 306, 314 and 330; and a CDR of LCVR, wherein LCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 42, 58, 90, 106, 154, 170, 194, 242, 258 and 274.

In some embodiments, the PD-1/PD-L1 signaling axis inhibitor comprises an antibody or antigen-binding fragment thereof that specifically binds to PD-1. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and REGN2810. In some other embodiments, the PD-1/PD-L1 signaling axis inhibitor comprises an antibody or antigen binding fragment thereof that specifically binds PD-L1. In one embodiment, the anti-PD-L1 antibody is atezolizumab. In one embodiment, the anti-PD-L1 antibody comprises HCVR according to SEQ ID NO: 82 and LCVR according to SEQ ID NO: 90.

IV. Examples.

Certain embodiments of the present disclosure are illustrated by the following non-limiting examples.

Example 1: Generation of human antibodies against PD-L1.

Human antibodies against PD-L1, including those shown in table. 1 were prepared and characterized as described in US Patent Publication No. US 2015-0203580 A1, which is incorporated herein by reference in its entirety. Briefly, human anti-PD-L1 antibodies were generated using a fragment of PD-L1 that varies over approximately 19,239 amino acids of PD-L1 (Genbank accession number NP_054862.1). The immunogen was administered directly, with an adjuvant to stimulate the immune response, to VELOCIMMUNE® mice containing DNA encoding the variable regions of the heavy and kappa light chains of human immunoglobulin. The antibody-mediated immune response was monitored using a PD-L1-specific immunoassay. When the desired immune response was achieved, the splenocytes were collected and fused with mouse myeloma cells to maintain their viability and form a hybridoma cell line. Hybridoma cell lines were screened and selected to identify cell lines that produce PD-L1-specific antibodies. Using this technique and the immunogen described above, several anti-PD-L1 chimeric antibodies (ie, antibodies having human variable domains and murine constant domains) were produced; exemplary antibodies generated by this method are designated H2M8306N, H2M8307N, H2M8309N, H2M8310N, H2M8312N, H2M8314N, H2M8316N, H2M8317N, H2M8321N, H2M8323N, H2M8718N, H2M8718N 2 and H2M8719N.

Anti-PD-L1 antibodies have also been isolated directly from antigen-positive B cells without fusion with myeloma cells, as described in US Patent No. US 2007/0280945A1, specifically incorporated herein by reference in its entirety. Using this method, several full human antibodies against PD-L1 (ie, antibodies having human variable domains and human constant domains) were obtained; exemplary antibodies generated by this method are designated H1H9323P, H1H9327P, H1H9329P, H1H9336P, H1H9344P2, H1H9345P2, H1H9351P2, H1H9354P2, H1H9364P2, H1H9373P2, H1H9382P2, H 1H9387P2 and H1H9396P2.

Example 2: Conjugation of anti-PD-L1 antibody H4H8314N to p-SCN-Bn-DFO.

To modify the parent anti-PD-L1 antibody H4H8314N and the isotype control antibody to be suitable for radiolabeled immuno-PET studies, the chelator p-SCN-bn-deferochamine (DFO; Macrocylics, cat. no. B-) was added to the antibody. 705).

To modify H4H8314N, the histidine buffer was first exchanged to PBS, Ph 7.2, by dialysis at 4°C overnight (Slide-A-Lyzer Dialysis Cassette G2 10k MWCO; ThermoScientific), then the buffer was exchanged again using a PD-10 column ( GE Healthcare, Cat. No. 17-0851-01) into a buffer consisting of 50 mM carbonate buffer, 150 mM NaCl, pH 9.0 (conjugation buffer). To determine the concentration after buffer exchange, samples were measured on a Nanodrop 2000 UV/VIS spectrometer (Thermo Scientific) using a MacVector sequence-based extinction coefficient of 1.46 g/L (see Table 2). In a 15 mL polyethylene tube, 773.9 μL of H4H8314N (12.5 mg) was added to 1676.1 μL of conjugation buffer. In a separate vial, 29.3 μL of DMSO was added to 20.7 μL of DFO. This DFO solution was added one-quarter at a time to the H1H8314N solution, stirring gently with a pipette up and down each time. The final solution was 5 mg/ml H4H8314N in conjugation buffer, 2% DMSO with a 6-fold molar excess of DFO. This solution was allowed to incubate in a water bath at 37°C without further stirring.

After 30 min at 37°C, the solution was rapidly passed through a PD-10 desalting column (GE Healthcare, part no. 17-0851-01) pre-equilibrated with a buffer containing 250 mM NaAcO at pH 5.4 (formulation buffer). The final solution was sterile filtered through a syringe filter (13 mm Acrodisc syringe filter, Pall Corporation, catalog no. 4602). The concentration and DFO to antibody ratio (DAR) were then measured using UV/VIS spectroscopy. To measure absorbance, DFO-conjugated antibody was measured against formulation buffer at 252 nm (A252), 280 nm (A280), and 600 nm (A600). For calculations, the background was corrected at each absorbance value using the equation:

Αχ = A ~ Aoo.

The antibody conjugate was tested for aggregation using SEC chromatography, with 25 μg of sample applied to a Superdex 200 column (GE Healthcare, catalog no. 17-5175-01), monitored at 280 nm using a PBS mobile phase (0.75 ml/ min). Antibody integrity was assessed using 4-20% Tris/Gly SDS-PAGE ready gel (Novex) with 2 μg of sample loaded. The gel is shown in Fig. 1. Antibody concentration, conjugate concentration and DAR were calculated using the equations below:

antibody concentration calculation

Conc. inAb (mi/ml) — —— calculation of conjugate concentration

END CONUS (MiMil .. — —— — DAR calculation

DAR =

625274-280 - £280^252

ШОА^ - 28700A' _28G

table 2

Molar extinction coefficients and molar mass

Antibody MW (g mol' ¹ ) ε280 (l Hem' ¹ ) ε252 (l g' ¹ cm' ¹ ) H4H8314N 144984 1.46 0.553

Table 3

UV DAR, percentage of aggregate and concentration after DFO attachment

Antibody UV DAR Concentration (mg/ml) % of aggregate Η4Η8314Ν 1.2 3.34 <1%

Example 3: Chelation of ⁸⁹ Zr DFO-Conjugated Monoclonal Antibody.

For use in in vivo immuno-PET studies, the DFO-conjugated anti-PD-L1 antibody, H4H8314N, and the DFO-conjugated isotype control antibody were radiolabeled with ⁸⁹ Zr.

DFO-conjugated antibody (250 or 750 μg) was first adjusted to 1.25 mg/ml in 1 M HEPES, pH 7.2. The composition of the DFO-Ab conjugate solution for each study is given in Table. 4. Separately, a solution ^{of 89} Zr was prepared using the composition for each respective study shown in Table. 5. Stock solution ⁸⁹ Zr-oxalic acid was obtained from PerkinElmer or 3D Imaging. If the radioactivity concentration of the mother solution was low (see Table 5), then a neutralization step was performed using 1 M borate, pH 9.0. The final radioactivity of the solution was first confirmed using a Capintec CRC-25R dose calibrator (Capintec, no. 520), then immediately combined with the DFO-Ab conjugate solution, mixed gently (pipetted), and then incubated for 45 min at room temperature.

After incubation, a small sample of each reaction mixture was taken for iTLC (instant thin layer liquid chromatography) to determine radiolabeling of the reaction output, and the remaining reaction mixtures were transferred to pre-equilibrated PD-10 columns (Vendor) with 250 mM sodium acetate at pH 5.4 for desalting with gravity feed. Each PD-10 column held no more than 1.2 mL of reaction mixture (otherwise multiple columns were used). After the contents of the reaction mixture entered the column bed, 1.6 ml of 250 mM sodium acetate at pH 5.4 (formulation buffer) was added; the flow-through fraction was discarded. An additional 1.8 mL of formulation buffer was added to the column and the eluate was collected from each column. Approximately 500 µl of each solution was then analyzed using a Nanodrop spectrophotometer (ThermoScientific). The final Ab concentration was calculated using the corresponding extinction coefficient and absorbance at 280 nm using the equation: concentration in mg/mL = absorbance at 280 nm - extinction coefficient at 280 nm (found in Table 6).

The final mass, measured in grams, is recorded in the table. 4. Then the radioactivity was measured using a dose calibrator and presented in table. 5. The final material, along with the material prior to PD-10 column treatment, was then analyzed by iTLC. For this analysis, 1 μL of each solution was added to iTLC-SG-Glass microfiber chromatography paper impregnated with silica gel (Agilent Technologies, part no. SG10001) processed in a TLC chamber with 20 mM citric acid buffer solution. The final material was also analyzed using SECHPLC with UV 280 and a radioisotope detector coupled in series (Agilent 1260 with Lablogic Radio-TLC/HPLC Detector, SCAN-RAM), using a Superdex 200 column with a PBS mobile phase at a flow rate of 0.75 ml/ min. Radiotrace was used to determine radiochemical purity by comparing the integration of the protein peak (~10-16 min) and the free ⁸⁹ Zr peak (~25 min). Monomer purity was determined by comparing the integration of the oligomeric peak (10 min to ∼15 min) with the monomer (∼16 min).

The specific activity and protein recovery (%) of each radiolabeled conjugate were determined using the following equations:

a) mass of conjugate in mg = concentration in mg/ml x mass of solution in grams;

b) specific activity in mCi/mg = vessel activity in mCi - mass of conjugate in mg;

c) protein recovery = initial mass of conjugate (mg) - mass of conjugate in mg.

Finally, the appearance was noted and recorded in the table. 7. Both UV280 and iTLC labeling were determined on the purified product.

The results are shown in table. 7. Radio-SEC-HPLC chromatograms are shown in FIG. 2-4. An example SEC UV280 HPLC and radio-iTLC chromatogram is shown in FIG. 5 for ⁸⁹ Zr radiolabeling, study 1. SEC UV280-HPLC chromatogram confirms high monomeric product (99%). The radio-iTLC trace was processed using a 7-point binomial smoothing function. The onset and solvent front were approximately 16 and 100 mm, respectively. No detectable ⁸⁹ Zr was detected beyond 22 mm, confirming the radiochemical purity determined by SEC radio-SEC-HPLC in FIG. 2B.

Table 4

Receipt. DFO-antibody conjugate for radioactive labeling

No. of radioactive labeling Study No. Radiolabeled batches Concentration (mg/ml) DA R* Weight of conjugate a (mg) Total volume (µl) Final concentration (mg/ml) 1 1 Isotype DFO- ⁸⁹ Zr 3.7 1.6 250 200 1.25 2 1 H4H8314N- DFO- ⁸⁹ Zr 3.34 1.2 250 200 1.25 3 2 H4H8314N- DFO- ⁸⁹ Zr 3.34 1.2 750 600 1.25 4 3 Isotype DFO- ⁸⁹ Zr 3.7 1.6 250 200 1.25 5 3 H4H8314N- DFO- ⁸⁹ Zr 3.34 1.2 250 200 1.25

*DAR is defined as the ratio of DFO to antibody.

Table 5

Preparation of ⁸⁹ Zr reaction solution for radioactive labeling

Tagging for active marking Study no. Batches of radioactive labeling ⁸⁹ Zr oxa lat (µl) Additional addition of 1 M oxalic acid (µl) Addition of 1 M borate , pH 9.0 (µl) 1 m NERE S, pH 7.2 (µl) Final volume (µL) Final activity (µCi) Specific activity (µCi/µl) 1 1 Isotype DFO- ⁸⁹ Zr 50 50 400 500 1000 1009 1.01

- 28 045226

2 1 Н4Н8314 N-DFO ⁸⁹ Zr 50 50 400 500 1000 1000 1 3 2 Н4Н8314 N-DFO ⁸⁹ Zr 150 150 1200 1500 3000 3070 1.02 4 3 Isotype DFO- ⁸⁹ Zr ~1 0 0 1000 1000 1680 1.68 5 3 Н4Н8314 N-DFO ⁸⁹ Zr ~1 0 0 1000 1000 1640 1.64

Table 6

Extinction coefficients for batches of conjugates

Radiolabeling batch £280 (AU ML MT ¹ CM' ¹ ) Isotype-DF O- ⁸⁹ Zr 1.71 H4H8314N-DFO- ⁸⁹ Zr 1.61

Table 7

Brief description of ⁸⁹ Zr labeled DFO-Ab conjugates for in vivo imaging and ____________ biodistribution studies_______________

Labeling with a radioactive label Research No. Conjugato batches Appearance Radiokh imicheka purity a* (%) Monodimensional I am pure* (%) Protein recovery (%) Concentration (mg/ml) Specific active value (mCi/mg) 1 1 Isotype DFO- ⁸⁹ Zr Transparent >99% >95% 60% 0.106 3.35 2 1 H4H8341N- DFO- ⁸⁹ Zr Transparent >99% >95% 63% 0.121 2.75 3 2 H4H8341N- DFO- ⁸⁹ Zr Transparent >99% >95% 62% 0.134 3.58 4 3 Isotype DFO- ⁸⁹ Zr Transparent >99% >95% 66% 0.074 5.38 5 3 H4H8341N- DFO- ⁸⁹ Zr Transparent >99% >95% 74% 0.084 5.13

* via radio-SEC-HPLC.

Example 4. Immunoreactivity.

Immunoreactivity (IR) of radiolabeled anti-PD-L1 antibody and isotype control antibody was measured as follows. C3 8-cOVA/eGFP-mPD-L 1 ^-/- hPD-L 1 'g cells were used for initial studies, and then LOX-IMVI cells (see example 5 for detailed description of cell lines) were also used in a further study. In these assays, 20 ng of the corresponding ⁸⁹ Zr-labeled antibodies were added to 15x106 MC38-cOVA/eGFP-mPD-L1 ^-/- hPD-L1 ^Tg cells or 30x106 LOX-IMVI cells in a final volume of 1 ml. Samples were incubated for 45 min with continuous agitation and then subjected to 3 washes with media to remove any unbound antibody. The radioactivity of the test cell masses was then counted using an automatic gamma counter (Wizard 2470, Perkin Elmer) against 2 reference standards containing the same 20 ng of ⁸⁹ Zr-labeled antibody. Percentage immunoreactivity was determined for samples using the average of the standards as a measure of total activity.

As can be seen from table. 8, ⁸⁹ Zr labeled anti-PD-L1 antibody remained immunoreactive after conjugation and radiolabeling, with %IR varying from 88 to 98% across studies. Binding specificity is evident with control antibodies having a baseline %IR of less than 1%.

- 29 045226

Table 8

Immunoreactivity of DFO-conjugates with chelated ⁸⁹ Zr

Study Study 1 Study 2 Study 3 Cell line MC38-cOVA/eGFPmPD-LlVPD-LV MC38-cOVA/eGFPmPD-LlXiPD-LV MC38-cOVA/eGFPmPD-LlXiPD-LV LOX-IMVI Antibody ⁸⁹ Antibody against PD-L1 ⁸⁹ Z-control ⁸⁹ Antibody against PD-L1 ⁸⁹ Z-control ⁸⁹ Antibody against PD-L1 ⁸⁹ Z-control ⁸⁹ Anti LO against PD-L1 ⁸⁹ Zrcont role Cell mass activity 4048.4 29.6 8311.9 No data 6262.4 68 5587.5 4 65.4 Average standard activity 4536.5 6432.4 8567.2 No data 6386.6 9544.8 6386.6 9544.8 Percent IR 89.2 0.5 97.0 No data 98.1 0.7 87.5 0.7

Example 5 In Vitro and Ex Vivo Characterization of Human PD-L1 Expression in Tumor Cell Lines Several tumor cell lines were examined to evaluate the level of human PD-L1 expression to identify human PD-L1 endogenously expressed by tumors in vivo in male NCr nude mice (Taconic, Hudson NY) or in mice modified to homozygosity to express human PD-L1 extracellular domain instead of mouse PD-L1 extracellular domain (PDL1 HumIn mice) on a 75% C57/B16/25% 129 strain background using VelociGene technology ® (Valenzuela et al. 2003, Nat. Biotechnol. 21: 652-659; US Patent Application Publication No. US 2016/0157469).

Cell lines used in this study include: 1) the mouse colon carcinoma cell line MC38 (obtained from the NCI Frederick, MD, Laboratory of Tumor Immunology and Biology), which was engineered to knock out mouse PD-L1 but overexpresses full-length human PD-L1 and full-length chicken ovalbumin fused to eGFP, thus referred to herein as MC38-cOVA/eGFP-mPD-L1- ^/ -hPD-L1 ^Tg ); 2) several lines of human tumor cells: human melanoma cell line LOX-IMVI (endogenous PD-L1 positive line obtained from NCI at Frederick, MD, Division of Cancer Treatment and Diagnosis, Tumor Repository), human breast cancer cell lines MDA-MB -231 (endogenous PD-L1 positive cell line) and SK-Br-3 (PD-L1 negative cell line) (both obtained from ATCC). In some cases, human PD-L1 was assessed directly without any induction in vitro; in some cases, human PD-L1 expression was assessed by overnight treatment with mouse or human IFNy (100 ng/ml) (obtained from Peprotech); in some cases, human PD-L1 was assessed ex vivo on enzymatically dissociated tumor cells isolated from tumor-bearing nude mice or humanized mice. All surface staining for human PD-L1 was performed according to a standard protocol. Briefly, tumor cells were washed once with PBS, washed once with ice-cold staining buffer, stained with commercially available directly fluorochrome-conjugated anti-human PD-L1 antibody (eBioscience, clone MIH1) in staining buffer for 30 min on ice in the dark, and then washed again with 2 ml PBS. The elimination dye eFluor506 was also included according to the manufacturer's protocol (eBioscience, catalog no. 17-5983). Samples were acquired on a BD FACSCanto II™ IVD10 equipped with DIVA v8. Data were further analyzed using FlowJo v10.0.6 or higher.

Expression of PD-L1 by MC38-cOVA/eGFP-mPD-L1- ^/- hPD-L1 ^Tg cells before implantation and several days after implantation in nude mice is shown in Table 1. 9.

Table 9

Percentage of human PD-L1 positive MC38-cOVA/eGFP-mPD-L1- ^/ -hPD-L1 ^Tg cells before implantation and 7 days after implantation in nude mice

Isotype staining hPD-Ll staining Before implantation 0.6% 94.7% After implantation 1.09% 74.0%

Before implantation, the vast majority of MC38-cOVA/eGFP-mPD-L1- ^/- hPD-L1 ^Tg cells were positive for human PD-L1 compared to isotype control staining. Several days after implantation in nude mice and after enzymatic and mechanical treatment to dissociate the tumor, -70% of MC38-cOVA/eGFP-mPD-L1' ^/ 'hPD-L1 ^Tg cells were still positive for human PD-L1.

Expression of PD-L1 by MC38-cOVA/eGFP-mPD-L1' ^/ 'hPD-L1 ^Tg cells before implantation and fourteen days after implantation in PD-L1 humanized mice is shown in Table 1. 10.

Table 10

Percentage of human PD-L1 positive MC38-cOVA/eGFP-mPD-L1' ^/ 'hPD-L1 ^Tg cells before implantation and 14 days after implantation in PD-L1 humanized mice

Isotype staining hPD-Ll staining Before implantation 0.2% 92.5% After implantation 3.6 46.2%

Before implantation, the vast majority of MC38-cOVA/eGFP-mPD-L1 ^-/ 'hPD-L1 ^Tg cells were positive for human PD-L1 compared to isotype control staining. Fourteen days after implantation in PD-1/PD-L1 doubly humanized mice and after enzymatic and mechanical treatments to dissociate the tumor, ~50% of MC38-cOVA/eGFP-mPD-L1 ^/- hPD-L1 ^Tg cells were still PD positive -L1 person.

Expression of PD-L1 by multiple tumor cell lines in vitro is shown in FIG. 6. To assess how comparable the level of PD-L1 expression is using an engineered cell line (MC38-cOVA/eGFP-mPD-L1 ^{- /} 'hPD-L1 ^Tg ) and other human tumor cell lines (melanoma cells LOX-IMVI, MDA-MB-231 breast cancer cells and SK-Br-3 breast cancer cells) dose titration of anti-PD-L1 antibody staining was performed. In fig. Figure 6 shows that MC38-cOVA/eGFP-mPD-L1 ^-/ 'hPD-L1 ^Tg cells had the highest level of human PD-L1 expression (Figure 6A), and SK-Br-3 cells had the lowest expression without detecting PD -L1 (Figure 6D), whereas PD-L1 expression by LOX-IMVI and MDA-MB-231 cells was moderate (approximately 5-fold lower than in MC38-cOVA/eGFP-mPD-L1 ^-/ 'hPD- cells L1 ^Tg ) (Figs. 6B and 6C).

In the second experiment, further comparison between LOX-IMVI and MC38-cOVA/eGFPmPD-Lr ^/- hPD-L1 ^Tg cells was performed with or without in vitro treatment with 100 ng/ml hIFNy/mIFNy overnight, respectively. In fig. Figure 7 shows that the average fluorescence intensity of PD-L1 reached a plateau at ~150 nM anti-PD-L1 antibody used for staining. At baseline, PD-L1 expression by LOX-IMVI cells was moderate (approximately 6-7 times lower than that of MC38-cOVA/eGFP-mPD-L1 ^-/ 'hPD-L1 ^Tg cells). After mIFNy treatment, no change in PD-L1 staining was observed in MC38-cOVA/eGFP-mPD-L1 ^-/ 'hPD-L1 ^Tg cells, whereas a 3-fold increase in human PD-L1 staining was observed in LOX-IMVI cells after treatment with hIFNy.

Ex vivo expression of PD-L1 by LOX-IMVI and MC38-cOVA/eGFP-mPD-L1 ^-/ 'hPD-L1 ^Tg cells approximately three weeks after implantation in nude mice was shown in Table 1. 11 and 12.

Table 11

Percentage of PD-L1 positive LOX-IMVI and MC38-cOVA/eGFP-mPD-L1 ^-/ 'hPD-L1 ^{Tg cells} ~3 weeks after implantation in nude mice

Isotype staining hPD-Ll staining LOX-IMVI 0.2% 56.6% MS 3 8-cO VA/eGFP-mPD-L 1 OTPD-L 1 ^Tg 0.2% 96.2%

Table 12

Average PD-L1 fluorescence intensity in LOX-IMVI and MC38-cOVA/eGFP-mPD-L1' ^/ 'hPDL1 ^Tg cells ~3 weeks post-implantation in nude mice

Tumor 1 Tumor 2 LOX-IMVI 8479.1 12121.5 MSZ 8-сО VA/eGFP-mPD-L 1 APD-L 1 ^Tg 49589.1 51445.0

After enzymatic and mechanical treatment to ensure tumor dissociation, cells were stained with anti-PD-L1 antibody (20 μg/ml). The expression level of PD-L1 in LOX-IMVI cells was approximately 5-fold lower than that in MC38-cOVA/eGFP-mPD-L1' ^/ 'hPD-L1 ^Tg tumor cells.

Example 6 Selective localization of radiolabeled anti-PD-L1 antibody in hPD-L1 positive tumors in nude mice.

To determine the in vivo localization of anti-PD-L1 antibody, a zirconium-89 labeled DFO-antibody conjugate was administered intravenously to nude mice bearing PD-L1 positive tumors.

The tumor line used for the study was a mouse colon carcinoma cell line called MC38-cOVA/eGFP-mPD-L1 ^-/ 'hPD-L1 ^Tg , which was engineered to knock out mouse PD-L1 from wild-type MC38 but overexpressed full size PD- 31 045226

Human L1 and full-length chicken ovalbumin fused to eGFP. For a second study of tumors with endogenous human PD-L1 expression, the human melanoma cell line LOX-IMVI was used to establish in vivo tumors for subsequent antibody localization studies against

PD-L1.

An exemplary radiolabeled anti-PD-L1 antibody used for this study was H1H8314N, comprising HCVR/LCVR of SEQ ID NO: 82/90.

For the first study, 1 × 10 ⁶ MC38-cOVA/eGFP-mPD-L1-/-hPD-L1 ^Tg cells were implanted subcutaneously in the left flank of 8-10 week old male NCr nude mice (Taconic, Hudson NY). For LOX-IMVI tumors, 1 x ¹⁰⁶ cells were implanted subcutaneously into the left flank of 8-10 week old male NCr nude mice. Once tumors reached a mean volume of 50-150 mm ³ (days ~7-10), mice were randomized to receive either an ⁸⁹ Zr-labeled DFO-anti-PD-L1 conjugate (H1H8314N) or ^{an 89} Zr-labeled non-binding isotype control DFO-antibody conjugate. Nude mice bearing MC38-cOVA/eGFP-mPD-L1-/-hPD-L1 ^Tg tumors received 50 ± 1 μCi of ⁸⁹ Zr-labeled antibody with a protein dose of ~0.6 mg/kg. In a study using mice bearing LOXIMVI tumors, mice received 35 ± 1 μCi of ⁸⁹ Zr-labeled antibody with a final antibody dose of 0.3 or 1 mg/kg.

PET imaging of antibody localization was assessed 6 days after antibody administration. A Sofie Biosciences G8 PET/CT (Sofie Biosciences and Perkin Elmer) was used for imaging. The instrument was pre-calibrated to detect ⁸⁹ Zr before imaging. The energy window varied from 150 to 650 keV with a recovered resolution of 1.4 mm at the center of the field of view. Mice were inductively anesthetized using isoflurane and kept under a continuous flow of isoflurane during imaging. Static 10-min images were acquired using G8 software and then reconstructed using preconfigured settings. The image data were corrected for decay and other parameters. CT images were obtained after PET scanning and then co-registered with the PET images. Images were prepared using VivoQuant post-processing software (inviCRO Imaging Services).

For biodistribution, mice were euthanized at the final time point (5-6 days post-dose) and blood was collected by cardiac puncture. Tumors and normal tissues were then excised and placed in counting tubes. The mass for each sample was measured and recorded. Count data for ⁸⁹ Zr in CPM were then collected by measuring samples on an automatic gamma counter (Wizard 2470, Perkin Elmer). The percent injected dose per gram (% ID/g) was calculated for each sample using standards prepared from the injected material.

The average % ID/g for each antibody is presented in Table. 13.

Table 13

Average % ID/g in analyzed tissues

Sample ⁸⁹ Ζγ-Η1Η8314Ν ⁸⁹ 2g-antibody isotype control Average ID/r % Art. deviation %ID/r Average %ID/r Art. deviation %ID/r Liver 3.1 0.4 0.9 0.9 Spleen 4.4 1.1 1.5 1.3 Bud 4.0 0.7 1.4 1.6 Bone 5.1 2.6 1.7 1.6 Lung 5.1 1.1 2.5 h,o Heart 2.4 0.2 1.3 1.4 Blood 7.6 1.6 3.8 4.6 Thymus 5.3 3.0 2.8 2.2 MC38-cOVA/eGFPmPD-Ll+hPD-Ll^ 55.3 12.2 h,o z, z Small intestine 1.5 o, s 0.6 0.6

Based on this, the apparent high uptake in MC38-cOVA/eGFP-mPD-L1-/-hPD-L1 ^Tg tumors was evident compared with other normal tissues, with tumor uptake being 55.3%ID/g, which is significantly exceeded the next highest uptake of 5.3% ID/g observed in the thymus. Uptake in the tumor was 7.3 times and 17.8 times higher than the activity in the blood and liver, respectively. Tumor specificity of anti-PD-L1 antibody uptake (55.3% ID/g) was evident compared with the significantly reduced tumor uptake of 3% observed for the non-binding isotype control antibody. Pilot PET imaging performed in this case demonstrated clear localization of the ⁸⁹ Zr-labeled DFO-antibody conjugate against

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PD-L1 in MC38-cOVA/eGFP-mPD-L1-/-hPD-L1 ^Tg tumors. At the time point on day 6 post-dose, a weak background signal was observed in the animals. In contrast to the clear tumor localization that was evident with the anti-PD-L1 antibody, only weak background activity was visible when imaging with the control antibody in this model. Imaging clearly indicates high specific uptake of anti-PD-L1 antibody in PD-LI-positive human tumor and shows localization of ⁸⁹ Zr radiolabeled anti-PD-L1 antibody in MC38cOVA/eGFP-mPD-L1-/-hPD-L1 ^Tg tumor naked mouse NCr.

The second study assessed the ability of an anti-PD-L1 antibody to selectively target tumors expressing endogenous levels of human PD-L1 antigen. Here, mice bearing human LOX-IMVI melanoma tumors received ⁸⁹ Zr-labeled antibody at doses of 0.3 and 1 mg/kg. Blood, tumor and tissue were collected again on day 6 post-injection and % ID/g was calculated for the samples. The average % ID/g for each antibody is presented in Table. 14.

Table 14

Average value value % ID Sample Liver Spleen Bud Bone Lung Heart Blood Thymus >/g in analysis ⁸⁹ Zr-DFO-I 0.3 mg/kg Average value % ID/r 2.9 4.2 4.3 3.2 5.7 3.2 8.1 5.3 zirovannye τι ΠΗ8314Ν, Art. deviation %ID/r o, s 0.2 0.4 0.6 1.0 0.8 1.4 2.3 < any from v' ⁸⁹ Zr-DFO-I 1 mg/kg Average value % ID/r з, з 4.3 4.3 2.7 6.6 3.2 9.5 5.6 which iss ΠΗ8314Ν, Art. rejected not % ID/r 0.2 0.9 0.8 0.5 1.6 0.4 1.0 0.7 ' research ⁸⁹ Ζγ-Αητηί CONTROLLED isotype, 1 Average value % ID/r 3.9 4.2 3.4 3.6 5.9 2.9 I.1 4.9 i(gelo tumors [OGO mg/kg Art. deviation % ID/r o, s 0.7 0.4 0.4 1.2 0.6 6.2 1.4 LOX-IMVI) LOX-IMVI tumor 20.6 2.7 10.6 2.6 12.0 1.8 Small intestine 1.5 0.2 1.8 0.4 2.0 o, s

At the lower dose of 0.3 mg/kg, clear targeting of the tumor rather than normal tissue was observed, with 20.6% ID/g observed in LOX-IMVI tumors. When mice received the higher dose of 1 mg/kg, a 10.6% ID/g reduction in tumor uptake was observed relative to the 0.3 mg/kg level. This suggests that the higher protein dose and possibly the subsequent higher proportion of unlabeled antibody resulted in blocking tumor uptake of ⁸⁹ Zr labeled anti-PD-L1 antibody. Consistent with this, PET imaging performed immediately prior to the biodistribution study also showed that the uptake of anti-PD-L1 antibody at 1 mg/kg was approximately equivalent to that of control antibody. At the lower dose of 0.3 mg/kg, a clear increase in tumor localization of anti-PD-L1 antibody was evident compared to control antibody. Overall, PET imaging and biodistribution data demonstrate tumor-specific targeting of LOX-IMVI at a dose of 0.3 mg/kg anti-PD-L1 antibody.

Example 7 Selective localization of radiolabeled anti-PD-L1 antibody in hPD-L1 positive tumors in mice.

This example describes the in vivo localization of a zirconium-89-labeled anti-PD-L1 DFO antibody conjugate in PD-L1 humanized mice. An exemplary antibody used in this example was H1H8314N, comprising HCVR/LCVR of SEQ ID NO: 82/90.

PD-L1 humanized mice were modified using VelociGene® technology (Valenzuela et al. 2003, Nat. Biotechnol. 21: 652-659; US Patent Application Publication No. US2016/0157469).

The tumor line used was a mouse colon carcinoma cell line called MC38-cOVA/eGFP-mPD-L1 ^-/- hPD-L1 ^Tg , engineered to express full-length chicken ovalbumin fused to eGFP and to knock out mouse PD-L1 from MC38 wild type but overexpressing full-length human PD-L1.

χ 10 ⁶ MC38-cOVA/eGFP-mPD-L1 ^-/- hPD-L1 ^Tg cells were implanted subcutaneously into the left flank of male PD-L1 humanized mice. Once tumors reached a mean volume of 50-150 mm ³ (day ~7), mice were randomized and dosed with either ⁸⁹ Zr-labeled DFO-anti-PD-L1 conjugate or ⁸⁹ Zr-labeled DFO-non-binding isotype control antibody conjugate . Mice received 50 ± 1 μCi of ⁸⁹ Zr-labeled antibody with a final protein dose of 1 or 3 mg/kg.

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PET imaging of antibody localization was assessed 6 days after antibody administration. A Sofie Biosciences G8 PET/CT (Sofie Biosciences and Perkin Elmer) was used for imaging. The instrument was pre-calibrated to detect ⁸⁹ Zr before imaging. The energy window varied from 150 to 650 keV with a recovered resolution of 1.4 mm at the center of the field of view. Mice were inductively anesthetized using isoflurane and kept under a continuous flow of isoflurane during imaging. Static 10-min images were acquired using G8 software and then reconstructed using preconfigured settings. The image data were corrected for decay and other parameters. CT images were obtained after PET scanning and then co-registered with the PET images. Images were prepared using VivoQuant post-processing software (inviCRO Imaging Services).

For biodistribution, mice were euthanized at the final time point (5-6 days post-dose) and blood was collected by cardiac puncture. Tumors and normal tissues were then excised and placed in counting tubes. The mass for each sample was measured and recorded. Count data for ⁸⁹ Zr in CPM were then collected by measuring samples on an automatic gamma counter (Wizard 2470, Perkin Elmer). The percent injected dose per gram (% ID/g) was calculated for each sample using standards prepared from the injected material.

Results.

PD-L1 humanized mice bearing MC38-cOVA/eGFP-mPD-L1' ^/ 'hPD-L1 ^Tg tumors were treated with ⁸⁹ Zr-labeled DFO-anti-PD-L1 conjugate at a final antibody dose of 1 or 3 mg/kg. Blood, tumor, and tissue were collected and weighed on day 6 post-injection, and % ID/g was calculated for the samples based on the number of counts from each sample. The average value of % ID/g for doses of 1 and 3 mg/kg is presented in table. 15 and table. 16, respectively.

Table 15

Average value % ID/g in an Sample Liver Spleen Bud Bone Lung analyzed tissues for a Average value % ID/r 8.6 14.1 7.8 4.5 7.9 i antibodies against PD-L1 p Art. deviation %ID/r 1.5 1.1 1.0 1.4 h,o ri 1 mg/kg Heart 4.3 1.1 Blood 9.1 4.6 Thymus 9.7 3.5 MC38-cOVA/eGFPmPD-LFliPD-Ll^ 34.1 18.0 Small intestine 2.4 0.9

At the 1 mg/kg dose level, there was clear tumor targeting of MC38-cOVA/eGFP-mPD-L1 ⁷ · hPD-L1 ^Tg with a % ID/g of 34.1% despite PD-L1 expression in normal tissues of these humanized mice. At this dose, some localization of 89Zr-labeled anti-PD-L1 antibody was evident in the spleen, where antibody uptake of 14.1% ID/g was observed. This uptake is due to the normal expression of human PD-L1 instead of the expression of mouse PD-L1 and human PD-L1 in the spleen. At an antibody dose of 3 mg/kg, localization of the ⁸⁹ Zr-DFO-anti-PD-L1 antibody conjugate in the spleen was reduced as uptake now averaged 9.7% ID/g in mice treated with this dose

antibodies (Table 16). Table 16

Mean % ID/g in tissue analyzed for anti-PD-L1 antibody at 3 mg/kg

Sample Average %ID/r Art. deviation %ID/r Liver 6.7 1.4 Spleen 9.7 1.3 Bud 7.0 1.1 Bone 3.6 0.6 Lung and about 1.0 Heart 4.7 0.7

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Blood 12.4 2.1 Thymus 7.6 0.5 MSZ 8-cOVA/eGFP-mPD-L 1 ''hPD-L 1 ^Tg 28.7 13.1 Small intestine 0.4 0.2

At 3 mg/kg, clear tumor targeting was still observed with a mean of 28.7% ID/g taken by MC38-cOVA/eGFP-mPD-L1' ^/ 'hPD-L1 ^Tg tumors. Therefore, although decreased localization to normal tissue was evident on imaging at 3 mg/kg, clear localization of tagged anti-PD-L1 antibodies in MC38-cOVA/eGFP-mPD-L1' ^/- hPD-L1 ^Tg tumors remained apparent at this dose . Overall, these results indicate that clear targeting of MC38cOVA/eGFP-mPD-L1' ^/ 'hPD-L1 ^Tg tumors was possible in mice expressing PD-L1 at regular sites of normal tissue expression.

The results of the studies performed in this case clearly demonstrate that anti-PD-L1 antibody labeled with ⁸⁹ Zr can be significantly and specifically localized in tumors. One can envision a scenario in which an anti-PD-L1 antibody is used to select patients with PD-LI-positive tumors for subsequent treatment with inhibitors of the PD-1/PD-L1 signaling axis.

Example 8: Large-Scale Manufacturing Process for the Production of DFO-Anti-PD-L1 Antibody Conjugates.

This example details a large-scale manufacturing process for producing an anti-PD-L1 antibody suitable for radiolabeling by coupling p-SCNbn-deferoxamine (DFO) to the anti-PD-L1 antibody (mAb, H4H8314N) described herein: (1 ) ultrafiltration and diafiltration (UFDF) processes before mAb conjugation removed excipients that inhibit the conjugation process; (2) after UFDF pre-conjugation, mAb was conjugated with p-SCN-Bn-deferoxamine to obtain DFOmAb conjugates; and (3) UFDF after conjugation to remove residual salts ensured the appropriate concentration, excipient level, and pH of the conjugated monoclonal antibody. The resulting DFO-mAb conjugates were then provided in a buffered state with improved stability for subsequent formulation.

(1) Ultrafiltration and diafiltration (UFDF) pre-conjugation.

For 100 g of anti-PD-L1 antibody, buffer was exchanged to 5 mM acetate buffer solution having a pH of 5.50 using a Sius Prostream membrane (TangenX Technology Corporation) (membrane capacity <500 g/m ² ) to remove residual salts before conjugation. The process volume was reduced to further concentrate the antibody, then the antibody was sterile filtered using a Sartopore 2 membrane (Sartorius) having a pore size of 0.45/0.2 μm (heterogeneous bilayer PES) or equivalent pore size. The acetate buffer temperature was maintained at a target temperature of 20±5°C. The solutions were mixed well.

(2) Conjugation.

The concentrated and filtered antibody (20 g) was transferred to a conjugation vessel containing a carbonate buffer system without amine (56 mM carbonate, 167 mM sodium chloride, pH 9.40), which resulted in negligible levels of residual acetate. DFO (25 mM p-SCN-Bn-deferoxamine) was solubilized in DMSO and added to the conjugation vessel along with additional DMSO such that DMSO was present at a final amount of 5%. DFO was added in molar excess at a DFO to mAb ratio of 4.5:1. The total reaction volume was 2.0 L. The buffer system was mixed throughout the addition of reaction ingredients and throughout the reaction time.

The reaction temperature was controlled over time using an equation that relates temperature to reaction time. In this case, the reaction temperature was maintained at 18°C for 120 min. The reaction was quenched by adding 2M acetic acid (23 ml/L), resulting in a solution pH of 6.

(3) UFDF after conjugation.

After the conjugation step, the quenched DFO-mAb conjugation solution was buffer exchanged with histidine buffer (10 mM histidine, pH 5.50, supplemented with 0.0005% (w/v) super-refined polysorbate 80 as a shear protectant) to remove residual process salts, DMSO and unreacted DFO. After diafiltration, the solution was concentrated and then compounded. Histidine buffer was chosen for long-term protein storage at -80°C. The same Sius Prostream membrane mentioned in step (1) was used in the last UFDF step. The resulting concentrated DFO-mAb conjugate solution was sterile filtered using the Sartopore 2 filter mentioned above.

UV-DAR (target 1.5) and protein concentration determination were performed as described in Example 2.

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Table 17

Molar extinction coefficients and molecular weight

Antibody MW (g mol' ¹ ) ε280 (l Hem' ¹ ) ε252 (l g' ¹ cm' ¹ ) H4H8314N 144984 211480 80172

Example 9: Predicted whole body and tissue exposure to radioactivity in human subjects receiving IV dose of ⁸⁹ Zr-DFO-anti-PD-L1 conjugate.

The purpose of the following experiment was to evaluate the predicted whole body and tissue exposure in human subjects to an intravenous (IV) dose of the ⁸⁹ Zr-DFO-anti-PD-L1 antibody conjugate. An example of an anti-PD-L1 antibody used in a radiolabeled conjugate was H4H8314N.

Characteristics of radioimmunoconjugates.

Anti-PD-L1 antibody immunoconjugate (DFO-Ab) and isotype control immunoconjugate (DFO-IgG4 ^P control) were radiolabeled and purified for use in in vivo imaging and biodistribution studies. SEC-HPLC analysis and in vitro analysis based on MC38/mPD-L1' ^/- hPD-L1 cells (MC38 murine colon adenocarcinoma cells engineered for mouse PD-L1 knockout and stable expression of human PD-L1) were performed to characterize the resulting radioimmunoconjugates.

Monomeric and radiochemical purity.

To evaluate monomeric and radiochemical purity, SEC-HPLC was performed using UV and γ emission detectors. The results for the radioimmunoconjugate preparations of the ⁸⁹ Zr-DFOantibody anti-PD-L1 conjugate and the isotype control radioimmunoconjugate ⁸⁹ Zr-DFO- ^IgG4P are shown in FIG. 8.

Analysis of absorbance chromatograms at 280 nm was performed to estimate the relative amounts of high molecular weight (HMW) and monomeric protein in the radioimmunoconjugate preparations. As shown in table. 18, monomer peaks (monomeric purity read) account for 99.6, 99.2 and 98.6%, respectively, of the total protein peak area for the conjugate preparations ⁸⁹ Zr-DFO-anti-PD-L1 antibody and ⁸⁹ Zr-DFO-IgG4 ^P isotype control; low levels of HMW compounds were also detected (0.4, 0.8 and 1.4%, respectively). Low molecular weight (LMW) compounds were not observed for any of the samples tested.

Analysis of γ-emission radiochromatograms was performed to evaluate the relative amounts of ⁸⁹ Zr incorporated into radioimmunoconjugates compared to unincorporated ⁸⁹ Zr (such as free ⁸⁹ Zr or ⁸⁹ Zr chelated with free DFO derivatives). As briefly described in Table. 18, the peaks for unincorporated ⁸⁹ Zr are <1.1% of the total γ-emission peak area, while the combined peaks for radiolabeled monomeric and HMW compounds (radiochemical purity readout) are 98.9, 99.5, and 99 .5%, respectively, of the total area of γ89 89 P emission peaks for the Zr-DFO-antibody conjugate preparations against PD-L1 and Zr-DFO-IgG4 control isotype.

Table 18

SEC-HPLC Data Summary

Peak number Compound Approximate retention time (minutes) Peak area (%) UV chromatogram Radiochromatograph mma Study 1 ⁸⁹ Zr-DFO-H4H8314N 1 H.M.W. 13 0.4 1.1 2 Monomer 16 99.6 97.8 3 Not included ⁸⁹ Zr 26 no data 1.1 Study 2 ⁸⁹ Zr-DFO-H4H8314N 1 H.M.W. 14 0.8 1.3 2 Monomer 16 99.2 98.2 3 Not included ⁸⁹ Zr 26 no data 0.5 Control ⁸⁹ Zr-DFO-IgG4 ^p 1 H.M.W. 13 1.4 1.5 2 Monomer 16 98.6 98.0 3 Not included ⁸⁹ Zr 26 no data 0.5

The numerical values for the SEC-HPLC analysis are graphically presented in FIG. 8. UV chromatogram

- 36 045226 is an absorbance chromatogram at 280 nm and the radio chromatogram is a γ-emission intensity chromatogram. HMW: high molecular weight; no data: not determined.

Immunoreactivity.

Immunoreactivity, a measure of the percentage of radiolabeled conjugated antibody capable of binding its antigen, was determined by incubating the ⁸⁹ Zr-DFO-anti-PD-L1 antibody conjugate with MC38/mPD-Lr ^/ 7hPD-L1 cells. 2 batches of ⁸⁹ Zr-DFO-anti-PD-L1 conjugate tested showed immunoreactivity of 84.5 and 88.8% on MC38/mPD-L1' ^/ '/hPD-L1 cells (Table 19). A background nonspecific immunoreactivity of 8.8% was observed for the isotype control radioimmunoconjugate.

Table 19

Immunoreactivity of ⁸⁹ Zr-labeled DFO-antibody conjugate against PD-L1 and ⁸⁹ Zr-DFO-IgG4 ^P isotype control

Radioimmunoconjugate Immunoreactivity conjugate ⁸ Ag-ORO-antibody against PD-L1 (lot 1) 84.5% conjugate ⁸ Ag-ORO-antibody against PD-L1 (lot 2) 88.8% ⁸⁹ Zr-DFO-IgG4 ^p isotype control 8.8%

In conclusion, two separate batches of ⁸⁹ Zr-DFO-anti-PD-L1 conjugate showed high immunoreactivity, monomer percentage, and radiochemical purity.

Biodistribution of ⁸⁹ Zr-DFO anti-PD-L1 antibodies in mice.

This experiment assessed the biodistribution of the human PD-L1 antibody radioimmunoconjugate, ⁸⁹ Zr-DFO-anti-PD-L1 antibody conjugate, over time following administration of a single intravenous (IV) dose of 50 μCi (1 mg/kg) PD-L1/PD-1 -humanized mice (PD-1hu/huPD-L1hu/hu). Because H4H8314N does not bind mouse PD-L1, the portion of the mouse PD-L1 gene encoding the PD-L1 ectodomain was replaced with the corresponding human sequence in PD-1hu/hu-PD-L1hu/hu mice. In this strain, the mouse PD-1 ectodomain is similarly humanized. These mice have not been subjected to an immuno/inflammatory challenge and are therefore expected to have unstimulated levels of PD-L1 expression in immune cells. Two groups of 8 animals each were sacrificed 6 days (144 hours) or 10 days (240 hours) after dosing, blood was collected, and the following tissues were collected: heart, lungs, liver, spleen, kidneys, stomach, small intestine, cecum , large intestine, bone (femur), thymus, muscle, bladder and brain. The percentage of radioactivity in the total injected dose (% ID) localized to specific tissues or blood was determined and reported as the average % ID per gram (% ID/g) of tissue. Before sacrifice, immuno-PET/computed tomography (CT) images were obtained at 1, 24, 48, 72, 144, 192 (10-day group only) and 240 hours (10-day group only) post-dose from the same animals.

Relative to blood levels of ⁸⁹ Zr, uptake of the ⁸⁹ Zr-DFO-anti-PD-L1 conjugate in certain tissues was negligible during the 10-day study period, as assessed by ex vivo tissue analysis (Table 20 and FIG. 9) and imaging. in vivo. Compared to blood (9.4±2.2% ID/g), all collected tissues except spleen showed lower ^89Zr levels (<6.7% ID/g) at day 6 post-dose. A small degree of target-mediated uptake of the ⁸⁹ Zr-DFO-anti-PD-L1 conjugate (10.2 ± 1.9% ID/g) was observed in the spleen, consistent with PD-L1 expression in splenocytes as demonstrated by flow cytometry . At 10 days post-dose, ⁸⁹ Zr blood levels were reduced 7.8-fold compared with day 6 post-dose, indicating a human anti-mouse antibody (MAHA) response influencing ⁸⁹ Zr-DFO-anti-antibody conjugate levels. PD-L1. This observed MAHA response is likely due to the fact that the target, PD-L1, is expressed on antigen-presenting cells (Francisco, 2010), leading to presentation of the human antibody to the mouse immune system and subsequent formation of MAHA. In parallel, hepatic ⁸⁹ Zr levels were increased 4.1-fold on day 10 compared with day 6 post-dose, likely resulting from the formation of the MAHA ⁸⁹ Zr-DFO-antibody anti-PD-L1 immune complex (IC) and subsequent mediated liver clearance of IC (Rojko, 2014). Whole-animal in vivo PET imaging did not reveal significant tissue-specific uptake of the ⁸⁹ Zr-DFO-anti-PD-L1 conjugate beyond the low signal for the spleen and MAHA-mediated accumulation in the liver described above.

Thus, the marked target-mediated uptake of the ⁸⁹ Zr-DFO-anti-PD-L1 conjugate into certain tissues above blood ⁸⁹ Zr levels was not observed over a 6-day period in PD-L1/PD-1 humanized mice given a single dose of IV dose of 1 mg/kg (50 μCi) of ⁸⁹ Zr-DFO-anti-PD-L1 conjugate, except in the spleen, where a small degree of target-mediated uptake was observed, consistent with demonstrated PD-L1 expression in splenocytes. Data collected after day 6 until the end of the study on day 10 post-dose were influenced by MAHA response.

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Table 20

Average ex vivo biodistribution data

Textile ⁸⁹ Zr levels on day 6 post-dose (% ID/r) ⁸⁹ Zr levels on day 10 post-dose (%ID/r) Average value SD Average value SD Blood 9.4 2.2 1.2 1.4 Heart 3.1 0.6 1.2 0.4 Lungs 5.9 0.7 2.6 0.7 Liver 4.9 1.9 20.2 7.8 Spleen 10.2 1.9 12.1 h,o Kidneys 5.3 1.1 3.9 1.3 Stomach 0.9 o, s 0.4 0.1 Small intestine 1.5 o, s 0.9 0.1 Cecum 1.0 0.2 0.6 0.2 Thick 1.4 o, s 0.7 0.2 Bone (femur) 6.3 2.1 6.9 1.4 Thymus 6.7 1.6 5.3 1.1 Muscle 0.9 0.1 0.5 0.1 Bladder 4.3 2.1 1.7 0.9 Brain 0.4 0.1 0.2 0.1

Abbreviation: %ID/g = percentage of injected dose per gram (of tissue).

Whole body and tissue exposure assessments in humans This experiment used PET/CT imaging data from four male PD-1/PD-L1 humanized mice and four female PD-1/PD-L1 humanized mice imaged at 1. 24, 48, 72, 144, 192 and 240 hours after a single IV dose of 50 μCi (1 mg/kg) of the ⁸⁹ Zr-DFO-αanti-PD-L1 conjugate. Data obtained from this clinically relevant dose were used to calculate estimates of human exposure to radioactivity. Tissue concentration data were determined using volume of interest (VOI) analysis.

To evaluate radiation dosimetry, the mean dwell time was determined for the following sites: brain, stomach contents, heart contents, kidneys, liver, lungs, muscle, red bone marrow, spleen, bladder contents, and the rest of the body. These average residence times were used as input into OLINDA EXM 1.1 software to estimate average tissue absorbed doses and effective dose in humans.

The human effective dose of the ⁸⁹ Zr-DFO-anti-PD-L1 conjugate was estimated to be 0.513 mSv/MBq (millisievert/megabecquerel) in an adult male and 0.622 mSv/MBq in an adult female. The organs predicted to have the highest absorbed dose in humans were the spleen and liver. The estimated absorbed dose to the spleen was 0.856 mSv/MBq in an adult male and 1.12 mSv/MBq in an adult female. The estimated absorbed dose to the liver was 0.764 mSv MBq in an adult male and 0.974 mSv/MBq in an adult female.

The mean decay-corrected percent injection dose per ml (DC % ID/ml) for male and female mice (n=4 male, n=4 female) for each VOI is shown in Table 1. 21.

- 38 045226

Table 21

Biodistribution data

Mean decay-corrected percentage of injection dose per mL (DC % ID/mL) (DC % ID/mL) ± SD Time watch) 1 24 48 72 144 Floor Zhenek IY Male oh Zhenek IY Male oh Zhenek IY Male oh Zhenek IY Male oh Zhenek IY Male oh Brain 1.365 ± 0.115 1.190 ± 0.050 0.903 ± 0.115 0.538 ± 0.071 0.640 ± 0.079 0.548 ± 0.218 0.685 ± 0.096 0.623 ± 0.224 0.465 ± 0.231 0.398 ± 0.073 Lungs 12,503 £1,146 £12,4980.414 8.293 ± 0.635 7.155 ± 1.175 6.715 ± 0.370 5.888 ± 0.990 6.060 ± 0.708 5.558 ± 0.385 4.863 ± 0.316 4.585 ± 0.339 Liver 12,298 £0.664 12,078 £0.372 9.058 ± 0.793 7.200 ± 0.499 8.113 ± 0.969 6.125 ± 0.858 7.838 ± 0.932 6.203 ± 0.483 9.423 ± 1.885 6.208 ± 1.428 Heart £27,688 £1,942 £25,695 0.934 15,685 ± 1.223 £13,323 £1,133 12,088 ±0.883 10.25 ± 1.335 11,740 £1,553 9.915 ± 0.171 8.140 ± 0.598 7.463 ± 0.768 Kidneys 11,430 £0.387 12,100 £0.872 7.345 ± 0.322 6.783 ± 0.811 6.418 ± 0.761 5.565 ± 0.680 6.475 ± 0.493 5.568 ± 0.550 5.643 ± 0.222 4.815 ± 0.450 Spleen £15,263 £2,166 15,860 £0.974 14.135 ±2.010 £11,265 1,706 13.675 ±2.195 9.388 ± 1,389 £13,655 £3,606 9.920 ± 1.414 15.105 ±2.959 £10,303 1,102 Bladder 6.045 ± 3,910 9.688 ± 4,991 1.653 ± 0.107 1.820 ± 0.283 1.443 ± 0.205 1.403 ± 0.160 1.318 ± 0.108 1.710 ± 0.346 1.115 ± 0.224 1.293 ± 0.430 Muscle 1.608 ± 0.182 1.435 ± 0.198 2.608 ± 0.196 1.780 ± 0.137 2.368 ± 0.259 1.955 ± 0.339 2.408 ± 0.181 2.148 ± 0.176 2.095 ± 0.168 1.918 ± 0.144 Stomach 3.238 ± 1.063 3.978 ± 0.632 2.875 ± 0.921 3.073 ± 0.566 2.478 ± 0.296 2.238 ± 0.487 2.260 ± 0.306 2.233 ± 0.491 2.380 ± 0.405 1.665 ± 0.148 Bone 3.683 ± 1.418 3.023 ± 0.244 3.310 ± 0.330 2.738 ± 0.171 4.600 ± 0.511 3.493 ± 0.716 4.850 ± 1,292 4.658 ± 1,399 8.993 ± 1.057 7.635 ± 0.872

The estimated values of the mean residence time (MRT) in humans are presented in Table. 22 for each of the source organs. MRI in the rest of the body was obtained by subtracting the sum of all residence times in the source organ from the reciprocal of the decay constant of ⁸⁹ Zr (Huang et al., Biodistribution, toxicity and radiation dosimetry studies of the serotonin transporter radioligand 4-[18F]-ADAM in rats and monkeys. Eur J Nucl Med Mol Imaging 2010;37:545-555). This is a conservative estimate of cumulative tissue radioactivity.

Table 22

Average residence time (hours) in a person

Physical decay ¹ Biexponential fit ² Organ/Tissue Female Male Female Male Brain 0.398 0.364 0.372 0.344 Stomach contents 0.511 0.476 0.492 0.480 Contents of the Heart 2.433 2,279 2,290 2,154 Kidneys 0.868 0.818 0.832 0.794 Liver 5,902 5,919 8,240 5.938 Lungs 2,508 2,772 2,411 2,642 Muscles 17,635 23,677 13,348 17,182 Red bone marrow 2,777 2,024 2,613 1,913 Spleen 0.996 0.871 1.053 0.910 Contents of urinary bubble 0.299 0.491 0.315 0.405 Other parts body 78,794 73,430 81,157 80,361

¹ Average residence time was calculated based only on physical decay after time point day 6.

² Average residence time was calculated by biexponential fitting of the data.

The estimated tissue absorbed doses for all target organs for the adult male and adult female simulators according to OLINDA/EXM 1.1 are presented in Table. 23. Effective

-39045226 dose, as defined by the International Commission on Radiological Protection. 1990 Recommendations of the International Commission on Radiological Protection. ICRP Publication 60, Pergamon Press, New York, 1991, is a value calculated by multiplying the absorbed dose for a given organ by the stochastic risk weighting factor and adding all weighted doses together. The estimated effective doses are given at the end of the table. 23. These values represent a conservative estimate of the absorbed radioactive doses.

Table 23

Estimated absorbed doses to human tissue and effective dose

Organ/Tissue Physical decay ¹ Biexponential fit ² Adult male (mSv/MBq) Adult female (mSv/MBq) Adult male (mSv/MBq) Adult female (mSv/MBq) Adrenal glands 0.561 0.702 0.567 0.726 Brain 0.179 0.237 0.182 0.234 Mammary gland 0.366 0.459 0.379 0.466 Gallbladder wall 0.601 0.692 0.610 0.751 LLI wall 0.519 0.652 0.530 0.651 Small intestine 0.563 0.600 0.582 0.605 Stomach wall 0.575 0.714 0.584 0.718 Wall ULI 0.553 0.685 0.571 0.700 Heart wall 0.789 0.973 0.781 0.964 Bud 0.650 0.773 0.641 0.774 Liver 0.764 0.974 0.764 1,220 Lungs 0.575 0.705 0.561 0.700 Muscle 0.396 0.481 0.381 0.464 Ovaries 0.533 0.645 0.542 0.642 Pancreas 0.597 0.743 0.606 0.765 Red bone marrow 0.480 0.591 0.483 0.587 Osteogenic cells 0.604 0.777 0.625 0.779 Leather 0.291 0.373 0.297 0.374 Spleen 0.856 1.120 0.876 1,160 Testicles 0.399 N.A. 0.407 N.A. Thymus 0.481 0.605 0.484 0.601 Thyroid 0.417 0.484 0.423 0.480 Bladder wall 0.580 0.496 0.559 0.494 Uterus 0.545 0.638 0.554 0.636 Whole body 0.440 0.550 0.440 0.554 Effective dose 0.513 0.622 0.516 0.625

¹ Absorbed doses calculated from MRI assuming only physical decay after time point day 6.

² Absorbed doses calculated from MRI with biexponential fitting of data.

Abbreviations: LLI = lower colon, ULI = upper colon, NA = not determined.

The estimated human tissue absorbed doses and the effective human dose (Table 23) by physical decay and biexponential fitting methods were similar. The physical degradation method was chosen to obtain a final set of estimated human tissue absorbed doses and effective dose due to the apparent MAHA response in this mouse model. Therefore, the effective human dose for the ⁸⁹ Zr-DFO-anti-PD-L1 conjugate was estimated to be 0.513 mSv/MBq in an adult male and 0.622 mSv/MBq in an adult female. The organs predicted to have the highest absorbed dose in humans are the spleen and liver. The estimated absorbed dose to the spleen was 0.856 mSv/MBq in an adult male and 1.12 mSv/MBq in an adult female. The estimated absorbed dose to the liver was 0.764 mSv/MBq in an adult male and 0.974 mSv/MBq in an adult female.

- 40 045226

Example 10 Immuno-PET imaging of PD-L1 in tumors using the ⁸⁹ Zr-DFO anti-PD-L1 antibody conjugate in patients with advanced thoracic malignancies.

The primary objective of this study is to determine the safety and tolerability of the ⁸⁹ Zr-DFO-anti-PD-L1 antibody conjugate, in which the anti-PD-L1 antibody used in the radiolabeled conjugate is H4H8314N. The secondary objectives of the study are:

Part A of the study only: establishing the appropriate mass dose of the ⁸⁹ Zr-DFOantibody anti-PD-L1 conjugate and the optimal timing of post-infusion imaging assessed by imaging and blood sampling after tracer infusion;

Part B of the study only: establishing test/retest reliability of PET measurements assessed from two separate tracer infusions at the optimal mass dose and imaging time point as defined in Part A;

Characterization of the pharmacokinetic (PK) profile of the ⁸⁹ Zr-DFO-anti-PD-L1 antibody conjugate based on plasma activity concentration of the labeled substance.

This is an open-label, 2-part study designed to evaluate the safety and tolerability of ^{the 89} Zr-DFO anti-PD-L1 antibody. Part A of the study will establish the appropriate mass dose, the activity dose of ⁸⁹ Zr-DFO-anti-PD-L1 antibody, and the optimal post-infusion imaging time. Test/retest variability of ⁸⁹ Zr-DFO-anti-PD-L1 antibody will be assessed in Part B.

All patients will undergo screening procedures. Patients who meet the eligibility criteria will undergo ¹⁸ F-fluorodeoxyglucose ( ¹⁸ F-FDG) PET/computed tomography (CT) and diagnostic CT scans to assess lesion viability, location and size. These scans will not be required if suitable quality images are available, obtained within 28 days of the intended first dose of ⁸⁹ Zr-DFO-anti-PD-L1 antibody.

Part A

Three sequentially dosed cohorts are planned to be treated open-label with ^{the 89} Zr-DFO anti-PD-L1 antibody at 5, 10 or 20 mg.

After infusion of ⁸⁹ Zr-DFO-anti-PD-L1 antibody, patients will undergo PET/CT scans for ⁸⁹ Zr-DFO-anti-PD-L1 antibody on day 1, day 4±1 and day 7±1. Additional imaging may be performed up to day 10. Patients will undergo safety assessments and provide samples for hematology, chemistry, immune safety, pharmacokinetic, antidrug antibody, and biomarker analyses.

Patients will continue to undergo safety assessments, including physical examination, vital signs, and documentation of adverse events (AEs), until day 21 after infusion of labeled ⁸⁹ Zr-DFO-anti-PD-L1 antibody.

Dose escalation decisions to determine the appropriate dose will be based on safety and tolerability data and assessment of immunopositron emission tomography (iPET) positivity and plasma tracer activity concentrations, as described below.

Part A dose cohorts.

Up to 3 escalating mass dose cohorts are planned. Each mass-dose cohort will be dosed with a minimum 48-dose interval for each patient. Upon completion of the PET/CT scan on day 7±1 for the second patient at a given mass dose, all available imaging data, plasma concentrations of tracer activity, clinical dosimetry, and safety will be reviewed. Based on this review, a decision will be made on:

expansion of the 6-patient cohort if there is a positive tumor uptake/tumor location finding in at least 1 patient, as defined by a tumor-to-blood ratio >1;

moving to the next mass-dose cohort if there is inappropriate tumor uptake and plasma tracer activity concentration, with an appropriate standardized blood uptake value (SUV) range of 1 to 5 at the optimal imaging time point;

moving to the next mass-dose cohort at a lower mass dose based on insufficient tumor uptake and appropriate plasma concentration of tracer activity.

If the tumor site is inappropriate in at least 2 patients at all three proposed mass dose levels, and it is determined that this is due to low signal-to-noise ratio in the image, the activity dose will be increased to a maximum of 185 MBq to further expand previously tested recipients mass dose cohorts.

- 41 045226

Part B

Part B of the study will begin after Part A has determined the appropriate dose of ⁸⁹ ZrDFO anti-PD-L1 antibody and optimal imaging time. On Day 1 of Part B, patients will receive a mass dose of labeled substance. After receiving the tracer, patients will be scanned at the optimal time determined in Part A. Patients in Part B will receive a second dose of the tracer and will be scanned after a dosing interval of 14 to 28 days. The actual timing of the second dose of labeled substance after the interval will be determined based on the results obtained in Part A.

Patients will undergo safety assessments including physical examination, vital signs, and documentation of adverse events (AEs) during and after visits at which the ⁸⁹ Zr-DFO-labeled anti-PD-L1 antibody is administered. During these visits, patients will provide samples for PK, hematology, chemistry, and immune safety testing. For both Part A and Part B, patients will continue to undergo safety assessments, including physical examination, vital signs, and documentation of AEs, until 21 days after the last infusion of labeled ⁸⁹ Zr-DFO-anti-PD-L1 antibody.

Duration of the study.

For Part A, patients will undergo a screening period of up to 28 days (4 weeks) and a follow-up period of up to 21 days (approximately 3 weeks) after the labeled dose infusion. The duration of Part A of the study will be approximately 7 weeks, including the screening period.

For Part B, patients will undergo a screening period of up to 28 days (4 weeks) with an interval between infusions of up to 28 days (4 weeks) and a 21-day (3 weeks) follow-up safety assessment period that includes a second scanning period. The total duration of the study for each patient will be up to 11 weeks, including the screening period.

The end of study for this study was defined as the last visit of the last patient.

For Part A, the study plans 3 sequential dose levels of up to 6 patients per cohort, with 3 cohorts planned for a total of up to 18 patients. For Part B of the study, up to 10 patients will be included. For the entire study, enrollment of a maximum of 28 patients per study site is planned.

Target patient population.

The target population will consist of patients 18 years of age or older with advanced thoracic malignancies and an IHC PD-L1 score on diagnostic or follow-up biopsy >1% (positive IHC PD-L1 score by 22C3 PharmDx assay, Dako North America Inc.).

For Part A, thoracic malignancies will be limited to NSCLC, gastroesophageal junction adenocarcinoma, and gastric cancer with a PD-L1 score >1% by IHC.

All patients with advanced chest malignancies and a PD-L1 score >1% by IHC will be eligible for Part B. Patients must also have had stable disease according to RECIST 1.1 between the last two imaging studies. All patients requiring therapy should undergo standard therapy.

Treatment.

⁸⁹ Zr-DFO anti-PD-L1 antibody is a radioimmunoconjugate formed by covalently conjugating a bifunctional chelator (p-SCN-Bn-DFO) to H4H8314N (an anti-PD-L1 monoclonal antibody) and radiolabeling ⁸⁹ Zr to this compound. ⁸⁹ Zr-DFO anti-PD-L1 antibody is supplied in an aqueous buffer vehicle.

For Part A ^{, 89} Zr-DFO anti-PD-L1 antibody will be administered IV on day 1 (baseline). For Part B ^{, 89} Zr-DFO anti-PD-L1 antibody will be administered IV on day 1 and day 7±3. The actual timing of the second dose in Part B will be determined by the results in Part A.

The labeled substance, ^{the 89} Zr-DFO anti-PD-L1 antibody, will be administered at a dose level significantly below the estimated cumulative exposure levels in humans based on PK models, and lower than the levels at which currently available anti-PD-L1 antibody treatments PD-1 is used to treat malignant tumors. This study will exclude patients currently being treated with an anti-PD-L1 antibody to avoid competition for the target.

Endpoints.

The primary endpoint of the study is the incidence and severity of treatment-emergent adverse events (TEAEs) up to day 21 after the last dose of tracer infusion in patients with chest cancer who were dosed with ^{the 89} Zr-DFO anti-PD-L1 antibody.

For Part A only: the study will establish the appropriate mass dose and activity dose of ⁸⁹ ZrDFO anti-PD-L1 antibody, as well as the optimal post-infusion imaging time, and by

- 42 045226 blood draws and imaging on days 1, 4 and 7 after the tracer infusion will determine the following:

standardized absorbance value of ⁸⁹ Zr-DFO-anti-PD-L1 antibody in the blood pool followed by calculation of tumor-to-blood ratios at the time of imaging;

standardized uptake values (SUV) by tumor region of interest (ROI);

maximum SUV (SUVmax) with tumor ROI;

concentration _of activity of the labeled substance in plasma, expressed as SUV, with calculation of the area under the curve up to day 7 (AUC _0.7 ).

For Part B only: the study will establish test/retest reliability from PET measurements ^{of 89} Zr-DFO-anti-PD-L1 antibody, and from measurements of 2 separate tracer infusions at the appropriate mass dose and optimal imaging time points determined from Part A. will determine the following:

SUV of the blood pool followed by calculation of the tumor-to-blood ratio;

SUV by tumor ROI;

SUVmax in tumor ROI;

biodistribution of ⁸⁹ Zr-DFO-anti-PD-L1 antibody.

The resulting data will demonstrate the safety and tolerability of ^{the 89} Zr-DFO anti-PD-L1 antibody in humans.

The embodiments and examples described above are for illustrative purposes only and are not limiting. Those skilled in the art will recognize or be able to determine, using no more than routine experimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are deemed to come within the scope of the appended claims and are covered thereby.

Sequence Listing <110> Regeneron Pharmaceuticals, Inc.

Kelly Marcus

Ma Dangshe

Olson William

Thurston Gavin <12 0> RADIO LABELED ANTI-PD-L1 ANTIBODIES FOR IMMUNO-PET IMAGING <130> 10305WO01 <140> TBD <141> 2017-12-01 <160>344 <170> Patent version 3.5 <210>1 < 211>363 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>1 gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc60 tcctgtgcag cctctggatt cacctttagt aggttttgga tgagctgggt ccgccaggct120 ccagggaagg ggctggagtg ggtggccaac ataa accaag atggaactga gaaatactat180 gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat240 ctgcaaatga acagcctgag agccggggac acggctgtgt attactgtgc gaatacgtat300

- 43 045226 tacgattttt ggagtggtca ctttgactac tggggccagg gaaccctggt caccgtctcc

360

363 tca <210> 2 <211> 121 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 2

Glu Val Gln Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gln Pro Gly Gly

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Arg Phe 25 30

Trp Met Ser Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Asn Ile Asn Gln Asp Gly Thr

55

Glu Lys Tyr Tyr Val Asp Ser Val 60

Lys Gly Arg Phe Thr Ile Ser Arg 65 70

Asp Asn Ala Lys Asn Ser Leu Tyr

80

Leu Gln Met Asn Ser Leu Arg Ala 85

Gly Asp Thr Ala Val Tyr Tyr Cys

95

Ala Asn Thr Tyr Tyr Asp Phe Trp

100

Ser Gly His Phe Asp Tyr Trp Gly

105 110

Gln Gly Thr Leu Val Thr Val Ser

115120

Ser <210>3 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>3 ggattcacct ttagtaggtt ttgg <210>4 <211>8 <212> PRT <213> Artificial sequence

- 44 045226 <220>

< 223> synthetic < 400>4

Gly Phe Thr Phe Ser Arg Phe Trp 15 < 210>5 < 211>24 < 212> DNA < 213> Artificial sequence <220>

< 223> synthetic < 400>5 ataaaccaag atggaactga gaaa24 < 210>6 < 211>8 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400>6

Ile Asn Gln Asp Gly Thr Glu Lys 15 < 210>7 < 211>42 < 212> DNA < 213> Artificial sequence <220>

<223> synthetic <400>7 gcgaatacgt attacgattt ttggagtggt cactttgact ac42 <210>8 <211>14 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>8

Ala Asn Thr Tyr Tyr Asp Phe Trp Ser Gly His Phe Asp Tyr

510 <210>9

- 45 045226 <211> 321 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 9

gacatccaga tgaccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc atcacttgtc gggccagtca gagtattagt aattggttgg cctggtatca gcagaaacca gggaaagccc ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca aggttcagcg gcagtggatc tggacagaa ttcactctca ccatcagcag cctgcagcct gatgattttg caacttatta ctgccaacag tatcatagtt attcgtacac ttttggccag gggaccaagc tggagatcaa a

120

180

240

300

321 <210> 10 <211> 107 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 10

Asp Ile Gln Met

Thr Gln Ser Pro 5

Ser Thr Leu Ser

Ala Ser Val Gly 15

Asp Arg Val Thr

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Ala Ser Gln Ser 25

Ile Ser Asn Trp 30

Leu Ala Trp Tyr

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Gly Lys Ala Pro

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Tyr Lys Ala Ser 50

Ser Leu Glu Ser

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Arg Phe Ser Gly

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Thr Glu Phe Thr

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Thr Tyr Tyr Cys 85

Gln Gln Tyr His 90

Ser Tyr Ser Tyr 95

Thr Phe Gly Gln

100

Gly Thr Lys Leu

Glu Ile Lys 105 <210> 11 <211> 18 <212> DNA <213> Artificial sequence

- 46 045226 <220>

<223> synthetic <400>11 cagagtatta gtaattgg <210>12 <211>6 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>12

Gln Ser Ile Ser Asn Trp < 210>13 < 211>9 < 212> DNA < 213> Artificial sequence <220>

< 223> synthetic < 400>13 aaggcgtct < 210>14 < 211>3 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400>14

Lys Ala Ser <210>15 <211>27 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 15 caacagtatc atagttattc gtacact <210> 16 <211> 9 <212> PRT

- 47 045226 <213> Artificial sequence <220>

<223> synthetic <400>16

Gln Gln Tyr His Ser Tyr Ser Tyr Thr 15 <210>17 <211>363 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 17

caggagcacc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgaag cgtctggatt caccttcagt aactttggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagct ttatggtctg atggaagtaa taaatactat 180 gcagactccg tgaagggtcg agtcaccatc tccagagaca attccaagaa cacactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtct attackgtgc gagagggaga 300 ggagccccccg gtattccgat ttttgggtac tggggccagg gaaccctggt caccgtctcc 360

tca 363 <210> 18 <211> 121 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 18

Gln Glu His Leu Val Glu Ser Gly 1 5

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Ser Leu Arg Leu Ser Cys Glu Ala 20

Ser Gly Phe Thr Phe Ser Asn Phe 25 30

Gly Met His Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Ala Leu Trp Ser Asp Gly Ser

55

Asn Lys Tyr Tyr Ala Asp Ser Val 60

Lys Gly Arg Val Thr Ile Ser Arg 65 70

Asp Asn Ser Lys Asn Thr Leu Tyr

80

- 48 045226

Leu Gln Met Asn Ser Leu Arg Ala 85

Glu Asp Thr Ala Val Tyr Tyr Cys

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100

Gly

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105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115120 <210>19 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>19 ggattcacct tcagtaactt tggc <210>20 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>20

Gly Phe Thr Phe Ser Asn Phe Gly <210>21 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>21 ttatggtctg atggaagtaa taaa <210>22 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400> 22

Leu Trp Ser Asp Gly Ser Asn Lys

- 49 045226 <210>23 <211>42 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>23 gcgagaggga gaggagcccc cggtattccg atttttgggt ac <210>24 <211>14 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>24

Ala Arg Gly Arg Gly Ala Pro Gly Ile Pro Ile Phe Gly Tyr 1,510 <210>25 <211>321 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 25 gacatccaga atcacttgcc gggaaagccc agattcagcg gaagattttg gggaccaagg tgacccagtc gggcaagtca ctaagcgcct gcagtggatc caacttatta tggcgatcaa tccatcctcc gggcattaga gatctatact tgggacagaa ctgtctaca a a ctgtctgcat aatgatttag gcatccagtt ttcactctca cataatagtt ctgttggaga gctggtatca tgcaaagtgg caatcagcag accctctcac cagagtcacc gcagaaacca ggtcccatca cctacagcct attcggcgga

120

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321 <210> 26 <211> 107 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 10 15

- 50 045226

Asp Arg Val Thr 20

Ile Thr Cys Arg

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Gln Gln Lys Pro 40

Gly Lys Ala Pro

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Tyr Thr Ala Ser 50

Ser Leu Gln Ser

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Arg Phe Ser Gly

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Thr Glu Phe Thr

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100

Gly Thr Lys Val

Ala Ile Lys 105 <210> 27 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>27 cagggcatta gaaatgat <210>28 <211>6 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>28

Gln Gly Ile Arg Asn Asp <210>29 <211>9 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 29 actngcatcc

- 51 045226 <210> 30 <211> 3 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>30

Thr Ala Ser < 210 > 31 < 211 > 27 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400>31 ctacaacata atagttaccc tctcaca <210>32 <211>9 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>32

Leu Gln His Asn Ser Tyr Pro Leu Thr <210>33 <211>390 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 33

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60 tcctgtgcag cctctggatt cactttcagt aacgcctgga tgagctggggt ccgccaggct 120 ccagggaagg ggctggagtg ggttggccgt attaaaagga aaactgatgg tgggacaaca 180 gactacgctg cacccgtgaa aggcagattc accatctcaa gagatgattc aaaaaatacg 240 ctgcatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaccaca 300 gatgatattg tagttgtacc agctgttatg agggaatact acttcggtat ggacgtctgg 360 ggccaaggga ccacggtcac cgtctcctca 390

- 52 045226 <210> 34 <211> 130 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 34

Glu Val 1

Gln Leu

Val Glu 5

Ser Gly

Gly Gly 10

Leu Val

Lys Pro

Ser Leu

Arg Leu 20

Ser Cys

Ala Ala

Ser Gly 25

Phe Thr

Phe Ser

Trp Met

Ser Trp 35

Val Arg

Gln Ala

Pro Gly

Lys Gly

Leu Glu 45

Gly Arg 50

Ile Lys

Arg Lys

Thr Asp 55

Gly Gly

Thr Thr

Asp Tyr

Pro Val 65

Lys Gly

Arg Phe 70

Thr Ile

Ser Arg

Asp Asp 75

Ser Lys

Leu His

Leu Gln

Met Asn 85

Ser Leu

Lys Thr 90

Glu Asp

Thr Ala

Tyr Cys

Thr Thr

100

Asp Asp

Ile Val

Val Val 105

Pro Ala

Val Met

110

Tyr Tyr

Phe Gly 115

Met Asp

Val Trp

120

Gly Gln

Gly Thr

Thr Val 125

Gly Gly 15

Asn Ala

Trp Val

Ala Ala

Asn Thr 80

Val Tyr 95

Arg Glu

Thr Val

Ser Ser

130 <210> 35 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 35 ggattcactt tcagtaacgc ctgg <210> 36 <211> 8 <212> PRT

- 53 045226 <213> Artificial sequence <220>

<223> synthetic <400>36

Gly Phe Thr Phe Ser Asn Ala Trp <210>37 <211>30 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>37 attaaaagga aaactgatgg tgggacaaca <210>38 <211>10 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>38

Ile Lys Arg Lys Thr Asp Gly Gly Thr Thr

510 <210>39 <211>63 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>39 accacagatg atattgtagt tgtaccagct gttatgaggg aatactactt cggtatggac gtc <210>40 <211>21 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 40

Thr Thr Asp Asp Ile Val Val Val Pro Ala Val Met Arg Glu Tyr Tyr

5 10 15

- 54 045226

Phe Gly Met Asp Val 20 <210> 41 <211> 321 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>41 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc60 atcacttgcc ggacaagtca gggcattaga aatgatttag gctggtatca gcagaaacca120 gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaag tgg ggtcccatca180 aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct240 gaagattttg caacttatta ctgtctacag cataataatt acccgtacac ttttggccag300 gggaccaagc tggagatcaa a321 <210>42 <211>10 7 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400>42

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 1015

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Gly Ile Arg Asn Asp 20 2530

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 4045

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Asn Tyr Pro Tyr 85 9095

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

- 55 045226

100

105 <210> 43 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>43 cagggcatta gaaatgat18 <210>44 <211>6 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>44

Gln Gly Ile Arg Asn Asp 15 < 210>45 < 211>9 < 212> DNA < 213> Artificial sequence <220>

< 223> synthetic < 400>45 gctgcatcc < 210>46 < 211>3 < 212> PRT < 213> Artificial sequence <220>

<223> synthetic <400>46

Ala Ala Ser <210>47 <211>27 <212> DNA <213> Artificial sequence <220>

< 223> synthetic

- 56 045226 <400>47 ctacagcata ataattaccc gtacact <210>48 <211>9 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>48

Leu Gln His Asn Asn Tyr Pro Tyr Thr 15 < 210 > 49 < 211 > 363 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400>49 caggtgcaat tggtgcagtc tggggcggag gtgaagaagc ctggggcctc agtgcaggtc60 tcctgcaagg cttctggata ctccttcacc ggctactata tacactgggt gcgacaggcc120 cctggacaag gacttgagtg gatgggatgg atcaacc cta acagtggcac caaaaagtat180 gcacacaagt ttcagggcag ggtcaccatg accagggaca cgtccatcga cacagcctac240 atgattttga gcagtctgat atccgacgac acggccgtgt attactgtgc gagagatgag300 gactggaact ttgggagctg gttcgactcc tgggg ccagg gaaccctggt caccgtctcc360 tca363 <210>50 < 211>121 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>50

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro GlyAla

5 1015

Ser Val Gln Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr GlyTyr

2530

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 4045

- 57 045226

Gly Trp Ile Asn 50

Pro Asn Ser Gly 55

Thr Lys Lys Tyr 60

Ala His Lys Phe

Gln Gly Arg Val 65

Thr Met Thr Arg 70

Asp Thr Ser Ile 75

Asp Thr Ala Tyr 80

Met Ile Leu Ser

Ser Leu Ile Ser 85

Asp Asp Thr Ala 90

Val Tyr Tyr Cys

Ala Arg Asp Glu

100

Asp Trp Asn Phe

Gly Ser Trp Phe 105

Asp Ser Trp Gly 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115120 <210>51 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>51 ggatactcct tcaccggcta ctat <210>52 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>52

Gly Tyr Ser Phe Thr Gly Tyr Tyr <210>53 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>53 atcaacccta acagtggcac caaa <210>54 <211>8 <212> PRT <213> Artificial sequence

- 58 045226 <220>

<223> synthetic <400>54

Ile Asn Pro Asn Ser Gly Thr Lys < 210>55 < 211>42 < 212> DNA < 213> Artificial sequence <220>

<223> synthetic <400>55 gcgagagatg aggactggaa ctttgggagc tggttcgact cc42 <210>56 <211>14 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>56

Ala Arg Asp Glu Asp Trp Asn Phe Gly Ser Trp Phe Asp Ser

510 < 210> 57 < 211> 336 < 212> DNA < 213> Artificial sequence <220>

< 223> synthetic < 400>57 gatattgtga tgacccagac tccactctcc tcacctgtca cccttggaca gccggcctcc60 atctcctgca ggtctagtca aaccctcgta cacggtgatg gaaacacgta cttgagttgg120 attcagcaga ggccaggcca gcctccgaga ctcctcat tt ataaggtttc taatcagttc180 tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc240 agcaggtgg aagctgagga tgtcgggctt tatttctgca tgcaagctac acattttccg300 atcaccttcg gccaagggac acgactggag attaaa 336 < 210>58 < 211>112 < 212> PRT < 213> Artificial sequence

- 59 045226 <220>

<223> synthetic <400> 58

Asp Ile 1

Val Met

Thr Gln 5

Thr Pro

Leu Ser

Ser Pro

Val Thr

Glin Pro

Ala Ser

Ile Ser

CysArg

Ser Ser 25

Gln Thr

Leu Val

Asp Gly

Asn Thr 35

Tyr Leu

Ser Trp 40

Ile Gln

Gln Arg

Pro Gly 45

Pro Arg 50

Leu Leu

Ile Tyr

Lys Val 55

Ser Asn

Gln Phe

Ser Gly

Asp Arg 65

Phe Ser

Gly Ser 70

Gly Ala

Gly Thr

Asp Phe 75

Thr Leu

Ser Arg

Val Glu

Ala Glu 85

Asp Val

Gly Leu 90

Tyr Phe

Cys Met

Thr His

Phe Pro

100

Ile Thr

Phe Gly

Gln Gly 105

Thr Arg

Leu Glu

110

Leu Gly 15

His Gly

Glin Pro

Val Pro

Lys Ile 80

Gln Ala 95

Ile Lys <210> 59 <211> 33 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>59 caaaccctcg tacacggtga tggaaacacg tac <210>60 <211>11 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>60

Gln Thr Leu Val His Gly Asp Gly Asn Thr Tyr

510 <210>61 <211>9 <212> DNA <213> Artificial sequence

- 60 045226 <220>

<223>

synthetic <400>

aaggtttct <210> 62 <211> 3 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>62

Lys Val Ser < 210 > 63 < 211 > 27 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400>63 atgcaagcta cacattttcc gatcacc27 <210>64 <211>9 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>64

Met Gln Ala Thr His Phe Pro Ile Thr 15 < 210 > 65 < 211 > 363 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400>65 caggtacacc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc60 tcctgcaagg cttctggata caccttcacc ggctactata tacattgggt gcgacaggcc120 cctggacacg ggcttgagtg gatgggatgg ctcaacccta atactggtac cacaaagtat180

- 61 045226

atacagaact ttcaggggcag ggtcaccatg accagggaca cgtccagcag cacagcctac 240 atggagctga ccaggctgag atctgacgac acggccgtgt attackgtgc gagagatgag 300 gactggaatt atgggagctg gttcgacacc tggggccagg gaaccctggt cacagtctcc 360 tca 363

<210> 66 <211> 121 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>66

Gln Val His Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

5 1015

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 2530

Tyr Ile His Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met 35 4045

Gly Trp Leu Asn Pro Asn Thr Gly Thr Thr Lys Tyr Ile Gln Asn Phe 50 5560

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 7580

Met Glu Leu Thr Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 9095

Ala Arg Asp Glu Asp Trp Asn Tyr Gly Ser Trp Phe Asp Thr Trp Gly

100 105110

Gln Gly Thr Leu Val Thr Val Ser Ser

115120 <210>67 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 67 ggatacacct tcaccggcta ctat

- 62 045226 <210> 68 <211> 8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>68

Gly Tyr Thr Phe Thr Gly Tyr Tyr <210>69 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>69 ctcaacccta atactggtac caca <210>70 <211>8 <212>PRT <213>Artificial sequence <220>

< 223> synthetic < 400>70

Leu Asn Pro Asn Thr Gly Thr Thr <210>71 <211>42 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>71 gcgagagatg aggactggaa ttatgggagc tggttcgaca cc42 <210>72 <211>14 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 72

Ala Arg Asp Glu Asp Trp Asn Tyr Gly Ser Trp Phe Asp Thr

- 63 045226 <210> 73 <211> 336 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>73 gatattgtaa tgacccagac tccactctcc tcacctgtca cccttggaca gccggcctcc60 atctcctgca ggtctagtcc aagcctcgta cacagtgatg gaaacaccta cttgagttgg120 cttcagcaga ggccaggcca gcctccaaga ctcctaattt a taagatttc taaccgattc180 tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac gctgaaaatc240 agcaggtgg aagctgagga tgtcggggtt tattactgca tgcaagctac acattttccg300 atcaccttcg gccaagggac acgactggag attaga336 <210> 74 <211>112 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 74

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Pro Ser Leu Val His Ser

Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro

Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro

Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

Thr His Phe Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Arg

100

105

110

- 64 045226 <210> 75 <211> 33 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>75 ccaagcctcg tacacagtga tggaaacacc tac <210>76 <211>11 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>76

Pro Ser Leu Val His Ser Asp Gly Asn Thr Tyr

510 < 210>77 < 211>9 < 212> DNA < 213> Artificial sequence <220>

< 223> synthetic < 400>77 aagatttct < 210>78 < 211>3 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400>78

Lys Ile Ser < 210 > 79 < 211 > 27 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400> 79 atgcaagcta cacattttcc gatcacc

- 65 045226 <210> 80 <211> 9 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>80

Met Gln Ala Thr His Phe Pro Ile Thr <210>81 <211>360 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 81

gaggtgcagc tggtggaatc tgggggaggt gtggtgcggc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacttttgat gattatggca tgacctgggt ccgccaagct 120 ccagggggg gcctggaatg ggtctctggt attcattggc atggtaaacg cacaggttat 180 gcagactctg tgaagggccg attcaccata tccagagaca acgccaagaa atccctgtat 240 ctgcaaatga acagtctgaa aggcgaggac acggccttgt atcattgtgt gaggggggga 300 atgagtacag gggactggtt cgacccctgg ggccagggaa ccctggtcat cgtctcctca 360

<210> 82 <211> 120 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400> 82

Glu Val Gln Leu Val Glu Ser Gly 1 5

Gly Gly Val Val Arg Pro Gly Gly

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Asp Asp Tyr 25 30

Gly Met Thr Trp Val Arg Gln Ala

40

Pro Gly Arg Gly Leu Glu Trp Val 45

Ser Gly Ile His Trp His Gly Lys

55

Arg Thr Gly Tyr Ala Asp Ser Val 60

- 66 045226

Lys 65 Gly Arg Phe Thr Ile 70 Ser Arg Asp Asn Ala 75 Lys Lys Ser Leu Tyr 80 Leu Gln Met Asn Ser 85 Leu Lys Gly Glu Asp 90 Thr Ala Leu Tyr His 95 Cys Val Arg Gly Gly 100 Met Ser Thr Gly Asp 105 Trp Phe Asp Pro Trp 110 Gly Gln Gly Thr Leu 115 Val Ile Val Ser Ser 120

<210> 83 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>83 ggattcactt ttgatgatta tggc <210>84 <211>8 <212>PRT <213>Artificial sequence <220>

< 223> synthetic < 400>84

Gly Phe Thr Phe Asp Asp Tyr Gly <210>85 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>85 attcattggc atggtaaacg caca <210>86 <211>8 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 86

- 67 045226

Ile His Trp His Gly Lys Arg Thr 1 5 <210> 87 <211> 39 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 87 gtgagggggg gaatgagtac aggggactgg ttcgacccc <210> 88 <211> 13 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 88

Val Arg Gly Gly Met Ser Thr Gly Asp Trp Phe Asp Pro 1 5 10

324

DNA

Artificial synthetic <210>

<211>

<212>

<213>

<220>

<223>

<400>

gacatccaga atcacttgcc gggaaagccc aggttcagtg gaagattttg caagggacac sequence tgacccagtc gggcaagtca ctaaactcct gcagtggatc caacttacta gactggagat tccatcctcc gagcattaac gatctatgtt tgggacagaa ctgtcaacag taaa ctgtctgcat agtt atttaa gcatccagtt ttcactctca agttacagta ctctaggaga attggtatca tgcaaagtgg ccatcagcaa cccctccgat cagagtcacc gcagaaacca ggtcccatca tctgcaacct caccttcggc

120

180

240

300

324 <210> 90 <211> 108 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 90

- 68 045226

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 1015

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Tyr 20 2530

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 4045

Tyr Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 9095

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100105 <210>91 <211>18 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>91 cagagcatta acagttat <210>92 <211>6 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>92

Gln Ser Ile Asn Ser Tyr <210>93 <211>9 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 93

- 69 045226 gttgcatcc <210> 94 <211> 3 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>94

Val Ala Ser < 210 > 95 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400>95 caacagagtt acagtacccc tccgatcacc <210>96 <211>10 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>96

Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr

510 < 210 > 97 < 211 > 360 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400> 97

gaggtgcagc tggtggagtc tgggggaggt gtggtacggc cggggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttgat gattatggca tgacctgggt ccgccaagtt 120 ccagggaagg ggctggagtg ggtctctggt attcattgga gtggtagaag cacaggttat 180 gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240 ctgcaaatga acagtctgag agccgaggac acggccttgt attackgtgc gaggggggga 300 atgagtacgg gggactggtt cgacccctgg ggccagggaa ccctggtcac cgtctcctca 360

- 70 045226 <210> 98 <211> 120 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>98

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly

5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 2530

Gly Met Thr Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ser Gly Ile His Trp Ser Gly Arg Ser Thr Gly Tyr Ala Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 7580

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 9095

Ala Arg Gly Gly Met Ser Thr Gly Asp Trp Phe Asp Pro Trp Gly Gln

100 105110

Gly Thr Leu Val Thr Val Ser Ser

115120 <210>99 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>99 ggattcacct ttgatgatta tggc <210>100 <211>8 <212> PRT <213> Artificial sequence <220>

<223> synthetic

- 71 045226 <400>100

Gly Phe Thr Phe Asp Asp Tyr Gly 15 < 210>101 < 211>24 < 212> DNA < 213> Artificial sequence <220>

<223> synthetic <400>101 attcattgga gtggtagaag caca24 <210>102 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>102

Ile His Trp Ser Gly Arg Ser Thr < 210>103 < 211>39 < 212> DNA < 213> Artificial sequence <220>

<223> synthetic <400>103 gcgagggggg gaatgagtac gggggactgg ttcgacccc39 <210>104 <211>13 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>104

Ala Arg Gly Gly Met Ser Thr Gly Asp Trp Phe Asp Pro

510 <210>105 <211>324 <212> DNA <213> Artificial sequence

- 72 045226

<220> <223> synthetic <400> 105 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca gggaaagccc ctaagctcct gatctatgtt gcatccagtt tgcaaagtgg ggtcccatca aggttcagtg gcagtggatc tggacagat ttcactctca ccatcagcag tctgcaacct gaagatttg caactacta ctgtcaacag agttacagta cccctccgat caccttcggc caagggacac gactggagat taaa

120

180

240

300

324 <210> 106 <211> 108 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 106

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 10 15

Asp Arg Val Thr Ile Thr Cys Arg 20

Ala Ser Gln Ser Ile Ser Ser Tyr 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro

40

Gly Lys Ala Pro Lys Leu Leu Ile 45

Tyr Val Ala Ser Ser Leu Gln Ser

55

Gly Val Pro Ser Arg Phe Ser Gly 60

Ser Gly Ser Gly Thr Asp Phe Thr 65 70

Leu Thr Ile Ser Ser Leu Gln Pro

80

Glu Asp Phe Ala Thr Tyr Tyr Cys 85

Gln Gln Ser Tyr Ser Thr Pro Pro

95

Ile Thr Phe Gly Gln Gly Thr Arg

100

Leu Glu Ile Lys 105 <210> 107 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> synthetic

- 73 045226 <400>107 cagagcatta gcagctat <210>108 <211>6 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>108

Gln Ser Ile Ser Ser Tyr < 210>109 < 211>9 < 212> DNA < 213> Artificial sequence <220>

< 223> synthetic < 400>109 gttgcatcc < 210>110 < 211>3 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400>110

Val Ala Ser < 210 > 111 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400>111 caacagagtt acagtacccc tccgatcacc <210>112 <211>10 <212> PRT <213> Artificial sequence <220>

- 74 045226 <223> synthetic <400>112

Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 510 <210>113 <211>345 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 113 gaggtgcagt tcctgtgcag ccagggaagg gattccgtga caaatgagca ggtctggacg tggtggagtc cctctgggtt gactggagtg agggccgatt gcctgagacc tctggggcca tggaggaggc caccgtcggt ggtctcagtt caccatctcc t gaggacacg agggaccacg ttggtccagc agtaactaca atttatagtg agactcactt gccgtgtatt gtcaccgtct ctggggggtc tgaactgggt gtggtagtac ccaagaacac attgtgcgag cttca cctaagactc ccgtcaggct atactacgca actgtatctt agggattagg

120

180

240

300

345 <210> 114 <211> 115 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>114

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Gly Ser Asn 20 2530

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 5560

Gly Arg Phe Thr Ile Ser Arg Leu Thr Ser Lys Asn Thr Leu Tyr Leu 65 70 7580

Gln Met Ser Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 9095

- 75 045226

Arg Gly Ile Arg Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr

100 105110

Val Ser Ser

115 <210>115 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>115 gggttcaccg tcggtagtaa ctac <210>116 <211>8 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>116

Gly Phe Thr Val Gly Ser Asn Tyr <210>117 <211>21 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>117 atttatagtg gtggtagtac a <210>118 <211>7 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400> 118

Ile Tyr Ser Gly Gly Ser Thr 1 5 <210> 119 <211> 27

- 76 045226 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>119 gcgagaggga ttaggggtct ggacgtc <210>120 <211>9 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>120

Ala Arg Gly Ile Arg Gly Leu Asp Val 15 < 210>121 < 211>324 < 212> DNA < 213> Artificial sequence <220>

<223> synthetic <400>121 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc60 atcacttgcc gggcaagtca gaccattaac atctatttaa attggtatca gcagaaacca120 gggagagccc ctaggctcct gatctatgct gcatccagtt tgcaaagt gg ggtcccatca180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct240 gaagattttg caacttacta ctgtcaccag agttacagta cccctccgat caccttcggc300 caagggacac gactggagat taaa324 <210>122 <211 >108 < 212> PRT <213> Artificial sequence <220>

<223> synthetic <400>122

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 1015

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Asn Ile Tyr 20 2530

- 77 045226

Leu Asn Trp 35 Tyr Gln Gln Lys Pro 40 Gly Arg Ala Pro Arg 45 Leu Leu Ile Tyr Ala 50 Ala Ser Ser Leu Gln 55 Ser Gly Val Pro Ser 60 Arg Phe Ser Gly Ser 65 Gly Ser Gly Thr Asp 70 Phe Thr Leu Thr Ile 75 Ser Ser Leu Gln Pro 80 Glu Asp Phe Ala Thr 85 Tyr Tyr Cys His Gln 90 Ser Tyr Ser Thr Pro 95 Pro

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100105 <210>123 <211>18 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>123 cagaccatta acatctat <210>124 <211>6 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>124

Gln Thr Ile Asn Ile Tyr <210>125 <211>9 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>125 gctgcatcc <210>126 <211>3 <212> PRT <213> Artificial sequence

- 78 045226 <220>

<223> synthetic <400>126

Ala Ala Ser <210>127 <211>30 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>127 caccagagtt acagtacccc tccgatcacc <210>128 <211>10 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>128

His Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 510 <210>129 <211>345 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 129

gaggaacggt tggtggagtc tggagaggagac ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggcat caccgtcggt actaattata tgaactgggt ccgccaggct 120 ccagggaagg gactggagtg ggtctcagtt atttctagcg gtggtaatac acactacgca 180 gactccgtga aggggccgatt cattatgtcc agacaaactt ccaaaaacac gctgtatctt 240 cagatgaata gcctggaaac tgaggacacg gccgtatatt attgtgcgag ggggatcaga 300 ggtttggacg tctggggcca aggaccatg gtcaccgtct cctca 345

<210> 130 <211> 115 <212> PRT <213> Artificial sequence

- 79 045226 <220>

<223> synthetic <400> 130

Glu Glu 1

Arg Leu

Val Glu 5

Ser Gly

Gly Asp

Leu Val

Glin Pro

Ser Leu

Arg Leu 20

Ser Cys

Ala Ala

Ser Gly 25

Ile Thr

Val Gly 30

Tyr Met

Asn Trp 35

Val Arg

Gln Ala

Pro Gly

Lys Gly

Leu Glu 45

Ser Val

Ile Ser

Ser Gly

Gly Asn 55

Thr His

Tyr Ala 60

Asp Ser

Gly Gly 15

Thr Asn

Trp Val

Val Lys

Gly Arg 65

Phe Ile

Met Ser

Arg Gln

Thr Ser

Lys Asn 75

Thr Leu

Tyr Leu 80

Gln Met

Asn Ser

Leu Glu 85

Thr Glu

Asp Thr

Ala Val

Tyr Tyr

Cys Ala 95

Arg Gly

Ile Arg

100

Gly Leu

Asp Val

Trp Gly 105

Gln Gly

Thr Met

110

Val Thr

Val Ser Ser

115 <210> 131 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>131 ggcatcaccg tcggtactaa ttat <210>132 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400> 132

Gly Ile Thr Val Gly Thr Asn Tyr

5

- 80 045226 <210> 133 <211> 21 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>133 atttctagcg gtggtaatac a21 <210>134 <211>7 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>134

Ile Ser Ser Gly Gly Asn Thr <210>135 <211>27 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>135 gcgaggggga tcagaggttt ggacgtc27 <210>136 <211>9 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>136

Ala Arg Gly Ile Arg Gly Leu Asp Val <210>137 <211>324 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 137 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

- 81 045226

atcacttgcc gggcaagtca gagcatgagc agctatttaa attggtatca gcagaaacca 120 ggggagagccc ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagatttg caactacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaa 324

<210> 138 <211> 108 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 138

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 10 15

Asp Arg Val Thr Ile Thr Cys Arg 20

Ala Ser Gln Ser Met Ser Ser Tyr 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro

40

Gly Arg Ala Pro Lys Leu Leu Ile 45

Phe Ala Ala Ser Ser Leu Gln Ser

55

Gly Val Pro Ser Arg Phe Ser Gly 60

Ser Gly Ser Gly Thr Asp Phe Thr 65 70

Leu Thr Ile Ser Ser Leu Gln Pro

80

Glu Asp Phe Ala Thr Tyr Tyr Cys 85

Gln Gln Ser Tyr Ser Thr Pro Pro

95

Ile Thr Phe Gly Gln Gly Thr Arg

100

Leu Glu Ile Lys 105 <210> 139 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>139 cagagcatga gcagctat <210>140 <211>6

- 82 045226 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>140

Gln Ser Met Ser Ser Tyr < 210>141 < 211>9 < 212> DNA < 213> Artificial sequence <220>

< 223> synthetic < 400>141 gctgcatcc < 210>142 < 211>3 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400>142

Ala Ala Ser < 210 > 143 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400>143 caacagagtt acagtacccc tccgatcacc <210>144 <211>10 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400> 144

Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr

5 10

- 83 045226 <210> 145 <211> 354 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 145 caggtccagc tcctgcaagg cctggacaag gcacagaagt atggagctga ggattaggct tggtgcagtc cttctggagg ggcttgaatg tccaggacag gcagcctgaa ctttttatat tggggctgag catcttcagc gatggggagag agtcatattt atctgg ggac ctggggccaa gtgaagatgc agttctacta atcatccctg accgcggacg acggccgtat gggacaatgg ctgggtcctc tcagttgggt tctttggtac aatctacgac atttctgtgc tcaccgtctc ggtgaggtc gcgacaggcc agtaaactac tacagcctac gcgaaattgg t tca

120

180

240

300

354 <210> 146 <211> 118 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 146

Gln Val Gln Leu Val Gln Ser Gly 1 5

Ala Glu Val Lys Met Pro Gly Ser

15

Ser Val Arg Val Ser Cys Lys Ala 20

Ser Gly Gly Ile Phe Ser Ser Ser 25 30

Thr Ile Ser Trp Val Arg Gln Ala

40

Pro Gly Gln Gly Leu Glu Trp Met 45

Gly Glu Ile Ile Pro Val Phe Gly

55

Thr Val Asn Tyr Ala Gln Lys Phe 60

Gln Asp Arg Val Ile Phe Thr Ala 65 70

Asp Glu Ser Thr Thr Thr Ala Tyr

80

Met Glu Leu Ser Ser Leu Lys Ser 85

Gly Asp Thr Ala Val Tyr Phe Cys

95

Ala Arg Asn Trp Gly Leu Gly Ser

100

Phe Tyr Ile Trp Gly Gln Gly Thr

105 110

Met Val Thr Val Ser Ser

115

- 84 045226 <210> 147 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>147 ggaggcatct tcagcagttc tact24 <210>148 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>148

Gly Gly Ile Phe Ser Ser Ser Thr <210>149 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>149 atcatccctg tctttggtac agta24 <210>150 <211>8 <212>PRT <213>Artificial sequence <220>

< 223> synthetic < 400>150

Ile Ile Pro Val Phe Gly Thr Val <210>151 <211>33 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 151

- 85 045226 gcgcgaaatt ggggattagg ctctttttat atc <210> 152 <211> 11 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>152

Ala Arg Asn Trp Gly Leu Gly Ser Phe Tyr Ile

510 <210>153 <211>324 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 153 gaaattgtgt ctctcctgca cctggccagg gacaggttca cctgaagatt caagggacca tgacgcagtc gggccagtca ctcccagact gtggcagtgg ttggagtgtt aggtggaaat tccaggcacc gagttttaac cctcatctat gtctgggaca ttatt gtcag caaa ctgtctttgt ttcaactact ggtgcatcca gacttcactc cagtatgaaa ctccagggga tagcctggta gcagggccac tcaccatcaa gcgcaccttg aagagccacc ccagcagaaa tggcatccca caggctggag gacgttcggc

120

180

240

300

324 <210>

<211>

<212>

<213>

154

108

PRT

Artificial sequence <220>

<223> synthetic <400> 154

Glu Ile Val Leu 1

Thr Gln Ser Pro 5

Gly Thr Leu Ser 10

Leu Ser Pro Gly 15

Glu Arg Ala Thr 20

Leu Ser Cys Arg

Ala Ser Gln Ser 25

Phe Asn Phe Asn

Tyr Leu Ala Trp

Tyr Gln Gln Lys 40

Pro Gly Gln Ala

Pro Arg Leu Leu 45

Ile Tyr Gly Ala 50

Ser Ser Arg Ala

Thr Gly Ile Pro 60

Asp Arg Phe Ser

- 86 045226

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75

Ile Asn Arg Leu Glu 80

Pro Glu Asp Phe Gly Val Phe Tyr Cys Gln Gln 85 90

Tyr Glu Ser Ala Pro 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105

Lys <210> 155 <211> 21 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 155 cagagtttta acttcaacta c <210> 156 <211> 7 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400> 156

Gln Ser Phe Asn Phe Asn Tyr

5 <210> 157 <211> 9 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 157 ggtgcatcc <210> 158 <211> 3 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400> 158

Gly Ala Ser

- 87 045226 <210> 159 <211> 27 <212> DNA <213> Artificial sequence <220>

< 223> synthetic <400>159 cagcagtatg aaagcgcacc ttggacg <210>160 <211>9 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>160

Gln Gln Tyr Glu Ser Ala Pro Trp Thr 15 < 210 > 161 < 211 > 345 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400> 161

gaggtgcagc ttgtagagtc tggggggagac ttggtacatc ctggcaggtc cctgagactc 60 tcctgtgcag cctctggttt cccctttgat gagtatgcca tgcactgggt ccggcaagtt 120 ccagggaagg gcctggagtg ggtctcaggt attagttgga gtaataataa cataggctat 180 gcggactctg tgaagggccg attcaccatc tccagagaca acgccaaaaa ctccctgtat 240 ctacaaatga acagtctgag acctgaggac acggcctttt attackgtgc aaaatctgga 300 atctttgact cctggggcca gggaaccctg gtcaccgtct cctca 345

< 210> 162 < 211> 115 < 212> PRT < 213> Artificial sequence < 220>

< 223> synthetic < 400> 162

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val His Pro Gly Arg

5 10 15

- 88 045226

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Pro Phe Asp Glu Tyr 25 30

Ala Met His Trp Val Arg Gln Val

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ser Gly Ile Ser Trp Ser Asn Asn

55

Asn Ile Gly Tyr Ala Asp Ser Val 60

Lys Gly Arg Phe Thr Ile Ser Arg 65 70

Asp Asn Ala Lys Asn Ser Leu Tyr

80

Leu Gln Met Asn Ser Leu Arg Pro 85

Glu Asp Thr Ala Phe Tyr Tyr Cys

95

Ala Lys Ser Gly Ile Phe Asp Ser

100

Trp Gly Gln Gly Thr Leu Val Thr

105 110

Val Ser Ser

115 <210> 163 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>163 ggtttcccct ttgatgagta tgcc <210>164 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>164

Gly Phe Pro Phe Asp Glu Tyr Ala <210>165 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic

- 89 045226 <400>165 attagttgga gtaataataa cata <210>166 <211>8 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>166

Ile Ser Trp Ser Asn Asn Asn Ile <210>167 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>167 gcaaaatctg gaatctttga ctcc <210>168 <211>8 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>168

Ala Lys Ser Gly Ile Phe Asp Ser <210>169 <211>315 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 169

gacatccaga tgaccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaagctcc tgatctatgc tgcatccagt ttgcaaagtg gggtccatc acggttcagt 180 ggcggtggat ctggacaga tttcactctc accatcagca gtctgcgacc tgaagatttt 240 gcaacttact actgtcaaca gagttactgt acccctccga tcaccttcgg ccaagggaca 300

- 90 045226 cgactggaga ttaaa

315 <210> 170 <211> 105 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>170

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 1015

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 2530

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Leu Leu Ile Tyr Ala Ala 35 4045

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Gly Gly Ser 50 5560

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Arg Pro Glu Asp Phe 65 70 7580

Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Cys Thr Pro Pro Ile Thr Phe 85 9095

Gly Gln Gly Thr Arg Leu Glu Ile Lys

100105 <210>171 <211>18 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>171 cagagcatta gcagctat <210>172 <211>6 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 172

- 91 045226

Gln Ser Ile Ser Ser Tyr < 210> 173 < 211> 9 < 212> DNA < 213> Artificial sequence <220>

< 223> synthetic < 400>173 gctgcatcc < 210>174 < 211>3 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400>174

Ala Ala Ser < 210 > 175 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400>175 caacagagtt actngtacccc tccgatcacc <210>176 <211>10 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>176

Gln Gln Ser Tyr Cys Thr Pro Pro Ile Thr

510 <210>177 <211>354 <212> DNA <213> Artificial sequence <220>

- 92 045226 <223> synthetic

<400> 177 gaggtgcagc tggtggagtc cgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agttatggca tgcactgggt ccgccaggct 120 ccaggcaagg gactggagtg ggtgacactt atatcatatg agggaaggaa taaatactat 180 gcagactccg tgaagggccg attcaccatt tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtat attackgtgc gaaagatagg 300 accctttacg gtatggacgt ctggggccaa ggaaccacgg tcaccgtctc ctca 354

<210> 178 <211> 118 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>178

Glu 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Val Val Gln Pro Gly 15 Arg Ser Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Phe Ser 30 Ser Tyr Gly Met His 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Val Thr Leu 50 Ile Ser Tyr Glu Gly 55 Arg Asn Lys Tyr Tyr 60 Ala Asp Ser Val Lys 65 Gly Arg Phe Thr Ile 70 Ser Arg Asp Asn Ser 75 Lys Asn Thr Leu Tyr 80 Leu Gln Met Asn Ser 85 Leu Arg Ala Glu Asp 90 Thr Ala Val Tyr Tyr 95 Cys Ala Lys Asp Arg 100 Thr Leu Tyr Gly Met 105 Asp Val Trp Gly Gln 110 Gly Thr

Thr Val Thr Val Ser Ser

115 <210>179 <211>24 <212> DNA <213> Artificial sequence

- 93 045226 <220>

<223> synthetic <400>179 ggattcacct tcagtagtta tggc <210>180 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>180

Gly Phe Thr Phe Ser Ser Tyr Gly <210>181 <211>24 <212> DNA <213> Artificial sequence <220>

< 223> synthetic < 400>181 atatcatatg agggaaggaa taaa24 < 210>182 < 211>8 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400>182

Ile Ser Tyr Glu Gly Arg Asn Lys < 210>183 < 211>33 < 212> DNA < 213> Artificial sequence <220>

<223> synthetic <400>183 gcgaaagata ggacccttta cggtatggac gtc33 <210>184 <211>11 <212> PRT <213> Artificial sequence

- 94 045226 <220>

<223> synthetic <400> 184

Ala Lys Asp Arg Thr Leu Tyr Gly Met Asp Val

5 10 <210>

<211>

<212>

<213>

185

363

DNA

Artificial sequence <220>

<223>

synthetic <400>

185

caggtcacct tgagggagtc tggtcctgcg ctggtgaaaa ccacacagac cctcacactg 60 acctgcacct tctctggggtt ctcactcagc actaatagaa tgtgtgtgac ctggatccgt 120 cagcccccag ggaaggccct ggagtggctt gcgcgcattg attggatgg tgttaaatac 180 tacaacacat ctctgaagac caggctcacc atctccaagg acacctccaa aaaccaggtg 240 gtccttacaa tgaccaacat ggaccctgtg gacacagcca ctttttactg tgcacggtcg 300 acttcgttga ctttttacta ctttgactac tggggccagg gaaccctggt caccgtctcc 360 tca 363

<210>

<211>

<212>

<213>

186

121

PRT

Artificial sequence <220>

<223>

synthetic <400>

186

Gln 1

Val

Thr

Leu

Arg 5

Glu

Ser

Gly

Pro

Ala 10

Leu

Val

Lys

Thr

Thr 15

Gln

Thr

Leu

Thr

Leu 20

Thr

Cys

Thr

Phe

Ser 25

Gly

Phe

Ser

Leu

Ser 30

Thr

Asn

Arg

Met

Cys 35

Val

Thr

Trp

Ile

Arg 40

Gln

Pro

Pro

Gly

Lys 45

Ala

Leu

Glu

Trp

Leu 50

Ala

Arg

Ile

Asp

Trp 55

Asp

Gly

Val

Lys

Tyr 60

Tyr

Asn

Thr

Ser

Leu 65

Lys

Thr

Arg

Leu

Thr 70

Ile

Ser

Lys

Asp

Thr 75

Ser

Lys

Asn

Gln

Val 80

- 95 045226

Val Leu Thr Met Thr Asn Met Asp 85

Pro Val Asp Thr Ala Thr Phe Tyr

Cys Ala Arg Ser Thr

100

Ser Leu Thr Phe Tyr Tyr Phe Asp Tyr Trp Gly

105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 187 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 187 gggttctcac tcagcactaa tagaatgtgt <210> 188 <211> 10 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 188

Gly Phe Ser Leu Ser Thr Asn Arg Met Cys

5 10 <210> 189 <211> 21 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 189 attgattggg atggtgttaa a <210> 190 <211> 7 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 190

Ile Asp Trp Asp Gly Val Lys 1 5

- 96 045226 <210> 191 <211> 39 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>191 gcacggtcga cttcgttgac tttttactac tttgactac <210>192 <211>13 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>192

Ala Arg Ser Thr Ser Leu Thr Phe Tyr Tyr Phe Asp Tyr 1 510 <210>193 <211>324 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 193 gacatccaga atcacttgcc gggaaagccc aggttcagtg gaagattttg caagggacac tgacccagtc gggcaagtca ctaagctcct gcagtggatc caacttacta gactggagat tccatcctcc gagcattagc gatctatgct tgggacagat ctgtcaacag taaa ctgtctgcat agctatttaa gcatccagtt ttcactctca agttacagta ctgtaggaga attggtatca tgcaaagtgg ccatcagcag cccctccgat cagagtcacc gcagaaacca ggtcccatca tctgcaacct caccttcggc

120

180

240

300

324 <210> 194 <211> 108 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 194

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 10 15

- 97 045226

Asp Arg Val Thr Ile Thr Cys Arg 20

Ala Ser Gln Ser Ile Ser Ser Tyr 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro

40

Gly Lys Ala Pro Lys Leu Leu Ile 45

Tyr Ala Ala Ser Ser Leu Gln Ser

55

Gly Val Pro Ser Arg Phe Ser Gly 60

Ser Gly Ser Gly Thr Asp Phe Thr 65 70

Leu Thr Ile Ser Ser Leu Gln Pro

80

Glu Asp Phe Ala Thr Tyr Tyr Cys 85

Gln Gln Ser Tyr Ser Thr Pro Pro

95

Ile Thr Phe Gly Gln Gly Thr Arg

100

Leu Glu Ile Lys 105 <210> 195 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>195 cagagcatta gcagctat <210>196 <211>6 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>196

Gln Ser Ile Ser Ser Tyr <210>197 <211>9 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 197 gctgcatcc

- 98 045226 <210> 198 <211> 3 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>198

Ala Ala Ser <210>199 <211>30 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>199 caacagagtt acagtacccc tccgatcacc <210>200 <211>10 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>200

Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 510 <210>201 <211>345 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 201 gaggtgcagc tcctgtgcag ccagggaagg gactccgtga caaatgaaca ggtatggacg tggtggagtc cctctgagtt gactggagtg agggccgatt gcctgacagc tctggggcca tggaggaggc caccgtcggt ggtctcagtt caccatctcc agaggacacg agggaccacg ttggtccagc accaaccaca atttatagcg agacacactt gccgtatatt gtcaccgtct cgggggggtc tgaactgggt gtggtaacac ccaagaacac actgtgcgcg cctca cctgagactc ccgccaggct attctacgca gctgtatctt aggattgggg

120

180

240

300

345 <210> 202

- 99 045226 <211> 115 < 212> PRT < 213> Artificial sequence <220>

<223> synthetic <400>202

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly

5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Val Gly ThrAsn

2530

His Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ser Val Ile Tyr Ser Gly Gly Asn Thr Phe Tyr Ala Asp Ser Val Lys 50 5560

Gly Arg Phe Thr Ile Ser Arg His Thr Ser Lys Asn Thr Leu TyrLeu

70 7580

Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr CysAla

9095

Arg Gly Leu Gly Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 100 105110

Val Ser Ser

115 <210>203 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>203 gagttcaccg tcggtaccaa ccac <210>204 <211>8 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 204

- 100 045226

Glu Phe Thr Val Gly Thr Asn His <210> 205 <211> 21 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>205 atttatagcg gtggtaacac a21 <210>206 <211>7 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>206

Ile Tyr Ser Gly Gly Asn Thr <210>207 <211>27 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>207 gcgcgaggat tggggggtat ggacgtc27 <210>208 <211>9 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>208

Ala Arg Gly Leu Gly Gly Met Asp Val <210>209 <211>321 <212> DNA <213> Artificial Sequence <220>

<223> synthetic

- 101 045226 <400> 209

gacatccaga tgaccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcgagtca ggtcattagc aattatttag cctggtatca gcagaaacca 120 gggaaagttc ctaggctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180 cgtttcagtg gcagtggatc tggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagatgttg caacttatta ctgtcaaaag tataacagtg cccctcggac gttcggccaa 300 gggaccaagg tggaaatcaa a 321

<210> 210 <211> 107 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>210

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 1015

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Ser Asn Tyr 20 2530

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Arg Leu Leu Ile 35 4045

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 7580

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Arg 85 9095

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100105 <210>211 <211>18 <212>DNA <213>Artificial sequence <220>

< 223> synthetic < 400> 211 caggtcatta gcaattat

- 102 045226 <210> 212 <211> 6 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>212

Gln Val Ile Ser Asn Tyr <210>213 <211>9 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>213 gctgcatcc9 <210>214 <211>3 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>214

Ala Ala Ser <210>215 <211>27 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>215 caaaagtata acagtgcccc tcggacg <210>216 <211>9 <212>PRT <213>Artificial sequence <220>

<223> synthetic <400> 216

- 103 045226

Gln Lys Tyr Asn Ser Ala Pro Arg Thr 15 <210>217 <211>360 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 217

gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ggggggagtc cctgagactt 60 tactgtgcag cctctggatt cacctttagt aaatattgga tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtggccaac aaaagggag atggaagtga gaaatactat 180 gtggactctg tgaagggccg gttcaccatc tccagagaca acgccaagaa ctaccatat 240 ctacaaatga acagcctgag agccgaggac acggctgttt attackgtgc gagagattat 300 tggggatcag gctactactt tgacttctgg ggccagggaa ccctggtcac cgtctcctca 360

<210> 218 <211> 120 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 218

Glu Val Gln Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gln Arg Gly Glu

15

Ser Leu Arg Leu Tyr Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Lys Tyr 25 30

Trp Met Asn Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Asn Ile Lys Gly Asp Gly Ser

55

Glu Lys Tyr Tyr Val Asp Ser Val 60

Lys Gly Arg Phe Thr Ile Ser Arg 65 70

Asp Asn Ala Lys Asn Ser Leu Tyr

80

Leu Gln Met Asn Ser Leu Arg Ala 85

Glu Asp Thr Ala Val Tyr Tyr Cys

95

Ala Arg Asp Tyr Trp Gly Ser Gly

100

Tyr Tyr Phe Asp Phe Trp Gly Gln

105 110

- 104 045226

Gly Thr Leu Val Thr Val Ser Ser

115120 <210>219 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>219 ggattcacct ttagtaaata ttgg24 <210>220 <211>8 <212>PRT <213>Artificial sequence <220>

< 223> synthetic < 400>220

Gly Phe Thr Phe Ser Lys Tyr Trp < 210 > 221 < 211 > 24 < 212 > DNA < 213 > Artificial sequence < 220 >

< 223> synthetic < 400>221 ataaagggag atggaagtga gaaa24 < 210>222 < 211>8 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400>222

Ile Lys Gly Asp Gly Ser Glu Lys <210>223 <211>39 <212> DNA <213> Artificial sequence

- 105 045226 gcgagagatt attggggatc aggctactac tttgacttc <220>

<223> synthetic <400>223 <210>224 <211>13 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>224

Ala Arg Asp Tyr Trp Gly Ser Gly Tyr Tyr Phe Asp Phe

510 <210>225 <211>321 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 225 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc atcacttgcc gggcaagtca gaacattaac aactatttaa attggtatca gcagaaacca gggaaagccc ctaaactcct gatctatgct gcatccagtt tccaaaatgc ggtcccatca aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct gaagattttg caacttacta ctgtcaacag agttacaata ccccgctcac tttcggcggg gggaccaagg tggagatcaa a

120

180

240

300

321 <210>

<211>

<212>

<213>

226

107

PRT

Artificial sequence <220>

<223> synthetic <400>226

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValGly

5 1015

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asn AsnTyr

2530

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 4045

- 106 045226

Tyr Ala 50 Ala Ser Ser Phe Gln 55 Asn Ala Val Pro Ser 60 Arg Phe Ser Gly Ser Gly 65 Ser Gly Thr Asp 70 Phe Thr Leu Thr Ile 75 Ser Ser Leu Gln Pro 80 Glu Asp Phe Ala Thr 85 Thr Phe Gly Gly Gly 100 <210> 227 <211> 18 <212> DNA <213> Artificial <220> <223> synthetic <400> 227 cagaacatta acaactat Tyr Tyr Cys Gln Gln 90 Thr Lys Val Glu Ile 105 subsequence Ser Lys Tyr Asn Thr Pro 95 Leu 18

<210> 228 <211> 6 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>228

Gln Asn Ile Asn Asn Tyr <210>229 <211>9 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>229 gctgcatcc <210>230 <211>3 <212> PRT <213> Artificial sequence <220>

<223> synthetic

- 107 045226 <400>230

Ala Ala Ser 1 <210>231 <211>27 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>231 caacagagtt acaatacccc gctcact <210>232 <211>9 <212>PRT <213>Artificial sequence <220>

<223> synthetic <400>232

Gln Gln Ser Tyr Asn Thr Pro Leu Thr 15 <210>233 <211>390 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 233 gaggtgcagc tcctgtgcag ccagggaagg gtggactctg ctgcaaatga attgtagtag ggccaaggga tggtggagtc cctctggatt ggctggagtg tgaagggccg acagcctgag taccagctcc ccacggtcac tgggggaggc cacctttagt ggtgg ccaac attcaccatc agccgatgac tatgggatat cgtctcctca ttggtccagt agctattgga ataaagcaag tccagagaca acggctgtgt tactactact ctggggggtc tgagctgggt atggaagtga acgccaagaa attactgtgc acttcggtat cctgagactc ccgccaggct gaaatactat ctcact gtat gagagatgat ggacgtctgg

120

180

240

300

360

390 <210> 234 <211> 130 <212> PRT <213> Artificial sequence <220>

- 108 045226 <223> synthetic <400> 234

Glu Val 1

Gln Leu

Val Glu 5

Ser Gly

Gly Gly

Leu Val

Gln Ser

Ser Leu

Arg Leu 20

Ser Cys

Ala Ala

Ser Gly 25

Phe Thr

Phe Ser

Trp Met

Ser Trp 35

Val Arg

Gln Ala

Pro Gly

Lys Gly

Leu Glu 45

Ala Asn

Ile Lys

Gln Asp

Gly Ser 55

Glu Lys

Tyr Tyr 60

Val Asp

Lys Gly 65

Arg Phe

Thr Ile

Ser Arg

Asp Asn

Ala Lys 75

Asn Ser

Leu Gln

Met Asn

Ser Leu 85

Arg Ala

Asp Asp

Thr Ala

Val Tyr

Ala Arg

Asp Asp

100

Ile Val

Val Val

Pro Ala 105

Pro Met

Gly Tyr

110

Tyr Tyr

Phe Gly 115

Met Asp

Val Trp

120

Gly Gln

Gly Thr

Thr Val 125

Gly Gly 15

Ser Tyr

Trp Val

Ser Val

Leu Tyr 80

Tyr Cys 95

Tyr Tyr

Thr Val

Ser Ser

130 <210> 235 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>235 ggattcacct ttagtagcta ttgg <210>236 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400> 236

Gly Phe Thr Phe Ser Ser Tyr Trp

- 109 045226 <210> 237 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>237 ataaagcaag atggaagtga gaaa <210>238 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>238

Ile Lys Gln Asp Gly Ser Glu Lys <210>239 <211>69 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>239 gcgagagatg atattgtagt agtaccagct cctatgggat attactacta ctacttcggt atggacgtc <210>240 <211>23 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>240

Ala Arg Asp Asp Ile Val Val Val Pro Ala Pro Met Gly Tyr Tyr Tyr

5 1015

Tyr Tyr Phe Gly Met Asp Val 20 <210>241 <211>321

- 110 045226 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 241

gacatccaga tgaccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tggacagaa ttcactctca caatcagcag cctgcagcct 240 gaagatttg caacttatta ctgtctacag cataatagtt acccgtacac ttttggccag 300 gggaccaagc tggagatcaa a 321

<210> 242 <211> 107 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>242

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 1015

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 2530

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 4045

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Tyr 85 9095

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100105 <210>243 <211>18 <212> DNA <213> Artificial sequence

- 111 045226 <220>

<223> synthetic <400>243 cagggcatta gaaatgat <210>244 <211>6 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>244

Gln Gly Ile Arg Asn Asp 15 < 210>245 < 211>9 < 212> DNA < 213> Artificial sequence <220>

< 223> synthetic < 400>245 gctgcatcc < 210>246 < 211>3 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400>246

Ala Ala Ser < 210 > 247 < 211 > 27 < 212 > DNA < 213 > Artificial sequence < 220 >

< 223 > synthetic < 400 > 247 ctacagcata atagttaccc gtacact < 210 > 248 < 211 > 9 < 212 > PRT < 213 > Artificial sequence

- 112 045226 <220>

<223> synthetic <400>248

Leu Gln His Asn Ser Tyr Pro Tyr Thr 15 <210>249 <211>369 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>249 gaagtgcagc tggtggagtc tgggggaggc ttggttcagc ctggcaggtc cctgagactc60 tcctgtgcag cctctggatt cacctttgat gattttgcca tgcactgggt ccgacaagct120 ccagggaagg gcctggagtg ggtctcaggt attagttgga ctggtggtaa catggactat180 gcgaactctg tgaagggccg attcaccatc tccagagagg acgccaagaa ttccctgtat240 ctgcaaatga acagtctgag agctgcggac acggccttgt attactgtgt aaaagatata300 agggggatag tggctacggg gggggctttt gatatct ggg gccgagggac aatggtcacc360 gtctcttca369 <210>250 < 211>123 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 250

Glu Val Gln Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gln Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Asp Asp Phe 25 30

Ala Met His Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ser Gly Ile Ser Trp Thr Gly Gly

55

Asn Met Asp Tyr Ala Asn Ser Val 60

Lys Gly Arg Phe Thr Ile Ser Arg 65 70

Glu Asp Ala Lys Asn Ser Leu Tyr

80

- 113 045226

Leu Gln Met Asn Ser 85 Leu Arg Ala Ala Asp 90 Thr Ala Leu Tyr Tyr 95 Cys Val Lys Asp Ile 100 Arg Gly Ile Val Ala 105 Thr Gly Gly Ala Phe 110 Asp Ile Trp Gly Arg 115 Gly Thr Met Val Thr 120 Val Ser Ser

<210> 251 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>251 ggattcacct ttgatgattt tgcc24 <210>252 <211>8 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>252

Gly Phe Thr Phe Asp Asp Phe Ala <210>253 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>253 attagttgga ctggtggtaa catg24 <210>254 <211>8 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 254

Ile Ser Trp Thr Gly Gly Asn Met 1 5

- 114 045226 <210> 255 <211> 48 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>255 gtaaaagata taagggggat agtggctacg gggggggctt ttgatatc <210>256 <211>16 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>256

Val Lys Asp Ile Arg Gly Ile Val Ala Thr Gly Gly Ala Phe Asp Ile

5 1015 < 210>257 < 211>321 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 257

gacatccaga tgaccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atctcttgcc gggcaagtca gaccattagc acttatttaa attggtttca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgtt gtgtccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagatttg caacttatta ctgtcaacag agttacagta ccccattcac tttcggccct 300 gggaccaaag tggatatcaa a 321

<210> 258 <211> 107 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 258

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 10 15

- 115 045226

Asp Arg Val Thr Ile Ser Cys Arg 20

Ala Ser Gln Thr Ile Ser Thr Tyr 25 30

Leu Asn Trp Phe Gln Gln Lys Pro

40

Gly Lys Ala Pro Lys Leu Leu Ile 45

Tyr Val Val Ser Ser Leu Gln Ser

55

Gly Val Pro Ser Arg Phe Ser Gly 60

Ser Gly Ser Gly Thr Asp Phe Thr 65 70

Leu Thr Ile Ser Ser Leu Gln Pro

80

Glu Asp Phe Ala Thr Tyr Tyr Cys 85

Gln Gln Ser Tyr Ser Thr Pro Phe

95

Thr Phe Gly Pro Gly Thr Lys Val

100

Asp Ile Lys 105 <210> 259 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>259 cagaccatta gcacttat <210>260 <211>6 <212>PRT <213>Artificial sequence <220>

< 223> synthetic < 400>260

Gln Thr Ile Ser Thr Tyr <210>261 <211>9 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 261 gttgtgtcc

- 116 045226 <210> 262 <211> 3 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>262

Val Val Ser <210>263 <211>27 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>263 caacagagtt acagtacccc attcact <210>264 <211>9 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>264

Gln Gln Ser Tyr Ser Thr Pro Phe Thr <210>265 <211>345 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 265

gaggtgcagc tggtggagtc tggaggaggc ttggtccagc cggggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccgtcggt accaactaca tgaactgggt ccgccaggct 120 ccagggaagg gactggagtg gatctcagtt atttatagcg gtggtagcac attctacgca 180 gactccgtga aggggccgatt caccattctcc agacagactt cccagaacac gctgtatctt 240 caaatgaaca gcctgagacc tgaggacacg gccgtatatt actgtgcgag aggtatacgt 300 ggttttgata tctggggcca agggacaatg gtcaccgtct cttca 345

<210> 266

- 117 045226 <211> 115 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>266

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly

5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Gly ThrAsn

2530

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 4045

Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys 50 5560

Gly Arg Phe Thr Ile Ser Arg Gln Thr Ser Gln Asn Thr Leu TyrLeu

70 7580

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr CysAla

9095

Arg Gly Ile Arg Gly Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr 100 105110

Val Ser Ser

115 <210>267 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>267 ggattcaccg tcggtaccaa ctac <210>268 <211>8 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 268

- 118 045226

Gly Phe Thr Val Gly Thr Asn Tyr 15 <210>269 <211>21 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>269 atttatagcg gtggtagcac a21 <210>270 <211>7 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>270

Ile Tyr Ser Gly Gly Ser Thr <210>271 <211>27 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>271 gcgagaggta tacgtggttt tgatatc27 <210>272 <211>9 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>272

Ala Arg Gly Ile Arg Gly Phe Asp Ile <210>273 <211>324 <212> DNA <213> Artificial sequence <220>

<223> synthetic

- 119 045226 <400> 273

gacatccaga tgaccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180 aggttcagtg gcagtggatc tggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagatttg caactacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaa 324

<210> 274 <211> 108 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400> 274

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

5 10 15

Asp Arg Val Thr Ile Thr Cys Arg 20

Ala Ser Gln Ser Ile Ser Ser Tyr 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro

40

Gly Lys Ala Pro Lys Leu Leu Ile 45

Tyr Ala Ala Ser Ser Leu Gln Ser

55

Gly Val Pro Ser Arg Phe Ser Gly 60

Ser Gly Ser Gly Thr Asp Phe Thr 65 70

Leu Thr Ile Ser Ser Leu Gln Pro

80

Glu Asp Phe Ala Thr Tyr Tyr Cys 85

Gln Gln Ser Tyr Ser Thr Pro Pro

95

Ile Thr Phe Gly Gln Gly Thr Arg

100

Leu Glu Ile Lys 105 <210> 275 <211> 18 <212> DNA <213> Artificial sequence <220>

< 223> synthetic < 400> 275 cagagcatta gcagctat

- 120 045226 <210> 276 <211> 6 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>276

Gln Ser Ile Ser Ser Tyr < 210 > 277 < 211 > 9 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400>277 gctgcatcc <210>278 <211>3 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>278

Ala Ala Ser <210>279 <211>30 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>279 caacagagtt acagtacccc tccgatcacc <210>280 <211>10 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 280

- 121 045226

Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 510 <210>281 <211>345 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 281

gaggtgcagc tggtggagtc tggaggaggc ttggtccagc cggggggggtc cctgagactc 60 tcctgtgcag cctctggggtt taccatcagt accaactaca tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtcgcagtt atttatagca gtggttccac atactatatc 180 gactccgtga aggggccgatt caccattctcc agactcactt ccaagaacac ggtgtatctt 240 caaatgagca gcctgaattc tgaagacacg gccgtgtatt actgtgcgag ggggatcagg 300 ggttttgata tttggggcca agggacaatg gtcaccgtct cttca 345

<210> 282 <211> 115 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 282

Glu Val Gln Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gln Pro Gly Gly

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Ile Ser Thr Asn 25 30

Tyr Met Asn Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Val Ile Tyr Ser Ser Gly Ser

55

Thr Tyr Tyr Ile Asp Ser Val Lys 60

Gly Arg Phe Thr Ile Ser Arg Leu 65 70

Thr Ser Lys Asn Thr Val Tyr Leu

80

Gln Met Ser Ser Leu Asn Ser Glu

Asp Thr Ala Val Tyr Tyr Cys Ala

95

Arg Gly Ile Arg Gly Phe Asp Ile

100

Trp Gly Gln Gly Thr Met Val Thr

105 110

- 122 045226

Val Ser Ser

115 <210> 283 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>283 gggtttacca tcagtaccaa ctac24 <210>284 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>284

Gly Phe Thr Ile Ser Thr Asn Tyr <210>285 <211>21 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>285 atttatagca gtggttccac a21 <210>286 <211>7 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>286

Ile Tyr Ser Ser Gly Ser Thr <210>287 <211>27 <212> DNA <213> Artificial sequence

- 123 045226 <220>

<223> synthetic <400> 287 gcgaggggga tcaggggttt tgatatt <210> 288 <211> 9 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 288

Ala Arg Gly Ile Arg Gly Phe Asp Ile 1 5

289

372

DNA

Artificial synthetic

289 <210>

<211> <212>

<213>

<220> <223>

<400>

gaagtgcagc tcctgtgcag ccagggaagg gcggactctg ctgcaaatga aggggcaact actgtctcct sequence tggtggagtc cctctggatt gcctggagtg tgaggggccg acagtctgag ggaactacgg ca ggggggaggc caccattgat ggtctcaggt attcaccatc agttgaggac gggaaactgg ttggtacagc gatagtgcca attagttgga tccagagaca acggccttgt ttcgacccct ctggcaggtc tgcactgggt aaagtggtag acgccaagaa attactgtgt ggggccaggg cctgagactc ccggcaaact cataggttat ttccctctat aaaagatata aaccctggtc

120

180

240

300

360

372 <210> 290 <211> 124 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>290

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser 2025

Gly Phe Thr Ile Asp Asp Ser

- 124 045226

Ala Met

Ser Gly 50

Arg Gly 65

Leu Gln

His Trp 35

Ile Ser

Arg Phe

Met Asn

Val Arg

Trp Lys

Thr Ile

Ser Leu 85

Val Lys

Asp Ile

100

Arg Gly

Gln Thr

Ser Gly 55

Ser Arg

Arg Val

Asn Trp

Pro Gly

Ser Ile

Asp Asn

Glu Asp 90

Asn Tyr 105

Lys Gly

Gly Tyr 60

Ala Lys 75

Thr Ala

Gly Gly

Leu Glu 45

Ala Asp

Asn Ser

Leu Tyr

Asn Trp

110

Trp Val

Ser Val

Leu Tyr 80

Tyr Cys 95

Phe Asp

ProTrp

Gly Gln 115

Gly Thr

Leu Val

120

Thr Val

Ser Ser <210> 291 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>291 ggattcacca ttgatgatag tgcc <210>292 <211>8 <212>PRT <213>Artificial sequence <220>

< 223> synthetic < 400>292

Gly Phe Thr Ile Asp Asp Ser Ala <210>293 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 293 attagttgga aaagtggtag cata <210> 294

- 125 045226 <211> 8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>294

Ile Ser Trp Lys Ser Gly Ser Ile <210>295 <211>51 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>295 gtaaaagata taaggggcaa ctggaactac gggggaaact ggttcgaccc c <210>296 <211>17 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>296

Val Lys Asp Ile Arg Gly Asn Trp Asn Tyr Gly Gly Asn Trp Phe Asp

5 1015

Pro <210>297 <211>345 <212> DNA <213> Artificial sequence <220>

< 223> synthetic

<400> 297 gaggtgcagc tggtggagtc tggaggaggc ttggtccagc ctggggggtc cctgagactc 60 tcatgtgaag cctctggggtt caccgtcggt gtcaaccaca tgaactgggt ccgccaggct 120 ccagggaagg gtctggagtg ggtctcagtt attttcagta gtggtaggac attctacgga 180 gactacgtga aggggcgatt aaccatcttc agacaaacct cccagaacac ggtgtatctt 240 caaatgaata gcctgagaag tgaggacacg gccatatatt actgtgcgag aggggattggc 300 ggtttggaca tctggggccg agggacaatg gtcaccgtct cttca 345

- 126 045226 <210> 298 <211> 115 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 298

Glu Val Gln Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gln Pro Gly Gly

15

Ser Leu Arg Leu Ser Cys Glu Ala 20

Ser Gly Phe Thr Val Gly Val Asn 25 30

His Met Asn Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ser Val Ile Phe Ser Ser Gly Arg

55

Thr Phe Tyr Gly Asp Tyr Val Lys 60

Gly Arg Leu Thr Ile Phe Arg Gln 65 70

Thr Ser Gln Asn Thr Val Tyr Leu

80

Gln Met Asn Ser Leu Arg Ser Glu 85

Asp Thr Ala Ile Tyr Tyr Cys Ala

95

Arg Gly Ile Gly Gly Leu Asp Ile

100

Trp Gly Arg Gly Thr Met Val Thr

105 110

Val Ser Ser

115 <210> 299 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>299 gggttcaccg tcggtgtcaa ccac <210>300 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic

- 127 045226 <400>300

Gly Phe Thr Val Gly Val Asn His 15 <210>301 <211>21 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>301 attttcagta gtggtaggac a21 <210>302 <211>7 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>302

Ile Phe Ser Ser Gly Arg Thr <210>303 <211>27 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>303 gcgagaggga ttggcggttt ggacatc27 <210>304 <211>9 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>304

Ala Arg Gly Ile Gly Gly Leu Asp Ile <210>305 <211>369 <212> DNA <213> Artificial sequence

- 128 045226 <220>

<223> synthetic <400>305 gaagtgcagc tggtggagtc tgggggaggc ttggttcagc ctggcaggtc cctaagactc60 tcctgtgcag cctctggatt cacctttgat gattatgcct tgcactgggt ccggcaagct120 ccagggaagg gcctggagtg ggtctcaggt attagttgga ctggtggtac tatagactat180 gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat240 ctgcaaatga gcagtctgag aactgaggac acggccatat attactgtac aagagatatc300 cgggggaact ggaagtacgg aggctggttc gacccctggg g ccagggaac cctggtcacc360 gtctcctca369 <210> 306 < 211> 123 < 212> PRT < 213> Artificial sequence <220>

< 223> synthetic < 400> 306

Glu Val Gln 1

Leu Val Glu Ser Gly Gly 5

Gly Leu Val Gln Pro Gly Arg

15

Ser Leu Arg

Leu Ser Cys Ala Ala Ser

25

Gly Phe Thr Phe Asp Asp Tyr 30

Ala Leu His

Trp Val Arg Gln Ala Pro 40

Gly Lys Gly Leu Glu Trp Val 45

Ser Gly Ile 50

Ser Trp Thr Gly Gly Thr 55

Ile Asp Tyr Ala Asp Ser Val 60

Lys Gly Arg Phe Thr 65

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

75 80

Leu Gln Met Ser Ser

Leu Arg Thr Glu Asp Thr Ala Ile Tyr Tyr Cys 90 95

Thr Arg Asp Ile Arg

100

Gly Asn Trp Lys Tyr Gly Gly Trp Phe Asp Pro

105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 307

- 129 045226 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>307 ggattcacct ttgatgatta tgcc24 <210>308 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>308

Gly Phe Thr Phe Asp Asp Tyr Ala <210>309 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>309 attagttgga ctggtggtac tata24 <210>310 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>310

Ile Ser Trp Thr Gly Gly Thr Ile <210>311 <211>48 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 311 acaagagata tccgggggaa ctggaagtac ggaggctggt tcgacccc 48

- 130 045226 <210> 312 <211> 16 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>312

Thr Arg Asp Ile Arg Gly Asn Trp Lys Tyr Gly Gly Trp Phe Asp Pro

5 1015 <210>313 <211>360 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>313 caggtgcagc tggtgcagtc tgggactgag gtgaagaagc ctggggcctc agtgaaggtc60 tcctgcaagg cttctggata caccttcacc gcctactata tgcactgggt gcgacaggcc120 cctggtcaag gacttgactg gatgggatgg atcag cccta acagtggttt cacaaactat180 gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcaa cacattttat240 atggagctga gtggactgag atctgacgac acggccgtat attactgtgc gcgagagggt300 tctactcacc acaattcttt cgacccctgg ggccagggaa ccctggtcac cgtctcctca360 < 210>314 < 211 >120 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400> 314

Gln Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ala

5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Met

Gly Trp Ile Ser Pro Asn Ser Gly Phe Thr Asn Tyr Ala Gln Lys Phe

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Phe Tyr

- 131 045226

70

80

Met Glu Leu Ser Gly Leu Arg Ser 85

Asp Asp Thr Ala Val Tyr Tyr Cys

95

Ala Arg Glu Gly Ser Thr His His

100

Asn Ser Phe Asp Pro Trp Gly Gln

105 110

Gly Thr Leu Val Thr Val Ser Ser

115120 <210>315 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>315 ggatacacct tcaccgccta ctat <210>316 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>316

Gly Tyr Thr Phe Thr Ala Tyr Tyr <210>317 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>317 atcagcccta acagtggttt caca <210>318 <211>8 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 318

- 132 045226

Ile Ser Pro Asn Ser Gly Phe Thr <210> 319 <211> 39 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>319 gcgcgagagg gttctactca ccacaattct ttcgacccc <210>320 <211>13 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400>320

Ala Arg Glu Gly Ser Thr His His Asn Ser Phe Asp Pro

510 <210>321 <211>342 <212> DNA <213> Artificial sequence <220>

< 223> synthetic

<400> 321 gaggtgcagc tggtggagtc tggaggaggc ttggtccaac cggggggggtc cctgaggctc 60 tcctgtgcag cctctggggtt caccgtcggt actaacttca tgaattgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atttatagcg gtggtaccgc taactacgca 180 gactccgtga aggggccgatt caccatttcc agagacactt caggaacac gctgtatctt 240 caaatgaaca gcctgagaac tgaggacacg gccgtttatt attgtgcgag aggggggggt 300 atggacgtct ggggccaagg gaccacggtc accgtctcct ca 342

< 210> 322 < 211> 114 < 212> PRT < 213> Artificial sequence < 220>

<223> synthetic <400> 322

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

- 133 045226

Ser Leu

Arg Leu 20

Ser Cys

Ala Ala

PheMet

Asn Trp 35

Val Arg

Gln Ala

Ser Ala

Ile Tyr

Ser Gly

Gly Thr 55

Gly Arg 65

Phe Thr

Ile Ser

Arg Asp

Gln Met

Asn Ser

Leu Arg 85

Thr Glu

Arg Gly

Gly Gly

100

Met Asp

Val Trp

Ser Gly 25

Pro Gly

Ala Asn

Thr Ser

Asp Thr 90

Gly Gln 105

Phe Thr

Lys Gly

Tyr Ala 60

Arg Asn 75

Ala Val

Gly Thr

Val Gly 30

Leu Glu 45

Asp Ser

Thr Leu

Tyr Tyr

Thr Val

110

Thr Asn

Trp Val

Val Lys

Tyr Leu 80

Cys Ala 95

Thr Val

Ser Ser <210> 323 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>323 gggttcaccg tcggtactaa cttc <210>324 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>324

Gly Phe Thr Val Gly Thr Asn Phe <210>325 <211>21 <212> DNA <213> Artificial sequence <220>

- 134 045226 <223> synthetic <400>325 atttatagcg gtggtaccgc t <210>326 <211>7 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>326

Ile Tyr Ser Gly Gly Thr Ala 15 < 210 > 327 < 211 > 24 < 212 > DNA < 213 > Artificial sequence < 220 >

<223> synthetic <400>327 gcgagagggg ggggtatgga cgtc <210>328 <211>8 <212>PRT <213>Artificial sequence <220>

< 223> synthetic < 400>328

Ala Arg Gly Gly Gly Met Asp Val <210>329 <211>354 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 329

caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcaac acctatgttc tcagctggggt gcgacaggcc 120 cctggacaag ggcttgagtg gatggggagag atcatcccta tcttaggtgc agcaaactac 180 gcacagaact tccaggggcag agtcactttt accacggacg aatccacgaa tacagcctac 240

- 135 045226 atggacctga gcagcctaag atctgaggac acctccgggg ggttcgaccc ctggggccag acggccgtgt attactgtgc gagagatcgg ggaaccctgg tcactgtctc ctca

300

354 <210> 330 <211> 118 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 330

Gln Val Gln Leu Val Gln Ser Gly 1 5

Ala Glu Val Lys Lys Pro Gly Ser

15

Ser Val Lys Val Ser Cys Lys Ala 20

Ser Gly Gly Thr Phe Asn Thr Tyr 25 30

Val Leu Ser Trp Val Arg Gln Ala

40

Pro Gly Gln Gly Leu Glu Trp Met 45

Gly Glu Ile Ile Pro Ile Leu Gly

55

Ala Ala Asn Tyr Ala Gln Asn Phe 60

Gln Gly Arg Val Thr Phe Thr Thr 65 70

Asp Glu Ser Thr Asn Thr Ala Tyr

80

Met Asp Leu Ser Ser Leu Arg Ser 85

Glu Asp Thr Ala Val Tyr Tyr Cys

95

Ala Arg Asp Arg Thr Ser Gly Gly

100

Phe Asp Pro Trp Gly Gln Gly Thr

105 110

Leu Val Thr Val Ser Ser

115 <210> 331 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>331 ggaggcacct tcaacaccta tgtt <210>332 <211>8 <212> PRT <213> Artificial sequence

- 136 045226 <220>

<223> synthetic <400>332

Gly Gly Thr Phe Asn Thr Tyr Val 15 <210>333 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>333 atcatcccta tcttaggtgc agca24 <210>334 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>334

Ile Ile Pro Ile Leu Gly Ala Ala <210>335 <211>33 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>335 gcgagagatc ggacctccgg ggggttcgac ccc33 <210>336 <211>11 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 336

Ala Arg Asp Arg Thr Ser Gly Gly Phe Asp Pro

5 10 <210> 337

- 137 045226

<211> 357 <212>DNA < 213> Artificial sequence <220> < 223> synthetic <400> 337 caggttcagc tggtgcagtc tggagctgag gtggagaagc ctggggcctc agtgaaggtc tcctgcaagg cttctggtta catctttacc cactatggta tcagctggggt gcgacaggcc cctggacaag gacttgagtg ggtggggctgg atcagccctt acaatggtta cacagactat gcacagaaac tccaggggcag agtcaccttg accacagaca catccacgac cacagcctac atggagctga ggaacctgag atctgacgac acggccatgt attackgttc gagagggagg ggcccttact ggtccttcga tctctggggc cgtggcaccc tggtcaccgt ctcctca

120

180

240

300

357 <210> 338 <211> 119 <212> PRT <213> Artificial sequence <220>

<223> synthetic <400> 338

Gln Val Gln Leu Val Gln Ser Gly 1 5

Ala Glu Val Glu Lys Pro Gly Ala

15

Ser Val Lys Val Ser Cys Lys Ala 20

Ser Gly Tyr Ile Phe Thr His Tyr 25 30

Gly Ile Ser Trp Val Arg Gln Ala

40

Pro Gly Gln Gly Leu Glu Trp Val 45

Gly Trp Ile Ser Pro Tyr Asn Gly

55

Tyr Thr Asp Tyr Ala Gln Lys Leu 60

Gln Gly Arg Val Thr Leu Thr Thr 65 70

Asp Thr Ser Thr Thr Thr Ala Tyr

80

Met Glu Leu Arg Asn Leu Arg Ser 85

Asp Asp Thr Ala Met Tyr Tyr Cys

95

Ser Arg Gly Arg Gly Pro Tyr Trp

100

Ser Phe Asp Leu Trp Gly Arg Gly

105 110

Thr Leu Val Thr Val Ser Ser

115

- 138 045226 <210> 339 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>339 ggttacatct ttacccacta tggt24 <210>340 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>340

Gly Tyr Ile Phe Thr His Tyr Gly <210>341 <211>24 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400>341 atcagccctt acaatggtta caca24 <210>342 <211>8 <212> PRT <213> Artificial sequence <220>

< 223> synthetic < 400>342

Ile Ser Pro Tyr Asn Gly Tyr Thr <210>343 <211>36 <212> DNA <213> Artificial sequence <220>

<223> synthetic <400> 343 tcgagaggga ggggccctta ctggtccttc gatctc 36

- 139 -

Claims (36)

<210> 344 <211> 12 <212> PRT <213> Artificial sequence <220> < 223> synthetic < 400> 344 Ser Arg Gly Arg Gly Pro Tyr Trp Ser Phe Asp Leu 1 5 10 CLAIM 1. A radiolabeled antibody conjugate comprising an antibody or an antigen-binding fragment thereof that binds a monomeric human programmed cell death ligand 1 (PD-L1), a chelating moiety, and a positron emitter, wherein the antibody or antigen-binding fragment thereof contains three heavy chain complementarity-determining regions (HCDR) (HCDR1, HCDR2 and HCDR3, respectively) within the heavy chain variable region (HCVR) SEQ ID NO: 82 and three light chain complementarity determining regions (LCDR) (LCDR1, LCDR2 and LCDR3, respectively) within the light chain variable region circuits (LCVR) SEQ ID NO: 90. 2. The radiolabeled conjugate of claim 1, wherein said conjugate comprises an antibody or antigen binding fragment thereof that binds PD-L1, wherein said antibody or antigen binding fragment thereof is covalently linked to one or more fragments of formula (A) -L-Mz (A), in which L represents a chelating moiety; M represents a positron emitter; and z is independently equal to 0 or 1 in each case; and at least one of z is equal to 1. 3. The radiolabeled conjugate according to claim 1 or 2, wherein the chelating moiety includes desferrioxamine. 4. The radiolabeled conjugate according to any one of claims 1 to 3, wherein the positron emitter is ⁸⁹ Zr. 5. The radiolabeled conjugate according to any one of claims 2 to 4, wherein -L-M represents in which Zr represents the positron emitter ⁸⁹ Zr. 6. A radiolabeled conjugate according to any one of claims 1 to 5, wherein the antibody or antigen binding fragment thereof is covalently linked to one, two or three moieties of formula (A). 7. The radiolabeled conjugate according to any one of claims 1 to 6, wherein the antibody has one or more properties selected from the group consisting of: (a) binding monomeric PD-L1 with an equilibrium binding dissociation constant (KD) of less than about 310 pM, as measured in a surface plasmon resonance assay at 37°C; (b) binding monomeric human PD-L1 to a KD of less than about 180 pM in a surface plasmon resonance assay at 25°C; (c) binding dimeric human PD-L1 with a K _D of less than about 15 pM, as measured in a surface plasmon resonance assay at 37°C; and (d) binding dimeric human PD-L1 to a K _D of less than about 8 pM in a surface plasmon resonance assay at 25°C. - 140 045226 8. The radiolabeled conjugate according to any one of claims 1 to 7, wherein the antibody contains a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 82 and the light chain variable region (LCVR) containing the amino acid sequence SEQ ID NO: 90. 9. The radiolabeled conjugate according to any one of claims 1 to 7, wherein the antibody comprises the HCVR heavy chain amino acid sequence of SEQ ID NO: 82 and the LCVR light chain amino acid sequence of SEQ ID NO: 90. 10. A method of imaging tissue that expresses PD-L1, comprising introducing a radiolabeled antibody conjugate according to any one of claims 1 to 9 into the tissue and visualizing the expression of PD-L1 using positron emission tomography (PET) imaging. 11. Use of a radiolabeled conjugate according to any one of claims 1 to 9 in a method for treating a tumor, comprising: (a) selecting a subject with a solid tumor; (b) determining that the solid tumor is PD-LI positive by administering a radiolabeled antibody conjugate to the subject; and visualizing the expression of the radiolabeled antibody conjugate in the tumor using positron emission tomography (PET) imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is PD-LI positive; and (c) administering one or more doses of a PD-1/PD-L1 signaling axis inhibitor to a subject in need thereof. 12. Use according to claim 11, wherein the subject is administered 0.1-10 mg/kg of the radiolabeled antibody conjugate. 13. Use according to claim 11 or 12, wherein the radiolabeled antibody conjugate is administered to the subject subcutaneously or intravenously. 14. Use according to claims 11-13, wherein PET imaging is performed 2-7 days after administration of the radiolabeled antibody conjugate. 15. Use according to any one of claims 11 to 14, wherein step (b) is performed before treating the subject with an inhibitor of the PD-1/PD-L1 signaling axis. 16. Application according to any one of paragraphs. 11-14, additionally providing: (a) administering a radiolabeled antibody conjugate after treating the subject with at least one dose of a PD-1/PD-L1 signaling axis inhibitor; and (b) visualizing the expression of the radiolabeled antibody conjugate in the tumor using PET imaging, with a decrease from baseline at the site of radiolabeled antibody conjugate expression in the tumor indicating tumor regression. 17. The use of claim 16, wherein the subject is administered the radiolabeled antibody conjugate 1 to 20 weeks after administration of the PD-1/PD-L1 signaling axis inhibitor. 18. Use according to any one of claims 11 to 17, wherein the tumor is selected from the group consisting of blood cancer, brain cancer, renal cell cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, cancer bone, colon cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, mesothelioma, B-cell lymphoma and melanoma. 19. Use according to any one of claims 11 to 18, wherein the inhibitor of the PD-1/PD-L1 signaling axis is an anti-PD-1 antibody or an antigen-binding fragment thereof. 20. The use of claim 19, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab and REGN2810. 21. Use according to any one of claims 11 to 18, wherein the inhibitor of the PD-1/PD-L1 signaling axis is an anti-PD-L1 antibody or an antigen-binding fragment thereof. 22. The use of claim 21, wherein the anti-PD-L1 antibody is atezolizumab. 23. The use of claim 21, wherein the anti-PD-L1 antibody or antigen binding fragment thereof comprises HCVR according to SEQ ID NO: 82 and LCVR according to SEQ ID NO: 90. 24. The use of claim 21, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 contains the amino acid sequence according to SEQ ID NO : 84; HCDR2 contains the amino acid sequence according to SEQ ID NO: 86; HCDR3 contains the amino acid sequence according to SEQ ID NO: 88; LCDR1 contains the amino acid sequence according to SEQ ID NO: 92; LCDR2 contains the amino acid sequence of SEQ ID NO: 94, and LCDR3 contains the amino acid sequence of SEQ ID NO: 96. 25. Use of a radiolabeled conjugate according to any one of claims 1 to 9 for monitoring the effectiveness of antitumor therapy, comprising (a) administering the radiolabeled conjugate to a subject in need thereof; and (b) visualization of PD-L1 expression using positron emission tomography (PET) imaging. 26. Application according to claim 25, wherein the monitoring further includes (c) visualization of the - 141 045226 Holi using computed tomography (CT). 27. The use of claim 25, wherein the antitumor therapy comprises a PD1/PD-L1 signaling axis inhibitor, a LAG3 inhibitor, or a CTLA4 inhibitor. 28. The use of claim 25, wherein the subject is administered 0.1-10 mg/kg of the radiolabeled antibody conjugate. 29. The use of claim 28, wherein the radiolabeled antibody conjugate is formulated for subcutaneous or intravenous administration. 30. Use according to claim 25, wherein PET imaging is performed 2-7 days after administration of the radiolabeled antibody conjugate. 31. The use of claim 25, wherein the baseline PD-L1 expression level is determined prior to treatment of the subject with anticancer therapy. 32. Use according to any one of claims 25 to 31, wherein the tumor is selected from the group consisting of blood cancer, brain cancer, renal cell cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, cancer bone, colon cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, mesothelioma, B-cell lymphoma and melanoma. 33. Use according to any one of claim 25, wherein a decrease in PD-L1 expression indicates tumor regression. 34. Compound of formula (III): wherein A is an antibody or antigen-binding fragment thereof that binds PD-L1, including: HCDR1 comprising SEQ ID NO: 84; HCDR2 comprising SEQ ID NO: 86; HCDR3 comprising SEQ ID NO: 88; LCDR1 comprising SEQ ID NO: 92; LCDR2 comprising SEQ ID NO: 94; And LCDR3 comprising SEQ ID NO: 96; and k is equal to an integer from 1 to 30. 35. A radiolabeled antibody conjugate comprising: wherein A is an antibody or antigen binding fragment thereof that binds PD-L1, comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 includes the amino acid sequence of SEQ ID NO: 84 ; HCDR2 includes the amino acid sequence SEQ ID NO: 86; HCDR3 includes the amino acid sequence SEQ ID NO: 88; LCDR1 includes the amino acid sequence SEQ ID NO: 92; LCDR2 includes the amino acid sequence SEQ ID NO: 94; and LCDR3 includes the amino acid sequence of SEQ ID NO: 96; and where k equals an integer from 1 to 30. 36. A radiolabeled antibody conjugate comprising an antibody or an antigen-binding fragment thereof that binds human programmed cell death ligand 1 (PD-L1), wherein the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs chains (LCDR1, LCDR2 and LCDR3), where HCDR1 includes the amino acid - 142 -