Patent Publication Number: US-2023143550-A1

Title: CD8A-Binding Fibronectin Type III Domains

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application Ser. No. 62/434,017, filed 14 Dec. 2016. The entire contents of the aforementioned application are incorporated herein by reference in its entirety. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 5, 2017, is named JBI5112USNP_SL.txt and is 256,587 bytes in size. 
     TECHNICAL FIELD 
     The present invention relates to fibronectin type III (FN3) domains that specifically bind to cluster of differentiation 8a (CD8a). Such FN3 domains may be used for example, for medical imaging, diagnostics, and pharmaceutical therapy. Methods for the production of such molecules and diagnostic agents comprising them are also provided. 
     BACKGROUND 
     The rapidly evolving fields of cancer immunotherapy have recently led to the FDA approval of several new immunotherapies, with many more therapies presently in clinical trials for a variety of cancers. Furthermore, cellular, small molecule, antibody-based immunotherapies, and combinations thereof, are being rigorously tested preclinically for clinical translation. The dynamic tumor microenvironment and tumor heterogeneity have become important topics in both preclinical and clinical studies (Hanahan D, Weinberg R A. Cell 2011; 144:646-74; M antovani A, Allavena P, Sica A, Balkwill F. Nature 2008; 454:436-44; Schreiber R D, Old L J, Smyth M J. Science 2011; 331:1565-70.), but the ability to monitor changes in the immune status of primary lesions and metastatic cancers is limited. Current methods to monitor lymphocytes from whole blood or biopsies from heterogeneous tumors do not reflect the dynamic and spatial information likely required to monitor immune responses to therapeutic intervention, many of which elicit whole body changes in immune cell numbers and localization. Therefore, molecular imaging methods that can noninvasively monitor both systemic and intratumoral alterations in immune cell numbers or localization during experimental therapies have the ability to increase the understanding of the dynamics of immunotherapeutic mechanism with the potential to provide translatable methods for predicting and/or assessing clinical immunotherapeutic responses. 
     Analysis of tumor-infiltrating lymphocytes (TIL) has demonstrated the importance of tumor immune microenvironment and that the presence of cytotoxic CD8 +  T cells can predict overall survival in breast, lung, ovarian, melanoma, and colorectal cancers (reviewed in refs. Pages F, et al., Oncogene 2010; 29:1093-102. and Gooden M J, et al. Br J Cancer 2011; 105:93-103.). With the recent clinical successes of immunotherapies that alter the tumor immune microenvironment, including adoptive cell transfer (ACT) of T-cell receptor (TCR)- or chimeric antigen receptor-transduced cytotoxic T cells (Johnson L A, et al. Blood 2009; 114:535-46; Rosenberg S A. Sci Transl Med 2012; 4:127ps8.), agonistic antibodies targeting CD137 (4-1BB) and CD40 (Melero I, et al. Clin Cancer Res 2013; 19:997-1008; Melero I, et al. Nat Rev Cancer 2007; 7:95-106; Vinay D S, and Kwon B S. Mol Cancer Ther 2012; 11:1062-70.), and antibody blockade of the checkpoint inhibitors CTLA-4, PD-1, and PD-L1 (Callahan M K, and Wolchok J D. J Leukoc Biol 2013; 94:41-53; Shin D S, and Ribas A. Curr Opin Immunol 2015; 33C:23-35; Topalian S L, et al. Cancer Cell 2015; 27:450-61.), the ability to noninvasively monitor the tumor immune response to therapy has become of upmost importance. 
     SUMMARY 
     The present invention comprises CD8A-binding fibronectin type III (FN3) domains. Also described are related polynucleotides capable of encoding the provided FN3 domains, cells expressing the provided FN3 domains, as well as associated vectors. In addition, methods of using the provided FN3 domains are described. For example, the FN3 domains of the invention can be used to noninvasively and quantitatively monitor the presence and abundance of CD8+ T cells. 
     In some embodiments, the present invention comprises isolated FN3 domains, wherein the FN3 domains bind to a human CD8A comprising SEQ ID NO: 35. In other embodiments, the CD8A-specific FN3 domains bind to human CD8A and cynomolgus monkey CD8A. In yet other embodiments, the CD8A-specific FN3 domains are based on Tencon sequence of SEQ ID NO: 1. In further embodiments, the CD8A-specific FN3 domains are based on the Tencon27 sequence of SEQ ID NO: 4. In some embodiments, the albumin-specific FN3 domains are isolated from the library comprising the sequence of SEQ ID NOs: 2, 3, 5, 6, 7 or 8. In some embodiments, the CD8A-specific FN3 domains do not activate CD8+ T-cells in vitro as measured by the enzyme-linked immunospot (ELISPOT) assay. In some embodiments, the CD8A-specific FN3 domains bind human CD8A with an affinity (K D ) of between about 0.02 to about 6.6 nM as measured by surface plasmon resonance. In other embodiments, the CD8A-specific FN3 domains have a cysteine substitution at residue position 54 of SEQ ID NOs 79, 81, 83, 89, 122 and 68. In other embodiments, the CD8A-specific FN3 domains comprise the amino acid sequence of SEQ ID NOs: 40-269. In other embodiments, the CD8A-specific FN3 domains are conjugated to a detectable label. 
     In addition to the described CD8A-specific FN3 domains, also provided are polynucleotide sequences capable of encoding the described FN3 domains. Vectors comprising the described polynucleotides are also provided, as are cells expressing the CD8A-specific FN3 domains herein. Also described are cells capable of expressing the disclosed vectors. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacteria cells (such as  E. coli ). A process for the production of the described FN3 domains is also provided. 
     The present invention also comprises methods of conjugating or otherwise associating the described CD8A-specific FN3 domains to various molecules for diagnostic purposes. For example, Zr-89 or I-124 are ideal fusion partners for creation of diagnostic agents capable of detecting the presence of CD8+ T-cells. As such, the CD8A-specific FN3 domains have utility in cancer diagnostics using CD8A as a biomarker. 
     Another embodiment of the invention is a method of detecting CD8A-expressing cells in a biological sample comprising treating the biological sample with a diagnostic agent comprising the described CD8A-specific FN3 domains. These methods are provided in the EXAMPLES. 
     Within the scope of the invention are kits including the disclosed CD8A-specific FN3 domains. The kits may be used to carry out the methods of using the CD8A-specific FN3 domains provided herein, or other methods known to those skilled in the art. In some embodiments, the described kits may include the FN3 domains described herein and reagents for use in detecting the presence of human CD8A in a biological sample. The described kits may include one or more of the FN3 domains described herein and a vessel for containing the FN3 domains when not in use, instructions for use of the FN3 domains affixed to a solid support, and/or detectably labeled forms of the FN3 domains, as described herein. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG.  1 A- 1 D . The CD8S365-DFO conjugate does not activate T cells de novo and does not modulate the antigen dependent activation of T cells in a 24 hour INF EliSpot assay. CMV reactive T cells were treated with 365-DFO in the absence (A) or presence (B) of CMV peptides. A second M1 reactive donor was also tested in the absence (C) or presence (D) of M1 peptides. 
         FIGS.  2 A and  2 B . The CD8S365-DFO conjugate does not activate T cells de novo and does not modulate the antigen dependent activation of T cells in a 6 day INF γ  MSD assay. CMV reactive T cells were treated with 365-DFO in the absence (A) or presence (B) of CMV peptides. 
         FIG.  3   . Crude preparatory HPLC trace of [ 124 I]-IPEM. Preparatory HPLC was performed using a Waters 1525 Binary HPLC pump, a Waters 2489 dual wavelength UV/Visible Detector (λ=214 and 254 nm), a Bioscan Flow Count radiodetector (B-FC-2000) and a Atlantis T3, 100 Å, 5 μm, 150×4.6 mm HPLC column. The elution profile used was as follows: solvent A=H 2 O (0.1% AcOH (v/v)), Solvent B=MeCN (0.1% AcOH (v/v)), flow rate=1.5 mLmin −1 ; initial=80% A, 20 min=0% A (linear gradient). Multiple small molecule absorbance on UV-vis traces at 254 nm (top graph) and 214 nm (middle graph) indicate presence of impurities and by-products in the crude reaction mixture. Radiotrace (bottom graph) also shows expected baseline peaks due to radiolabeled impurities. 
         FIG.  4   . Analytical HPLC trace of [ 124 I]-IPEM. Analytical HPLC was performed using a Waters 1525 Binary HPLC pump, a Waters 2707 autosampler, a Waters 2489 dual wavelength UV/Visible Detector (λ=214 and 280 nm), a Bioscan Flow Count radiodetector (B-FC-2000) and a Phenomenex Kinetex 5 μm XB-C18 100 Å, 150×4.6 mm HPLC column. The elution profile used was as follows: solvent A=H 2 O (0.1% TFA (v/v)), Solvent B=MeCN (0.1% TFA (v/v)), flow rate=1 mLmin −1 ; initial=90% A, 15 min=0% A (linear gradient). Analytically pure [I-124] IPEM shows a single radiopeak (bottom graph) with a smooth baseline confirming successful purification. Please note that [I-124] IPEM is an organic small molecule; hence, lacking absorption at 280 nm (top graph) and 214 nm (middle graph). 
         FIG.  5   . Radio TLC of purified [ 124 I]-IPEM CD8S365. The iTLC-SG plate (Agilent, cat #SGI0001) was read on a Bioscan AR-2000 radio-TLC imaging scanner. The radio TLC plate ( FIG.  3   ) was co-spotted with 1 μL of NaI (0.1 M) and developed using citric acid (0.5 mM, pH=5) as eluent. The origin=20 mm and the solvent front=100 mm. The radio TLC eluent was prepared by dissolving 96 mg of citric acid (Spectrum cat #CI131) in 25 mL of Trace Select H 2 O and then Na 2 CO 3  was added (245 μL, 2 M); the pH was checked by strip (pH=5). 
         FIG.  6   . Analytical HPLC trace of purified [ 124 I]-IPEM CD8S 365. Analytical HPLC was performed using a Waters 1525 Binary HPLC pump, a Waters 2707 autosampler, a Waters 2489 dual wavelength UV/Visible Detector (λ=214 and 280 nm), a Bioscan Flow Count radiodetector (B-FC-2000) and a Phenomenex Kinetex 5 μm XB-C18 100 Å, 150×4.6 mm HPLC column. The elution profile used was as follows: solvent A=H 2 O (0.1% TFA (v/v)), Solvent B=MeCN (0.1% TFA (v/v)), flow rate=1 mLmin −1 ; initial=90% A, 15 min=0% A (linear gradient). Biomolecule (CD8S) absorbance at 280 nm (top graph) and small molecule (I124-IPEM) absorbance at 214 nm (middle graph) confirms successful conjugation reaction. UV and radio traces (bottom graph) indicate an analytically pure sample. 
         FIG.  7   . MALDI-MS of IPEM CD8S365 (theoretical MW=10786.12). The MALDI-MS analysis was performed at the Biointerfaces Institute using a Bruker UltrafleXtreme MALDI TOF/TOF in positive ion mode (linear detector). A saturated solution of sinapinic acid was prepared in TA30 solvent (30:70 (v/v) MeCN:0.1% TFA in water). The sample (c=0.397 mgmL −1 ) was mixed in a 1:1 ratio with the matrix solution and 1 μL was spotted on the plate. A protein solution was used as an external standard. 
         FIG.  8   . Co-injection of [ 124 I]-IPEM CD8S365 with cold standard. Analytical HPLC was performed using a Waters 1525 Binary HPLC pump, a Waters 2707 autosampler, a Waters 2489 dual wavelength UV/Visible Detector (λ=214 and 280 nm), a Bioscan Flow Count radiodetector (B-FC-2000) and a Phenomenex Kinetex 5 μm XB-C18 100 Å, 150×4.6 mm HPLC column. The elution profile used was as follows: solvent A=H 2 O (0.1% TFA (v/v)), Solvent B=MeCN (0.1% TFA (v/v)), flow rate=1 mLmin −1 ; initial=90% A, 15 min=0% A (linear gradient). Co-injection with cold sample leads to complete overlap of UV peaks (top and middle graphs), confirming the product&#39;s molecular identity (i.e. Cold and radiolabeled conjugates are identical except for the replacement of Iodine by Iodine-124) 
         FIG.  9   . Representative PET image showing CD8S365-IPEM5 radiolabeled with I-124, taken at 2 h post-injection. The image is a maximum intensity projection (anterior-posterior), with the spleen centered on the cross-hairs. The organs below the spleen are the kidneys, and the image is oriented to show the head at the top. The uptake in the thyroid is evidence of some de-iodination of the protein. 
         FIG.  10   . Time-activity curves for blood radioactivity in non-human primate for each anti-CD8A FN3 domain labeled with either Zr-89 or I-124. 
         FIGS.  11 A and  11 B . Time-activity curves for organ radioactivity in NHP for each centyrin labeled with either Zr-89 or I-124.  FIG.  11 A  includes kidneys, liver and spleen, while  FIG.  11 B  is focused on the spleen. The 24 h time point for [ 124 I]-IPEM CD8S365 is missing due to a technical issue. The high uptake of Zr-89 in kidneys due to residualization of the isotope is largely absent from the I-124 data. 
         FIG.  12 A- 12 C . Confirmation of CD8 T cell depletion by Day 3 in blood taken from a non-human primate ( 12 A). Also shown are changes in CD4 ( 12 B) and CD3 T cells ( 12 C). 
         FIG.  13   . Representative PET image showing the 365 anti-CD8A FN3 domain radiolabeled with I-124, taken at 2 h post-injection in a CD8-depleted animal. The image is a maximum intensity projection (anterior-posterior). This is to be compared against the non-depleted animal in  FIG.  9   , where the spleen is clearly visible above the kidney. 
         FIG.  14   . Time-activity curves for blood radioactivity in cynomolgus monkeys for both depleted and non-depleted animals after administration of [ 124 I]-IPEM CD8S365. 
         FIGS.  15 A and  15 B . Time-activity curves for organ radioactivity in cynomolgus monkeys for both depleted and non-depleted animals.  15 A includes kidneys, liver and spleen, while  15 B is focused on the spleen. 
         FIG.  16   . Representative PET image of a two idenitally treated mice showing the CD8S365-IPEM radiolabeled with I-124, taken at 3 h post-injection. The image is a 3D maximum intensity projection, overlaid on a CT scan. Tumor (formed from HEK-293-luc transfected to over-express huCD8+) and other organs are indicated by arrows. The uptake in the thyroid is evidence of some de-iodination of the protein. 
         FIG.  17   . Time-activity curve for blood radioactivity in mice bearing either HEK-293-luc CD8+ or HEK-293 parental tumors. 
         FIG.  18   . Time-activity curve for tumor radioactivity in mice bearing either HEK-293-luc CD8+ or HEK-293 parental tumors. 
         FIG.  19   . Uptake of the I-124 labeled CD8S365 in the HEK293 CD8 overexpressing cells, as a function of the number of implanted cells. 
     
    
    
     DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 
     Definitions 
     Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein. 
     As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. 
     The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. 
     Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. 
     “Isolated” means a biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides and proteins can be part of a composition and still be isolated if such composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An “isolated” FN3 domain, as used herein, is intended to refer to an FN3 domain which is substantially free of other FN3 domains having different antigenic specificities (for instance, an isolated FN3 domain that specifically binds to human serum albumin is substantially free of FN3 domains that specifically bind antigens other than human serum albumin). An isolated FN3 domain that specifically binds to an epitope, isoform or variant of human serum albumin may, however, have cross-reactivity to other related antigens, for instance from other species (such as serum albumin species homologs). 
     The term “fibronectin type III (FN3) domain” (FN3 domain) as used herein refers to a domain occurring frequently in proteins including fibronectins, tenascin, intracellular cytoskeletal proteins, cytokine receptors and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:15659-15665, 1990). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fibronectin (FN), and non-natural synthetic FN3 domains as described for example in U.S. Pat. No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, e.g., the 3rd FN3 domain of tenascin (TN3), or the 10th FN3 domain of fibronectin (FN10). 
     “Centyrin” as used herein refers to a FN3 domain that is based on the consensus sequence of the 15 different FN3 domains present in human tenascin C. 
     The term “capture agent” refers to substances that bind to a particular type of cells and enable the isolation of that cell from other cells. Examples of capture agents include but are not limited to magnetic beads, ferrofluids, encapsulating reagents and the like. 
     The term “biological sample” refers to blood, tissue, marrow, sputum and the like. 
     The term “diagnostic reagent” refers to any substance that may be used to analyze a biological sample, whether or not such substance is distributed as a single substance or in a combination with other substances in a diagnostic kit. 
     The term “substituting” or “substituted” or ‘mutating” or “mutated” as used herein refers to altering, deleting of inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence. 
     The term “randomizing” or “randomized” or “diversified” or “diversifying” as used herein refers to making at least one substitution, insertion or deletion in a polynucleotide or polypeptide sequence. 
     “Variant” as used herein refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions or deletions. 
     The term “specifically binds” or “specific binding” as used herein refers to the ability of the FN3 domain of the invention to bind to a predetermined antigen with a dissociation constant (K D ) of about 1×10 −6  M or less, for example about 1×10 −7 M or less, about 1×10 −8 M or less, about 1×10 −9 M or less, about 1×10 −10  M or less, about 1×10 −11  M or less, about 1×10 −12 M or less, or about 1×10 −13  M or less. Typically the FN3 domain of the invention binds to a predetermined antigen (i.e. human CD8A) with a K D  that is at least ten fold less than its K D  for a nonspecific antigen (for example BSA or casein) as measured by surface plasmon resonance using for example a Proteon Instrument (BioRad). The isolated FN3 domain of the invention that specifically binds to human CD8A may, however, have cross-reactivity to other related antigens, for example to the same predetermined antigen from other species (orthologs), such as  Macaca Fascicularis  (cynomolgous monkey, cyno) or  Pan troglodytes  (chimpanzee). 
     The term “library” refers to a collection of variants. The library may be composed of polypeptide or polynucleotide variants. 
     As used herein, the terms “CD8A” or “CD8” specifically include the human CD8 alpha protein, for example, as described in NCBI Reference Sequence: NP_001139345.1, NP_0011759.3, and NP_741969.1. CD8A is also known in the scientific literature as CD8a molecule, MAL, p32, Leu2, T-cell surface glycoprotein CD8 alpha chain, CD8 antigen, alpha polypeptide (p32), Leu2 T-lymphocyte antigen, OKT8 T-cell antigen, T-cell antigen Leu2, T-lymphocyte differentiation antigen T8/Leu-2, and T8 T-cell antigen. 
     “Tencon” as used herein refers to the synthetic fibronectin type III (FN3) domain having the sequence shown in SEQ ID NO: 1 and described in U.S. Pat. Publ. No. US2010/0216708. 
     The term “vector” means a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these. 
     The term “expression vector” means a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector. 
     The term “polynucleotide” means a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double and single-stranded DNAs and RNAs are typical examples of polynucleotides. 
     The term “polypeptide” or “protein” means a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than about 50 amino acids may be referred to as “peptides”. 
     The term “in combination with” as used herein means that two or more therapeutics can be administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order. 
     Compositions of Matter 
     The present invention provides human CD8A binding FN3 domains and CD8A binding FN3 domains conjugated to detectable labels. The present invention provides polynucleotides encoding the FN3 domains of the invention or complementary nucleic acids thereof, vectors, host cells, and methods of making and using them. 
     CD8A Binding Molecules 
     The present invention provides fibronectin type III (FN3) domains that bind specifically to CD8A, optionally conjugated to a detectable label. These molecules may be widely used in preclinical applications and in cancer diagnostics using CD8A as a biomarker. The present invention provides polynucleotides encoding the FN3 domains of the invention or complementary nucleic acids thereof, vectors, host cells, and methods of making and using them. 
     The FN3 domains of the invention bind CD8A with high affinity and can localize CD8-expressing cells, thereby providing an efficient way to deliver diagnostic reagents into tumor microenvironment. 
     One embodiment of the invention an isolated FN3 domain that specifically binds a human CD8A comprising the amino acid sequence of SEQ ID NO: 35. 
     In some embodiment of the invention described herein, the FN3 domain of the invention cross-reacts with cynomolgus monkey CD8A having the amino acid sequence of SEQ ID NO: 271. 
     The FN3 domain of the invention may bind human,  Macaca Fascicularis  and/or  Pan troglodytes  CD8A with a dissociation constant (K D ) of less than about 1×10 −7  M, for example less than about 1×10 −8  M, less than about 1×10 −9  M, less than about 1×10 −10  M, less than about 1×10 −11  M, less than about 1×10 −12  M, or less than about 1×10 −13  M as determined by surface plasmon resonance, as practiced by those of skill in the art. The measured affinity of a particular FN3 domain-antigen interaction can vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., K D , K on , K off ) are made with standardized solutions of protein scaffold and antigen, and a standardized buffer, such as the buffer described herein. 
     In some embodiments, the CD8A binding FN3 domains comprise an initiator methionine (Met) linked to the N-terminus of the molecule. 
     In some embodiments, the CD8A binding FN3 domains comprise a cysteine (Cys) linked to the FN3 domain. 
     The addition of the N-terminal Met and/or the Cys may facilitate expression and/or conjugation of second molecules. 
     Another embodiment of the invention is an isolated FN3 domain that specifically binds human CD8A and wherein the CD8A-specific FN3 domain does not activate CD8+ T-cells in vitro. CD8+ T cell activation may be measured using standard methods. For example, the enzyme-linked immunospot (ELISPOT) assay may be used. The ELISPOT assay employs the sandwich enzyme-linked immunosorbent assay (ELISA) technique. The interferon-gamma antibody is pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells (cells+peptides, FN3 domains, etc) are pipetted into the wells and the microplate is placed into a humidified 37° C. CO 2  incubator for a specified period of time. During this incubation period, the immobilized interferon-gamma antibody, in the immediate vicinity of the secreting cells, binds the secreted interferon gamma. After washing away any cells and unbound substances, a second biotinylated interferon-gamma antibody is added to the wells. Following a wash to remove any unbound biotinylated antibody, alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. A blue-black colored precipitate forms and appears as spots at the sites of interferon-gamma localization, with each individual spot representing an individual interferon gamma-secreting cell. The spots can be counted with an automated ELISpot reader system or manually, using a stereomicroscope. The isolated CD8A binding FN3 domains of the invention do not activate CD8+ T-cells in vitro when tested at 1 μM concentrations as described in the EXAMPLES. 
     In some embodiments of the invention described herein, the isolated FN3 domain comprises the amino acid sequence of SEQ ID NOs: 40-269. 
     In some embodiments of the invention described herein, the CD8A-specific FN3 domain has a cysteine substitution at residue position 54 of SEQ ID NOs 79, 81, 83, 89, 122 and 68. 
     Substitutions resulting in introduction of cysteine into a protein sequence may be utilized to chemically conjugate small molecules such as cytotoxic agents, detectable labels, half-life extension molecules, chelators, polyethylene glycol and/or nucleic acids to the FN3 domain using standard chemistry. 
     In some embodiments, the FN3 domain specifically binding human CD8A competes for binding to human CD8A with the FN3 domain of SEQ ID NOs: 229-234. FN3 domains may be evaluated for ther competition with a reference molecule for binding human CD8A using well known in vitro methods. In an exemplary method, HEK cells recombinantly expressing human CD8A may be incubated with unlabeled reference molecule for 15 min at 4° C., followed by incubation with an excess of fluorescently labeled test FN3 domain for 45 min at 4° C. After washing in PBS/BSA, fluorescence may be measured by flow cytometry using standard methods. In another exemplary method, extracellular portion of human CD8A may be coated on the surface of an ELISA plate. Excess of unlabelled reference molecule may be added for about 15 minutes and subsequently biotinylated test FN3 domains may be added. After washes in PBS/Tween, binding of the test biotinylated FN3 domain may be detected using horseradish peroxidase (HRP)-conjugated streptavidine and the signal detected using standard methods. It is readily apparent that in the competition assays, reference molecule may be labelled and the test FN3 domain unlabeled. The test FN3 domain may compete with the reference molecule when the reference molecule inhibits binding of the test FN3 domain, or the test FN3 domain inhibits binding of the reference molecule by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%. 
     In some embodiments, the isolated FN3 domain that specifically binds human CD8A of the invention is conjugated to a chelator that can bind to a radioactive metal and may be used as an imaging agent to evaluate tumor distribution, diagnosis for the presence of CD8-T cells inside tumors and/or efficacy of cancer treatment. 
     In some embodiments, the CD8A-specific FN3 domains are removed from the blood via renal and/or liver clearance. 
     Isolation of CD8A Binding FN3 Domains from a Library Based on Tencon Sequence 
     Tencon (SEQ ID NO: 1) is a non-naturally occurring fibronectin type III (FN3) domain designed from a consensus sequence of fifteen FN3 domains from human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; U.S. Pat. Publ. No. 2010/0216708). The crystal structure of Tencon shows six surface-exposed loops that connect seven beta-strands as is characteristic to the FN3 domains, the beta-strands referred to as A, B, C, D, E, F, and G, and the loops referred to as AB, BC, CD, DE, EF, and FG loops (Bork and Doolittle, Proc Natl Acad Sci USA 89:8990-8992, 1992; U.S. Pat. No. 6,673,901). These loops, or selected residues within each loop, may be randomized in order to construct libraries of fibronectin type III (FN3) domains that may be used to select novel molecules that bind CD8A. Table 1 shows positions and sequences of each loop and beta-strand in Tencon (SEQ ID NO: 1). 
     Library designed based on Tencon sequence may thus have randomized FG loop, or randomized BC and FG loops, such as libraries TCL1 or TCL2 as described below. The Tencon BC loop is 7 amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may be randomized in the library diversified at the BC loop and designed based on the Tencon sequence. The Tencon FG loop is 7 amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may be randomized in the library diversified at the FG loop and designed based on Tencon sequence. Further diversity at loops in the Tencon libraries may be achieved by insertion and/or deletions of residues at loops. For example, the FG and/or BC loops may be extended by 1-22 amino acids, or decreased by 1-3 amino acids. The FG loop in Tencon is 7 amino acids long, whereas the corresponding loop in antibody heavy chains ranges from 4-28 residues. To provide maximum diversity, the FG loop may be diversified in sequence as well as in length to correspond to the antibody CDR3 length range of 4-28 residues. For example, the FG loop can further be diversified in length by extending the loop by additional 1, 2, 3, 4 or 5 amino acids. 
     A library designed based on the Tencon sequence may also have randomized alternative surfaces that form on a side of the FN3 domain and comprise two or more beta strands, and at least one loop. One such alternative surface is formed by amino acids in the C and the F beta-strands and the CD and the FG loops (a C-CD-F-FG surface). A library design based on Tencon alternative C-CD-F-FG surface is is described in U.S. Pat. Publ. No. US2013/0226834. Library designed based on Tencon sequence also includes libraries designed based on Tencon variants, such as Tencon variants having substitutions at residues positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO: 1), and which variants display improve thermal stability. Exemplary Tencon variants are described in US Pat. Publ. No. 2011/0274623, and include Tencon27 (SEQ ID NO: 4) having substitutions E11R, L17A, N46V and E86I when compared to Tencon of SEQ ID NO: 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Tencon topology 
               
            
           
           
               
               
               
            
               
                   
                   
                 Tencon 
               
               
                   
                 FN3 domain 
                 (SEQ ID NO: 1) 
               
               
                   
               
               
                   
                 A strand 
                  1-12 
               
               
                   
                 AB loop 
                 13-16 
               
               
                   
                 B strand 
                 17-21 
               
               
                   
                 BC loop 
                 22-28 
               
               
                   
                 C strand 
                 29-37 
               
               
                   
                 CD loop 
                 38-43 
               
               
                   
                 D strand 
                 44-50 
               
               
                   
                 DE loop 
                 51-54 
               
               
                   
                 E strand 
                 55-59 
               
               
                   
                 EF loop 
                 60-64 
               
               
                   
                 F strand 
                 65-74 
               
               
                   
                 FG loop 
                 75-81 
               
               
                   
                 G strand 
                 82-89 
               
               
                   
               
            
           
         
       
     
     Tencon and other FN3 sequence based libraries may be randomized at chosen residue positions using a random or defined set of amino acids. For example, variants in the library having random substitutions may be generated using NNK codons, which encode all 20 naturally occurring amino acids. In other diversification schemes, DVK codons may be used to encode amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys. Alternatively, NNS codons may be used to give rise to all 20 amino acid residues and simultaneously reducing the frequency of stop codons. Libraries of FN3 domains with biased amino acid distribution at positions to be diversified may be synthesized for example using Slonomics® technology (http:_//www_sloning_com). This technology uses a library of pre-made double stranded triplets that act as universal building blocks sufficient for thousands of gene synthesis processes. The triplet library represents all possible sequence combinations necessary to build any desired DNA molecule. The codon designations are according to the well known IUB code. 
     The FN3 domains specifically binding human CD8A of the invention may be isolated by producing the FN3 library such as the Tencon library using cis display to ligate DNA fragments encoding the scaffold proteins to a DNA fragment encoding RepA to generate a pool of protein-DNA complexes formed after in vitro translation wherein each protein is stably associated with the DNA that encodes it (U.S. Pat. No. 7,842,476; Odegrip et al., Proc Natl Acad Sci USA 101, 2806-2810, 2004), and assaying the library for specific binding to CD8A by any method known in the art and described in the Example. Exemplary well known methods which can be used are ELISA, sandwich immunoassays, and competitive and non-competitive assays (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley &amp; Sons, Inc., New York). The identified FN3 domains specifically binding CD8A are further characterized for their inhibition of CD8A activity, internalization, stability, and other desired characteristics. 
     The FN3 domains specifically binding human CD8A of the invention may be generated using any FN3 domain as a template to generate a library and screening the library for molecules specifically binding human CD8A using methods provided within. Exemplar FN3 domains that may be used are the 3rd FN3 domain of tenascin C (TN3) (SEQ ID NO: 145), Fibcon (SEQ ID NO: 146), and the 10th FN3 domain of fibronectin (FN10) (SEQ ID NO: 147). Standard cloning and expression techniques are used to clone the libraries into a vector or synthesize double stranded cDNA cassettes of the library, to express, or to translate the libraries in vitro. For example ribosome display (Hanes and Pluckthun, Proc Natl Acad Sci USA, 94, 4937-4942, 1997), mRNA display (Roberts and Szostak, Proc Natl Acad Sci USA, 94, 12297-12302, 1997), or other cell-free systems (U.S. Pat. No. 5,643,768) can be used. The libraries of the FN3 domain variants may be expressed as fusion proteins displayed on the surface for example of any suitable bacteriophage. Methods for displaying fusion polypeptides on the surface of a bacteriophage are well known (U.S. Pat. Publ. No. 2011/0118144; Int. Pat. Publ. No. WO2009/085462; U.S. Pat. Nos. 6,969,108; 6,172,197; 5,223,409; 6,582,915; 6,472,147). 
     In some embodiments of the invention described herein, the FN3 domain specifically binding human CD8A is based on Tencon sequence of SEQ ID NO: 1 or Tencon27 sequence of SEQ ID NO: 4, the SEQ ID NO: 1 or the SEQ ID NO: 4, optionally having substitutions at residues positions 11, 14, 17, 37, 46, 73, and/or 86. 
     The FN3 domains specifically binding human CD8A of the invention may be modified to improve their properties such as improve thermal stability and reversibility of thermal folding and unfolding. Several methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparison to highly similar thermostable sequences, design of stabilizing disulfide bridges, mutations to increase alpha-helix propensity, engineering of salt bridges, alteration of the surface charge of the protein, directed evolution, and composition of consensus sequences (Lehmann and Wyss, Curr Opin Biotechnol, 12, 371-375, 2001). High thermal stability may increase the yield of the expressed protein, improve solubility or activity, decrease immunogenicity, and minimize the need of a cold chain in manufacturing. Residues that may be substituted to improve thermal stability of Tencon (SEQ ID NO: 1) are residue positions 11, 14, 17, 37, 46, 73, or 86, and are described in US Pat. Publ. No. 2011/0274623. Substitutions corresponding to these residues may be incorporated to the FN3 domain containing molecules of the invention. 
     Measurement of protein stability and protein lability can be viewed as the same or different aspects of protein integrity. Proteins are sensitive or “labile” to denaturation caused by heat, by ultraviolet or ionizing radiation, changes in the ambient osmolarity and pH if in liquid solution, mechanical shear force imposed by small pore-size filtration, ultraviolet radiation, ionizing radiation, such as by gamma irradiation, chemical or heat dehydration, or any other action or force that may cause protein structure disruption. The stability of the molecule can be determined using standard methods. For example, the stability of a molecule can be determined by measuring the thermal melting (“T m ”) temperature, the temperature in ° Celsius (° C.) at which half of the molecules become unfolded, using standard methods. Typically, the higher the T m , the more stable the molecule. In addition to heat, the chemical environment also changes the ability of the protein to maintain a particular three dimensional structure. 
     In one embodiment, the FN3 domains specifically binding human CD8A of the invention may exhibit increased stability by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more compared to the same domain prior to engineering measured by the increase in the T m . 
     Chemical denaturation can likewise be measured by a variety of methods. Chemical denaturants include guanidinium hydrochloride, guanidinium thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride); reducing agents (e.g. dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene, and hydrides, such as sodium borohydride), non-ionic and ionic detergents, acids (e.g. hydrochloric acid (HCl), acetic acid (CH 3 COOH), halogenated acetic acids), hydrophobic molecules (e.g. phosopholipids), and targeted denaturants. Quantitation of the extent of denaturation can rely on loss of a functional property, such as ability to bind a target molecule, or by physiochemical properties, such as tendency to aggregation, exposure of formerly solvent inaccessible residues, or disruption or formation of disulfide bonds. 
     The FN3 domains of the invention may be generated as monomers, dimers, or multimers, for example, as a means to increase the valency and thus the avidity of target molecule binding, or to generate bi- or multispecific scaffolds simultaneously binding two or more different target molecules. The dimers and multimers may be generated by linking monospecific, bi- or multispecific protein scaffolds, for example, by the inclusion of an amino acid linker, for example a linker containing poly-glycine, glycine and serine, or alanine and proline. Exemplary linker include (GS) 2 , (SEQ ID NO: 148), (GGGS) 2  (SEQ ID NO: 149), (GGGGS) 5  (SEQ ID NO: 150), (AP) 2  (SEQ ID NO: 151), (AP) 5  (SEQ ID NO: 152), (AP) 10  (SEQ ID NO: 153), (AP) 20  (SEQ ID NO: 154) and A(EAAAK) 5 AAA (SEQ ID NO: 142). The dimers and multimers may be linked to each other in a N- to C-direction. The use of naturally occurring as well as artificial peptide linkers to connect polypeptides into novel linked fusion polypeptides is well known in the literature (Hallewell et al.,  J Biol Chem  264, 5260-5268, 1989; Alfthan et al.,  Protein Eng.  8, 725-731, 1995; Robinson &amp; Sauer,  Biochemistry  35, 109-116, 1996; U.S. Pat. No. 5,856,456). 
     Diagnostic Agents 
     According to the invention, a CD8A-specific FN3 domain of the invention may comprise a detectable label. In an embodiment, the detectable label may be complexed with a chelating agent that is conjugated to the FN3 domain. In another embodiment, the detectable label may be complexed with a chelating agent that is conjugated to a linker that is conjugated to the FN3 domain. In still another embodiment, the detectable label may be coupled to a linker that is conjugated to the FN3 domain. In still yet another embodiment, a detectable label may be indirectly attached to a peptide of the invention by the ability of the label to be specifically bound by a second molecule. One example of this type of an indirectly attached label is a biotin label that can be specifically bound by the second molecule, streptavidin. Single, dual or multiple labeling may be advantageous. As used herein, a “detectable label” is any type of label which, when attached to an FN3 domain of the invention renders the FN3 domain detectable. A detectable label may also be toxic to cells or cytotoxic. In general, detectable labels may include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescent quenching agents, colored molecules, radioisotopes, radionuclides, cintillants, massive labels such as a metal atom (for detection via mass changes), biotin, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, Grb2, polyhistidine, Ni 2+ , Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors, acridinium esters, and colorimetric substrates. In a specific embodiment, the detectable label is a radionuclide. The skilled artisan would readily recognize other useful labels that are not mentioned above, which may be employed in the operation of the present invention. 
     A detectable label emits a signal that can be detected by a signal transducing machine. In some cases, the detectable label can emit a signal spontaneously, such as when the detectable label is a radionuclide. In other cases, the detectable label emits a signal as a result of being stimulated by an external field such as when the detectable label is a relaxivity metal. Examples of signals include, without limitation, gamma rays, X-rays, visible light, infrared energy, and radiowaves. Examples of signal transducing machines include, without limitation, gamma cameras including SPECT/CT devices, PET scanners, fluorimeters, and Magnetic Resonance Imaging (MRI) machines. As such, the detectable label comprises a label that can be detected using magnetic resonance imaging, scintigraphic imaging, ultrasound, or fluorescence. 
     Suitable fluorophores include, but are not limited to, fluorescein isothiocyante (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes. An FN3 domain of the invention can be labeled for fluorescence detection by labeling the agent with a fluorophore using techniques well known in the art (see, e.g., Lohse et al., Bioconj Chem 8:503-509 (1997)). For example, many known dyes are capable of being coupled to NH 2 -terminal amino acid residues. Alternatively, a fluorochrome such as fluorescein may be bound to a lysine residue of the peptide linker. 
     A radionuclide may be a γ-emitting radionuclide, Auger-emitting radionuclide, γ-emitting radionuclide, an alpha-emitting radionuclide, or a positron-emitting radionuclide. A radionuclide may be a detectable label and/or a cytotoxic agent. Non-limiting examples of suitable radionuclides may include carbon-11, nitrogen-13, oxygen-15, fluorine-18, fluorodeoxyglucose-18, phosphorous-32, scandium-47, copper-64, 65 and 67, gallium-67 and 68, bromine-75, 77 and 80m, rubidium-82, strontium-89, zirconium-89, yttrium-86 and 90, ruthenium-95, 97, 103 and 105, rhenium-99m, 101, 105, 186 and 188, technetium-99m, rhodium-105, mercury-107, palladium-109, indium-111, silver-111, indium-113m, lanthanide-114m, tin-117m, tellurium-121 m, 122m and 125m, iodine-122, 123, 124, 125, 126, 131 and 133, praseodymium-142, promethium-149, samarium-153, gadolinium-159, thulium-165, 167 and 168, dysprosium-165, holmium-166, lutetium-177, rhenium-186 and 188, iridium-192, platinum-193 and 195m, gold-199, thallium-201, titanium-201, astatine-21 1, bismuth-212 and 213, lead-212, radium-223, actinium-225, and nitride or oxide forms derived there from. In a specific embodiment, a radionuclide is selected from the group consisting of copper-64, zirconium-89, yttrium-90, indium-111, and lutetium-177. In another specific embodiment, a radionuclide is selected from the group consisting of yttrium-90, indium-111, and lutetium-177. In an exemplary embodiment, a radionuclide is zirconium-89. 
     A variety of metal atoms may be used as a detectable label. The metal atom may generally be selected from the group of metal atoms comprised of metals with an atomic number of twenty or greater. For instance, the metal atoms may be calcium atoms, scandium atoms, titanium atoms, vanadium atoms, chromium atoms, manganese atoms, iron atoms, cobalt atoms, nickel atoms, copper atoms, zinc atoms, gallium atoms, germanium atoms, arsenic atoms, selenium atoms, bromine atoms, krypton atoms, rubidium atoms, strontium atoms, yttrium atoms, zirconium atoms, niobium atoms, molybdenum atoms, technetium atoms, ruthenium atoms, rhodium atoms, palladium atoms, silver atoms, cadmium atoms, indium atoms, tin atoms, antimony atoms, tellurium atoms, iodine atoms, xenon atoms, cesium atoms, barium atoms, lanthanum atoms, hafnium atoms, tantalum atoms, tungsten atoms, rhenium atoms, osmium atoms, iridium atoms, platinum atoms, gold atoms, mercury atoms, thallium atoms, lead atoms, bismuth atoms, francium atoms, radium atoms, actinium atoms, cerium atoms, praseodymium atoms, neodymium atoms, promethium atoms, samarium atoms, europium atoms, gadolinium atoms, terbium atoms, dysprosium atoms, holmium atoms, erbium atoms, thulium atoms, ytterbium atoms, lutetium atoms, thorium atoms, protactinium atoms, uranium atoms, neptunium atoms, plutonium atoms, americium atoms, curium atoms, berkelium atoms, californium atoms, einsteinium atoms, fermium atoms, mendelevium atoms, nobelium atoms, or lawrencium atoms. In some embodiments, the metal atoms may be selected from the group comprising alkali metals with an atomic number greater than twenty. In other embodiments, the metal atoms may be selected from the group comprising alkaline earth metals with an atomic number greater than twenty. In one embodiment, the metal atoms may be selected from the group of metals comprising the lanthanides. In another embodiment, the metal atoms may be selected from the group of metals comprising the actinides. In still another embodiment, the metal atoms may be selected from the group of metals comprising the transition metals. In yet another embodiment, the metal atoms may be selected from the group of metals comprising the poor metals. In other embodiments, the metal atoms may be selected from the group comprising gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms. In preferred embodiments, the metal atoms may be selected from the group comprising metals with an atomic number of 53 (i.e. iodine) to 83 (i.e. bismuth). In an alternative embodiment, the metal atoms may be atoms suitable for magnetic resonance imaging. In another alternative embodiment, the metal atoms may be selected from the group consisting of metals that have a K-edge in the x-ray energy band of CT. Preferred metal atoms include, but are not limited to, manganese, iron, gadolinium, gold, and iodine. 
     The metal atoms may be metal ions in the form of +1, +2, or +3 oxidation states. For instance, non-limiting examples include Ba 2+ , Bi 3+ , Cs + , Ca 2+ , Cr 2+ , Cr 3+ , Cr 6+ , Co 2+ , Co 3+ , Cu + , Cu 2+ , Cu 3+ , Ga 3+ , Gd 3+ , Au + , Au 3+ , Fe 2+ , Fe 3+ , F 3+ , Pb 2+ , Mn 2+ , Mn 3+ , Mn 4+ , Mn 7+ , Hg 2+ , Ni 2+ , Ni 3+ , Ag + , Sr 2+ , Sn 2+ , Sn 4+ , and Zn 2+ . The metal atoms may comprise a metal oxide. For instance, non-limiting examples of metal oxides may include iron oxide, manganese oxide, or gadolinium oxide. Additional examples may include magnetite, maghemite, or a combination thereof. 
     According to the invention, an FN3 domain comprising a chelating agent may incorporate a radionuclide or metal atom. Incorporation of the radionuclide or metal atom with an FN3 domain-chelating agent complex may be achieved by various methods common in the art of coordination chemistry. 
     Half-Life Extending Moieties 
     The FN3 domain specifically binding human CD8A of the invention may incorporate other subunits for example via covalent interaction. In one aspect of the invention, the FN3 domain of the invention further comprises a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin-binding proteins and/or domains, transferrin and fragments and analogues thereof, and Fc regions. 
     Additional moieties may be incorporated into the FN3 domain of the invention such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties. These moieties may be direct fusions with the protein scaffold coding sequences and may be generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the moieties to recombinantly produced molecules of the invention. 
     A pegyl moiety may for example be added to the FN3 domain of the invention by incorporating a cysteine residue to the C-terminus of the molecule, or engineering cysteines into residue positions that face away from the human CD8A binding face of the molecule, and attaching a pegyl group to the cysteine using well known methods. FN3 domain of the invention incorporating additional moieties may be compared for functionality by several well known assays. For example, altered properties due to incorporation of Fc domains and/or Fc domain variants may be assayed in Fc receptor binding assays using soluble forms of the receptors, such as the FcγRI, FcγRII, FcγRIII or FcRn receptors, or using well known cell-based assays measuring for example ADCC or CDC, or evaluating pharmacokinetic properties of the molecules of the invention in in vivo models. 
     Polynucleotides, Vectors, Host Cells 
     The invention provides for nucleic acids encoding the FN3 domains specifically binding human CD8A of the invention as isolated polynucleotides or as portions of expression vectors or as portions of linear DNA sequences, including linear DNA sequences used for in vitro transcription/translation, vectors compatible with prokaryotic, eukaryotic or filamentous phage expression, secretion and/or display of the compositions or directed mutagens thereof. Certain exemplary polynucleotides are disclosed herein, however, other polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the FN3 domains of the invention are also within the scope of the invention. 
     One embodiment of the invention is an isolated polynucleotide encoding the FN3 domain specifically binding human CD8A comprising the amino acid sequence of SEQ ID NOs: 40-269. 
     The polynucleotides of the invention may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer and assembled into complete single or double stranded molecules. Alternatively, the polynucleotides of the invention may be produced by other techniques such a PCR followed by routine cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art. 
     The polynucleotides of the invention may comprise at least one non-coding sequence, such as a promoter or enhancer sequence, intron, polyadenylation signal, a cis sequence facilitating RepA binding, and the like. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids that encode for example a marker or a tag sequence such as a histidine tag or an HA tag to facilitate purification or detection of the protein, a signal sequence, a fusion protein partner such as RepA, Fc or bacteriophage coat protein such as pIX or pIII. 
     Another embodiment of the invention is a vector comprising at least one polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the polynucleotides of the invention into a given organism or genetic background by any means. Such vectors may be expression vectors comprising nucleic acid sequence elements that can control, regulate, cause or permit expression of a polypeptide encoded by such a vector. Such elements may comprise transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of encoded polypeptides in a given expression system. Such expression systems may be cell-based, or cell-free systems well known in the art. 
     Another embodiment of the invention is a host cell comprising the vector of the invention. The FN3 domain specifically binding human CD8A of the invention may be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley &amp; Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley &amp; Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley &amp; Sons, NY, NY, (1997-2001). 
     The host cell chosen for expression may be of mammalian origin or may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2/0, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell may be selected from a species or organism incapable of glycosylating polypeptides, e.g. a prokaryotic cell or organism, such as BL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174 (DE3), and any of the natural or engineered  E. coli  spp,  Klebsiella  spp., or  Pseudomonas  spp strains. 
     Another embodiment of the invention is a method of producing the isolated FN3 domain specifically binding human CD8A of the invention, comprising culturing the isolated host cell of the invention under conditions such that the isolated FN3 domain specifically binding human CD8A is expressed, and purifying the FN3 domain. 
     The FN3 domain specifically binding human CD8A may be purified from recombinant cell cultures by well-known methods, for example by protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography, or high performance liquid chromatography (HPLC). 
     Kits for Detecting Human CD8A 
     Provided herein are kits for detecting CD8A in a biological sample. These kits include one or more of the CD8A-specific FN3 domains described herein and instructions for use of the kit. 
     The provided CD8A-specific FN3 domain may be in solution; lyophilized; affixed to a substrate, carrier, or plate; or detectably labeled. 
     The described kits may also include additional components useful for performing the methods described herein. By way of example, the kits may comprise means for obtaining a sample from a subject, a control or reference sample, e.g., a sample from a subject having slowly progressing cancer and/or a subject not having cancer, one or more sample compartments, and/or instructional material which describes performance of a method of the invention and tissue specific controls or standards. 
     The means for determining the level of CD8A can further include, for example, buffers or other reagents for use in an assay for determining the level of CD8A. The instructions can be, for example, printed instructions for performing the assay and/or instructions for evaluating the level of CD8A. 
     The described kits may also include means for isolating a sample from a subject. These means can comprise one or more items of equipment or reagents that can be used to obtain a fluid or tissue from a subject. The means for obtaining a sample from a subject may also comprise means for isolating blood components, such as serum, from a blood sample. Preferably, the kit is designed for use with a human subject. 
     Uses of Human CD8A Binding FN3 Domains of the Invention 
     The FN3 domains specifically binding human CD8A of the invention may be used to diagnose human disease or specific pathologies in cells, tissues, organs, fluid, or, generally, a host, using CD8A as a biomarker. The methods of the invention may be used in an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals. 
     EXAMPLES 
     The following examples are provided to supplement the prior disclosure and to provide a better understanding of the subject matter described herein. These examples should not be considered to limit the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within, and can be made without departing from, the true scope of the invention. 
     Example 1. Construction of Tencon Libraries with Randomized Loops 
     Tencon (SEQ ID NO: 1) is an immunoglobulin-like scaffold, fibronectin type III (FN3) domain, designed from a consensus sequence of fifteen FN3 domains from human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; U.S. Pat. No. 8,278,419). The crystal structure of Tencon shows six surface-exposed loops that connect seven beta-strands. These loops, or selected residues within each loop, can be randomized in order to construct libraries of fibronectin type III (FN3) domains that can be used to select novel molecules that bind to specific targets. Tencon: 
                            LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVG                   EAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLS                   AEFTT (SEQ ID NO 1):            
Various libraries were generated using the tencon scaffold and various design strategies. In general, libraries TCL1 and TCL2 produced good binders. Generation of TCL1 and TCL2 libraries are described in detail in Int. Pat. Publ. No. WO2014081944A2.
 
     Construction of TCL1 Library 
     A library designed to randomize only the FG loop of Tencon (SEQ ID NO: 1), TCL1, was constructed for use with the cis-display system (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012). In this system, a double-stranded DNA incorporating sequences for a Tac promoter, Tencon library coding sequence, RepA coding sequence, cis-element, and ori element is produced. Upon expression in an in vitro transcription/translation system, a complex is produced of the Tencon-RepA fusion protein bound in cis to the DNA from which it is encoded. Complexes that bind to a target molecule are then isolated and amplified by polymerase chain reaction (PCR), as described below. 
     Construction of the TCL1 library for use with cis-display was achieved by successive rounds of PCR to produce the final linear, double-stranded DNA molecules in two halves; the 5′ fragment contains the promoter and Tencon sequences, while the 3′ fragment contains the repA gene and the cis- and ori elements. These two halves are combined by restriction digest in order to produce the entire construct. The TCL1 library was designed to incorporate random amino acids only in the FG loop of Tencon, KGGHRSN (SEQ ID NO: 32). NNS codons were used in the construction of this library, resulting in the possible incorporation of all 20 amino acids and one stop codon into the FG loop. The TCL1 library contains six separate sub-libraries, each having a different randomized FG loop length, from 7 to 12 residues, in order to further increase diversity. 
                            TCL1 library           (SEQ ID NO: 2)           LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVG                   EAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX 7-12 PLSAE                   FTT;            
wherein
 
X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7  is any amino acid; and
 
X 8 , X 9 , X 10 , X 11  and X 12  are any amino acid or deleted
 
     Construction of TCL2 Library 
     TCL2 library was constructed in which both the BC and the FG loops of Tencon were randomized and the distribution of amino acids at each position was strictly controlled. Table 2 shows the amino acid distribution at desired loop positions in the TCL2 library. The designed amino acid distribution had two aims. First, the library was biased toward residues that were predicted to be structurally important for Tencon folding and stability based on analysis of the Tencon crystal structure and/or from homology modeling. For example, position 29 was fixed to be only a subset of hydrophobic amino acids, as this residue was buried in the hydrophobic core of the Tencon fold. A second layer of design included biasing the amino acid distribution toward that of residues preferentially found in the heavy chain HCDR3 of antibodies, to efficiently produce high-affinity binders (Birtalan et al., J Mol Biol 377:1518-28, 2008; Olson et al., Protein Sci 16:476-84, 2007). Towards this goal, the “designed distribution” in Table 1 refers to the distribution as follows: 6% alanine, 6% arginine, 3.9% asparagine, 7.5% aspartic acid, 2.5% glutamic acid, 1.5% glutamine, 15% glycine, 2.3% histidine, 2.5% isoleucine, 5% leucine, 1.5% lysine, 2.5% phenylalanine, 4% proline, 10% serine, 4.5% threonine, 4% tryptophan, 17.3% tyrosine, and 4% valine. This distribution is devoid of methionine, cysteine, and STOP codons. 
     
       
         
           
               
            
               
                 TCL2 library 
               
               
                 (SEQ ID NO: 3) 
               
               
                 LPAPKNLVVSEVTEDSLRLSWX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 SFLIQYQ 
               
               
                   
               
               
                 ESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX 9 X 10   
               
               
                   
               
               
                 X 11 X 12 X 13 SX 14 X 15 LSAEFTT; 
               
               
                 wherein 
               
               
                 X 1  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr 
               
               
                 or Val; 
               
               
                 X 2  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr 
               
               
                 or Val; 
               
               
                 X 3  Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, 
               
               
                 Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
               
               
                 X 4  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr 
               
               
                 or Val; 
               
               
                 X 5  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr 
               
               
                 or Val; 
               
               
                 X 6  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr 
               
               
                 or Val; 
               
               
                 X 7  is Phe, Ile, Leu, Val or Tyr; 
               
               
                 X 8  is Asp, Glu or Thr; 
               
               
                 X 9  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr 
               
               
                 or Val; 
               
               
                 X 10  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr 
               
               
                 or Val; 
               
               
                 X 11  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, 
               
               
                 Tyr or Val; 
               
               
                 X 12  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, 
               
               
                 Tyr or Val; 
               
               
                 X 13  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, 
               
               
                 Tyr or Val; 
               
               
                 X 14  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr 
               
               
                 or Val; 
               
               
                 and 
               
               
                 X 15  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, 
               
               
                 Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, 
               
               
                 Tyr or Val. 
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Residue 
                   
                   
               
               
                 Position* 
                 WT residues 
                 Distribution in the TCL2 library 
               
               
                   
               
             
            
               
                 22 
                 T 
                 designed distribution 
               
               
                 23 
                 A 
                 designed distribution 
               
               
                 24 
                 P 
                 50% P + designed distribution 
               
               
                 25 
                 D 
                 designed distribution 
               
               
                 26 
                 A 
                 20% A + 20% G + designed distribution 
               
               
                 27 
                 A 
                 designed distribution 
               
               
                 28 
                 F 
                 20% F, 20% I, 20% L, 20% V, 20% Y 
               
               
                 29 
                 D 
                 33% D, 33% E, 33% T 
               
               
                 75 
                 K 
                 designed distribution 
               
               
                 76 
                 G 
                 designed distribution 
               
               
                 77 
                 G 
                 designed distribution 
               
               
                 78 
                 H 
                 designed distribution 
               
               
                 79 
                 R 
                 designed distribution 
               
               
                 80 
                 S 
                 100% S 
               
               
                 81 
                 N 
                 designed distribution 
               
               
                 82 
                 P 
                 50% P + designed distribution 
               
               
                   
               
               
                 *residue numbering is based on Tencon sequence of SEQ ID NO: 1 
               
            
           
         
       
     
     Subsequently, these libraries were improved by various ways, including building of the libraries on a stabilized Tencon framework (U.S. Pat. No. 8,569,227) that incorporates substitutions E11R/L17A/N46V/E86I (Tencon27; SEQ ID NO: 4) when compared to the wild type tencon as well as altering of the positions randomized in the BC and FG loops. Tencon27 is described in Int. Pat. Appl. No. WO2013049275. From this, new libraries designed to randomize only the FG loop of Tencon (library TCL9), or a combination of the BC and FG loops (library TCL7) were generated. These libraries were constructed for use with the cis-display system (Odegrip et al., Proc Natl Acad Sci USA 101: 2806-2810, 2004). The details of this design are shown below: 
                    Stabilized Tencon (Tencon27)       (SEQ ID NO: 4)       LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVP               GSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT               TCL7 (randomized FG and BC loops)       (SEQ ID NO: 5)       LPAPKNLVVSRVTEDSARLSWX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 FDSFLIQYQES               EKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX 10 X 11 X 12 X 13                 X 14 X 15 X 16 X 17 X 18 X 19 SNPLSAIFTT;            
wherein
 
X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15  and X 16  is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and
 
X 7 , X 8 , X 9 , X 17 , X 18  and X 19 , is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.
 
                    TCL9 (randomized FG loop)       (SEQ ID NO: 6)       LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVP               GSERSYDLTGLKPGTEYTVSIYGVX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11                 X 12 SNPLSAIFTT;            
X 1 , X 2 , X 3 , X 4 , X 5 , X 6  and X 7 , is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and
 
X 8 , X 9 , X 10 , X 11  and X 12  is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.
 
     For library construction, DNA fragments encoding randomized BC loops (lengths 6-9 positions) or FG loops (lengths 7-12 positions) were synthesized using Slonomics technology (Sloning Biotechnology GmbH) so as to control the amino acid distribution of the library and to eliminate stop codons. Two different sets of DNA molecules randomizing either the BC loop or the FG loops were synthesized independently and later combined using PCR to produce the full library product. 
     Construction of FG Loop Libraries (TCL9) 
     A set of synthetic DNA molecules consisting of a 5′ Tac promoter followed by the complete gene sequence of Tencon with the exception of randomized codons in the FG loop was produced (SEQ ID NOs: 26-31). For FG loop randomization, all amino acids except cysteine and methionine were encoded at equal percentages. The lengths of the diversified portion are such that they encode for 7, 8, 9, 10, 11, or 12 amino acids in the FG loop. Sub-libraries of each length variation were synthesized individually at a scale of 2 ug and then amplified by PCR using oligos Sloning-FOR (SEQ ID NO: 9) and Sloning-Rev (SEQ ID NO: 10). 
     The 3′ fragment of the library is a constant DNA sequence containing elements for display, including a PspOMI restriction site, the coding region of the repA gene, and the cis- and ori elements. PCR reactions were performed to amplify this fragment using a plasmid (pCR4Blunt) (Invitrogen) as a template with M13 Forward and M13 Reverse primers. The resulting PCR products were digested by PspOMI overnight and gel-purified. To ligate the 5′ portion of library DNA to the 3′ DNA containing repA gene, 2 pmol (˜540 ng to 560 ng) of 5′ DNA was ligated to an equal molar (˜1.25 μg) of 3′ repA DNA in the presence of NotI and PspOMI enzyme and T4 ligase at 37° C. overnight. The ligated library product was amplified by using 12 cycles of PCR with oligos POP2250 (SEQ ID NO: 11) and DigLigRev (SEQ ID NO: 12). For each sub-library, the resulting DNA from 12 PCR reactions were combined and purified by Qiagen spin column. The yield for each sub-library of TCL9 ranged from 32-34 μg. 
     Construction of FG/BC Loop Libraries (TCL7) 
     The TCL7 library provides for a library with randomized Tencon BC and FG loops. In this library, BC loops of lengths 6-9 amino acids were mixed combinatorially with randomized FG loops of 7-12 amino acids in length. Synthetic Tencon fragments BC6, BC7, BC8, and BC9 (SEQ ID No. 13-16) were produced to include the Tencon gene encoding for the N-terminal portion of the protein up to and including residue VX such that the BC loop is replaced with either 6, 7, 8, or 9 randomized amino acids. These fragments were synthesized prior to the discovery of L17A, N46V and E831 mutations (CEN5243) but these mutations were introduced in the molecular biology steps described below. In order to combine this fragment with fragments encoding for randomized FG loops, the following steps were taken. 
     First, a DNA fragment encoding the Tac promoter and the 5′ sequence of Tencon up to the nucleotide endoding for amino acid A17 (130mer-L17A, SEQ ID No. 17) was produced by PCR using oligos POP2222ext (SEQ ID No. 18) and LS1114 (SEQ ID No. 19). This was done to include the L17A mutation in the library (CEN5243). Next, DNA fragments encoding for Tencon residues R18-V75 including randomized BC loops were amplified by PCR using BC6, BC7, BC8, or BC9 as a templates and oligos LS1115 (SEQ ID No. 20) and LS1117 (SEQ ID No. 21). This PCR step introduced a BsaI site at the 3′ end. These DNA fragments were subsequently joined by overlapping PCR using oligos POP2222ext and LS1117 as primers. The resulting PCR product of 240 bp was pooled and purified by Qiagen PCR purification kit. The purified DNA was digested with BsaI-HF and gel purified. 
     Fragments encoding the FG loop were amplified by PCR using FG7 (SEQ ID No. 31), FG8 (SEQ ID No. 30), FG9 (SEQ ID No. 29), FG10 (SEQ ID No. 28), FG11 (SEQ ID No. 27), and FG12 (SEQ ID No. 26) as templates with oligonucleotides SDG10 (SEQ ID No. 22) and SDG24 (SEQ ID No. 23) to incorporate a BsaI restriction site and N46V and E86I variations (CEN5243). 
     The digested BC fragments and FG fragments were ligated together in a single step using a 3-way ligation. Four ligation reactions in the 16 possible combinations were set up, with each ligation reaction combining two BC loop lengths with 2 FG loop lengths. Each ligation contained ˜300 ng of total BC fragment and 300 ng of the FG fragment. These 4 ligation pools were then amplified by PCR using oligos POP2222 (SEQ ID No. 24) and SDG28 (SEQ ID No. 25). 7.5 μg of each reaction product were then digested with NotI and cleaned up with a Qiagen PCR purification column. 5.2 μg of this DNA, was ligated to an equal molar amount of RepA DNA fragment (˜14 μg) digested with PspOMI and the product amplified by PCR using oligos POP2222. 
     Example 2: Generation of Tencon Libraries Having Alternative Binding Surfaces 
     The choice of residues to be randomized in a particular library design governs the overall shape of the interaction surface created. X-ray crystallographic analysis of an FN3 domain containing scaffold protein selected to bind maltose binding protein (MBP) from a library in which the BC, DE, and FG loops were randomized was shown to have a largely curved interface that fits into the active site of MBP (Koide et al., Proc Natl Acad Sci USA 104: 6632-6637, 2007). In contrast, an ankyrin repeat scaffold protein that was selected to bind to MBP was found to have a much more planar interaction surface and to bind to the outer surface of MBP distant from the active (Binz et al., Nat Biotechnol 22: 575-582, 2004). These results suggest that the shape of the binding surface of a scaffold molecule (curved vs. flat) may dictate what target proteins or specific epitopes on those target proteins are able to be bound effectively by the scaffold. Published efforts around engineering protein scaffolds containing FN3 domains for protein binding has relied on engineering adjacent loops for target binding, thus producing curved binding surfaces. This approach may limit the number of targets and epitopes accessible by such scaffolds. 
     Tencon and other FN3 domains contain two sets of CDR-like loops lying on the opposite faces of the molecule, the first set formed by the BC, DE, and FG loops, and the second set formed by the AB, CD, and EF loops. The two sets of loops are separated by the beta-strands that form the center of the FN3 structure. If the image of the Tencon is rotated by 90 degrees, an alternative surface can be visualized. This slightly concave surface is formed by the CD and FG loops and two antiparallel beta-strands, the C and the F beta-strands, and is herein called the C-CD-F-FG surface. The C-CD-F-FG surface can be used as a template to design libraries of protein scaffold interaction surfaces by randomizing a subset of residues that form the surface. Beta-strands have a repeating structure with the side chain of every other residue exposed to the surface of the protein. Thus, a library can be made by randomizing some or all surface exposed residues in the beta strands. By choosing the appropriate residues in the beta-strands, the inherent stability of the Tencon scaffold should be minimally compromised while providing a unique scaffold surface for interaction with other proteins. 
     Library TCL14 (SEQ ID NO: 7), was designed into Tencon27 scaffold (SEQ ID NO: 4). 
     A full description of the methods used to construct this library is described in US. Pat. Publ. No. 2013/0226834. 
     
       
         
           
               
            
               
                 TCL14 library (SEQ ID NO: 7): 
               
               
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 1 IX 2 YX 3 EX 4 X 5 X 6 X 7 GE 
               
               
                   
               
               
                 AIVLTVPGSERSYDLTGLKPGTEYX 8 VX 9 IX 10 GVKGGX 11 X 12 SX 13 PL 
               
               
                   
               
               
                 SAIFTT; 
               
            
           
         
       
     
     Wherein 
     X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 10 , X 11 , X 12  and X 13  are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or M. 
     The two beta strands forming the C-CD-F-FG surface in Tencon27 have a total of 9 surface exposed residues that could be randomized; C-strand: S30, L32, Q34, Q36; F-strand: E66, T68, S70, Y72, and V74, while the CD loop has 6 potential residues: S38, E39, K40, V41, G42, and E43 and the FG loop has 7 potential residues: K75, G76, G77, H78, R79, S80, and N81. Select residues were chosen for inclusion in the TCL14 design due to the larger theoretical size of the library if all 22 residues were randomized. 
     Thirteen positions in Tencon were chosen for randomizing: L32, Q34 and Q36 in C-strand, S38, E39, K40 and V41 in CD-loop, T68, S70 and Y72 in F-strand, H78, R79, and N81 in FG-loop. In the C and F strands S30 and E66 were not randomized as they lie just beyond the CD and FG loops and do not appear to be as apparently a part of the C-CD-F-FG surface. For the CD loop, G42 and E43 were not randomized as glycine, providing flexibility, can be valuable in loop regions, and E43 lies at the junction of the surface. The FG loop had K75, G76, G77, and S80 excluded. The glycines were excluded for the reasons above while careful inspection of the crystal structures revealed S80 making key contacts with the core to help form the stable FG loop. K75 faces away from the surface of the C-CD-F-FG surface and was a less appealing candidate for randomization. Although the above mentioned residues were not randomized in the original TCL14 design, they could be included in subsequent library designs to provide additional diversity for de novo selection or for example for an affinity maturation library on a select TCL14 target specific hit. 
     Subsequent to the production of TCL14, 3 additional Tencon libraries of similar design were produced. These two libraries, TCL19, TCL21 and TCL23, are randomized at the same positions as TCL14 (see above) however the distribution of amino acids occurring at these positions is altered (Table 2). TCL19 and TCL21 were designed to include an equal distribution of 18 natural amino acids at every position (5.55% of each), excluding only cysteine and methionine. TCL23 was designed such that each randomized position approximates the amino acid distribution found in the HCDR3 loops of functional antibodies (Birtalan et al., J Mol Biol 377: 1518-1528, 2008) as described in Table 2. As with the TCL21 library, cysteine and methionine were excluded. 
     A third additional library was built to expand potential target binding surface of the other libraries library. In this library, TCL24, 4 additional Tencon positions were randomized as compared to libraries TCL14, TCL19, TCL21, and TCL23. These positions include N46 and T48 from the D strand and S84 and 186 from the G strand. Positions 46, 48, 84, and 86 were chosen in particular as the side chains of these residues are surface exposed from beta-strands D and G and lie structurally adjacent to the randomized portions of the C and F strand, thus increasing the surface area accessible for binding to target proteins. The amino acid distribution used at each position for TCL24 is identical to that described for TCL19 and TCL21 in Table 2. 
                    TCL24 Library       (SEQ ID NO: 8)       LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 1 IX 2 YX 3 EX 4 X 5 X 6 X 7 GE               AIX 8 LX 9 VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 GVKGGX 13 X 14 S               X 15 PLX 16 AX 17 FTT;            
wherein
 
X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 10 , X 11 , X 12  and X 13  are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W.
 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Amino acid frequency (%) at each randomized  
               
               
                 position for TCL21, TCL23, and TCL24. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Amino Acid 
                 TCL19 
                 TCL21  
                 TCL23  
                 TCL24 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Ala 
                 5.6 
                 5.6 
                 6.0 
                 5.6 
               
               
                   
                 Arg 
                 5.6 
                 5.6 
                 6.0 
                 5.6 
               
               
                   
                 Asn 
                 5.6 
                 5.6 
                 3.9 
                 5.6 
               
               
                   
                 Asp 
                 5.6 
                 5.6 
                 7.5 
                 5.6 
               
               
                   
                 Cys 
                 0.0 
                 0.0 
                 0.0 
                 0.0 
               
               
                   
                 Gln 
                 5.6 
                 5.6 
                 1.5 
                 5.6 
               
               
                   
                 Glu 
                 5.6 
                 5.6 
                 2.5 
                 5.6 
               
               
                   
                 Gly 
                 5.6 
                 5.6 
                 15.0 
                 5.6 
               
               
                   
                 His 
                 5.6 
                 5.6 
                 2.3 
                 5.6 
               
               
                   
                 Ile 
                 5.6 
                 5.6 
                 2.5 
                 5.6 
               
               
                   
                 Leu 
                 5.6 
                 5.6 
                 5.0 
                 5.6 
               
               
                   
                 Lys 
                 5.6 
                 5.6 
                 1.5 
                 5.6 
               
               
                   
                 Met 
                 0.0 
                 0.0 
                 0.0 
                 0.0 
               
               
                   
                 Phe 
                 5.6 
                 5.6 
                 2.5 
                 5.6 
               
               
                   
                 Pro 
                 5.6 
                 5.6 
                 4.0 
                 5.6 
               
               
                   
                 Ser 
                 5.6 
                 5.6 
                 10.0 
                 5.6 
               
               
                   
                 Thr 
                 5.6 
                 5.6 
                 4.5 
                 5.6 
               
               
                   
                 Trp 
                 5.6 
                 5.6 
                 4.0 
                 5.6 
               
               
                   
                 Tyr 
                 5.6 
                 5.6 
                 17.3 
                 5.6 
               
               
                   
                 Val 
                 5.6 
                 5.6 
                 4.0 
                 5.6 
               
               
                   
               
            
           
         
       
     
     Generation of TCL21, TCL23, and TCL24 Libraries 
     The TCL21 library was generated using Colibra library technology (Isogenica) in order to control amino acid distributions. TCL19, TCL23, and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control amino acid distributions. PCR was used to amplify each library following initial synthesis followed by ligation to the gene for RepA in order to be used in selections using the CIS-display system (Odegrip et al., Proc Natl Acad Sci USA 101: 2806-2810, 2004) as described above for the loop libraries. 
     Example 3: Selection of Fibronectin Type III (FN3) Domains that Bind Cd8A 
     Design and Production of Human CD8 Alpha Antigens: 
     Two human CD8 alpha (Swiss Prot P01732) constructs were expressed and purified from HEK cells to produce recombinant protein for CIS-Display panning (Table 3). 
                     TABLE 3                  CD8A constructs generated for use as antigens                         Construct   SEQID No.   Description               CD8W7   35   Human CD8 alpha residues 22-167                fused to Fc fragment of human IgG1       CD8W13   36   Human CD8 alpha residues 22-182                fused to Fc fragment of human IgG1                    
Each construct was designed to include a murine IgG Kappa secretion signal (SEQ ID No 3) and was fused to the Fc fragment of human IgG1 (SEQID No. 4). The CD8 alpha and Fc fragment sequences were connected by a linker containing a flag and polyhistidine tag sequence (SEQID No 5.)
 
     Plasmids encoding these proteins were transfected into HEK 293-Expi cells by transient transfection and culture supernatants were harvested by centrifugation at 6000×g and clarified with a 0.2 micron filter. Supernatants were loaded onto a HiTrap Mabsure Select column (GE Healthcare) and CD8A proteins eluted in 0.1 M Na-Acetate pH 3.5 and neutralized by addition of 2M Tris pH 7. Each sample was then dialyzed into PBS pH 7.4 for biotinylation with a No Weigh EZ-Link-Sulfo-NHS-LC-Biotin biotinylation kit (Thermo Scientific). 
     Library Screening 
     Cis-display was used to select human CD8 alpha-binding domains from the TCL18, TCL19, TCL21, TCL23, and TCL24 libraries. Biotinylated CD8W7 and CD8W13 were used for panning. For in vitro transcription and translation (ITT), 3 μg of library DNA were incubated with 0.1 mM complete amino acids, 1×S30 premix components, and 15 μL of S30 extract (Promega) in a total volume of 50 μL and incubated at 30° C. After 1 hour, 375 μL of blocking solution ((0.1% Casein (Thermo Fisher, Rockford, Ill.), 100 mg/ml Herring Sperm DNA (Promega, Madison, Wis.), 1 mg/mL heparin (Sigma-Aldrich, St. Louis, Mo.)) was added and the reaction was incubated on ice for 15 minutes. For selection, biotinylated antigen was added at concentrations of 400 nM (Round 1), 200 nM (Rounds 2 and 3) and 100 nM (Rounds 4 and 5). Bound library members were recovered using neutravidin magnetic beads (Thermo Fisher, Rockford, Ill.) (Rounds 1, 3, and 5) or streptavidin magnetic beads (Promega, Madison, Wis.) (Rounds 2 and 4) and unbound library members were removed by washing the beads 5-14 times with 500 μL PBST followed by 2 washes with 500 μL PBS. Additional selection rounds were performed in order to identify scaffold molecules with improved affinities. Briefly, outputs from round 5 were prepared as described above and subjected to additional iterative rounds of selection with the following changes: the biotinylated target concentration decreased to 25 nM (Rounds 6 and 7) or 2.5 nM (Rounds 8 and 9), and an additional 1 hour wash was performed in the presence of an excess of non-biotinylated target protein. The goal of these changes was to simultaneously select for binders with a potentially faster on-rate and a slower off-rate yielding a substantially lower K D . 
     Following panning, selected FN3 domains were amplified by PCR using oligos Tcon6 (SEQID NO: 33) and Tcon5shortE86I (SEQID NO: 34), subcloned by annealing into a pET15-LIC and transformed into BL21-GOLD (DE3) cells (Agilent, Santa Clara, Calif.) for soluble expression in  E. coli  using standard molecular biology techniques. Single clones were picked and grown to saturation in 1 mL LB with ampicillin in 96 deepwell plates at 37° C. The following day, 25 uL was transferred to fresh 1 mL LB-Amp media in 96 deepwell plates and grown at 37° C. for 2 hours. IPTG was added at 1 mM final concentration and protein expression was induced at 30° C. for 16 hours. The cells were harvested by centrifugation and subsequently lysed with Bugbuster HT (EMD Chemicals, Gibbstown, N.J.) supplemented with 0.2 mg/mL final Chicken Egg White Lysozyme (Sigma-Aldrich, St. Louis, Mo.). Cells were harvested approximately 16 hours later by centrifugation and frozen at −20° C. Cell lysis was achieved by incubating each pellet in 0.6 mL of BugBuster® HT lysis buffer (Novagen EMD Biosciences) with shaking at room temperature for 45 minutes. 
     Selection of FN3 Domains that Bind CD8A 
     Neutravidin-coated plates were blocked for 1 hour in Starting Block T20 (Pierce) and then coated with biotinylated CD8W7 or CD8W13 (same antigen as in panning) or negative control (human Fc) for 1 hour. Plates were rinsed with TBST and diluted lysate was applied to plates for 1 hour. Following additional rinses, wells were treated with HRP-conjugated anti-FN3 domain antibody (PAB25) for 1 h and then assayed with POD (Roche). FN3 domain molecules with signals at least 10-fold above background were selected for further analysis. 
     Small Scale Expression and Purification of Identified FN3 Domains Binding CD8A 
     Isolated clones from unique hits identified by biochemical binding ELISA were combined into a single hit plate for growth in 96-well block plates; clones grew in 1 mL cultures (LB media supplemented with kanamycin for selection) at 37° C. overnight with shaking. For protein expression in 96-block plates, 1 mL TB media supplemented with kanamycin was inoculated with 50 uL of the overnight culture and grown at 37° C. with continual shaking at 300 rpm until OD 600 =0.6-1. Once the target OD was reached, protein expression was induced with addition of IPTG to 1 mM; plates were transferred to 30° C. (300 rpm) for overnight growth. Overnight cultures were centrifuged to harvest the cells; bacterial pellets were stored at −80° C. until ready for use. Pellets were lysed with BugBuster® HT lysis buffer (Novagen EMD Biosciences) and His-tagged Centyrins purified from the clarified lysates with His MultiTrap™ HP plates (GE Healthcare) and eluted in buffer containing 20 mM sodium phosphate, 500 mM sodium chloride, and 250 mM imidazole at pH 7.4. Purified samples were exchanged into PBS pH 7.4 for analysis using PD MultiTrap™ G-25 plates (GE Healthcare). 
     Size Exclusion Chromatography Analysis 
     Size exclusion chromatography was used to determine the aggregation state of anti-CD8 alpha FN3 domain molecules. Aliquots (10 μL) of each purified FN3 domain were injected onto a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min in a mobile phase of PBS pH 7.4. Elution from the column was monitored by absorbance at 280 nm. Wild-type Tencon was included in each run as a control. Agilent ChemStation software was used to analyse the elution profiles. Only those proteins with elution profiles similar to that of the tenascin consensus protein in the same run were considered for further characterization. After panning, ELISA screening and size exclusion chromatographic analysis, a total of 190 unique anti-human CD8 alpha FN3 domains were isolated that bound to recombinant human CD8 alpha greater 5 than 10-fold over background and were free of aggregates by SEC (Table 4, SEQID no. 40-228, and 70). 
                     TABLE 4                  Summary of CD8A-binding FN3 domains identified from ELISA screens                                                 Human           Cyno T-                   T-cell   Human T-       cell               Binding   cell Binding       Binding   Cyno T-cell           SEQ ID   2 uM   0.2 uM       2 uM   Binding 0.2       Clone ID   NO:   (MFI)   (MFI)   kd (1/s)   (MFI)   uM (MFI)                                                 P282AR9P1356_A10   40   4258   2093   2.91E−04   10584   3122       P282AR9P1356_A4   41   16674   4380   8.61E−05   26447   8632       P282AR9P1356_A6   42   10835   3441   9.73E−05   31432   5783       P282AR9P1356_B9   43   17158   3670   2.95E−04   36397   5437       P282AR9P1356_D3   44   5963   2403   1.58E−04   13852   3365       P282AR9P1356_H1   45   14696   3234   1.14E−04   46317   5699       P282AR9P1356_H6   46   6646   2642   8.08E−05   14393   3205       P282BR9P1357_A9   47   3117   1074   5.90E−04   7281   1940       P282BR9P1357_B2   48   5931   2875   1.00E−04   17974   3841       P282BR9P1357_C10   49   9779   2901   4.58E−04   24476   5110       P282BR9P1357_C4   50   16809   4224   1.27E−04   41586   7064       P282BR9P1357_D12   51   15269   3899   8.76E−05   40450   7364       P282BR9P1357_D2   52   9606   1568   1.05E−03   25843   2525       P282BR9P1357_E5   53   6726   2587   2.10E−04   10563   4101       P282BR9P1357_G9   54   12733   2803   3.04E−04   41492   4635       P282BR9P1357_H3   55   11142   3033   2.85E−04   27090   5701       P282CR9P1358_C2   56   10086   1059   1.13E−03   55786   7047       P282CR9P1358_C5   57   2706   511   9.99E−04   25688   1831       P282CR9P1358_D10   58   28650   2764   3.11E−04   74051   4072       P282CR9P1358_F11   59   6420   749   1.35E−03   62412   6585       P282CR9P1358_F5   60   24427   3072   6.37E−04   85691   13667       P282DR9P1359_A12   61   32222   5952   8.12E−05   88032   15491       P282DR9P1359_A7   62   38382   8764   7.54E−04   83943   22803       P282DR9P1359_A8   63   21124   2113   6.38E−04   70263   7766       P282DR9P1359_B2   64   22228   2726   6.38E−04   60866   4472       P282DR9P1359_C10   65   27822   2879   9.91E−04   98481   15134       P282DR9P1359_C11   66   18176   1288   2.16E−03   19916   457       P282DR9P1359_C12   67   15106   944   9.78E−04   66538   3636       P282DR9P1359_C5   68   31017   5551   1.74E−04   95679   14183       P282DR9P1359_D12   69   4540   542   1.93E−03   37139   1746       P282DR9P1359_E11   70   40607   7578   2.65E−04   104291   33144       P282DR9P1359_E2   71   28491   4824   2.06E−03   77725   10939       P282DR9P1359_E3   72   4307   349   2.63E−03   52426   1625       P282DR9P1359_E5   73   24100   1954   1.01E−03   81183   13601       P282DR9P1359_E6   74   20507   1262   1.71E−03   61734   5065       P282DR9P1359_E8   75   26074   2919   1.19E−03   80973   16948       P282DR9P1359_F11   76   35639   6592   5.54E−04   86740   16146       P282DR9P1359_F2   77   18415   3047   7.22E−04   38228   4031       P282DR9P1359_F3   78   6343   646   1.06E−03   48861   3084       P282DR9P1359_F5   79   48931   8483   9.02E−05   113733   34709       P282DR9P1359_F6   80   19937   3782   3.89E−04   73219   10680       P282DR9P1359_F7   81   38323   6932   3.65E−04   96456   26331       P282DR9P1359_G4   82   26568   2670   5.17E−04   78619   6006       P282DR9P1359_G7   83   37626   6129   1.14E−04   69085   8769       P282DR9P1359_H5   84   919   278   4.49E−03   2252   500       P282ER9P1360_A9   85   23379   5344   1.33E−04   64694   8732       P282ER9P1360_C1   86   25874   6291   1.81E−04   64813   9679       P282ER9P1360_C4   87   19202   3459   1.07E−03   33427   3896       P282ER9P1360_C6   88   25942   5079   1.75E−04   52783   7579       P282ER9P1360_C8   89   30578   6013   1.56E−04   66829   10220       P282ER9P1360_D11   90   36755   3210   1.42E−04   76564   1937       P282ER9P1360_E4   91   26889   5030   1.91E−04   60757   6867       P282ER9P1360_F11   92   22442   3863   2.25E−04   48653   4407       P282ER9P1360_G10   93   26951   7046   2.07E−03   62701   22641       P282ER9P1360_G7   94   25438   5869   2.21E−04   69709   9921       P282ER9P1360_H10   95   2513   506   1.04E−03   27063   1887       P282ER9P1360_H2   96   15165   3479   2.69E−04   44563   4535       P282ER9P1360_H3   97   19992   4271   2.42E−04   65994   6441       P282FR9P1361_A3   98   7670   1661   7.57E−04   8476   740       P282FR9P1361_A5   99   32752   5213   1.92E−04   63541   8108       P282FR9P1361_C7   100   8538   1575   2.24E−03   11639   896       P282FR9P1361_D3   101   6881   1028   5.02E−03   14762   764       P282FR9P1361_E12   102   15794   1130   1.09E−03   63536   15052       P282FR9P1361_F1   103   5498   801   1.26E−03   9869   1392       P282FR9P1361_F11   104   2189   382   2.13E−03   2289   384       P282FR9P1361_F2   105   4610   498   4.96E−03   10883   462       P282FR9P1361_F3   106   5157   674   1.07E−02   9709   513       P282FR9P1361_F7   107   7001   1107   1.14E−03   1705   353       P282FR9P1361_G9   108   859   297   6.53E−03   3746   666       P282FR9P1361_H4   109   13056   3736   3.17E−04   26273   2504       P282FR9P1361_H5   110   5730   698   5.77E−03   11794   637       P283AR9P1362_A3   111   5535   1400   1.53E−03   17345   3533       P283AR9P1362_A4   112   6314   2539   3.02E−04   21218   4402       P283AR9P1362_B10   113   15380   3703   1.39E−04   35686   8380       P283AR9P1362_B2   114   13649   3505   1.60E−04   38828   6479       P283AR9P1362_B8   115   5737   1576   6.48E−04   12886   2271       P283AR9P1362_C12   116   7064   2616   9.94E−05   14808   3832       P283AR9P1362_C6   117   15955   4147   1.09E−03   17494   5690       P283AR9P1362_C7   118   10957   2792   1.86E−04   19690   5515       P283AR9P1362_D2   119   8650   2758   2.53E−04   17182   5333       P283AR9P1362_D3   120   9498   3484   1.25E−04   34619   6052       P283AR9P1362_D4   121   9832   2977   9.72E−05   25671   4101       P283AR9P1362_D6   122   13686   3664   2.64E−05   33547   7721       P283AR9P1362_D7   123   17327   3354   1.18E−04   27491   4849       P283AR9P1362_E9   124   6178   2010   3.27E−04   15869   2837       P283AR9P1362_F12   125   8970   2623   7.28E−05   26333   3794       P283AR9P1362_F2   126   9619   1366   2.11E−03   26443   5518       P283AR9P1362_F8   127   9195   3167   1.12E−04   23735   4571       P283AR9P1362_G11   128   12690   3531   1.02E−04   32484   6826       P283AR9P1362_G3   129   18512   4307   9.45E−05   35268   9198       P283AR9P1362_H11   130   5734   2268   1.80E−04   11588   3655       P283BR9P1363_A10   131   7886   2753   3.60E−04   27790   4105       P283BR9P1363_A8   132   11285   2536   3.53E−04   24234   3453       P283BR9P1363_B2   133   8358   2399   2.08E−04   14846   2819       P283BR9P1363_B6   134   14534   3453   2.69E−04   37691   6839       P283BR9P1363_C4   135   9073   2247   4.09E−04   23387   3266       P283BR9P1363_C8   136   16541   3739   3.35E−04   37175   9082       P283BR9P1363_D11   137   8692   2890   4.95E−04   20572   11630       P283BR9P1363_E4   138   10790   2498   3.29E−04   17702   2469       P283BR9P1363_E6   139   8239   2079   1.36E−03   16784   3715       P283BR9P1363_F2   140   14473   3274   2.88E−04   33286   5278       P283BR9P1363_F4   141   11933   2963   1.55E−04   20245   4479       P283BR9P1363_F6   142   10632   3229   8.21E−05   31568   4571       P283BR9P1363_G2   143   9640   3226   1.22E−04   15899   5383       P283BR9P1363_G5   144   14798   3307   1.40E−04   24945   4430       P283BR9P1363_G7   145   4639   2340   4.01E−05   7212   3022       P283DR9P1364_A4   146   9491   1024   1.09E−03   48337   6653       P283DR9P1364_A7   147   8985   435   1.97E−03   39870   2641       P283DR9P1364_B1   148   1477   666   1.56E−03   8617   746       P283DR9P1364_B11   149   4255   451   1.30E−03   22852   1590       P283DR9P1364_B4   150   45452   6062   1.09E−04   96492   20238       P283DR9P1364_C10   151   4936   649   1.29E−03   34234   2713       P283DR9P1364_D11   152   32293   4223   5.14E−04   70431   16240       P283DR9P1364_D8   153   656   244   6.61E−03   2484   365       P283DR9P1364_D9   154   42285   5245   4.30E−04   88300   19979       P283DR9P1364_E3   155   1285   317   2.53E−03   9128   887       P283DR9P1364_E5   156   17625   1269   8.25E−04   55654   5091       P283DR9P1364_E7   157   5394   442   2.43E−03   28732   2241       P283DR9P1364_E8   158   14321   1181   7.56E−04   59328   5510       P283DR9P1364_E9   159   4295   548   1.90E−03   19688   2096       P283DR9P1364_F2   160   39164   6252   1.61E−04   91474   16946       P283DR9P1364_F6   161   17215   1831   1.00E−03   33767   3161       P283DR9P1364_F8   162   6305   458   1.74E−03   36659   1302       P283DR9P1364_G10   163   6291   409   2.53E−03   10920   769       P283DR9P1364_G9   164   9892   401   7.79E−03   47097   2796       P283DR9P1364_H1   165   29248   3033   6.13E−04   54014   10610       P283DR9P1364_H11   166   11479   834   9.64E−04   60609   9459       P283DR9P1364_H6   167   2623   268   2.30E−03   6002   418       P283DR9P1364_H9   168   32763   4057   2.71E−04   54593   4556       P283ER9P1365_A1   169   25512   3862   4.67E−04   9676   1365       P283ER9P1365_A7   170   18513   1315   7.86E−04   36568   2960       P283ER9P1365_B6   171   22998   3397   2.88E−04   30081   2692       P283ER9P1365_C1   172   8004   644   1.15E−03   23975   1884       P283ER9P1365_E2   173   20011   2867   3.11E−04   17177   1905       P283ER9P1365_F4   174   24065   2596   2.16E−04   43243   2038       P283ER9P1365_G1   175   1280   318   3.67E−03   489   383       P283ER9P1365_G3   176   12481   2916   2.50E−03   3480   1470       P283ER9P1365_H3   177   17965   953   3.75E−04   19560   436       P283FR9P1366_A1   178   8782   516   2.26E−03   39384   1650       P283FR9P1366_A5   179   27649   3598   5.85E−04   67839   10945       P283FR9P1366_A9   180   1717   252   3.94E−03   8809   580       P283FR9P1366_B7   181   11365   899   1.15E−03   51186   4668       P283FR9P1366_C2   182   40957   4319   4.91E−04   89242   19288       P283FR9P1366_C3   183   1823   407   2.07E−03   4628   1044       P283FR9P1366_C4   184   33821   3754   5.36E−04   63373   10200       P283FR9P1366_C6   185   4541   483   1.43E−03   26242   1675       P283FR9P1366_D12   186   27793   1528   1.76E−03   87643   8143       P283FR9P1366_D6   187   32924   4554   5.09E−04   79621   10399       P283FR9P1366_D7   188   7517   566   3.54E−04   41434   2581       P283FR9P1366_D8   189   3394   413   1.34E−03   28181   2296       P283FR9P1366_E11   190   4594   567   1.41E−03   14194   1469       P283FR9P1366_F5   191   6880   720   1.04E−03   46414   4695       P283FR9P1366_F8   192   3970   369   4.03E−03   26970   2269       P283FR9P1366_F9   193   33559   6295   4.94E−04   84279   24622       P283FR9P1366_G1   194   3605   650   8.72E−04   39796   4981       P283FR9P1366_G5   195   8450   261   7.05E−04   36380   369       P283FR9P1366_G8   196   6857   574   1.08E−03   37144   3126       P283FR9P1366_H10   197   25020   2414   6.30E−04   75192   13854       P283FR9P1366_H11   198   18896   2331   1.39E−03   37386   3659       P283FR9P1366_H3   199   7671   632   1.21E−03   40770   3173       P283FR9P1366_H5   200   3137   252   3.18E−03   5091   477       P283FR9P1366_H6   201   43937   7129   2.05E−04   81542   18993       P283FR9P1366_H7   202   13778   567   1.77E−03   24435   1238       P283FR9P1366_H8   203   24942   4544   1.75E−04   61256   17144       P283FR9P1366_H9   204   8570   693   1.98E−03   36501   2877       P283GR7P1367_A11   205   11326   1029   6.35E−04   66691   5666       P283GR7P1367_B4   206   8302   446   5.18E−03   396   367       P283GR7P1367_B7   207   10865   739   1.27E−03   37518   3134       P283GR7P1367_B9   208   11242   1092   1.16E−03   2924   442       P283GR7P1367_C9   209   10989   896   2.21E−03   66977   5553       P283GR7P1367_E5   210   10014   1333   1.24E−03   3189   533       P283GR7P1367_F5   211   4565   601   1.08E−03   28950   2051       P283GR7P1367_G8   212   1463   450   3.85E−03   21031   1421       P283GR7P1367_H2   213   1621   390   2.35E−03   4207   864       P283GR7P1367_H8   214   5269   303   9.74E−03   20918   930       P283GR7P1367_H9   215   1714   434   1.47E−03   6121   918       P283HR7P1368_A10   216   13632   3233   5.13E−04   42326   4772       P283HR7P1368_B12   217   13399   1538   4.53E−05   18650   826       P283HR7P1368_C3   218   12727   2215   3.49E−04   13326   1306       P283HR7P1368_D1   219   14077   2312   1.66E−03   7850   1408       P283HR7P1368_D2   220   15246   1907   1.30E−03   11132   950       P283HR7P1368_D4   221   28979   6850   2.35E−04   52999   23549       P283HR7P1368_F10   222   18836   2661   1.65E−04   16121   1019       P283HR7P1368_F6   223   14325   3510   1.80E−04   20580   3541       P283HR7P1368_G1   224   31276   4940   2.15E−03   69817   11559       P283HR7P1368_G10   225   8122   753   1.45E−03   23790   2660       P283HR7P1368_G11   226   19305   2647   3.73E−04   14857   1343       P283HR7P1368_H1   227   15389   2460   5.52E−04   17285   1974       P283HR7P1368_H8   228   22758   1612   7.63E−04   35932   4888       Tencon25-His   270   341   219       337   336                    
Screen for Binding to T-Cells from Human and Cynomolgus Monkey Donors
 
     Binding of the 190 ELISA hits to human and cynomologous monkey primary CD8 T cells was assessed by flow cytometry. The FN3 domain molecules were diluted to 2 μM and 0.2 μM in PBS and incubated with human or cynomologous monkey CD8+ T cells in 96-well format. After 1 hour at 4° C., the cells were washed once with PBS and then resuspended with an anti-FN3 domain antibody (PAB25) solution. Following this incubation, the cells were washed twice with PBS and a PE conjugated secondary antibody and a viability dye were added. Finally, cells were washed and resuspended in PBS for flow cytometric analysis using a BD Canto Instrument. Cells were gating on live cells and median fluorescence intensity of the bound Centyrins (PE channel) was calculated using Cytobank software. Results are summarized in Table 4. 
     Off-Rate Analysis of Anti-Human CD8 Alpha Centyrins 
     Purified anti-CD8A FN3 domains were subjected to off-rate analysis using a Proteon surface plasmon resonance instrument in order to pick clones with the slowest off-rates for further characterization. Measured off-rates ranged from 2.64E-5 to 1.07E-2 sec −1  as shown in Table 4. 
     Goat anti-human Fc IgG (Jackson immunoresearch, Cat #109-005-098) was directly immobilized on a GLC sensor chip at 10 μg/ml, pH5.0 via amine coupling (pH 5.0) on all 6 ligand channels in vertical orientation on the chip with a flow rate of 30 μl/min in PBST (PBS, 0.005% Tween). The immobilized GAH-Fc IgG densities averaged about 6000 Response Units (Ru) with less than 1% variation among different channels. In house human CD8A-Fc was captured in vertical orientation at 3 different ligand densities, 10, 5, 2.5 μg/ml for 5 minutes at 30 ul/minute flowrate. All FN3 domains were normalized to a 3 μM concentration, and tested for binding in horizontal orientation. All 6 analyte channels were used for FN3 domains to maximize the screening throughput. The dissociation phase was monitored for 15 minutes at a flow rate of 100 μl/min using PBST as running buffer. Regeneration of the surface was achieved by a short pulse of 0.85% phosphoric acid (18 s contact time at 100 uL/min). Data analyses were performed using Bio-Rad ProteOn Manager software (version 3.1.0.6). Raw data were double referenced by subtraction of the interspot (empty chip surface, no protein immobilized or captured) signals to correct the non-specific binding of the FN3 domain to the pre-coated GAH-Fc IgG surface, followed by a double correction using empty channel L6 where no hCD8A-Fc was captured. The processed binding data were locally fit to a 1:1 simple Langmuir binding model to extract the koff for each FN3 domain binding to captured hCD8A-Fc. 
     Example 4: Engineering of Anti-CD8a FN3 Domains 
     A number of mutations were designed into top anti-CD8A candidates in order to eliminate post translational modification risks of oxidation (methionine, or tryptophan), deamidation (NS), isomerization (DG) and clipping (DP). Proline residues found in beta strands were also mutated as proline has a potential for destabilizing beta strands (Chiba T., et al. J Biol Chem. 2003; 278:47016-24). Only residues derived from FN3 domain library-designed positions were considered for mutation. Variant sequences were chosen to either mimic similar chemical properties of the parent molecule (example tryptophan to tyrosine) or to replace the PTM risk amino acid with an amino acid found in other CD8A FN3 domains at that position. A full list of engineered sequences is found in Table 5. The dissociation rate between each mutant and recombinant CD8 alpha was measured by surface plasmon resonance to estimate relative binding strengths. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Dissociation rates of CD8A Centyrin mutants. Mutants  
               
               
                 are grouped according to the parent molecule. 
               
            
           
           
               
               
               
               
            
               
                 Sample 
                 k d  (1/s) 
                 Mutations 
                 SEQ ID NO: 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 P282DR9P1359_C5 
                 1.47E−04 
                 Parent 
                 68 
               
               
                 CD8S402 
                 4.84E−05 
                 D40P 
                 266 
               
               
                 CD8S396 
                 1.52E−04 
                 W32Y 
                 260 
               
               
                 CD8S398 
                 4.43E−04 
                 W32S 
                 262 
               
               
                 CD8S397 
                 6.60E−04 
                 W32Q 
                 261 
               
               
                 CD8S399 
                 1.34E−03 
                 W38Y 
                 263 
               
               
                 CD8S401 
                 1.27E−02 
                 W38I 
                 265 
               
               
                 CD8S400 
                 2.26E−02 
                 W38L 
                 264 
               
               
                 CD8S404 
                 3.09E−02 
                 P36A 
                 268 
               
               
                 P282DR9P1359_F5 
                 5.78E−05 
                 Parent 
                 79 
               
               
                 CD8S371 
                 1.94E−04 
                 W48Y 
                 235 
               
               
                 CD8S377 
                 4.00E−04 
                 W81E 
                 241 
               
               
                 CD8S374 
                 4.03E−04 
                 W81Y 
                 238 
               
               
                 CD8S372 
                 5.71E−04 
                 W48L 
                 236 
               
               
                 CD8S375 
                 8.30E−04 
                 W81L 
                 239 
               
               
                 CD8S376 
                 8.46E−04 
                 W81S 
                 240 
               
               
                 CD8S373 
                 4.03E−03 
                 W48I 
                 237 
               
               
                 P282DR9P1359_G7 
                 1.06E−05 
                   
                 83 
               
               
                 CD8S379 
                 4.97E−05 
                 D43S 
                 243 
               
               
                 CD8S378 
                 5.80E−05 
                 D43E 
                 242 
               
               
                 CD8S388 
                 7.54E−05 
                 N81Q 
                 252 
               
               
                 CD8S387 
                 1.25E−04 
                 W83E 
                 251 
               
               
                 CD8S381 
                 2.00E−04 
                 W70F 
                 245 
               
               
                 CD8S383 
                 7.47E−04 
                 W74Y 
                 247 
               
               
                 CD8S380 
                 1.21E−03 
                 W70Y 
                 244 
               
               
                 CD8S382 
                 2.47E−01 
                 W70S 
                 246 
               
               
                 P282ER9P1360_C8 
                 1.79E−04 
                 Parent 
                 89 
               
               
                 CD8S390 
                 1.52E−04 
                 W68Y 
                 254 
               
               
                 CD8S389 
                 1.84E−04 
                 W68F 
                 253 
               
               
                 CD8S391 
                 3.20E−04 
                 W68H 
                 255 
               
               
                 CD8S405 
                 1.14E−03 
                 P48T 
                 269 
               
               
                 P282DR9P1359_F7 
                 3.39E−04 
                 Parent 
                 81 
               
               
                 CD8S403 
                 1.33E−04 
                 P36A 
                 267 
               
               
                 CD8S392 
                 1.55E−03 
                 W38Y 
                 256 
               
               
                 CD8S395 
                 1.89E−03 
                 W38H 
                 259 
               
               
                 CD8S393 
                 2.55E−03 
                 W38L 
                 257 
               
               
                 CD8S394 
                 3.55E−03 
                 W38I 
                 258 
               
               
                   
               
            
           
         
       
     
     From the data presented in Table 5, it is apparent that a number of mutations that reduce developability risks maintain dissociation rates similar to that of the parent molecule. Mutants CD8S402 (elimination of DP site), CD8S390 (elimination of Trp residue), and CD8S403 (removal of Pro from beta strand) resulted in slower dissociation rates than the parent appropriate molecule, indicative of tighter binding. A number of other mutations maintain binding similar to the parent molecule and thus might be preferred over the parent as these molecules pose less CMC related risks during development. 
     Example 5: Affinity Measurements of CD8a-Binding FN3 Domains 
     Nineteen anti-CD8A candidates were selected for full kinetic analysis of binding to recombinant human CD8 alpha. These candidates were selected from the above positive hits (Table 4) using the criteria of 1) strong relative binding to human T-cells, 2) strong relative binding to cyno T-cells, 3) minimal reduction in cell binding at 0.2 uM compared to 2 uM, 4) free of aggregates via SEC, 5) off-rates slower than 2.07E-3 sec-1, 6) sequence diversity with respect to sequence families, and 7) relative propensity for sequences with potential developability challenges (oxidation, deamidation, clipping and hydrophobicity). 
     Affinities of the top 19 candidates, later a repeat of the top 6 candidates, binding to hCD8A-Fc were measured on a ProteOn XPR36 instrument (Bio-Rad) using GLC sensor chips under similar conditions to those for koff screening. Goat anti-human Fc antibody was directly immobilized on the chip by standard amine coupling at 10 μg/ml, pH 5.0 on all 6 ligand channels in vertical orientation on the chip with a flow rate of 30 μl/min in PBST (PBS, 0.005% Tween), achieving an average of 6200 Rus on each ligand channel. Human CD8A-Fc was then captured at five surface densities ranging from 200 to 1200 response units, leaving the 6th channel as empty channel control for GAH-Fc IgG surface. Binding was measured by flowing five different concentrations of anti-CD8A FN3 domains (1 μM diluted in a 3-fold dilution series) as analytes simultaneously in the horizontal orientation over the captured hCD8A-Fc surfaces, with a sixth analyte channel containing only running buffer PBST. All interactions were measured at 100 uL/min flow rate with association and dissociation times being 4, 30 minutes respectively. Ligand surface regeneration was achieved by 1 short pulse of 0.85% phosphoric acid (18 s contact time at 100 uL/min). Data analyses were performed using Bio-Rad ProteOn Manager software (version 3.1.0.6). Raw data were double referenced by subtraction of the interspot (empty chip surface, no protein immobilized or captured) signals to correct the non-specific binding of the FN3 domain to the pre-coated GAH-Fc IgG surface, followed by a double referencing using the buffer blank response (to correct for any baseline drift resulting from ligand dissociation over time). It has been consistently observed in multiple analyses that the anti-CD8A FN3 domain binding data do not conform well to the 1:1 simple Langmuir binding model, implying either the reagents issues and/or the intrinsically complicated binding mechanisms that can&#39;t be accounted for using a simple 1:1 binding mode. Given that the GAH-Fc capture of hCD8A-Fc format is the least disruptive relative to other formats in introducing potential experimental artifacts (such as ligand activity loss and/or artificial eptiopes/heterogeneous ligand population due to amine coupling), it is considered that the results from the GAH-Fc capture experiments reported here represent the most reliable ProteOn SPR data, despite the non-conforming 1:1 Langmuir fits observed in many instances. A heterogeneous ligand model was chosen to fit the data assuming two different ligand species, either due to the heterogeneity in the ligand protein population or due to potential different mechanisms for each FN3 domain binding to the 2 hCD8A monomers in the Fc fusion protein. In this case, because each anti-CD8A FN3 domain would have separate affinities, the resultant sensorgram reflects the sum of two independent reactions with two sets of rate constants, which were reported for each FN3 domain binding. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Summary of kinetic affinities for top six anti-CD8A FN3 domain candidates. 
               
            
           
           
               
               
               
            
               
                 Sample 
                 Lower Affinity Population 
                 Higher Affinity Population 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 (SEQ ID NO:) 
                 k a  (1/Ms) 
                 k d  (1/s) 
                 K D (nM) 
                 k a  (1/Ms) 
                 k d  (1/s) 
                 K D  (nM) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 P282DR9P1359_F5 
                 3.48E+04 
                 6.60E−05 
                 6.6 
                 3.80E+05 
                 1.42E−05 
                 0.04 
               
               
                 (79) 
               
               
                 P282DR9P1359_F7 
                 4.03E+04 
                 3.65E−04 
                 12 
                 4.04E+05 
                 7.99E−05 
                 0.5 
               
               
                 (81) 
               
               
                 P282DR9P1359_G7 
                 6.84E+04 
                 5.51E−05 
                 2.1 
                 2.76E+05 
                 1.49E−05 
                 0.05 
               
               
                 (83) 
               
               
                 P282ER9P1360_C8 
                 3.09E+04 
                 9.52E−05 
                 4.1 
                 2.18E+05 
                 4.71E−05 
                 0.2 
               
               
                 (89) 
               
               
                 P283AR9P1362_D6 
                 5.62E+04 
                 3.12E−05 
                 0.98 
                 1.55E+05 
                 1.00E−06 
                 0.03 
               
               
                 (122) 
               
               
                 P282DR9P1359_C5 
                 1.92E+04 
                 1.27E−04 
                 6.5 
                 3.00E+05 
                 5.79E−06 
                 0.02 
               
               
                 (68) 
               
               
                   
               
               
                 Note: 
               
               
                 Affinity, K D  = kd/ka. 
               
            
           
         
       
     
     Example 6: Labeling of Anti-CD8a FN3 Domains with DFO and 89Zr 
     Anti-CD8A FN3 domains were modified to include a single cysteine residue for conjugation of maleimide containing chelators or PET labels. Synthetic plasmid DNA encoding clones P282DR9P1359_F5, P282DR9P1359_F7, P282DR9P1359_G7, P282ER9P1360_C8, P283AR9P1362_D6, and P282DR9P1359_C5 with a mutation of residue E54 to cysteine were synthesized at DNA2.0 (Table 7). E54 was chosen as the position for mutation based on earlier studies that demonstrated maintenance of binding affinity, stability, and expression levels for other FN3 domains mutated at this residue (Goldberg S. et al. Protein Engineering Design and Selection 2016 Epub ahead of print). 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Modified anti-CD8A FN3 domain molecules 
               
            
           
           
               
               
               
               
            
               
                 Original Clone 
                 SEQID NO 
                 Clone with E54C 
                 SEQID No 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 P282DR9P1359_F5 
                 79 
                 CD8S368 
                 229 
               
               
                 P282DR9P1359_F7 
                 81 
                 CD8S367 
                 230 
               
               
                 P282DR9P1359_G7 
                 83 
                 CD8S370 
                 231 
               
               
                 P282ER9P1360_C8 
                 89 
                 CD8S365 
                 232 
               
               
                 P283AR9P1362_D6 
                 122 
                 CD8S369 
                 233 
               
               
                 P282DR9P1359_C5 
                 68 
                 CD8S366 
                 234 
               
               
                   
               
            
           
         
       
     
     Anti-CD8A FN3 domins modified with a free cysteine were conjugated to Deferoxamine (DFO) in order to chelate radiometals. 0.5 mL of a 100-500 μM anti-CD8A FN3 domain solution was combined with 10 μL of 500 mM TCEP (Sigma, cat. #646547), gently flushed with nitrogen, and incubated for 1 hour at room temperature. 1.0 mL of saturated ammonium sulfate (4.02 M) was added to each tube to reach a final concentration of 3.2M before incubation on ice for 10 minutes and centrifugation at 16,000×g or higher to pellet the protein. The resulting pellet was resuspended and washed in 1.0 mL of 3.2 M ammonium sulfate supplemented with 100 mM sodium phosphate pH 7.2 and 1 mM EDTA before centrifuging again. After the second centrifugation step, the resulting pellet was dissolved in 100 mM sodium phosphate 7. 0, 1 mM EDTA and combined with 10 uL of 50 mM DFO solution to make a final molar ratio of 5:1 DFO to anti-CD8A. This reaction was allowed to proceed at room temperature for 30 minutes before quenching with 5.0 microliters of beta-mercaptoethanol. Excess DFO was finally removed by a variety of methods including a second round of ammonium sulfate precipitation as described above, passing through a desalting column such as Zeba 7k column (Pierce Cat #89889), or by purification with nickle-NTA resin (Qiagen #30450). Anti-CD8A FN3 domain-DFO conjugates were formulated in 1×PBS for further analysis. 
     Following conjugation to DFO, the binding of each anti-CD8A FN3 domain to recombinant human CD8 alpha was assessed by surface plasmon resonance as previously described. All samples retained tight binding to human CD8A following mutation of E54 to Cys and conjugation to DFO (Table 8). 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Binding affinity following DFO conjugation 
               
            
           
           
               
               
               
            
               
                   
                 Lower Affinity Population 
                 Higher Affinity Population 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Sample 
                 k a  (1/Ms) 
                 k d  (1/s) 
                 K D  (nM) 
                 k a  (1/Ms) 
                 k d  (1/s) 
                 K D  (nM) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 CD8S365-DFO 
                 4.41E+03 
                 4.29E−05 
                 9.73 
                 6.80E+04 
                 4.18E−05 
                 0.6 
               
               
                 CD8S366-DFO 
                 5.85E+03 
                 1.06E−04 
                 18.2 
                 7.95E+04 
                 7.01E−05 
                 0.9 
               
               
                 CD8S367-DFO 
                 1.09E+04 
                 9.75E−04 
                 89.1 
                 8.45E+04 
                 1.31E−04 
                 1.55 
               
               
                 CD8S368-DFO 
                 7.32E+03 
                 9.98E−05 
                 13.6 
                 1.08E+05 
                 2.53E−05 
                 0.23 
               
               
                 CD8S369-DFO 
                 2.87E+03 
                  ≤1E−05 
                 ≤3.4 
                 3.73E+04 
                  ≤1E−05 
                 ≤0.3 
               
               
                 CD8S370-DFO 
                 5.91E+03 
                 7.65E−05 
                 13 
                 4.64E+04 
                  ≤2E−05 
                 ≤0.3 
               
               
                   
               
            
           
         
       
     
     Example 7: Binding of Anti-CD8a FN3 Domains to Human and Cyno T-Cells 
     A full dose response binding curve was generated for the nineteen selected anti-CD8A FN3 domains. Each candidate was diluted to 20 μM in PBS followed by a 1:3 dilution series to generate either an 11-point or an 18-point dose response curve. Human or cyno CD8+ T cells were incubated with the diluted FN3 domain for 1 hour at 4° C. Cells were washed once with PBS and incubated with an anti-centyrin antibody (PAB25) for 1 hour at 4° C. The cells were washed twice with PBS, followed by incubation with a PE-secondary, anti-CD3-PacB, anti-CD4-APC, and a viability dye. Finally, cells were washed and resuspended in PBS for flow cytometric analysis using a BD Canto Instrument. CD8 T cells were defined as live CD3+CD4− cells. Median fluorescence intensity of the bound Centyrins (PE channel) and % of cells showing positive staining calculated using Cytobank software. Results were graphed using Prism and EC 50  values were calculated using the 4 parameter dose response variable slope equation. 
     A MesoScale Discovery-Cell Affinity Technology (MSD-CAT) based equilibrium cell-binding assay was performed to determine the affinity of the top six anti-CD8A candidates binding to primary human cytotoxic T cell surface CD8A receptors. Each anti-CD8A FN3 domain at a constant concentration of 50 pM was pre-incubated with 10 different concentrations of primary cytotoxic CD8 T cells (columns 2-11 in a row). Cell viability was checked prior to the binding measurements and a &gt;85% viability was desired for valid analysis. Since these cells were from different donors, in case of donor-to-donor variations, only cells of the same donors were combined together. Each individual anti-CD8A FN3 domain binding was measured in replicates using cells from the same donors. Cells and FN3 domains were incubated overnight at 4° C. on a rotator to reach equilibrium. Following the incubation the cells were spun down along with cell bound anti-CD8A FN3 domains and the unbound (free) anti-CD8A FN3 domains in the supernatants is quantified using MSD assays where biotinylated recombinant hCD8A-Fc protein was captured at 0.6 ug/mL in assay buffer to streptavidin MSD plates overnight ˜16 hours at 4° C. After blocking the plate, supernatant with free anti-CD8A FN3 domains was added to the plate and incubated for 1 hr, then followed by SulfoTag pAb139 (In-house) detection at 1.6 ug/ml. A buffer control without any FN3 domain and hCD8A (plate background binding control) in column 1 and FN3 domain alone control without hCD8A (100% free/unbound) in column 12 were inclubed. Mouse Anti-hCD8A mAb (mIgGlk, BD Biosciences, cat #555364, clone RPA-T8) was included as a postive control. Tencon27 was included in the initial assay validation as a negative control and no significant binding was observed, and therefore, was not included in the later cell binding due to the cell availability. Plates were read immediately on the MSD Sector Imager 6000™ Reader for luminescence levels after adding MSD Read Buffer by diluting 1:4 of stock into H2O. 
     Raw MSD data were exported and analysed in Prism using a non-linear fit with variable slope function to derive the Bmax and Hillslope values. Only those with converged Bmax values and hillslope within the range of −1.5˜−0.5 (ideal −1.0) will be considered for further analysis. Binding data were then normalized using the Bmax values to calculate the normalized % free FN3 domains. A surface CD8 density of 50,000 receptors per cell was used for the receptor concentration calculation. A saturation criterion of &lt;20% free Centyrin at highest CD8 cell concentrations was required to determine the affinity using a “Solution Affinity Equation for normalized data” for a 1:1 binding model. 
     Anti-CD8A FN3 domains bound to primary cells with affinities ranging from 0.167 to 2.81 nM (Table 9). 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Summary of EC 50  values for top six  
               
               
                 anti-CD8A FN3 domain candidates. 
               
            
           
           
               
               
               
               
            
               
                   
                 EC50 Binding to  
                 EC50 Binding to 
                 Affinity for 
               
               
                 Clone ID 
                 Human T-cells by 
                 cyno T-cells by  
                 Human T-cells by 
               
               
                 (SEQ ID  
                 Flow Cytometry 
                 Flow Cytometry  
                 MSD-CAT 
               
               
                 NO:) 
                 (nM) 
                 (nM) 
                 (nM) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 CD8S365 
                 556.0 
                 123.6 
                 0.167 
               
               
                 (232) 
                   
                   
                   
               
               
                 CD8S366 
                 162.7 
                 69.5 
                 0.123 
               
               
                 (234) 
                   
                   
                   
               
               
                 CD8S367 
                 194.5 
                 50.8 
                 0.225 
               
               
                 (230) 
                   
                   
                   
               
               
                 CD8S368 
                 154.7 
                 70.0 
                 0.459 
               
               
                 (229) 
                   
                   
                   
               
               
                 CD8S369 
                 124.2 
                 72.3 
                 2.81 
               
               
                 (233) 
                   
                   
                   
               
               
                 CD8S370 
                 208.7 
                 67.6 
                 0.869 
               
               
                 (231) 
                   
                   
                   
               
               
                   
               
            
           
         
       
     
     Example 8: Activation of Human T-Cells 
     De Novo Activation 
     In order to determine if the anti-CD8A FN3 domains activate T cells, a flow cytometry assay was performed to monitor changes in T cell activation markers. Six anti-CD8A FN3 domains were evaluated for T-cell activation. De novo activation was assessed by incubating the FN3 domains at either 1 μM or 10 nM in duplicate with human pan-T cells in media for 4 days. Two independent donors were tested. Plate bound anti-CD3 was used a positive control at 2 doses, 0.1 ug/mL and 0.01 ug/mL. PBS was used as a negative control. Cells were then stained with a viability dye and the following panel of antibodies: CD4-FITC, CD3-PerCP-Cy5.5, CD69-PacB, CD45RA-BV605, CD25-BV650, CD127-PE, and CD137-PE-Cy7. CD8+ cells were defined as live CD3+CD4-cells and were profiled for each T-cell activation marker. Median fluorescence intensity values were calculated using FlowJo software and replicate values were averaged. Results are summarized in Table 10A (donor 022) and 10B (donor 146). For 365, 366, 367, 368, and 370, small changes in the T cell activation markers were observed in only 1 out of the 2 donors tested at the highest dose level of 1 μM. These changes were absent in both donors at the 10 nM dose, suggesting the molecules do not activate T cells de novo at relevant concentrations. The 369 molecule does appear to significantly activate CD137 expression in both donors at the highest dose level. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Median Fluorescence Intensity (MFI) values for various T cells activation 
               
               
                 markers on CD8+ T cells for Donor 022 (A) and Donor 146 (B) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Anti- 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Sample 
                 CD8A 
               
               
                   
                 (SEQ ID 
                 FN3 conc 
                 Anti-CD3 
                 CD45RA 
                 CD25 
                 CD69 
                 CD127 
                 CD137 
               
               
                 Donor 
                 NO:) 
                 μM 
                 ug/mL 
                 MFI 
                 MFI 
                 MFI 
                 MFI 
                 MFI 
               
               
                   
               
            
           
           
               
            
               
                 A 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 022 
                 PBS 
                 0 
                 0 
                 12856 
                 571 
                 223 
                 651 
                 296 
               
               
                   
                 control 
               
               
                 022 
                 PBS 
                 0 
                 0.01 
                 13133 
                 707 
                 403 
                 517 
                 343 
               
               
                   
                 control 
               
               
                 022 
                 PBS 
                 0 
                 0.1 
                 11394 
                 1333 
                 1694 
                 158 
                 529 
               
               
                   
                 control 
               
               
                 022 
                 CD8S366 
                 1 
                 0 
                 15054 
                 949 
                 477 
                 425 
                 310 
               
               
                   
                 (234) 
               
               
                 022 
                 CD8S366 
                 0.01 
                 0 
                 13336 
                 814 
                 230 
                 586 
                 301 
               
               
                   
                 (234) 
               
               
                 022 
                 CD8S368 
                 1 
                 0 
                 12992 
                 858 
                 698 
                 367 
                 329 
               
               
                   
                 (229) 
               
               
                 022 
                 CD8S368 
                 0.01 
                 0 
                 15262 
                 677 
                 276 
                 489 
                 306 
               
               
                   
                 (229) 
               
               
                 022 
                 CD8S367 
                 1 
                 0 
                 15409 
                 796 
                 401 
                 511 
                 297 
               
               
                   
                 (230) 
               
               
                 022 
                 CD8S367 
                 0.01 
                 0 
                 13666 
                 723 
                 261 
                 502 
                 312 
               
               
                   
                 (230) 
               
               
                 022 
                 CD8S370 
                 1 
                 0 
                 12946 
                 916 
                 572 
                 376 
                 353 
               
               
                   
                 (231) 
               
               
                 022 
                 CD8S370 
                 0.01 
                 0 
                 14973 
                 776 
                 353 
                 435 
                 331 
               
               
                   
                 (231) 
               
               
                 022 
                 CD8S365 
                 1 
                 0 
                 13935 
                 904 
                 562 
                 367 
                 328 
               
               
                   
                 (232) 
               
               
                 022 
                 CD8S365 
                 0.01 
                 0 
                 15156 
                 697 
                 243 
                 504 
                 323 
               
               
                   
                 (232) 
               
               
                 022 
                 CD8S369 
                 1 
                 0 
                 13661 
                 783 
                 440 
                 441 
                 5122 
               
               
                   
                 (233) 
               
               
                 022 
                 CD8S369 
                 0.01 
                 0 
                 16513 
                 717 
                 251 
                 596 
                 416 
               
               
                   
                 (233) 
               
               
                 022 
                 TenCon 
                 1 
                 0 
                 14920 
                 702 
                 284 
                 447 
                 334 
               
            
           
           
               
            
               
                 B 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 146 
                 PBS 
                 0 
                 0 
                 7172 
                 627 
                 61 
                 1313 
                 500 
               
               
                 146 
                 PBS 
                 0 
                 0.01 
                 8076 
                 681 
                 153 
                 1296 
                 617 
               
               
                 146 
                 PBS 
                 0 
                 0.1 
                 5171 
                 1462 
                 1100 
                 139 
                 798 
               
               
                 146 
                 CD8S366 
                 1 
                 0 
                 8531 
                 673 
                 95 
                 1368 
                 589 
               
               
                   
                 (234) 
               
               
                 146 
                 CD8S366 
                 0.01 
                 0 
                 9414 
                 623 
                 74 
                 1615 
                 559 
               
               
                   
                 (234) 
               
               
                 146 
                 CD8S368 
                 1 
                 0 
                 8386 
                 691 
                 96 
                 1301 
                 561 
               
               
                   
                 (229) 
               
               
                 146 
                 CD8S368 
                 0.01 
                 0 
                 9147 
                 628 
                 82 
                 1424 
                 586 
               
               
                   
                 (229) 
               
               
                 146 
                 CD8S367 
                 1 
                 0 
                 8167 
                 660 
                 95 
                 1322 
                 581 
               
               
                   
                 (230) 
               
               
                 146 
                 CD8S367 
                 0.01 
                 0 
                 8734 
                 586 
                 77 
                 1479 
                 571 
               
               
                   
                 (230) 
               
               
                 146 
                 CD8S370 
                 1 
                 0 
                 8590 
                 737 
                 86 
                 1362 
                 583 
               
               
                   
                 (231) 
               
               
                 146 
                 CD8S370 
                 0.01 
                 0 
                 7934 
                 635 
                 71 
                 1526 
                 559 
               
               
                   
                 (231) 
               
               
                 146 
                 CD8S365 
                 1 
                 0 
                 8344 
                 813 
                 85 
                 1238 
                 586 
               
               
                   
                 (232) 
               
               
                 146 
                 CD8S365 
                 0.01 
                 0 
                 8460 
                 628 
                 80 
                 1355 
                 605 
               
               
                   
                 (232) 
               
               
                 146 
                 CD8S369 
                 1 
                 0 
                 8778 
                 681 
                 92 
                 1369 
                 5690 
               
               
                   
                 (233) 
               
               
                 146 
                 CD8S369 
                 0.01 
                 0 
                 7862 
                 591 
                 74 
                 1498 
                 784 
               
               
                   
                 (233) 
               
               
                 146 
                 TenCon 
                 1 
                 0 
                 7325 
                 609 
                 78 
                 1198 
                 574 
               
               
                 146 
                 TenCon 
                 0.01 
                 0 
                 7764 
                 596 
                 66 
                 1281 
                 530 
               
               
                   
               
            
           
         
       
     
     Pan T-Cell Activation 
     In order to determine if the anti-CD8A FN3 domains can affect markers of T cell activation in pan-actived T cells, the anti-CD8A FN3 domains were also evaluated in combination with a low dose of plate bound CD3. In this assay, a sub-optimal concentration (0.01 μg/mL) of plate bound anti-CD3 was used to activate the T cells in the presence of either 1 μM or 10 nM anti-CD8A. After 4 days, the cells were assessed using the same panel and gating strategy as described above. Two independent donors were tested. Median fluorescence intensity values were calculated using FlowJo software and replicate values were averaged. Results are summarized in Tables 11A (donor 022) and 11B (donor 146). 
     
       
         
           
               
             
               
                 TABLES 11A and B 
               
               
                   
               
               
                 Median Fluorescence Intensity (MFI) values for various T 
               
               
                 cells activation markers on CD8+ T cells for Donor 
               
               
                 022 (A) and Donor 146 (B) in the presence of plate bound CD3. 
               
               
                   
               
             
            
               
                 A 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Anti- 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Sample 
                 CD8A 
               
               
                   
                 (SEQ ID 
                 FN3 
                 Anti-CD3 
                 CD45RA 
                 CD25 
                 CD69 
                 CD127 
                 CD137 
               
               
                 Donor 
                 NO:) 
                 conc μM 
                 μg/mL 
                 MFI 
                 MFI 
                 MFI 
                 MFI 
                 MFI 
               
               
                   
               
               
                 022 
                 PBS 
                 0 
                 0 
                 12856 
                 571 
                 223 
                 651 
                 296 
               
               
                   
                 control 
               
               
                 022 
                 PBS 
                 0 
                 0.01 
                 13133 
                 707 
                 403 
                 517 
                 343 
               
               
                   
                 control 
               
               
                 022 
                 PBS 
                 0 
                 0.1 
                 11394 
                 1333 
                 1694 
                 158 
                 529 
               
               
                   
                 control 
               
               
                 022 
                 CD8S366 
                 1 
                 0.01 
                 11918 
                 892 
                 1005 
                 369 
                 376 
               
               
                   
                 (234) 
               
               
                 022 
                 CD8S366 
                 0.01 
                 0.01 
                 13417 
                 1068 
                 848 
                 384 
                 399 
               
               
                   
                 (234) 
               
               
                 022 
                 CD8S368 
                 1 
                 0.01 
                 11311 
                 1147 
                 1279 
                 260 
                 428 
               
               
                   
                 (229) 
               
               
                 022 
                 CD8S368 
                 0.01 
                 0.01 
                 13441 
                 760 
                 599 
                 499 
                 348 
               
               
                   
                 (229) 
               
               
                 022 
                 CD8S367 
                 1 
                 0.01 
                 13271 
                 1135 
                 1127 
                 367 
                 385 
               
               
                   
                 (230) 
               
               
                 022 
                 CD8S367 
                 0.01 
                 0.01 
                 14521 
                 960 
                 636 
                 483 
                 362 
               
               
                   
                 (230) 
               
               
                 022 
                 CD8S370 
                 1 
                 0.01 
                 15138 
                 1103 
                 890 
                 407 
                 378 
               
               
                   
                 (231) 
               
               
                 022 
                 CD8S370 
                 0.01 
                 0.01 
                 14230 
                 875 
                 612 
                 431 
                 355 
               
               
                   
                 (231) 
               
               
                 022 
                 CD8S365 
                 1 
                 0.01 
                 14395 
                 1112 
                 907 
                 380 
                 407 
               
               
                   
                 (232) 
               
               
                 022 
                 CD8S365 
                 0.01 
                 0.01 
                 14006 
                 1175 
                 1063 
                 297 
                 430 
               
               
                   
                 (232) 
               
               
                 022 
                 CD8S369 
                 1 
                 0.01 
                 13735 
                 877 
                 759 
                 464 
                 5457 
               
               
                   
                 (233) 
               
               
                 022 
                 CD8S369 
                 0.01 
                 0.01 
                 13864 
                 842 
                 617 
                 450 
                 498 
               
               
                   
                 (233) 
               
               
                 022 
                 TenCon 
                 1 
                 0.01 
                 14687 
                 791 
                 553 
                 408 
                 358 
               
               
                 022 
                 TenCon 
                 0.01 
                 0.01 
                 13090 
                 759 
                 630 
                 464 
                 368 
               
               
                   
               
            
           
           
               
               
            
               
                 B 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Anti- 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Sample 
                 CD8A 
                 Anti- 
               
               
                   
                 (SEQ ID 
                 FN3 
                 CD3, 
                 CD45RA 
                 CD25 
                 CD69 
                 CD127 
                 CD137 
               
               
                 Donor 
                 NO:) 
                 conc μM 
                 μg/mL 
                 MFI 
                 MFI 
                 MFI 
                 MFI 
                 MFI 
               
               
                   
               
               
                 146 
                 PBS 
                 0 
                 0 
                 12856 
                 571 
                 223 
                 651 
                 296 
               
               
                   
                 control 
               
               
                 146 
                 PBS 
                 0 
                 0.01 
                 13133 
                 707 
                 403 
                 517 
                 343 
               
               
                   
                 control 
               
               
                 146 
                 PBS 
                 0 
                 0.1 
                 11394 
                 1333 
                 1694 
                 158 
                 529 
               
               
                   
                 control 
               
               
                 146 
                 CD8S366 
                 1 
                 0.01 
                 6798 
                 876 
                 163 
                 1095 
                 632 
               
               
                   
                 (234) 
               
               
                 146 
                 CD8S366 
                 0.01 
                 0.01 
                 8589 
                 775 
                 158 
                 1077 
                 637 
               
               
                   
                 (234) 
               
               
                 146 
                 CD8S368 
                 1 
                 0.01 
                 6576 
                 945 
                 175 
                 1105 
                 662 
               
               
                   
                 (229) 
               
               
                 146 
                 CD8S368 
                 0.01 
                 0.01 
                 7608 
                 843 
                 200 
                 950 
                 678 
               
               
                   
                 (229) 
               
               
                 146 
                 CD8S367 
                 1 
                 0.01 
                 6447 
                 897 
                 173 
                 1088 
                 672 
               
               
                   
                 (230) 
               
               
                 146 
                 CD8S367 
                 0.01 
                 0.01 
                 7899 
                 801 
                 175 
                 1031 
                 655 
               
               
                   
                 (230) 
               
               
                 146 
                 CD8S370 
                 1 
                 0.01 
                 7327 
                 992 
                 169 
                 1055 
                 687 
               
               
                   
                 (231) 
               
               
                 146 
                 CD8S370 
                 0.01 
                 0.01 
                 8676 
                 790 
                 183 
                 946 
                 675 
               
               
                   
                 (231) 
               
               
                 146 
                 CD8S365 
                 1 
                 0.01 
                 6624 
                 977 
                 172 
                 1059 
                 670 
               
               
                   
                 (232) 
               
               
                 146 
                 CD8S365 
                 0.01 
                 0.01 
                 7902 
                 843 
                 193 
                 985 
                 659 
               
               
                   
                 (232) 
               
               
                 146 
                 CD8S369 
                 1 
                 0.01 
                 7660 
                 933 
                 165 
                 1149 
                 7114 
               
               
                   
                 (233) 
               
               
                 146 
                 CD8S369 
                 0.01 
                 0.01 
                 7892 
                 854 
                 187 
                 989 
                 842 
               
               
                   
                 (233) 
               
               
                 146 
                 TenCon 
                 1 
                 0.01 
                 8352 
                 829 
                 170 
                 1026 
                 658 
               
               
                 146 
                 TenCon 
                 0.01 
                 0.01 
                 7627 
                 761 
                 185 
                 1043 
                 673 
               
               
                   
               
            
           
         
       
     
     Cytokine Response 
     In order to determine if any of the changes observed in the activation markers resulting in changes in cytokine production, antigen-dependent T cell activation assays were also performed using two anti-CD8A FN3 domains. For one set of assays, either CMV reactive or M1 reactive human PBMCs were thawed and rested overnight at 37° C. in 6 well plates. The following day, the PBMCs were harvested by pipetting, counted, and plated onto IFNg Mabtech ELISpot plates in the presence or absence of 10 μg/mL peptide. 1 μM anti-CD8A FN3-DFO conjugate was added to the wells and plates were allowed to incubate at 37° C. for approximately 24 hours undisturbed. The cells were removed and the plates were washed 5 times with PBS. The supplied detection antibody was added and plates were incubated for 2 hours. The plates were again washed and the kit substrate was added to each well. Plates were developed for approximately 5 minutes before the reaction was stopped by running the plate under water. Plates were dried upside down overnight in the dark. Plates were read on the AID EliSpot Reader and spot counts were generated using the AID EliSpot Software. Results were graphed in Prism. Results are summarized in  FIG.  1   . In this assay, 365-DFO does not increase the number of IFNg spots compared to media alone or non-CD8A binding TenCon control in the absence of peptide ( FIG.  1 A,  1 C ). Peptide and CD3 are included as positive controls. In the presence of peptide, the 365-DFO does not change the number of IFNg spots compared to peptide alone or peptide with non-CD8A binding tencon ( FIG.  1 B,  1 D ). Media is included as a negative control and CD3 is included as a positive control. These results suggest that the centyrin does not affect T cell activation. 
     To confirm these results in a longer-term assay, IFN-gamma levels were also measured in a 6-Day activation assay. For this study, CMV reactive PBMCs were incubated in triplicate with anti-CD8A FN3 domains at 1 uM in the presence or absence of 0.25 μg/mL pp65 peptide. Cells were incubated for 6 days at 37° C. At each timepoint the cells were centrifuged and supernatant was harvested. Samples were stored at −80° C. until analyzed. Thawed samples were analyzed for IFN-gamma using a single-plex Meso Scale Discovery (MSD) based ELISA. For this assay, a standard curve was prepared as per manufacturer&#39;s instructions. Samples and standards were added to pre-coated 96 well MSD plates. After a 2 hour incubation, the kit detection antibody was added. After another 2 hour incubation, plates were washed three times followed by the addition of the supplied read buffer. Plates were read on MSD Sector Imager 6000 plate reader. Raw MSD data files were analyzed against the standard curves generated using the MSD Discovery Workbench software. The analyzed data graphed using the Tibco Spotfire program. Results are summarized in  FIG.  2   . In this assay, 365-DFO does not increase the secretion of IFNg into the media compared to media alone in the absence of peptide ( FIG.  2 A ). CMV peptide is included as a positive control. In the presence of peptide, the 365-DFO also does not change the amount of IFNg secretion compared to peptide alone ( FIG.  2 B ). Media is included as a negative control. These results suggest that the centyrin does not affect T cell activation. 
     Example 9: Labeling of Anti-CD8a FN3 Domains with 1124/1125 
     The current method to radiolabel CD8S365 with iodine-124 to produce [ 124 I]-IPEM CD8S 365 (Scheme 1) was adapted from literature procedures ( Bioconjugate Chem.  1991, 2, 435-440 ; Chemistry Open  2015, 4, 174-182). 
     
       
         
         
             
             
         
       
     
     To a 1.5 mL Eppendorf vial was added, in order, Na 124 I solution (≤13 μL, ≤2.5 mCi), AcOH (5 μL to acidify the solution), 1-(4-(tributylstannyl)phenethyl)-1H-pyrrole-2,5-dione (75 μL, 1.00 mg/mL in MeCN) and iodogen (5 μL, 1.00 mg/mL in MeCN) solution. The reaction was left for 5 min at room temperature. 
     The crude reaction mixture was diluted with 0.5 mL of 20% EtOH/H 2 O and was purified directly on preparatory HPLC, the retention time=14.4 min ( FIG.  3   ). The [ 124 I]-IPEM was collected in a 1 dram vial that had been pre-treated with Sigma-Cote′ (then rinsed with 3 mL of 70% EtOH, followed by 3 mL of H 2 O); total volume collected off preparatory HPLC &lt;750 μL. 
     An aliquot (˜5-25 μCi) of the purified fraction was then injected on analytical HPLC ( FIG.  4   , retention time=11.7 min). 
     The purified [ 124 ]-IPEM was then concentrated under vacuum at ambient temperature to a volume of &lt;100 μL. 
     Sodium phosphate buffer (1.0 M sodium phosphate, 1 mM EDTA, pH=6.86) was added (≥25 μL) to bring the pH to ˜6.5-7 (checked by strip). Lastly freshly reduced CD8S 365 (c ˜4.57 mg/mL in 100 mM sodium phosphate buffer, 1 mM EDTA, pH=6.86), was added in appropriate amount to achieve targeted specific activity (ie. if targeting specific activity of 25 mCi/mg and 2.0 mCi of [ 124 I]-IPEM was collected add 17.5 μL of centyrin at c ˜4.57 mg/mL). The conjugation reaction was left for 60 min at ambient temperature and the reaction progress was checked to verify that the conversion exceeded 90% by iTLC. 
     Purification consisted of diluting the reaction solution with PBS/10% EtOH (1 mL, pH=7) transferring the reaction solution from the 1 dram vial into a Vivaspin 6 5 kDa MWCO centrifugal filter (see appendix for the pre-conditioning). After the transfer, the reaction Eppendorf was rinsed with PBS/10% EtOH (2×1 mL, pH=7) and the washings were added to the filter. The crude reaction mixture was centrifuged at 4000 rpm, at 20° C. for 30 min. Following centrifugation &lt;500 μL of solution remained and was found to have a radiochemical purity (RCP)&gt;95% by radio TLC ( FIG.  5   ). The purified [ 124 I]-IPEM CD8S 365 was diluted to a volume of 500 μL with PBS/10% EtOH if the volume was &lt;500 μL and then filtered through a Millex-GV 0.22 μm hydrophilic Durapore (PVDF) membrane. 
     The radiochemical yield from the protocol is ˜50% with a radiochemical purity ≥95% RCP by radio TLC). Analytical reverse phase HPLC was used to determine the protein concentration and specific activity of the final product. The average integration of the peak at retention time=7.3 min in the UV at λ=280 nm was used to extrapolate the protein concentration from a calibration curve ( FIG.  6    for a representative example). A co-injection with the non-radioactive cold standard IPEM CD8S 365 (MALDI analysis shown in  FIG.  7   ) was also conducted (see  FIG.  8   ). The bacterial endotoxin concentration was measured using the Endosafe® portable test system using a 10× dilution with LAL reagent water. 
     Example 10: Detection of CD8 Expression in Cynomolgus Monkeys 
     Two anti-CD8A FN3 molecules (CD8S365 and CD8S368) were selected for PET imaging in non-human primates (NHP). The anti-CD8A molecules were radiolabeled with either Zr-89 (Zevacor, Somerset, N.J.) or I-124 (CPDC, Hamilton, Canada, and Zevacor, Somerset, N.J.). Approximately 1-2 mCi of radiolabeled anti-CD8A molecules was(were) injected into the saphenous vein of a female NHP (cynomolgus macaque), while anesthetized with isoflurane in oxygen. Each animal was scanned in a large-bore microPET Focus 220 PET scanner (Siemens, Knoxville, Tenn.), with the bed moved to accommodate the entire body of the animal (head to lower abdomen). Each scan lasted approximately 1 h, and scans were acquired at 15 min, 2 h, and 24 h after injection. PET images were reconstructed using a 2D maximum likelihood expectation maximization (ML-EM) algorithm, into 3D images of voxel size 1.898×1.898×0.796 mm, dimensions 128×128×475. Blood samples were obtained at multiple time points from the saphenous vein in the opposite leg to the injection, and the blood radioactivity counted in a well counter. 
     PET images were analyzed using PMOD v3.7 software (PMOD, Zurich, Switzerland). Regions-of-interest were drawn manually around organs such as spleen, kidneys and liver. Counts were converted to units of percent injected dose per gram of tissue (% ID/g), while blood radioactivity was presented as % ID. A representative PET image is shown in  FIG.  9   . 
     Blood kinetics for each NHP and each anti-CD8A FN3 domain molecule (labeled with either Zr-89 or I-124) are shown in Table 11, and summarized in  FIG.  10   . For the same animals and anti-CD8A molecules, the organ biodistributions are shown in Table 12 (units are % ID/g), and summarized in  FIG.  11   . The Zr-89 labeled molecules exhibited residualization of the radioisotope in the excretory organs, which caused a large background signal in the kidneys, potentially obscuring other nearby tissues. This was largely absent from the I-124 labeled molecules. The spleen uptake was very similar between the two different molecules and two different radioisotopes for all time points. 
                     TABLE 11                  Blood kinetics for each centyrin, radiolabeled       with either Zr-89 or I-124 (entries are % ID).                                             Time (h)   365 Zr-89   Time (h)   368 Zr-89   Time (h)   365 I-124   Time (h)   368 I-124                                                     0.38   32.49   0.25   75.64   0.40   73.53   0.33   50.56       0.62   17.62   0.50   44.01   0.65   44.59   0.57   37.07       1.18   9.03   0.75   32.06   0.92   29.79   0.87   19.33       2.00   6.12   1.00   24.20   1.13   24.51   1.17   16.01       3.70   3.77   1.50   18.82   2.00   17.14   1.37   12.84       24.00   1.36   2.00   14.22   3.33   10.92   2.07   12.18               3.33   8.40           3.88   8.20               24.00   1.94           23.03   1.24                    
For the same animals and anti-CD8A molecules, the organ biodistributions are shown in Table 12 (units are % ID/g), and summarized in  FIG.  11   .
 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Organ uptake for the different centyrins, labeled  
               
               
                 with either Zr-89 or I-124 (entries are % ID/g). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Time (h) 
                 Kidney 
                 Spleen 
                 Liver 
               
               
                   
               
            
           
           
               
            
               
                 365 Zr-89 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 h 
                 0.641 
                 0.0386 
                 0.0620 
               
               
                   
                   2 h 
                 0.624 
                 0.0218 
                 0.0513 
               
               
                   
                   24 h 
                 0.633 
                 0.0136 
                 0.0354 
               
            
           
           
               
            
               
                 368 Zr-89 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 h 
                 0.575 
                 0.0345 
                 0.0740 
               
               
                   
                   2 h 
                 0.664 
                 0.0307 
                 0.0688 
               
               
                   
                   24 h 
                 0.931 
                 0.0294 
                 0.0508 
               
            
           
           
               
            
               
                 365 I-124 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25h 
                 0.104 
                 0.0324 
                 0.0291 
               
               
                   
                   2 h 
                 0.065 
                 0.0222 
                 0.0142 
               
            
           
           
               
               
               
            
               
                   
                   24 h 
                 Not collected due to technical issue 
               
            
           
           
               
            
               
                 368 I-124 
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 h 
                 0.292 
                 0.0357 
                 0.0439 
               
               
                   
                   2 h 
                 0.140 
                 0.0271 
                 0.0241 
               
               
                   
                   24 h 
                 0.0089 
                 0.0022 
                 0.0029 
               
               
                   
               
            
           
         
       
     
     Example 11: Specificity of Anti-CD8a FN3 Domains in Cynomolgus Monkeys 
     In order to test specificity of the anti-CD8A molecules, the same monkeys were treated with a chimeric CD8-depleting antibody (CM-T807 mouse V/human Fc anti-CD8 antibody) to reduce CD8+ T cells prior to imaging. Animals were administered s.c. with 10 mg/kg CD8 depleting antibody 3 days prior to imaging. CD8 depletion was confirmed by measuring the percentage of CD8 T cells in blood samples taken from each animal before and after depletion ( FIG.  12   ). 
     Approximately 1-2 mCi of radiolabeled [I-124]CD8S365 anti-CD8 FN3 domain molecule was injected into the saphenous vein of a female NHP (cynomolgus macaque), while anesthetized with isoflurane in oxygen. Each animal was scanned in a large-bore microPET Focus 220 PET scanner (Siemens, Knoxville, Tenn.), with the bed moved to accommodate the entire body of the animal (head to lower abdomen). Each scan lasted approximately 1 h, and scans were acquired at 15 min, 2 h, and 24 h after injection. PET images were reconstructed using a 2D maximum likelihood expectation maximization (ML-EM) algorithm, into 3D images of voxel size 1.898×1.898×0.796 mm, dimensions 128×128×475. Blood samples were obtained at multiple time points from the saphenous vein in the opposite leg to the injection, and the blood radioactivity counted in a well counter. 
     PET images were analyzed using PMOD v3.7 software (PMOD, Zurich, Switzerland). Regions-of-interest were drawn manually around organs such as spleen, kidneys and liver. Counts were converted to units of percent injected dose per gram of tissue (% ID/g), while blood radioactivity was presented as % ID. A representative PET image is shown in  FIG.  13    for a depleted animal, showing a complete absence of the spleen signal seen in the non-depleted animal in  FIG.  9   . 
     Blood kinetics for each NHP, both depleted and non-depleted, are shown in  FIG.  14   , while the organ uptakes are shown in  FIG.  15   . There is little difference in blood kinetics between the depleted and non-depleted animals. Spleen uptake at the earliest time point are similar between depleted and non-depleted, since this is dominated by blood flow. However, at later time points (2 h) the spleen uptake in the depleted animals is less than half that seen in the non-depleted animals, and is essentially at background levels, demonstrating CD8A specificity of the radiolabeled centyrin. 
     Example 12: Sensitivity and Specificity of Pet Imaging in Cd8 Over-Expressing Tumors 
     In order to determine the lowest number of cells that can be detected with the anti-CD8A FN3 domain molecules and PET, a study was performed in mice using different numbers of CD8 overexpressing cells. Forty 4-5 week old female NOD-scid IL2rγ null  (NSG) mice (JAX Laboratory) were used, and acclimated for 7-10 days. Mice were group housed in IVC-cages under a 12-h light:dark cycle (lights on at 06:30 h) at a temperature of 19 to 22° C. Mice were fed a standard autoclaved laboratory chow and water ad libitum. Mice were ear-tagged and tails were tattooed 5-7 days prior to the start of the study to identify each animal. 
     HEK-293 parental and HEK-293-luc CD8+ over-expressing cell lines were maintained as 2D-cultures. Mice where implanted subcutaneously with a total of 10 6  tumor cells in a 1:1 medium to cultrex mix containing varying ratios of HEK-293-Luc CD8+ expressing cells and HEK-293 parental cells. Once tumors were palpable, approximately 10-14 days and 200-300 mm 3  in size, the human CD8+ cells were visualized using [I-124]CD8-S365. 
     Luciferase expression of HEK-293-Luc CD8+ cells was quantified in vivo using bioluminescence imaging in an IVIS Spectrum optical imager (Perkin Elmer). Dynamic optical imaging was performed immediately after injection of 150 mg/kg D-luciferin to identify the peak light emission. 
     Approximately 0.2-0.5 mCi of radiolabeled anti-CD8A FN3 domain molecules was injected into the tail vein while anesthetized with isoflurane in oxygen. Each animal was scanned in an Inveon microPET-CT scanner (Siemens, Knoxville, Tenn.) for 20 min static scan. Scans were acquired at 2-3 h post tracer injection. PET images were reconstructed using a 2D maximum likelihood expectation maximization (ML-EM) algorithm, into 3D images of voxel size 0.776×0.776×0.796 mm, dimensions 128×128×159. 
     PET images were analyzed using PMOD v3.7 software (PMOD, Zurich, Switzerland). Regions-of-interest were drawn manually around the tumor and other organs such as spleen, kidneys and liver. Counts were converted to units of percent injected dose per gram of tissue (% ID/g). A representative PET image is shown in  FIG.  16   . Luciferase expression was quantified by drawing regions-of-interest in Living Image v4.4 software (Perkin Elmer). Light emission was measured in units of photons/sec/cm 2 /steradian. 
     Time-activity curves of radiolabeled anti-CD8A FN3 domain molecules in the blood and tumor for both CD8+ HEK293 cells and parental cells are shown in  FIG.  17    and  FIG.  18   . There is a significant increase in anti-CD8A FN3-binding in the CD8-expressing cells compared to the parentals, while the blood activity is the same for both. Uptake of the anti-CD8A FN3 by the CD8+ HEK293 cells is shown in  FIG.  19   , as a function of number of implanted cells. Based on these data, it is estimated that the lowest level of detection is approximately 7.5×10 6  cells. 
     SEQUENCE LISTING 
       
                    SEQ ID No. 1 = Original Tencon Sequence       LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVP               GSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT               SEQ ID No. 2 = TCL1 library       LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVP               GSERSYDLTGLKPGTEYTVSIYGV(X) 7-12 PLSAEFTT;            
wherein
 
X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7  is any amino acid; and
 
X 8 , X 9 , X 10 , X 11  and X 12  are any amino acid or deleted
 
                    SEQ ID No. 3 = TCL2 library       LPAPKNLVVSEVTEDSLRLSWX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 SFLIQYQESEKVG               EAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX 9 X 10 X 11 X 12 X 13 SX 14                 X 15 LSAEFTT;            
wherein
 
     X 1  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 2  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 3  Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 4  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 5  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 6  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 7  is Phe, Ile, Leu, Val or Tyr; 
     X 8  is Asp, Glu or Thr; 
     X 9  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 10  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 11  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 12  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 13  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 14  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and 
     X 15  is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val. 
       
                    SEQ ID No. 4 = Stabilized Tencon (Tencon 27)       LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVP               GSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT               SEQ ID No. 5 = TCL7 (FG and BC loops)       LPAPKNLVVSRVTEDSARLSWX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 FDSFLIQYQES               EKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX 10 X 11 X 12 X 13                 X 14 X 15 X 16 X 17 X 18 X 19 SNPLSAIFTT;            
wherein
 
X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15  and X 16  are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and
 
X 7 , X 8 , X 9 , X 17 , X 18  and X 19 , are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted
 
                    SEQ ID No. 6 = TCL9 (FG loop)       LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVP               GSERSYDLTGLKPGTEYTVSIYGVX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11                 X 12 SNPLSAIFTT;            
wherein
 
X 1 , X 2 , X 3 , X 4 , X 5 , X 6  and X 7 , is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and
 
X 8 , X 9 , X 10 , X 11  and X 12  is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.
 
                    SEQ ID No. 7 = TCL14 library       LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 1 IX 2 YX 3 EX 4 X 5 X 6 X 7 GE               AIVLTVPGSERSYDLTGLKPGTEYX 8 VX 9 IX 10 GVKGGX 11 X 12 SX 13 PL               SAIFTT;            
wherein
 
X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 10 , X 11 , X 12  and X 13  are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or M.
 
                    SEQ ID No. 8 = TCL24 Library       TCL24 Library       (SEQ ID NO: 8)       LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 1 IX 2 YX 3 EX 4 X 5 X 6 X 7 GE               AIX 8 LX 9 VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 GVKGGX 13 X 14 S               X 15 PLX 16 AX 17 FTT;            
wherein
 
X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 10 , X 11 , X 12  and X 13  are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W.
 
     
       
         
           
               
            
               
                 SEQ ID No. 9 = Sloning-FOR 
               
               
                 GTGACACGGCGGTTAGAAC 
               
               
                   
               
               
                 SEQ ID No. 10 = Sloning-REV 
               
               
                 GCCTTTGGGAAGCTTCTAAG 
               
               
                   
               
               
                 SEQ ID No. 11 = POP2250 
               
               
                 CGGCGGTTAGAACGCGGCTACAATTAATAC 
               
               
                   
               
               
                 SEQ ID No. 12 = DigLigRev 
               
               
                 CATGATTACGCCAAGCTCAGAA 
               
               
                   
               
               
                 SEQ ID No. 13 = BC9 
               
               
                 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCC 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGA 
               
               
                   
               
               
                 TAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAA 
               
               
                   
               
               
                 ACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNN 
               
               
                   
               
               
                 NNNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCA 
               
               
                   
               
               
                 GGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTG 
               
               
                   
               
               
                 AACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTT 
               
               
                   
               
               
                 TCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC 
               
               
                   
               
               
                 SEQ ID No. 14 = BC8 
               
               
                 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCC 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGA 
               
               
                   
               
               
                 TAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAA 
               
               
                   
               
               
                 ACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNN 
               
               
                   
               
               
                 NNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGA 
               
               
                   
               
               
                 ATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAAC 
               
               
                   
               
               
                 GTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCT 
               
               
                   
               
               
                 ATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC 
               
               
                   
               
               
                 SEQ ID No. 15 = BC7 
               
               
                 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCC 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGA 
               
               
                   
               
               
                 TAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAA 
               
               
                   
               
               
                 ACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNN 
               
               
                   
               
               
                 NNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATC 
               
               
                   
               
               
                 TGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTT 
               
               
                   
               
               
                 CTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATC 
               
               
                   
               
               
                 TACGGTGTTCTTAGAAGCTTCCCAAAGGC 
               
               
                   
               
               
                 SEQ ID No. 16 = BC6 
               
               
                 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCC 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGA 
               
               
                   
               
               
                 TAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAA 
               
               
                   
               
               
                 ACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNN 
               
               
                   
               
               
                 NNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGA 
               
               
                   
               
               
                 AAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTT 
               
               
                   
               
               
                 ACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTAC 
               
               
                   
               
               
                 GGTGTTCTTAGAAGCTTCCCAAAGGC 
               
               
                   
               
               
                 SEQ ID No. 17 = 130mer-L17A 
               
               
                 CGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTG 
               
               
                   
               
               
                 ACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA 
               
               
                   
               
               
                 TTTCACACAGGAAACAGGATCTACCATGCTG 
               
               
                   
               
               
                 SEQ ID No. 18 = POP222ext 
               
               
                 CGG CGG TTA GAA CGC GGC TAC AAT TAA TAC 
               
               
                   
               
               
                 SEQ ID No. 19 = LS1114 
               
               
                 CCA AGA CAG ACG GGC AGA GTC TTC GGT AAC GCG AGA 
               
               
                   
               
               
                 AAC AAC CAG GTT TTT CGG CGC CGG CAG CAT GGT AGA 
               
               
                   
               
               
                 TCC TGT TTC 
               
               
                   
               
               
                 SEQ ID No. 20 = LS1115 
               
               
                 CCG AAG ACT CTG CCC GTC TGT CTT GG 
               
               
                   
               
               
                 SEQ ID No. 21 = LS1117 
               
               
                 CAG TGG TCT CAC GGA TTC CTG GTA CTG GAT CAG GAA 
               
               
                   
               
               
                 AGA GTC GAA 
               
               
                   
               
               
                 SEQ ID No. 22 = SDG10 
               
               
                 CATGCGGTCTCTTCCGAAAAAGTTGGTGAAGCGATCGTCCTGACCGTTCC 
               
               
                   
               
               
                 GGGT 
               
               
                   
               
               
                 SEQ ID No. 23 = SDG24 
               
               
                 GGTGGTGAAGATCGCAGACAGCGGGTTAG 
               
               
                   
               
               
                 SEQ ID No. 24 = POP2222 
               
               
                 CGGCGGTTAGAACGCGGCTAC 
               
               
                   
               
               
                 SEQ ID No. 25 = SDG28 
               
               
                 AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCACCGCCGGTGGTGA 
               
               
                   
               
               
                 AGATCGCAGAC 
               
               
                   
               
               
                 SEQ ID No. 26 = FG12 
               
               
                 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCC 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGA 
               
               
                   
               
               
                 TAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAA 
               
               
                   
               
               
                 ACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACC 
               
               
                   
               
               
                 GCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGA 
               
               
                   
               
               
                 AAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTT 
               
               
                   
               
               
                 ACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTAC 
               
               
                   
               
               
                 GGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCC 
               
               
                   
               
               
                 GCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCA 
               
               
                   
               
               
                 GCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC 
               
               
                   
               
               
                 SEQ ID No. 27 = FG11 
               
               
                 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCC 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGA 
               
               
                   
               
               
                 TAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAA 
               
               
                   
               
               
                 ACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACC 
               
               
                   
               
               
                 GCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGA 
               
               
                   
               
               
                 AAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTT 
               
               
                   
               
               
                 ACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTAC 
               
               
                   
               
               
                 GGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCT 
               
               
                   
               
               
                 GTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCG 
               
               
                   
               
               
                 GTTCTAGTCTAGCGGCCGCAACTGATCTTGGC 
               
               
                   
               
               
                 SEQ ID No. 28 = FG10 
               
               
                 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCC 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGA 
               
               
                   
               
               
                 TAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAA 
               
               
                   
               
               
                 ACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACC 
               
               
                   
               
               
                 GCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGA 
               
               
                   
               
               
                 AAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTT 
               
               
                   
               
               
                 ACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTAC 
               
               
                   
               
               
                 GGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTC 
               
               
                   
               
               
                 TGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTT 
               
               
                   
               
               
                 CTAGTCTAGCGGCCGCAACTGATCTTGGC 
               
               
                   
               
               
                 SEQ ID No. 29 = FG9 
               
               
                 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCC 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGA 
               
               
                   
               
               
                 TAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAA 
               
               
                   
               
               
                 ACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACC 
               
               
                   
               
               
                 GCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGA 
               
               
                   
               
               
                 AAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTT 
               
               
                   
               
               
                 ACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTAC 
               
               
                   
               
               
                 GGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGC 
               
               
                   
               
               
                 GATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTA 
               
               
                   
               
               
                 GTCTAGCGGCCGCAACTGATCTTGGC 
               
               
                   
               
               
                 SEQ ID No. 30 = FG8 
               
               
                 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCC 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGA 
               
               
                   
               
               
                 TAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAA 
               
               
                   
               
               
                 ACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACC 
               
               
                   
               
               
                 GCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGA 
               
               
                   
               
               
                 AAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTT 
               
               
                   
               
               
                 ACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTAC 
               
               
                   
               
               
                 GGTGTTNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGAT 
               
               
                   
               
               
                 CTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTC 
               
               
                   
               
               
                 TAGCGGCCGCAACTGATCTTGGC 
               
               
                   
               
               
                 SEQ ID No. 31 = FG7 
               
               
                 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCC 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGA 
               
               
                   
               
               
                 TAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAA 
               
               
                   
               
               
                 ACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACC 
               
               
                   
               
               
                 GCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGA 
               
               
                   
               
               
                 AAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTT 
               
               
                   
               
               
                 ACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTAC 
               
               
                   
               
               
                 GGTGTTNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTT 
               
               
                   
               
               
                 CACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAG 
               
               
                   
               
               
                 CGGCCGCAACTGATCTTGGC 
               
               
                   
               
               
                 FG loop of Tencon 
               
               
                 SEQ ID NO: 32 
               
               
                 KGGHRSN 
               
               
                   
               
               
                 SEQ ID No. 33 = Tcon 6 
               
               
                 AAGAAGGAGAACCGGTATGCTGCCGGCGCCGAAAAAC 
               
               
                   
               
               
                 SEQ ID No. 34 = Tcon5E86Ishort 
               
               
                 GAG CCG CCG CCA CCG GTT TAA TGG TGA TGG TGA TGG 
               
               
                   
               
               
                 TGA CCA CCG GTG GTG AAG ATC GCA GAC AG 
               
               
                   
               
               
                 CD8W7 
               
               
                 &gt;SEQ ID No. 35: 
               
               
                 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLL 
               
               
                   
               
               
                 YLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNS 
               
               
                   
               
               
                 IMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAGSGS 
               
               
                   
               
               
                 GSDYKDDDDKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT 
               
               
                   
               
               
                 CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH 
               
               
                   
               
               
                 QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK 
               
               
                   
               
               
                 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL 
               
               
                   
               
               
                 TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 CD8W13 
               
               
                 &gt;SEQ ID No 36: 
               
               
                 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLL 
               
               
                   
               
               
                 YLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNS 
               
               
                   
               
               
                 IMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA 
               
               
                   
               
               
                 VHTRGLDFACDGGGGSDYKDDDDKGGGGSHHHHHHDKTHTCPPCPAPELL 
               
               
                   
               
               
                 GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH 
               
               
                   
               
               
                 NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT 
               
               
                   
               
               
                 ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG 
               
               
                   
               
               
                 QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH 
               
               
                   
               
               
                 YTQKSLSLSPGK 
               
               
                   
               
               
                 mIgGK signal peptide 
               
               
                 &gt;SEQ ID No. 37: 
               
               
                 Metdtlllwvlllwvpgstg 
               
               
                   
               
               
                 Human Fc 
               
               
                 &gt;SEQ ID No. 38: 
               
               
                 Dkthtcppepapellggpsvflfppkpkdtlmisrtpevtcvvvdvshed 
               
               
                   
               
               
                 pevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeyk 
               
               
                   
               
               
                 ckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvk 
               
               
                   
               
               
                 gfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqg 
               
               
                   
               
               
                 nvfscsvmhealhnhytqkslslspgk 
               
               
                   
               
               
                 linker sequence 
               
               
                 &gt;SEQ ID No. 39: 
               
               
                 Ggggsdykddddkggggshhhhhh 
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                   
                 SEQ ID 
                   
               
               
                 Clone ID 
                 No 
                 Amino Acid Sequence 
               
               
                   
               
               
                 P282AR9P1356_A10 
                 40 
                 LPAPKNLVVSRVTEDSARLSWHTATNSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVDYNPTGRPVSSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P282AR9P1356_A4 
                 41 
                 LPAPKNLVVSRVTEDSARLSWVKRPNSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVVDYEGRPRWSNPLSAIFT 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 P282AR9P1356_A6 
                 42 
                 LPAPKNLVVSRVTEDSARLSWSKTDSSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVVYIEGNPVFSNPLSAIFT 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 P282AR9P1356_B9 
                 43 
                 LPAPKNLVVSRVTEDSARLSWPEGDRPFFDSFLIQYQESEKVGEAIV 
               
               
                   
                   
                 LTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P282AR9P1356_D3 
                 44 
                 LPAPKNLVVSRVTEDSARLSWTRHETSFDSFLIQYRESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVVVEYDAAGNPKYSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P282AR9P1356_H1 
                 45 
                 LPAPKNLVVSRVTEDSARLSWIPNPSSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVVFDPVGFPSHSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P282AR9P1356_H6 
                 46 
                 LPAPKNLVVSRVTEDSARLSWRKRANSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVHVEYDQHGRPRWSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P282BR9P1357_A9 
                 47 
                 LPAPKNLVVSRVTEDSARLSWKANRTTDLHFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVQYDGQQPLYSNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P282BR9P1357_B2 
                 48 
                 LPAPKNLVVSRVTEDSARLSWNPSEDPQRFDSFLIQYQESEKVGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSAI 
               
               
                   
                   
                 FTT 
               
               
                   
               
               
                 P282BR9P1357_C10 
                 49 
                 LPAPKNLVVSRVTEDSARLSWWSNDNRPIFDSFLIQYQESEKVGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSAI 
               
               
                   
                   
                 FTT 
               
               
                   
               
               
                 P282BR9P1357_C4 
                 50 
                 LPAPNNLVVSRVTEDSARLSWPFVSQNKPHFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P282BR9P1357_D12 
                 51 
                 LPAPKNLVVSRVTEDSARLSWGQYITAFSFDSFLIQYQESEKVGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYTVSIYGVDVAWFQGKPTWSNPLSAI 
               
               
                   
                   
                 FTT 
               
               
                   
               
               
                 P282BR9P1357_D2 
                 52 
                 LPAPKNLVVSRVTEDSARLSWIKDGHPRHFDSFLIQYQESEKVGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYTVSIYGVDVVYDRGQLISSNPLSAI 
               
               
                   
                   
                 FTT 
               
               
                   
               
               
                 P282BR9P1357_E5 
                 53 
                 LPAPKNLVVSRVTEDSARLSWWPRKYQRPFDSFLIQYQESEKVGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYTVSIYGVDIEWIGNRPIASNPLSAI 
               
               
                   
                   
                 FTT 
               
               
                   
               
               
                 P282BR9P1357_G9 
                 54 
                 LPAPKNLVVSRVTEDSARLSWPIASQIHSPFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLS 
               
               
                   
                   
                 AIFTT 
               
               
                   
               
               
                 P282BR9P1357_H3 
                 55 
                 LPAPKNLVVSRVTEDSARLSWKKREYQDPGFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P282CR9P1358_C2 
                 56 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIAYPEWPSNGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYAVFIWGVKGGAFSNPLSAIFTT 
               
               
                   
               
               
                 P282CR9P1358_C5 
                 57 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIAYPEWPDSGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYAVFIWGVKGGPLSHPLSAIFTT 
               
               
                   
               
               
                 P282CR9P1358_D10 
                 58 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLISYPEYPPPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIFGVKGGDTSWPLSAIFTT 
               
               
                   
               
               
                 P282CR9P1358_F11 
                 59 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIAYPEWPIFEGEAIV 
               
               
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFVVIYGVKGGEQSSPLSAIFTT 
               
               
                   
               
               
                 P282CR9P1358_F5 
                 60 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWISYPEWPPDGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIWGVKGGETSAPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_A12 
                 61 
                 LPAPKNLVVSRVTEDSARLSWTAPEAAFDSFQIAYPEWPPPREAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIQGVKGGEISWPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_A7 
                 62 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIGYPELEKLGYGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYWVIIWGVKGGENSWPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_A8 
                 63 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIAYPEWPVQGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGELSPPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_B2 
                 64 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIAYTEWPIPYEEAGQ 
               
               
                   
                   
                 EGEAIVLTVPGSERSYDLTGLKPGTEYWVSIYGVKGGPNSQPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P282DR9P1359_C10 
                 65 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIVYPEWPTDGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYAVFIWGVKGGNQSWPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_C11 
                 66 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIAYPEFPPPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYYVIIIGVKGGTDSWPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_C12 
                 67 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYISYPEWPVPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVVIYGVKGGALSVPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_C5 
                 68 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWITYPEWPDPGGEAIV 
               
               
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFVVIYGVKGGEIYSPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_D12 
                 69 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIAYPETATWGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGFESAPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_E11 
                 70 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYISYPEWPPVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIYGVKGGAISTPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_E2 
                 71 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIFYPEIVTWGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVNIVGVKGGDNSWPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_E3 
                 72 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYPELPLGGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGVESFPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_E5 
                 73 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAISYPEWPVPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGLYSAPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_E6 
                 74 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIAYPEWPVQGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIQGVKGGTPSWPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_E8 
                 75 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYPEWPVIGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIQGVKGGYTSWPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_F11 
                 76 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIFYPELPIHGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVNITGVKGGDFSWPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_F2 
                 77 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYPEALHPGYGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYWVIIGGVKGGQKSWPLSAIFTTGGH 
               
               
                   
                   
                 HHDHH 
               
               
                   
               
               
                 P282DR9P1359_F3 
                 78 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYITYPEWPVQGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIYGVKGGTESEPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_F5 
                 79 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYPEWPPPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIQGVKGGVESWPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_F6 
                 80 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPTTGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIWGVKGGDHSAPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_F7 
                 81 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYPEWPPQGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGSYSAPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_G4 
                 82 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIAYPEWPPPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGPEYFVVIQGVKGGDPSFPLSAIFTTGGNHH 
               
               
                   
                   
                 HHH 
               
               
                   
               
               
                 P282DR9P1359_G7 
                 83 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAITYIEKEHIEDGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYWVPIWGVKGGANSWPLSAIFTT 
               
               
                   
               
               
                 P282DR9P1359_H5 
                 84 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYPEALHPGYGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYFVVIYGVKGGTNSEPLSAIFTT 
               
               
                   
               
               
                 P282ER9P1360_A9 
                 85 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYYEPVDSGEAITL 
               
               
                   
                   
                 PIPGSERSYDLTGLKPGTEYWVVITGVKGGAPSTPLGAIFTT 
               
               
                   
               
               
                 P282ER9P1360_C1 
                 86 
                 LPAPKNLVVSRVTEDSARLSWTTPDAAFDSFGILYYEPVDSGEAITL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTEYWVVITGVKGGAPSTPLGAIFTT 
               
               
                   
               
               
                 P282ER9P1360_C4 
                 87 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGITYYEPNHGGEAISL 
               
               
                   
                   
                 SVPGSERSYDPTGLKPGTEYWVVITGVKGGAPSTPLGAIFTT 
               
               
                   
               
               
                 P282ER9P1360_C6 
                 88 
                 LSAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYYEPVDSGEAITL 
               
               
                   
                   
                 PIPGSERSYDLTGLKPGTEYWVVITGVKGGAPSTPLGAIFTT 
               
               
                   
               
               
                 P282ER9P1360_C8 
                 89 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYYEPVDSGEAITL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTEYWVVITGVKGGAPSTPLGTIFTT 
               
               
                   
               
               
                 P282ER9P1360_D11 
                 90 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYYEPVDSGEAITL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTEYFVIIVGVKGGYPSIPLGAAFTT 
               
               
                   
               
               
                 P282ER9P1360_E4 
                 91 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYYEPVDSGEAITL 
               
               
                   
                   
                 PVLGSERSYDLTGLKPGTEYWVVITGVKGGAPSTPLGAIFTT 
               
               
                   
               
               
                 P282ER9P1360_F11 
                 92 
                 LPAPKNLVVSRVTEDSARLSWIAPDAAFDSFSIAYVEAELVGEAIQL 
               
               
                   
                   
                 VVPGSERSYDLTGLKPGTEYWVVILGVKGGNPSNPLGASFTT 
               
               
                   
               
               
                 P282ER9P1360_G10 
                 93 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIWYVEQHPFGEAIPL 
               
               
                   
                   
                 FVPGSERSYDLTGLKPGTEYTVGIRGVKGGNFSTPLIAHFTT 
               
               
                   
               
               
                 P282ER9P1360_G7 
                 94 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYYEPVDSGEAITL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTEYWVVITGVKGGAPSTPLGAILTT 
               
               
                   
               
               
                 P282ER9P1360_H10 
                 95 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYPEWPFAGEAIGL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTEYFVVIYGVKGGELSEPLTAQFTT 
               
               
                   
               
               
                 P282ER9P1360_H2 
                 96 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYVEAELVGEAIQL 
               
               
                   
                   
                 VVPGSERSYDLTGLKPGTEYWVVILGVKGGNPSNPLGASFTTT 
               
               
                   
               
               
                 P282ER9P1360_H3 
                 97 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYVEAELVGEAIQL 
               
               
                   
                   
                 VVPGSERSYDLTGLKPGTEYWVVILGVKGGNPSNPLGASFTT 
               
               
                   
               
               
                 P282FR9P1361_A3 
                 98 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIWYAEYGYPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYDVAIVGVKGGNRSYPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_A5 
                 99 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYYEPVDSGEAITL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTEYWVVITGVKGGAPSTPLGAIFTT 
               
               
                   
               
               
                 P282FR9P1361_C7 
                 100 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIWYHEYGGDGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYDVAIWGVKGGDVSYPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_D3 
                 101 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIWYAEYGYPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLNPGTEYDVAISGVKGGPRSYPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_E12 
                 102 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSLGITYWESPYAGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYGVFILGVKGGYPSAPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_F1 
                 103 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIWYAEYGYSGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYDVAIWGVKGGVRSYPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_F11 
                 104 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIWYREYGGSGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYDVAIWGVKGGVRSYPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_F2 
                 105 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIWYAEYGYPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYDVAISGIKGGPRSYPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_F3 
                 106 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIWYAEYGYPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYDVAISGAKGGPRSYPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_F7 
                 107 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIWYREYATGEAIVLT 
               
               
                   
                   
                 VPGSERSYDLTGLKPGTEYDVVITGVKGGYPSYPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_G9 
                 108 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGITYWESPYAGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYGVFILGVKGGYPSAPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_H4 
                 109 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIWYAEYGYSGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYDVAIYGVKGGSPSYPLSAIFTT 
               
               
                   
               
               
                 P282FR9P1361_H5 
                 110 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIWYAEYGYPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYDVAISGVKGGPRSYPLSAIFTT 
               
               
                   
               
               
                 P283AR9P1362_A3 
                 111 
                 LPAPKNLVVSRVTEDSARLSWKRIDSPFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_A4 
                 112 
                 LPAPKNLVVSRVTEDSARLSWIGHDSGFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_B10 
                 113 
                 LPAPKNLVVSRVTEDSARLSWKRRWDSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVEWFNGLPHHSNPLSAIFT 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 P283AR9P1362_B2 
                 114 
                 LPAPKNLVVSRVTEDSARLSWAKHPNSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVVVNELNNPLFSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_B8 
                 115 
                 LPAPKNLVVSRVTEDSARLSWWTSPLPFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_C12 
                 116 
                 LPAPKNLVVSRVTEDSARLSWAKNLHSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_C6 
                 117 
                 LPAPKNLVVSRVTEDSARLSWYPSDPPFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVPNYHSRRSYYYSNPLSAIFT 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 P283AR9P1362_C7 
                 118 
                 LPAPKNLVVSRVTEDSARLSWVKRATSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVRYNEGQPIWSNPLSAIFT 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 P283AR9P1362_D2 
                 119 
                 LPAPKNLVVSRVTEDSARLSWQRPKSGFFDSFLIQYQESEKVGEAIV 
               
               
                   
                   
                 LTVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAI 
               
               
                   
                   
                 FTT 
               
               
                   
               
               
                 P283AR9P1362_D3 
                 120 
                 LPAPKNLVVSRVTEDSARLSWPVESNAFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVEYDQHGRPRWSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_D4 
                 121 
                 LPAPKNLVVSRVTEDSARLSWVREHDSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_D6 
                 122 
                 LPAPKNLVVSRVTEDSARLSWAKRPGAFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_D7 
                 123 
                 LPAPKNLVVSRVTEDSARLSWVKRATSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_E9 
                 124 
                 LPAPKNLVVSRVTEDSARLSWVPSPWGFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_F12 
                 125 
                 LPAPKNLVVSRVTEDSARLSWARNITSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_F2 
                 126 
                 LPAPKNLVVSRVTEDSARLSWRKKDHPFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_F8 
                 127 
                 LPAPKNLVVSRVTEDSARLSWGYYHGHFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSAIFT 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 P283AR9P1362_G11 
                 128 
                 LPAPKNLVVSRVTEDSARLSWRKEATSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 P283AR9P1362_G3 
                 129 
                 LPAPKNLVVSRVTEDSARLSWVKRATSFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSAIFT 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 P283AR9P1362_H11 
                 130 
                 LPAPKNLVVSRVTEDSARLSWPKIQGQHFDSFLIQYQESEKVGEAIV 
               
               
                   
                   
                 LTVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAI 
               
               
                   
                   
                 FTT 
               
               
                   
               
               
                 P283BR9P1363_A10 
                 131 
                 LPAPKNLVVSRVTEDSARLSWQRADDILPYFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLS 
               
               
                   
                   
                 AIFTT 
               
               
                   
               
               
                 P283BR9P1363_A8 
                 132 
                 LPAPKNLVVSRVTEDSARLSWVRSDTARFFDSFLIQYQESEKVGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P283BR9P1363_B2 
                 133 
                 LPAPKNLVVSRVTEDSARLSWASTVDPHPRFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLS 
               
               
                   
                   
                 AIFTT 
               
               
                   
               
               
                 P283BR9P1363_B6 
                 134 
                 LPAPKNLVVSRVTEDSARLSWQRHSDAHPLFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P283BR9P1363_C4 
                 135 
                 LPAPKNLVVSRVTEDSARLSWPIVNTPLHFDSFLIQYQESEKVGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYTVSIYGVDVQYTATGQPERSNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P283BR9P1363_C8 
                 136 
                 LPAPKNLVVSRVTEDSARLSWAKTSDLHPLFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P283BR9P1363_D11 
                 137 
                 LPAPKNLVVSRVTEDSARLSWNKKHDGQPTFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVVYEGSYPASSNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P283BR9P1363_E4 
                 138 
                 LPAPKNLVVSRVTEDSARLSWIKSETSQPAFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P283BR9P1363_E6 
                 139 
                 LPAPKNLVVSRVTEDSARLSWYARKFISPFDSFLIQYQESEKVGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSAI 
               
               
                   
                   
                 FTT 
               
               
                   
               
               
                 P283BR9P1363_F2 
                 140 
                 LPAPKNLVVSRVTEDSARLSWYRPDNRAGAFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLS 
               
               
                   
                   
                 AIFTT 
               
               
                   
               
               
                 P283BR9P1363_F4 
                 141 
                 LPAPKNLVVSRVTEDSARLSWERIVQ.TPHFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P283BR9P1363_F6 
                 142 
                 LPAPKNLVVSRVTEDSARLSWPEEAVTATSFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P283BR9P1363_G2 
                 143 
                 LPAPKNLVVSRVTEDSARLSWPKNQTNRHFDSFLIQYQESEKVGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSAI 
               
               
                   
                   
                 FTT 
               
               
                   
               
               
                 P283BR9P1363_G5 
                 144 
                 LPAPKNLVVSRVTEDSARLSWYRATTPAPHFDSFLIQYQESEKVGEA 
               
               
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSA 
               
               
                   
                   
                 IFTT 
               
               
                   
               
               
                 P283BR9P1363_G7 
                 145 
                 LPAPKNLVVSRVTEDSARLSWSAKKFPRHFDSFLIQYQESEKVGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYTVSIYGVDVKWEGNRPVASNPLSAI 
               
               
                   
                   
                 FTT 
               
               
                   
               
               
                 P283DR9P1364_A4 
                 146 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIAYPEWPVQGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGDWSEPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_A7 
                 147 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIAYPEWPVRGDAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIQGVKGGTDSFPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_B1 
                 148 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYITYPEIPLGGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGLLSSPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_B11 
                 149 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYISYPEWEQLGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGALSAPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_B4 
                 150 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAISYPEWPPPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIILGVKGGDQSWPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_C10 
                 151 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYPEWPKDGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYAVFIWGVKGGVYSNPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_D11 
                 152 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPPKGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGIHSAPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_D8 
                 153 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYPETPIQGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIHGVKGGITSFPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_D9 
                 154 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGISYPEWPPLGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIFGVKGGERSWPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_E3 
                 155 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIAYPELPIGGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIRGVKGGTLSPPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_E5 
                 156 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWISYPEWPVPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIQGVKGGKLSWPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_E7 
                 157 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYPEWPVRGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIYGVKGGDRSNPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_E8 
                 158 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYPEWPVHGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGVLSEPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_E9 
                 159 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPTKGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVINGVKGGWRSFPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_F2 
                 160 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIAYPEWPVPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIQGVKGGFGSFPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_F6 
                 161 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIAYPEREQDKWGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYWVIIQGVKGGRPSTPLSAILTT 
               
               
                   
               
               
                 P283DR9P1364_F8 
                 162 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYPEWPPGEAIVLT 
               
               
                   
                   
                 VPGSERSYDLTGLKPGTEYFVIIYGVKGGWTSPPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_G10 
                 163 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYPEWPGSGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIFGVKGGSQSWPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_G9 
                 164 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIWYPEWPVGGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVNISGVKGGEYSFPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_H1 
                 165 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQISYPEWPVHGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIWGVKGGRQSWPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_H11 
                 166 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPELPLGGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIWGVKGGDRSEPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_H6 
                 167 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIAYPETPVRGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIIGVKGGQESFPLSAIFTT 
               
               
                   
               
               
                 P283DR9P1364_H9 
                 168 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSISYIEYPEIPGGEAI 
               
               
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYWVPIWGVKGGIQSWPLSAIFTT 
               
               
                   
               
               
                 P283ER9P1365_A1 
                 169 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIAYVEWWHRGEAISL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTEYNVIITGVKGGIPSHPLGAIFTT 
               
               
                   
               
               
                 P283ER9P1365_A7 
                 170 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYWESEVYGEAIAL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTEYQVSIIGVKGGVYSQPLAAIFTT 
               
               
                   
               
               
                 P283ER9P1365_B6 
                 171 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIGYAEPVVTGEAISL 
               
               
                   
                   
                 SVPGSERSYDLTGLKPGTEYWVVIIGVKGGINSYPLGAIFTT 
               
               
                   
               
               
                 P283ER9P1365_C1 
                 172 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYWESEVYGEAIAL 
               
               
                   
                   
                 PVTGSERSYDLTGLKPGTEYQVSIIGVKGGVYSQPLAAIFTT 
               
               
                   
               
               
                 P283ER9P1365_E2 
                 173 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYRESEFRGEAIAL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTKYRVIIIGVKGGEFSQPLAAIFTT 
               
               
                   
               
               
                 P283ER9P1365_F4 
                 174 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYRESEFRGEAIAL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTKYSVIIIGVKGGEFSQPLGAIFTT 
               
               
                   
               
               
                 P283ER9P1365_G1 
                 175 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYRESEFRGEAIAL 
               
               
                   
                   
                 SVPGSERSYDLTGLKPGTKYRVIIIGVKGGEFSQPLGAIFTT 
               
               
                   
               
               
                 P283ER9P1365_G3 
                 176 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGISYYEWAPNGEAIQL 
               
               
                   
                   
                 SVPGSERSYDLTGLKPGTEYHVVIIGVKGGEPSHPLGAIFTT 
               
               
                   
               
               
                 P283ER9P1365_H3 
                 177 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYRESEFRGEAIAL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTKYRVIIIGVKGGEFSQPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_A1 
                 178 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWITYPEWPVPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYAVFIWGVKGGDASEPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_A5 
                 179 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIAYPEWPTRGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGSPSPPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_A9 
                 180 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYGEYPGPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVPIWGVKGGELSEPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_B7 
                 181 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWITYPEWPVNGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVVIWGVKGGVESPPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_C2 
                 182 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKISYPEWPPEGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYAVFIWCVKGGEHSWPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_C3 
                 183 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIAYPEWPDGGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGILSPPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_C4 
                 184 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYPEWPVRGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIIGVKGGEDSWPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_C6 
                 185 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYPEWPVYGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGNYSDPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_D12 
                 186 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPLGGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIILGVKGGDQSWPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_D6 
                 187 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIFYPELVFPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVNISGVKGGEHSWPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_D7 
                 188 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYPELPVKGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIWGVKGGTYSGPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_D8 
                 189 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIAYPEIPIAGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGDWSDPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_E11 
                 190 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPVPGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIKGVKGGNISWPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_F5 
                 191 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIGYPEWPIKGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIWGVKGGDRSEPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_F8 
                 192 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYPEIAKWGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGVHSFPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_F9 
                 193 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIFYPELPIAGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVNISGVKGGYESWPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_G1 
                 194 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYISYPELPVEGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIWGVKGGATSEPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_G5 
                 195 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYPEYPALGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIIGVKGGDESFPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_G8 
                 196 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPELPIGGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGIHSAPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_H10 
                 197 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYPEWPPEGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGHLSDPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_H11 
                 198 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIQYLETAPDGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYYVWIPGVKGGAFSPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_H3 
                 199 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYPEWPIKGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVVIYGVKGGVFSEPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_H5 
                 200 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIYIENKVNGEAIVLTV 
               
               
                   
                   
                 PGSERSYDLTGLKPGTEYHVTIGGVKGGTESNTLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_H6 
                 201 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPVTGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIFGVKGGERSWPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_H7 
                 202 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYPEYPALGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIAGVKGGIQSWPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_H8 
                 203 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYISYPEWPGSGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYAVFIWCVKGGWLSDPLSAIFTT 
               
               
                   
               
               
                 P283FR9P1366_H9 
                 204 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIAYPEWPVNGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVVIWGVKGGVNSYPLSAIFTT 
               
               
                   
               
               
                 P283GR7P1367_A11 
                 205 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPTDGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGSYSEPLSAIFTT 
               
               
                   
               
               
                 P283GR7P1367_B4 
                 206 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSILYYELPPSGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYTVSIFGVKGGDNSFPLSAIFTT 
               
               
                   
               
               
                 P283GR7P1367_B7 
                 207 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPTDGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGHWSYPLSAIFTT 
               
               
                   
               
               
                 P283GR7P1367_B9 
                 208 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIWYHEYHPRGEAIVL 
               
               
                   
                   
                 TVPSSERSYDLTGLKPGTEYDVVISGVKGGHWSYPLSAIFTT 
               
               
                   
               
               
                 P283GR7P1367_C9 
                 209 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIGYPEWPLGGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYWVIIYGVKGGEYSDPLSAIFTT 
               
               
                   
               
               
                 P283GR7P1367_E5 
                 210 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIWYHEYHPRGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYDVVISGVKGGHWSYPLSAIFTT 
               
               
                   
               
               
                 P283GR7P1367_F5 
                 211 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPTDGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVIIYGVKGGALSRPLSAIFTT 
               
               
                   
               
               
                 P283GR7P1367_G8 
                 212 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIAYPEYVWGGEATSL 
               
               
                   
                   
                 GEAIVLTVPGSERSYDLTGLKPGTEYFVVITGVKGGLGSYPLSAIFT 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 P283GR7P1367_H2 
                 213 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPTDGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGGRSYPLSAIFTT 
               
               
                   
               
               
                 P283GR7P1367_H8 
                 214 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSINYWEEDPAGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYRVLIGGVKGGHGSLPLSAIFTT 
               
               
                   
               
               
                 P283GR7P1367_H9 
                 215 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYPEWPTDGEAIVL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYFVVIYGVKGGGRSAPLSAIFTT 
               
               
                   
               
               
                 P283HR7P1368_A10 
                 216 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIFYLEPFPRGEAIPL 
               
               
                   
                   
                 EVPGSERSYDLTGLKPGTEYSVDIRGVKGGDHSDPLWAYFTT 
               
               
                   
               
               
                 P283HR7P1368_B12 
                 217 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIGYVEFTRAGEAISL 
               
               
                   
                   
                 SVPGSERSYDLTGLKPGTEYHVVIIGVKGGEPSHPLGAPFTT 
               
               
                   
               
               
                 P283HR7P1368_C3 
                 218 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIGYAEPAVTGEAISL 
               
               
                   
                   
                 SVPGSKRSYDLTGLKPGTEYWVVIIGVKGGINSYPLGASFTT 
               
               
                   
               
               
                 P283HR7P1368_D1 
                 219 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGISYYEWAPNGEAIQL 
               
               
                   
                   
                 SVPGSERSYDLTGLKPGTEYHVVIIGVKGGEPSHPLGAPFTT 
               
               
                   
               
               
                 P283HR7P1368_D2 
                 220 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFNSFGIGYAEPAVTGEAISL 
               
               
                   
                   
                 SVPGSERSYDLTGLKPGTEYWVVIIGVKGGINSYPLGASFTT 
               
               
                   
               
               
                 P283HR7P1368_D4 
                 221 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIWCVEPIPEGEAIPL 
               
               
                   
                   
                 FVPGSERSYDLTGLKPGTEYRVGIRGVKGGTFSSPLAAPFTT 
               
               
                   
               
               
                 P283HR7P1368_F10 
                 222 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYRESEFRGEAIAL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTKYRVIIIGVKGGEFSQPLGAIFTT 
               
               
                   
               
               
                 P283HR7P1368_F6 
                 223 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIGYIEWVHRGEAISL 
               
               
                   
                   
                 HVPGSERSYDLTGLKPGTEYVVAIVGVKGGEPSTPLGAPFTT 
               
               
                   
               
               
                 P283HR7P1368_G1 
                 224 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLITYWEIEPEGEAIFL 
               
               
                   
                   
                 GVPGSERSYDLTGLKPGTEYRVQINGVKGGTISYPLFAGFTT 
               
               
                   
               
               
                 P283HR7P1368_G10 
                 225 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIAYVEWWHRGEAISL 
               
               
                   
                   
                 PVPGSERSYDLTGLKPGTEYWVTILGVKGGIISTPLGASFTT 
               
               
                   
               
               
                 P283HR7P1368_G11 
                 226 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIGYAEPAVTGEAISL 
               
               
                   
                   
                 SVPGSERSYDLTGLKPGTEYWVVIIGVKGGINSYPLGASFTT 
               
               
                   
               
               
                 P283HR7P1368_H1 
                 227 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIAYIETARWGEAISL 
               
               
                   
                   
                 TVPGSERSYDLTGLKPGTEYNVVIIGVKGGTPSHPLGAPFTT 
               
               
                   
               
               
                 P283HR7P1368_H8 
                 228 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGITYLDPRNGEAISLN 
               
               
                   
                   
                 VPGSERSYDLTGLKPGTEYWVVIIGVKGGINSYPLGASFTT 
               
               
                   
               
               
                 CD8S368 
                 229 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYPEWPPPGEAIVL 
               
               
                   
                   
                 TVPGSCRSYDLTGLKPGTEYFVIIQGVKGGVESWPLSAIFTT 
               
               
                   
               
               
                 CD8S367 
                 230 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYPEWPPQGEAIVL 
               
               
                   
                   
                 TVPGSCRSYDLTGLKPGTEYFVVIYGVKGGSYSAPLSAIFTT 
               
               
                   
               
               
                 CD8S370 
                 231 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAITYIEKEHIEDGEAI 
               
               
                   
                   
                 VLTVPGSCRSYDLTGLKPGTEYWVPIWGVKGGANSWPLSAIFTT 
               
               
                   
               
               
                 CD8S365 
                 232 
                 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYYEPVDSGEAITL 
               
               
                   
                   
                 PVPGSCRSYDLTGLKPGTEYWVVITGVKGGAPSTPLGTIFTT 
               
               
                   
               
               
                 CD8S369 
                 233 
                 LPAPKNLVVSRVTEDSARLSWAKRPGAFDSFLIQYQESEKVGEAIVL 
               
               
                   
                   
                 TVPGSCRSYDLTGLKPGTEYTVSIYGVDVKYDIDSRPISSNPLSAIF 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 CD8S366 
                 234 
                 LPAPKNLWSRVTEDSARLSWTAPDAAFDSFWITYPEWPDPGGEAIVL 
               
               
                   
                   
                 TVPGSCRSYDLTGLKPGTEYFVVIYGVKGGEIYSPLSAIFTT 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 SEQ ID 
                   
                   
               
               
                 Clone 
                 No 
                 Parent 
                 Sequence 
               
               
                   
               
               
                 CD8S371 
                 235 
                 P282DR9P1359_F5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFQIAYPEYPPPGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 IIQGVKGGVESWPLSAIFTT 
               
               
                   
               
               
                 CD8S372 
                 236 
                 P282DR9P1359_F5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFQIAYPELPPPGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 IIQGVKGGVESWPLSAIFTT 
               
               
                   
               
               
                 CD8S373 
                 237 
                 P282DR9P1359_F5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFQIAYPEIPPPGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 IIQGVKGGVESWPLSAIFTT 
               
               
                   
               
               
                 CD8S374 
                 238 
                 P282DR9P1359_F5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFQIAYPEWPPPGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 IIQGVKGGVESYPLSAIFTT 
               
               
                   
               
               
                 CD8S375 
                 239 
                 P282DR9P1359_F5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFQIAYPEWPPPGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 IIQGVKGGVESLPLSAIFTT 
               
               
                   
               
               
                 CD8S376 
                 240 
                 P282DR9P1359_F5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFQIAYPEWPPPGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 IIQGVKGGVESSPLSAIFTT 
               
               
                   
               
               
                 CD8S377 
                 241 
                 P282DR9P1359_F5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFQIAYPEWPPPGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 IIQGVKGGVESEPLSAIFTT 
               
               
                   
               
               
                 CD8S378 
                 242 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIEEGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 WVPIWGVKGGANSWPLSAIFTT 
               
               
                   
               
               
                 CD8S379 
                 243 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIESGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 WVPIWGVKGGANSWPLSAIFTT 
               
               
                   
               
               
                 CD8S380 
                 244 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIEDGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 YVPIWGVKGGANSWPLSAIFTT 
               
               
                   
               
               
                 CD8S381 
                 245 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIEDGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 FVPIWGVKGGANSWPLSAIFTT 
               
               
                   
               
               
                 CD8S382 
                 246 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIEDGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 SVPIWGVKGGANSWPLSAIFTT 
               
               
                   
               
               
                 CD8S383 
                 247 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIEDGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 WVPIYGVKGGANSWPLSAIFTT 
               
               
                   
               
               
                 CD8S384 
                 248 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIEDGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 WVPIFGVKGGANSWPLSAIFTT 
               
               
                   
               
               
                 CD8S385 
                 249 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIEDGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 WVPISGVKGGANSWPLSAIFTT 
               
               
                   
               
               
                 CD8S386 
                 250 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIEDGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 WVPIWGVKGGANSYPLSAIFTT 
               
               
                   
               
               
                 CD8S387 
                 251 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIEDGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 WVPIWGVKGGANSEPLSAIFTT 
               
               
                   
               
               
                 CD8S388 
                 252 
                 P282DR9P1359_G7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAITYIEKEHIEDGEA 
               
               
                   
                   
                   
                 IVLTVPGSERSYDLTGLKPGTEY 
               
               
                   
                   
                   
                 WVPIWGVKGGAQSWPLSAIFTT 
               
               
                   
               
               
                 CD8S389 
                 253 
                 P282ER9P1360_C8 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFGILYYEPVDSGEAIT 
               
               
                   
                   
                   
                 LPVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 VITGVKGGAPSTPLGTIFTT 
               
               
                   
               
               
                 CD8S390 
                 254 
                 P282ER9P1360_C8 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFGILYYEPVDSGEAIT 
               
               
                   
                   
                   
                 LPVPGSERSYDLTGLKPGTEYYV 
               
               
                   
                   
                   
                 VITGVKGGAPSTPLGTIFTT 
               
               
                   
               
               
                 CD8S391 
                 255 
                 P282ER9P1360_C8 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFGILYYEPVDSGEAIT 
               
               
                   
                   
                   
                 LPVPGSERSYDLTGLKPGTEYHV 
               
               
                   
                   
                   
                 VITGVKGGAPSTPLGTIFTT 
               
               
                   
               
               
                 CD8S392 
                 256 
                 P282DR9P1359_F7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAIAYPEYPPQGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 VIYGVKGGSYSAPLSAIFTT 
               
               
                   
               
               
                 CD8S393 
                 257 
                 P282DR9P1359_F7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAIAYPELPPQGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 VIYGVKGGSYSAPLSAIFTT 
               
               
                   
               
               
                 CD8S394 
                 258 
                 P282DR9P1359_F7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAIAYPEIPPQGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 VIYGVKGGSYSAPLSAIFTT 
               
               
                   
               
               
                 CD8S395 
                 259 
                 P282DR9P1359_F7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAIAYPEHPPQGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 VIYGVKGGSYSAPLSAIFTT 
               
               
                   
               
               
                 CD8S396 
                 260 
                 P282DR9P1359_C5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFYITYPEWPDPGGEAI 
               
               
                   
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYF 
               
               
                   
                   
                   
                 VVIYGVKGGEIYSPLSAIFTT 
               
               
                   
               
               
                 CD8S397 
                 261 
                 P282DR9P1359_C5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFQITYPEWPDPGGEAI 
               
               
                   
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYF 
               
               
                   
                   
                   
                 VVIYGVKGGEIYSPLSAIFTT 
               
               
                   
               
               
                 CD8S398 
                 262 
                 P282DR9P1359_C5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFSITYPEWPDPGGEAI 
               
               
                   
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYF 
               
               
                   
                   
                   
                 VVIYGVKGGEIYSPLSAIFTT 
               
               
                   
               
               
                 CD8S399 
                 263 
                 P282DR9P1359_C5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFWITYPEYPDPGGEAI 
               
               
                   
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYF 
               
               
                   
                   
                   
                 VVIYGVKGGEIYSPLSAIFTT 
               
               
                   
               
               
                 CD8S400 
                 264 
                 P282DR9P1359_C5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFWITYPELPDPGGEAI 
               
               
                   
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYF 
               
               
                   
                   
                   
                 VVIYGVKGGEIYSPLSAIFTT 
               
               
                   
               
               
                 CD8S401 
                 265 
                 P282DR9P1359_C5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFWITYPEIPDPGGEAI 
               
               
                   
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYF 
               
               
                   
                   
                   
                 VVIYGVKGGEIYSPLSAIFTT 
               
               
                   
               
               
                 CD8S402 
                 266 
                 P282DR9P1359_C5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFWITYPEWPPPGGEAI 
               
               
                   
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYF 
               
               
                   
                   
                   
                 VVIYGVKGGEIYSPLSAIFTT 
               
               
                   
               
               
                 CD8S403 
                 267 
                 P282DR9P1359_F7 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFAIAYAEWPPQGEAIV 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYFV 
               
               
                   
                   
                   
                 VIYGVKGGSYSAPLSAIFTT 
               
               
                   
               
               
                 CD8S404 
                 268 
                 P282DR9P1359_C5 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFWITYAEWPDPGGEAI 
               
               
                   
                   
                   
                 VLTVPGSERSYDLTGLKPGTEYF 
               
               
                   
                   
                   
                 VVIYGVKGGEIYSPLSAIFTT 
               
               
                   
               
               
                 CD8S405 
                 269 
                 P282ER9P1360_C8 
                 LPAPKNLVVSRVTEDSARLSWTA 
               
               
                   
                   
                   
                 PDAAFDSFGILYYEPVDSGEAIT 
               
               
                   
                   
                   
                 LTVPGSERSYDLTGLKPGTEYWV 
               
               
                   
                   
                   
                 VITGVKGGAPSTPLGTIFTT 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                 Tencon25 
               
               
                 SEQ ID. No. 270 
               
               
                 LPAPKNLVVSEVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVP 
               
               
                   
               
               
                 GSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT 
               
               
                   
               
               
                 Cyno CD8alpha 
               
               
                 SEQ ID. NO: 271 
               
               
                 MRNQAPGRPKGATSPPPLPTGSRAPPVAPELRAEPRPGERVMAPPVTALL 
               
               
                   
               
               
                 LPLVLLLHAARPNQFRVSPLGRTWNLGETVELKCQVLLSNPTSGCSWLFQ 
               
               
                   
               
               
                 PRGTAARPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLRDFRQEN 
               
               
                   
               
               
                 EGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTTASQPLSL 
               
               
                   
               
               
                 RPEACRPAAGGSVNTRGLDFACDIYIWAPLAGACGVLLLSLVITLYCNHR 
               
               
                   
               
               
                 NRRRVCKCPRPVVKSGGKPSLSDRYV