Human cd8a-binding fibronectin type iii domains, diagnostic kit, capture agent and detection method

Abstract

Fibronectin type III (fn3) domains that specifically bind to CD8A, related polynucleotides capable of encoding CD8A-specific fn3 domains, cells expressing fn3 domains, as well as associated vectors, and detectably labeled fn3 domains are useful in therapeutic and diagnostic applications.

Description

1 / 165 “HUMAN CD8A BINDING FIBRONECTIN TYPE III DOMAINS, DIAGNOSTIC KIT, CAPTURE AGENT AND METHOD OF DETECTION SEQUENCE LISTING

[0001] This request contains a Listing of Sequences, which were submitted electronically in ASCII format and are incorporated herein by reference in their entirety. The ASCII copy, created on December 5, 2017, is named JBI5112WOPCT_SL.txt and is 266,978 bytes in size. FIELD OF TECHNIQUE

[0002] The present invention relates to fibronectin type III (FN3) domains that specifically bind to a differentiation group 8a (CD8a). Such FN3 domains can be used, for example, for musical imaging, diagnostics and pharmaceutical therapy. Methods for producing such molecules and diagnostic agents comprising them are also provided. FUNDAMENTALS OF THE INVENTION

[0003] The rapidly evolving fields of cancer immunotherapy have recently led to FDA approval of several new immunotherapies, with many more therapies currently in clinical trials for a variety of cancers. In addition, small molecule-based cellular immunotherapies... Petition 870260068423, dated 10 / 07 / 2026, page 16 / 201 2 / 165 antibodies, and their combinations, are being rigorously tested preclinically for clinical translation. The dynamic tumor microenvironment and tumor heterogeneity have become important themes in preclinical and clinical studies (Hanahan D, Weinberg RA. Cell 2011;144:646-74; Mantovani A, Allavena P, Sica A, Balkwill F. Nature 2008; 454:436-44; Schreiber RD, Old LJ, Smyth MJ. Science 2011; 331:1565-70.), but the ability to monitor changes in the immunological status of primary lesions and metastatic cancers is limited. Current methods for monitoring lymphocytes from whole blood or biopsies of heterogeneous tumors do not reflect the dynamic and spatial information likely needed to monitor immune responses to therapeutic intervention, many of which elicit whole-body changes in immune cell number and location.Therefore, molecular imaging methods that can non-invasively monitor systemic and intratumoral changes in immune cell number or location during experimental therapies have the ability to enhance understanding of the dynamics of the immunotherapeutic mechanism, with the potential to provide translatable methods for predicting and / or evaluating immunotherapeutic responses.

[0004] Infiltration lymphocyte analysis Petition 870260068423, dated 10 / 07 / 2026, page 17 / 201 The 3 / 165 tumor cell (TIL) study demonstrated the importance of the 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 MJ, et al. Br J Cancer 2011;105:93-103). With the recent clinical successes of immunotherapies that alter the tumor immune microenvironment, including T-cell receptor (TCR) adoptive cell transfer (ACT) – or chimeric antigen receptor-transduced cytotoxic T cells (Johnson LA, et al. Blood 2009;114:535-46; Rosenberg SA. 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;7:95106; Vinay DS, and Kwon BS. Mol Cancer Ther 2012;11:1062-70.), and antibody blockade of checkpoint inhibitors CTLA4, PD-1, and PD-L1 (Callahan MK, and Wolchok JD.J Leukoc Biol 2013;94:41-53; Shin DS, and Ribas A. Curr Opin Immunol 2015;33C:23-35; Topalian SL, et al. Cancer Cell 2015; 27:. 450-61.), the ability to non-invasively monitor the tumor's immune response to therapy has become extremely important. SUMMARY

[0005] The present invention comprises domains of Petition 870260068423, dated 10 / 07 / 2026, page 18 / 201 4 / 165 CD8A type III binding fibronectin (FN3). Related polynucleotides capable of encoding the provided FN3 domains, cells expressing the provided FN3 domains, and associated vectors are also described. Furthermore, methods for utilizing the provided FN3 domains are described. For example, the FN3 domains of the invention can be used to non-invasively and quantitatively monitor the presence and abundance of CD8+ T cells.

[0006] 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 still other embodiments, the CD8A-specific FN3 domains are based on the Tencon sequence of SEQ ID NO: 1. In other 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 domains CD8A-specific FN3 domains do not activate CD8+ T cells in vitro, as measured by the enzyme-linked immunospot assay (ELISPOT). In some embodiments, CD8A-specific FN3 domains bind to human CD8A with an affinity Petition 870260068423, dated 10 / 07 / 2026, page 19 / 201 5 / 165 (kD) between approximately 0.02 and approximately 6.6 nM, as measured by surface plasmon resonance. In other embodiments, the CD8A-specific FN3 domains have a cysteine ​​substitution at residue 54 of SEQ IDs 79, 81, 83, 89, 122, and 68. In other embodiments, the CD8A-specific FN3 domains comprise the amino acid sequence of SEQ IDs 40-269. In other embodiments, the CD8A-specific FN3 domains are conjugated with a detectable marker.

[0007] In addition to the CD8A-specific FN3 domains described, polynucleotide sequences capable of encoding the described FN3 domains are also provided. Vectors comprising the described polynucleotides are also provided, as are cells expressing the CD8A-specific FN3 domains of the present invention. Cells capable of expressing the disclosed vectors are also described. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacterial cells (such as E. coli). A process for producing the described FN3 domains is also provided.

[0008] The present invention also comprises methods of combining or otherwise associating the FN3 domains. Petition 870260068423, dated 10 / 07 / 2026, page 20 / 201 6 / 165 specific CD8A domains have been described for use in various molecules for diagnostic purposes. For example, Zr-89 or I-124 are ideal fusion partners for creating diagnostic agents capable of detecting the presence of CD8+ T cells. As such, CD8A-specific FN3 domains have utility in cancer diagnosis, using CD8A as a biomarker.

[0009] Another embodiment of the invention is a method for detecting cells expressing CD8A in a biological sample comprising treating the biological sample with a diagnostic agent comprising the CD8A-specific FN3 domains described. These methods are provided in the EXAMPLES.

[0010] Within the scope of the invention are kits including the specific CD8A FN3 domains described. The kits can be used to perform the methods of using the specific CD8A FN3 domains provided in this document, or other methods known to those skilled in the art. In some embodiments, the kits described may include the FN3 domains described in this document and reagents for use in detecting the presence of human CD8A in a biological sample. The kits described may include one or more of the FN3 domains described in this document and a container to hold the FN3 domains when not in use, instructions for using the FN3 domains affixed to a solid support and / or detectably marked forms of the Petition 870260068423, dated 10 / 07 / 2026, page 21 / 201 7 / 165 FN3 domains, as described in this document. BRIEF DESCRIPTION OF THE FIGURES

[0011] Fig. 1A-1D. The CD8S365-DFO conjugate does not activate T cells de novo and does not modulate antigen-dependent T cell activation in a 24-hour INFy EliSpot assay. CMV-reactive T cells were treated with 365-DFO in the absence (A) or presence (B) of CMV peptides. A second reactive M1 donor was also tested in the absence (C) or presence (D) of M1 peptides.

[0012] Fig. 2A and 2B. The CD8S365-DFO conjugate does not activate T cells de novo and does not modulate antigen-dependent T cell activation in a 6-day INFγ MSD assay. CMV-reactive T cells were treated with 365-DFO in the absence (A) or presence (B) of CMV peptides.

[0013] Fig. 3. Raw preparative HPLC screening of [124I] -IPEM. Preparative 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 an Atlantis T3, 100 A, 5 µm, 150 x 4.6 mm HPLC column. The elution profile used was as follows: solvent A = H2O (0.1% (v / v) AcOH), solvent B = MeCN (0.1% (v / v) AcOH), flow rate = 1.5 mL min⁻¹; initial = 80% A, 20 min = 0% A (linear gradient). Multiple absorbances of molecules Petition 870260068423, dated 10 / 07 / 2026, p. 22 / 201 8 / 165 small UV-vis screening at 254 nm (top graph) and 214 nm (middle graph) indicate the presence of impurities and byproducts in the crude reaction mixture. Radiotrace (bottom graph) also shows expected baseline peaks due to radiolabeled impurities.

[0014] Fig. 4. Analytical HPLC screening of [124I] -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), and a Bioscan radiodetector. Flow Count (B-FC-2000) and a Phenomenex Kinetex 5 pm HPLC column XB-C18 100 Å, 150 x 4.6 mm. The elution profile used was as follows: solvent A = H2O (0.1% TFA (v / v)), Solvent B = MeCN (0.1% TFA (v / v)), flow rate = 1 mL min⁻¹; initial = 90% A, 15 min = 0% A (linear gradient). Analytically pure IPEM [I-124] shows a single radio peak (lower graph) with a smooth baseline, confirming successful purification. Please note that [I-124] IPEM is a small organic molecule; therefore, it lacks absorption at 280 nm (upper graph) and 214 nm (middle graph).

[0015] Fig. 5. Purified [124I]-IPEM CD8S365 radio TLC. The iTLC-SG plate (Agilent, cat # SGI0001) was read on a Bioscan AR-2000 radio-TLC imaging scanner. Petition 870260068423, dated 10 / 07 / 2026, p. 23 / 201 9 / 165 The TLC radioplate (Fig. 3) was co-stained with 1 mL of Nai (0.1 M) and developed using citric acid (0.5 mM, pH = 5) as eluent. Origin = 20 mm and solvent front = 100 mm. The TLC radio eluent was prepared by dissolving 96 mg of citric acid (Spectrum cat # CI131) in 25 mL of Trace Select H2O and then Na2CO3 (245 pL, 2 M) was added; pH was checked using a strip (pH = 5).

[0016] Fig. 6. Analytical HPLC trace of purified [124I]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 pm XB-C18 100 Å, 150 x 4.6 mm HPLC column. The elution profile used was as follows: solvent A = H2O (0.1% TFA (v / v)), Solvent B = MeCN (0.1% TFA (v / v)), flow rate = 1 mL min-1; Initial = 90% A, 15 min = 0% A (linear gradient). The absorbance of the biomolecule (CD8S) at 280 nm (upper graph) and the absorbance of small molecules (I124-IPEM) at 214 nm (middle graph) confirm the successful conjugation reaction. UV and radio radiation scans (lower graph) indicate an analytically pure sample.

[0017] Fig. 7. MALDI-MS of IPEM CD8S365 (theoretical MW) Petition 870260068423, dated 10 / 07 / 2026, page 24 / 201 10 / 165 = 10786.12). 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 in TA30 solvent (MeCN 30:70 (v / v): 0.1% TFA in water) was prepared. The sample (c = 0.397 mgmL-1) was mixed in a 1:1 ratio with the matrix solution and 1 pL was stained onto the plate. A protein solution was used as an external standard.

[0018] Fig. 8. Co-injection of [124I]-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 pm XB-C18 100 Å, 150 x 4.6 mm HPLC column. The elution profile used was as follows: solvent A = H2O (0.1% TFA (v / v)), Solvent B = MeCN (0.1% TFA (v / v)), flow rate = 1 mL min-1; Initial = 90% A, 15 min = 0% A (linear gradient). Co-injection with a cold sample leads to complete overlap of the UV peaks (upper and middle graphs), confirming the molecular identity of the product (i.e., the cold and radiolabeled conjugates are identical, except for the substitution of Iodine with Iodine-124).

[0019] Fig. 9. Representative image of PET Petition 870260068423, dated 10 / 07 / 2026, page 25 / 201 Image 11 / 165 showing CD8S365-IPEM5 radioactively labeled with I-124, taken 2 h after injection. The image is a maximum intensity projection (anterior-posterior), with the spleen centered over the crosshairs. The organs below the spleen are the kidneys, and the image is oriented to show the head at the top. Uptake in the thyroid is evidence of some protein deiodization.

[0020] Fig. 10. Time-activity curves for blood radioactivity in non-human primates for each anti-CD8A FN3 domain labeled with Zr-89 or I-124.

[0021] Fig. 11A and 11B. Time-activity curves for organ radioactivity in NHP for each centrin labeled with Zr-89 or I-124. Fig. 11A includes kidneys, liver, and spleen, while Fig. 11B focuses on the spleen. The 24-hour point for [124I]-IPEM CD8S365 is missing due to a technical issue. The high absorption of Zr-89 in the kidneys due to isotope residualization is largely absent from the I-124 data.

[0022] Fig. 12A-12C. Confirmation of CD8 T cell depletion by Day 3 in blood taken from a non-human primate (12A). Changes in CD4 (12B) and CD3 (12C) T cells are also shown.

[0023] Fig. 13. Representative PET image showing the FN3-labeled anti-CD8A domain 365 Petition 870260068423, dated 10 / 07 / 2026, page 26 / 201 12 / 165 radioactively treated with I-124, taken 2 h after injection in an animal with CD8 depletion. 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.

[0024] Fig. 14. Time-activity curves for blood radioactivity in cynomolgus monkeys for depleted and non-depleted animals after administration of [124I] IPEM CD8S365.

[0025] Fig. 15A and 15B. Time-activity curves for organ radioactivity in cynomolgus monkeys for depleted and non-depleted animals. 15A includes kidneys, liver and spleen, while 15B is focused on the spleen.

[0026] Fig. 16. Representative PET image of two identically treated mice showing I-124 radiolabeled CD8S365-IPEM, taken 3 h post-injection. The image is a 3D maximum intensity projection superimposed on a computed tomography scan. The tumor (formed from HEK-293-luc transfected to overexpress huCD8+) and other organs are indicated by arrows. Uptake in the thyroid is evidence of some protein deiodization.

[0027] Fig. 17. Time-activity curve for blood radioactivity in mice bearing the parental tumors HEK-293-luc CD8+ or HEK-293. Petition 870260068423, dated 10 / 07 / 2026, page 27 / 201 13 / 165

[0028] Fig. 18. Time-activity curve for tumor radioactivity in mice carrying parental tumors HEK-293-luc CD8+ or HEK-293.

[0029] Fig. 19. Absorption of CD8S365 labeled with I124 in CD8 HEK293 overexpressing cells, as a function of the number of cells implanted. DETAILED DESCRIPTION OF THE ILLUSTRATIVE MODALITIES Definitions

[0030] Several terms relating to aspects of the description are used throughout the specification and claims. These terms should be given their usual meanings in the art, unless otherwise indicated. Other specifically defined terms should be interpreted in a manner consistent with the definition given in this document.

[0031] As used in this descriptive report and the accompanying claims, the singular forms a, an, and the include plural referents, unless the context clearly indicates otherwise. Thus, for example, the reference to “a cell” includes a combination of two or more cells and the like.

[0032] The term "approximately," as used in this document, when referring to a measurable value, such as a quantity, a duration of time, and the like, is intended to Petition 870260068423, dated 10 / 07 / 2026, p. 28 / 201 14 / 165 encompass variations of ±10% of the specified value, as these variations are appropriate for carrying out 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 descriptive report and claims, should be understood as being modified in all cases by the term approximately. Consequently, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired properties to be obtained by the present invention. At 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 interpreted in light of the reported number of significant digits and applying common rounding techniques.

[0033] Although the numerical ranges and parameters that establish 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 that necessarily result from the standard deviation found in its respective measurements of Petition 870260068423, dated 10 / 07 / 2026, page 29 / 201 15 / 165 test.

[0034] Isolated means that a biological component (such as a nucleic acid, peptide, or protein) has been substantially separated, produced separately, or purified from other biological components of the organism in which the component naturally occurs, i.e., other chromosomes and extrachromosomal DNA and RNA and proteins. The 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 may 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 encompasses 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 in this document, is intended to refer to an FN3 domain that is substantially free of other FN3 domains possessing different antigenic specificities (e.g., an isolated FN3 domain that specifically binds to human serum albumin is substantially free of FN3 domains). FN3 that bind specifically to antigens other than human serum albumin). An isolated FN3 domain that binds Petition 870260068423, dated 10 / 07 / 2026, page 30 / 201 16 / 165 specifically to an epitope, isoform or variant of human serum albumin may, however, have cross-reactivity with other related antigens, for example from other species (such as species homologs of serum albumin).

[0035] The term fibronectin type III (FN3) domain (FN3 domain) as used in this document refers to a domain that occurs 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 US Pat. No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, for example, the 3rd FN3 domain of tenascin (TN3) or the 10th FN3 domain of fibronectin (FN10).

[0036] Centirina, as used in this document, refers to an FN3 domain that is based on the consensus sequence of the 15 different FN3 domains present in Petition 870260068423, dated 10 / 07 / 2026, page 31 / 201 17 / 165 human tenascin C.

[0037] The term “capture agent” refers to substances that bind to a particular type of cell and allow that cell to be isolated from other cells. Examples of capture agents include, but are not limited to, magnetic beads, ferrofluids, encapsulation reagents, and the like.

[0038] The term “biological sample” refers to blood, tissue, bone marrow, sputum and the like.

[0039] The term “diagnostic reagent” refers to any substance that can be used to analyze a biological sample, whether that substance is distributed as a single substance or in combination with other substances in a diagnostic kit.

[0040] The term replacing or substituted or mutant or mutated as used in this document refers to the alteration, deletion or insertion of one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence.

[0041] The term randomization or randomized or diversifying or diversifying, as used in this document, refers to making at least one substitution, insertion or deletion in a polynucleotide or polypeptide sequence. Petition 870260068423, dated 10 / 07 / 2026, p. 32 / 201 18 / 165

[0042] Variant, as used in this document, 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.

[0043] The term "specific binding" or "specific binding" as used in this document refers to the ability of the FN3 domain of the invention to bind to a predetermined antigen with a dissociation constant (KD) of about 1x10⁻⁶ M or less, for example about 1x10⁻⁷ M or less, about 1x10⁻⁸ M or less, about 1x10⁻⁹ M or less, about 1x10⁻¹⁰ M or less, about 1x10⁻¹¹ M or less, about 1x10⁻¹² M or less, or about 1x10¹³ M or less. Typically, the FN3 domain of the invention binds to a predetermined antigen (i.e., human CD8A) with a KD that is at least ten times lower than its KD for a non-specific antigen (e.g., BSA or casein) measured by surface plasmon resonance using, for example, a Protein Instrument (BioRad).The isolated FN3 domain of the invention that specifically binds to human CD8A may, however, have cross-reactivity with other related antigens, for example, with the same predetermined antigen from other species (orthologs), such as Macaca Fascicularis (cynomolgus monkey) or Pan. Petition 870260068423, dated 10 / 07 / 2026, page 33 / 201 19 / 165 troglodytes (chimpanzee).

[0044] The term library refers to a collection of variants. The library may be composed of polypeptide or polynucleotide variants.

[0045] As used in this document, the terms CD8A or CD8 specifically include the human CD8 alpha protein, for example, as described in NCBI Reference Sequences: NP_001139345,1, NP_0011759,3 and NP_741969,1. CD8A is also known in the scientific literature as CD8a molecule, MAL, p32, Leu2, CD8 T-surface glycoprotein alpha chain, CD8 antigen, alpha polypeptide (p32), Leu2 T lymphocyte antigen, OKT8 T cell antigen, Leu2 T cell antigen, T8 / Leu-2 T lymphocyte differentiation antigen and T8 T cell antigen.

[0046] Tencon, as used in this document, refers to the synthetic fibronectin domain type III (FN3), having the sequence shown in SEQ ID NO: 1 and described in Pat. US Publ. No. US2010 / 0216708.

[0047] 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 signals, or selection markers that function to facilitate duplication or maintenance. Petition 870260068423, dated 10 / 07 / 2026, page 34 / 201 20 / 165 of these polynucleotides in a biological system. Examples of such biological systems may include cell, virus, animal, plant, and reconstructed biological systems using biological components capable of replicating a vector. The polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid thereof.

[0048] The term “expression vector” means a vector that can be used 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.

[0049] The term polynucleotide means a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemical structure. Double-stranded and single-stranded DNAs and RNAs are typical examples of polynucleotides.

[0050] The term polypeptide or protein means a molecule comprising at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides with fewer than about 50 amino acids may be referred to as peptides.

[0051] The term “in combination with” as used Petition 870260068423, dated 10 / 07 / 2026, page 35 / 201 21 / 165 in this document means that two or more therapeutic agents may be administered to a subject together in a mixture, simultaneously as single agents, or sequentially as single agents in any order. Compositions of matter

[0052] The present invention provides human CD8A-binding FN3 domains and CD8A-binding FN3 domains conjugated with detectable markers. The present invention provides polynucleotides encoding the FN3 domains of the invention or their complementary nucleic acids, vectors, host cells, and methods for producing and using them. CD8A binding molecules

[0053] The present invention provides fibronectin type III (FN3) domains that specifically bind to CD8A, optionally conjugated with a detectable marker. These molecules can 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 their complementary nucleic acids, vectors, host cells, and methods for producing and using them.

[0054] The FN3 domains of the invention bind to CD8A with high affinity and can target cells that express Petition 870260068423, dated 10 / 07 / 2026, page 36 / 201 22 / 165 CD8, thus providing an efficient way to deliver diagnostic reagents into the tumor microenvironment.

[0055] An embodiment of the invention is an isolated FN3 domain that specifically binds to a human CD8A comprising the amino acid sequence of SEQ ID NO: 35.

[0056] In some embodiments of the invention described in this document, the FN3 domain of the invention cross-reacts with monkey-cynomolgus CD8A having the amino acid sequence SEQ ID NO: 271.

[0057] The FN3 domain of the invention can bind to human Macaca Fascicularis and / or Pan troglodytes CD8A with a dissociation constant (KD) of less than about 1x10-7M, for example less than about 1x10-8M, less than about 1x10-9M, less than about 1x10-10M, less than about 1x10-11M, less than about 1x10-12M, or less than about 1x10-13M as determined by surface plasmonic resonance, as practiced by those skilled in the art. The measured affinity of a particular FN3 domain antigen interaction may vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, Kon, Koff) are made with standardized protein-antigen structure solutions and a standardized buffer. Petition 870260068423, dated 10 / 07 / 2026, page 37 / 201 23 / 165 as described in this document.

[0058] In some embodiments, the FN3 domains binding to CD8A comprise an initiator methionine (Met) attached to the N-terminus of the molecule.

[0059] In some embodiments, the FN3 domains binding to CD8A comprise a cysteine ​​(Cys) bound to the FN3 domain.

[0060] The addition of the N-terminal Met and / or Cys can facilitate the expression and / or conjugation of second molecules.

[0061] Another embodiment of the invention is an isolated FN3 domain that specifically binds to human CD8A and in which the CD8A-specific FN3 domain does not activate CD8+ T cells in vitro. Activation of CD8+ T cells can be measured using standard methods. For example, the enzyme-linked immunospot assay (ELISPOT) can be used. The ELISPOT assay employs the enzyme-linked immunosorbent assay (ELISA) sandwich technique. The interferon gamma antibody is pre-coated onto a PVDF (polyvinylidene difluoride)-coated microplate. Appropriately stimulated cells (cells + peptides, FN3 domains, etc.) are pipetted into the wells and the microplate is placed in a humidified CO2 incubator at 37°C for a specified period of time. During this incubation period, the immobilized interferon gamma antibody remains in the immediate vicinity of the cells. Petition 870260068423, dated 10 / 07 / 2026, page 38 / 201 24 / 165 secretory cells, binds to secreted interferon gamma. After washing away all unbound cells and substances, a second biotinylated interferon-gamma antibody is added to the wells. After washing to remove any unbound biotinylated antibody, streptavidin-conjugated alkaline phosphatase is added. The unbound enzyme is subsequently removed by washing and a substrate solution (BCIP / NBT) is added. A dark blue precipitate forms and appears as spots at the interferon gamma localization sites, with each individual spot representing an individual interferon-gamma secreting cell. The spots can be counted with an ELISpot automated 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 concentrations of 1 as described in the EXAMPLES.

[0062] In some embodiments of the invention described in this document, the isolated FN3 domain comprises the amino acid sequence SEQ ID NOs: 40-269.

[0063] In some embodiments of the invention described in this document, the specific FN3 domain of CD8A has a cysteine ​​substitution at residue position 54 of SEQ IDs 79, 81, 83, 89, 122 and 68.

[0064] The substitutions that result in the introduction Petition 870260068423, dated 10 / 07 / 2026, p. 39 / 201 25 / 165 of cysteine ​​in a protein sequence can be used to chemically conjugate small molecules, such as cytotoxic agents, detectable markers, half-life extension molecules, chelating agents, polyethylene glycol, and / or nucleic acids to the FN3 domain using standard chemistry.

[0065] In some embodiments, the FN3 domain that specifically binds to human CD8A competes for binding to human CD8A with the FN3 domain of SEQ ID NOS: 229-234. FN3 domains can be evaluated for their competition with a reference molecule for human CD8A binding using well-known in vitro methods. In one exemplary method, HEK cells recombinantly expressing human CD8A can be incubated with an 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 can be measured by flow cytometry using standard methods. In another exemplary method, the extracellular portion of human CD8A can be coated onto the surface of an ELISA plate. Excess unlabeled reference molecule can be added over approximately 15 minutes, and subsequently the FN3 test domains... Petition 870260068423, dated 10 / 07 / 2026, page 40 / 201 26 / 165 biotinylated compounds can be added. After washing in PBS / Tween, the binding of the biotinylated FN3 domain in the test can be detected using horseradish peroxidase-conjugated streptavidin (HRP), and the signal detected using conventional methods. It is readily apparent that in competition assays, the reference molecule can be labeled and the test FN3 domain unlabeled. The test FN3 domain can compete with the reference molecule when the reference molecule inhibits the binding of the test FN3 domain, or the test FN3 domain inhibits the binding of the reference molecule by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%.

[0066] In some embodiments, the isolated FN3 domain that specifically binds to human CD8A of the invention conjugated with a chelator that can bind to a radioactive metal can be used as an imaging agent to assess tumor distribution, diagnosis for the presence of CD8-T cells of internal tumors and / or cancer treatment efficacy.

[0067] In some modalities, the specific FN3 domains of CD8A are removed from the blood through renal and / or hepatic clearance. Isolation of FN3 domains for CD8A binding from a Tencon sequence-based library. Petition 870260068423, dated 10 / 07 / 2026, p. 41 / 201 27 / 165

[0068] 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; Publ. Pat. US No. 2010 / 0216708). The crystal structure of Tencon shows six surface-exposed loops connecting seven beta strands, as is characteristic of 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; Pat. US No. 6,673,901). These loops, or selected residues within each loop, can be randomized to construct libraries of fibronectin type III (FN3) domains that can be used to screen new molecules that bind to CD8A. Table 1 shows the positions and sequences of each loop and beta chain in Tencon (SEQ ID NO: 1).

[0069] The library designed based on the Tencon sequence can thus have a random FG loop, or randomly BC and FG loops, such as TCL1 or TCL2 libraries as described below. The Tencon BC loop is 7 amino acids long, so 1, 2, 3, 4, 5, 6 or 7 amino acids can be randomized in the diversified library in the BC loop and designed based on the Tencon sequence. The loop of Petition 870260068423, dated 10 / 07 / 2026, page 42 / 201 28 / 165 Tencon FG is 7 amino acids long, so 1, 2, 3, 4, 5, 6, or 7 amino acids can be randomized in the diversified library in the FG loop and designed based on the Tencon sequence. Further diversity of loops in Tencon libraries can be achieved by inserting and / or deleting residues in the loops. For example, FG and / or BC loops can be extended by 1-22 amino acids, or shortened by 1-3 amino acids. The FG loop in Tencon is 7 amino acids long, while the corresponding loop in antibody heavy chains ranges from 4 to 28 residues. To provide maximum diversity, the FG loop can be diversified in sequence as well as length to match the antibody CDR3 length range of 4-28 residues. For example, the FG loop can be further diversified in length by extending the loop by an additional 1, 2, 3, 4, or 5 amino acids.

[0070] A library designed based on the Tencon sequence may also have random alternative surfaces that form on one side of the FN3 domain and comprise two or more beta chains, and at least one loop. One such alternative surface is formed by amino acids in the beta chains C and F and in the loops CD and FG (a C-CD-F-FG surface). A library design based on the Tencon C-CD-F-FG alternative surface is described in Petition 870260068423, dated 10 / 07 / 2026, p. 43 / 201 29 / 165 Publ. Pat. US No. US2013 / 0226834. The library designed based on the Tencon sequence also includes libraries designed based on Tencon variants, such as Tencon variants possessing substitutions at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO: 1), and which variants exhibit improved thermal stability. Examples of Tencon variants are described in Publ. Pat. US No. 2011 / 0274623 and include Tencon27 (SEQ ID NO: 4) possessing the substitutions E11R, L17A, N46V, and E86I when compared to Tencon of SEQ ID NO: 1. Table 1. Tencon Topology FN3 Tencon Domain (SEQ ID NO: 1) Tape A 1-12 Loop AB 13-16 Tape B 17-21 Loop BC 22-28 Tape C 29-37 Loop CD 38-43 Tape D 44-50 Loop DE 51-54 Tape E 55-59 Petition 870260068423, dated 10 / 07 / 2026, p. 44 / 201 30 / 165 Loop EF 60-64 Tape F 65-74 Loop FG 75-81 Tape G 82-89

[0071] Tencon and other FN3 sequence-based libraries can be randomized at chosen residue positions using a random or defined set of amino acids. For example, variants in the library possessing random substitutions can be generated using NNK codons, which encode all 20 naturally occurring amino acids. In other diversification schemes, DVK codons can be used to encode the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys. Alternatively, NNS codons can be used to generate all 20 amino acid residues while simultaneously reducing the frequency of stop codons. FN3 domain libraries with amino acid distribution polarized at positions to be diversified can be synthesized, for example, using Slonomics® technology (http: / / www.sloning.com).This technology uses a library of pre-built double-stranded triplets that act as universal building blocks sufficient for thousands of gene synthesis processes. The triplet library represents all possible combinations. Petition 870260068423, dated 10 / 07 / 2026, p. 45 / 201 31 / 165 of the sequences needed to build any desired DNA molecule. The codon designations are in accordance with the well-known IUB code.

[0072] The FN3 domains that specifically bind to human CD8A of the invention can be isolated by producing the FN3 library, such as the Tencon library, using cis display to link DNA fragments encoding the support proteins to a DNA fragment encoding RepA to generate a pool of DNA-protein complexes formed after in vitro translation, in which 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 CD8A binding by any method known in the art and described in the Example. Well-known examples of methods that can be used include ELISA, sandwich immunoassays, and competitive and non-competitive assays (see, for example, Ausubel et al., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).The identified FN3 domains that specifically bind to CD8A are further characterized by their inhibition of CD8A activity, internalization, stability, and other desirable features.

[0073] The FN3 domains that bind specifically Petition 870260068423, dated 10 / 07 / 2026, page 46 / 201 The human CD8A of the invention can be generated using any FN3 domain as a template to generate a library and screening the library of molecules that specifically bind to human CD8a using methods provided within. Exemplary FN3 domains that can be used are the third 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 from the library, to express or translate the libraries in vitro. Ribosome display for example (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 (US Pat. No. 5,643,768) can be used. Libraries of FN3 domain variants can be expressed as fusion proteins displayed on the surface of, for example, any suitable bacteriophage. Methods for presenting fusion polypeptides on the surface of a bacteriophage are well known (Publ. Pat. US No. 2011 / 0118144; Publ. Pat. Int. No. WO2009 / 085462; Pat. US No. 6,969,108; Pat. US No. 6,172,197; Pat. US No. 5,223,409; Pat. US No. 6,582,915; Pat. Petition 870260068423, dated 10 / 07 / 2026, p. 47 / 201 33 / 165

[0074] In some embodiments of the invention described in this document, the FN3 domain that specifically binds to human CD8A is based on the Tencon sequence of SEQ ID NO: 1 or the Tencon27 sequence of SEQ ID NO: 4, optionally with substitutions at positions 11, 14, 17, 37, 46, 73 and / or 86 of the residues.

[0075] The FN3 domains that specifically bind to human CD8A of the invention can be modified to improve their properties, such as improving 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 with highly similar thermostable sequences, design of disulfide stabilizing bridges, mutations to increase alpha helix propensity, salt bridge engineering, alteration of protein surface charge, targeted evolution, and consensus sequence composition (Lehmann and Wyss, Curr Opin Biotechnol, 12, 371-375, 2001). High thermal stability can increase the yield of expressed protein, improve solubility or activity, decrease immunogenicity, and minimize the need for a cold chain in manufacturing.Waste products that can be substituted to improve the thermal stability of Tencon (SEQ ID NO:. Petition 870260068423, dated 10 / 07 / 2026, p. 48 / 201 34 / 165 1) are residue positions 11, 14, 17, 37, 46, 73 or 86, and are described in US Pat. Publ. No. 2011 / 0274623. The substitutions corresponding to these residues can be incorporated into the FN3 domain containing molecules of the invention.

[0076] Measuring protein stability and protein lability can be viewed as either the same or different aspects of protein integrity. Proteins are sensitive or labile to denaturation caused by heat, ultraviolet or ionizing radiation, changes in environmental osmolarity and pH if in liquid solution, mechanical shear force imposed by filtration of small portion sizes, ultraviolet radiation, ionizing radiation such as gamma irradiation, chemical or thermal dehydration, or any other action or force that may cause disruption of the protein structure. 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 temperature (Tm), the temperature in ° Celsius (°C) at which half of the molecules unfold, using standard methods. Typically, the higher the Tm, the more stable the molecule.In addition to heat, the chemical environment also alters the protein's ability to maintain a specific three-dimensional structure.

[0077] In one embodiment, the FN3 domains that connect Petition 870260068423, dated 10 / 07 / 2026, page 49 / 201 35 / 165 specifically the 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 before engineering, as measured by the increase in Tm.

[0078] Chemical denaturation can also be measured by a variety of methods. Chemical denaturants include guanidinium chloride, guanidinium thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitril), 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), nonionic and ionic detergents, acids (e.g., hydrochloric acid (HCl), acetic acid (CH3COOH), halogenated acetic acids), hydrophobic molecules (e.g., phospholipids) and specific denaturants.The quantification of the extent of denaturation can be based on the loss of a functional property, such as the ability to bind to a target molecule, or on physicochemical properties, such as a tendency to aggregate, exposure of previously inaccessible residues to the solvent, or the rupture or formation of disulfide bonds. Petition 870260068423, dated 10 / 07 / 2026, page 50 / 201 36 / 165

[0079] The FN3 domains of the invention can be generated as monomers, dimers or multimers, for example, as a means to increase the valence and thus the binding avidity of the target molecule, or to generate bi- or multispecific structures simultaneously by linking two or more different target molecules. Dimers and multimers can be generated by linking monospecific, bi- or multispecific protein structures, for example, by the inclusion of an amino acid linker, for example, a linker containing polyglycine, glycine and serine, or alanine and proline. Exemplary ligands 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)5AAA (SEQ ID NO: 142). Dimers and multimers can be linked to each other in an N-to-C direction.The use of natural and artificial peptide linkers to link polypeptides to new fusion-linked polypeptides is well known in the literature (Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer,). Biochemistry 35, 109-116, 1996; US Pat. No. 5,856,456). Diagnostic Agents

[0080] According to the invention, a specific FN3 domain of CD8A of the invention may comprise a marker. Petition 870260068423, dated 10 / 07 / 2026, page 51 / 201 37 / 165 detectable. In one embodiment, the detectable marker can be complexed with a chelating agent that is conjugated to the FN3 domain. In another embodiment, the detectable marker can be complexed with a chelating agent that is conjugated to a ligand that is conjugated to the FN3 domain. In yet another embodiment, the detectable marker can be coupled to a ligand that is conjugated to the FN3 domain. In yet another embodiment, a detectable marker can be indirectly linked to a peptide of the invention by the marker's ability to be specifically linked by a second molecule. An example of this type of indirectly linked marker is a biotin marker that can be specifically linked by the second molecule, streptavidin. Single, double, or multiple dialing can be advantageous. As used in this document, a detectable marker is any type of marker that, when linked to an FN3 domain of the invention, makes the FN3 domain detectable.A detectable marker can also be toxic to cells or cytotoxic. In general, detectable markers can include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescent quenching agents, colored molecules, radioisotopes, radionuclides, scintillators, massive labels such as a metallic atom (for detection via changes in...). Petition 870260068423, dated 10 / 07 / 2026, p. 52 / 201 38 / 165 mass), biotin, avidin, streptavidin, protein A, protein G, antibodies or their fragments, Grb2, polyhistidine, Ni2+, Flag markers, myc markers, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors / acceptors, acridinium esters, and colorimetric substrates. In one specific embodiment, the detectable marker is a radionuclide. Those skilled in the art would readily recognize other useful markers not mentioned above that may be employed in the operation of the present invention.

[0081] A detectable tracer emits a signal that can be detected by a signal transduction machine. In some cases, the detectable tracer may emit a signal spontaneously, such as when the detectable tracer is a radionuclide. In other cases, the detectable tracer emits a signal as a result of being stimulated by an external field, such as when the detectable tracer is a relaxivity metal. Examples of signals include, without limitation, gamma rays, X-rays, visible light, infrared energy, and radio waves. Examples of signal transduction machines include, without limitation, gamma cameras, including SPECT / CT devices, PET scanners, fluorimeters, and magnetic resonance imaging (MRI) machines. As such, a detectable tracer comprises a tracer that can be detected using Petition 870260068423, dated 10 / 07 / 2026, p. 53 / 201 39 / 165 magnetic resonance imaging, scintigraphic imaging, ultrasound or fluorescence.

[0082] Suitable fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near-infrared (NIR) fluorescent dyes (700-900 nm), 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, for example, Lohse et al., Bioconj Chem 8: 503-509 (1997)). For example, many known dyes are capable of being coupled to NH2-terminal amino acid residues. Alternatively, a fluorochrome such as fluorescein can be linked to a lysine residue of the peptide ligand.

[0083] A radionuclide can be a γ-emitting radionuclide, an electron-emitting radionuclide, a β-emitting radionuclide, an alpha-emitting radionuclide, or a positron-emitting radionuclide. A radionuclide can be a detectable tracer and / or a cytotoxic agent. Non-limiting examples of suitable radionuclides may include carbon-11, nitrogen-13, oxygen-15, fluorine-18, Petition 870260068423, dated 10 / 07 / 2026, page 54 / 201 40 / 165 fluorodeoxyglucose-18, phosphorus-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-121m, 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, forms of iridium-192, platinum-193 and 195m, gold-199, thallium-201, titanium-201, astatine-211, bismuth-212 and 213, lead-212, radium-223, actinium-225 and nitride or oxide derived from it. In one 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 one exemplary form, a radionuclide is zirconium-89.

[0084] A variety of metal atoms can be used as a detectable marker. The metal atom can usually be selected from the group of metal atoms made of metals with an atomic number of twenty or higher. For example, metal atoms can be atoms Petition 870260068423, dated 10 / 07 / 2026, p. 55 / 201 41 / 165 of calcium, scandium atoms, titanium atoms, vanadium atoms, chromium atoms, manganese atoms, iron atoms, cobalt atoms, nickel atoms, copper atoms, zinc atoms, gallium 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, atoms of tantalum, tungsten atoms, rhenium atoms, osmium 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, thulium atoms, ytterbium atoms, lutetium atoms, thorium atoms, protactinium atoms, uranium atoms, neptunium atoms, plutonium atoms, americium atoms, curium atoms, mendelenium atoms, mendelevium atoms, nobelium atoms, or lawrencium atoms. In some embodiments, the atoms of, Petition 870260068423, dated 10 / 07 / 2026, page 56 / 201 42 / 165 metals can be selected from the group comprising alkali metals with an atomic number greater than twenty. In other embodiments, metal atoms can be selected from the group comprising alkaline earth metals with an atomic number greater than twenty. In one embodiment, metal atoms can be selected from the group of metals comprising the lanthanides. In another embodiment, metal atoms can be selected from the group of metals comprising the actinides. In yet another embodiment, metal atoms can be selected from the group of metals comprising the transition metals. In yet another embodiment, metal atoms can be selected from the group of metals comprising the poor metals. In other embodiments, metal atoms can 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 from 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 yet another alternative embodiment, the metal atoms may be selected from the group consisting of metals that have a K-edge in the CT X-ray energy band. The preferred metal atoms... Petition 870260068423, dated 10 / 07 / 2026, p. 57 / 201 43 / 165 includes, but is not limited to, manganese, iron, gadolinium, gold, and iodine.

[0085] Metal atoms can be metal ions in the form of oxidation states +1, +2, or +3. For example, non-limiting examples include Ba2+, Bi3+, Cs+, Ca2+, Cr2+, Cr3+, Cr6+, Co2+, Co3+, Cu+, Cu2+, Cu3+, Ga3+, Gd3+, Au+, Au3+, Fe2+, Fe3+, F3+, Pb2+, Mn2+, Mn3+, Mn4+, Mn7+, Hg2+, Ni2+, Ni3+, Ag+, Sr2+, Sn2+, Sn4+, and Zn2+. The metal atoms may comprise a metal oxide. For example, non-limiting examples of metal oxides may include iron oxide, manganese oxide, or gadolinium oxide. Additional examples may include magnetite, magnesite, or a combination thereof.

[0086] According to the invention, an FN3 domain comprising a chelating agent can incorporate a radionuclide or a metal atom. The incorporation of the radionuclide or metal atom with an FN3 domain chelating agent complex can be achieved by several methods common in coordination chemistry techniques. Half-life extension halves

[0087] The FN3 domain that specifically binds to The human CD8A of the invention can incorporate other subunits, for example, through covalent interaction. In one aspect of the invention, the FN3 domain of the invention further comprises a half-life extending portion. Extending portions of Petition 870260068423, dated 10 / 07 / 2026, page 58 / 201 Examples of 44 / 165 half-lives include albumin, albumin variants, albumin-binding proteins and / or domains, transferrin and fragments and their analogues, and Fc regions.

[0088] Additional moieties may be incorporated into the FN3 domain of the invention, such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20000, 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 the desired properties. These moieties may be direct fusions with the protein structure coding sequences and may be generated by conventional cloning and expression techniques. Alternatively, well-known chemical coupling methods may be used to link the moieties to recombinantly produced molecules of the invention.

[0089] A PEGI moiety can, for example, be added to the FN3 domain of the invention by incorporating a cysteine ​​residue at the C-terminus of the molecule, or engineered cysteines at residue positions that extend away from the human CD8A binding face of the molecule and Petition 870260068423, dated 10 / 07 / 2026, page 59 / 201 45 / 165 linking a PEGI group to cysteine ​​using well-known methods.

[0090] The FN3 domain of the invention incorporating additional portions can be compared for functionality by several well-known assays. For example, altered properties due to the incorporation of Fc domains and / or variants of the Fc domain can be tested in Fc receptor binding assays using soluble forms of the receptors, such as FcyRI, FcyRII, FcyRIII or FcRn receptors, or using well-known cellulosic cells in assays that measure, for example, ADCC or CDC, or evaluating the pharmacokinetic properties of the molecules of the invention in in vivo models. Polynucleotides, Vectors and Host Cells

[0091] The invention provides nucleic acids encoding the FN3 domains that specifically bind to 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, prokaryote-compatible vectors, eukaryotic or filamentous phage expression, secretion and / or presentation of the compositions or their targeted mutagens. Certain exemplary polynucleotides are disclosed in this document, however, Petition 870260068423, dated 10 / 07 / 2026, page 60 / 201 46 / 165 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.

[0092] One embodiment of the invention is an isolated polynucleotide encoding the FN3 domain that specifically binds to human CD8A comprising the amino acid sequence SEQ ID NOS: 40-269.

[0093] The polynucleotides of the invention can be produced by chemical synthesis, such as solid-phase polynucleotide synthesis in an automated polynucleotide synthesizer, and assembled into complete single- or double-stranded molecules. Alternatively, the polynucleotides of the invention can be produced by other techniques, such as PCR, followed by routine cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art.

[0094] 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 that facilitates binding to RepA, and the like. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids that encode, for example, a Petition 870260068423, dated 10 / 07 / 2026, p. 61 / 201 47 / 165 marker or 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 coat protein or bacteriophage such as pIX or pIII.

[0095] 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 introducing the polynucleotides of the invention into a given organism or gene pool by any means. Such vectors may be expression vectors comprising nucleic acid sequence elements that can control, regulate, cause, or permit the expression of a polypeptide encoded by such a vector. Such elements may comprise transcription stimulator binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of polypeptides encoded in a given expression system. Such expression systems may be cell-based or cell-free systems well known in the art.

[0096] Another embodiment of the invention is a host cell comprising the vector of the invention. The FN3 domain Petition 870260068423, dated 10 / 07 / 2026, p. 62 / 201 The 48 / 165 antibody that specifically binds to human CD8A of the invention can optionally be produced by a single cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as is well known in the art. See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001).

[0097] 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, HeG2, SP2 / 0, HeLa, myeloma, lymphoma, yeast, insect, or plant cells, or any derivative, immortalized, or transformed of their cells. Alternatively, the host cell may be selected from a species or organism incapable of glycosylation of polypeptides, for example a prokaryotic cell or organism, such as BL21, BL21(DE3), BL21GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3), and any of the natural strains of E. coli spp., Klebsiella spp., or Petition 870260068423, dated 10 / 07 / 2026, page 63 / 201 49 / 165 strains of Pseudomonas spp.

[0098] Another embodiment of the invention is a method of producing the isolated FN3 domain that specifically binds to human CD8A of the invention, comprising culturing the isolated host cell of the invention under conditions such that the isolated FN3 domain that specifically binds to human CD8A is expressed and purifying the FN3 domain.

[0099] The FN3 domain that specifically binds to human CD8A can 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 Detection of Human CD8A

[0100] Kits are provided for the detection of CD8A in a biological sample. These kits include one or more of the CD8A-specific FN3 domains described in this document and instructions for using the kit.

[0101] The CD8A-specific FN3 domain provided may be in solution; lyophilized; attached to a substrate, Petition 870260068423, dated 10 / 07 / 2026, page 64 / 201 50 / 165 carrier or plate; or marked in a detectable manner.

[0102] The kits described may also include additional components useful for performing the methods described in this document. By way of example, the kits may comprise means for obtaining a sample from a subject, a control or reference sample, for example, a sample from a subject with slow-progressing cancer and / or a subject without cancer, one or more sample compartments, and / or instructional material describing the performance of a method of the invention and specific tissue controls or patterns.

[0103] The means for determining the CD8A level may also include, for example, buffers or other reagents for use in an assay to determine the CD8A level. Instructions may be, for example, printed instructions for performing the assay and / or instructions for assessing the CD8A level.

[0104] The kits described may also include means for isolating a sample from a subject. These means may comprise one or more items of equipment or reagents that can be used to obtain a fluid or tissue sample from a subject. The means for obtaining a sample from a subject may also comprise means for isolating blood components, Petition 870260068423, dated 10 / 07 / 2026, page 65 / 201 51 / 165 such as serum, from a blood sample. Preferably, the kit was designed for use with a human subject. Uses of the FN3 domains for binding to human CD8A of the invention

[0105] The FN3 domains that specifically bind to human CD8A of the invention can be used to diagnose human disease or specific pathologies in cells, tissues, organs, fluids, or generally, a host, using CD8A as a biomarker. The methods of the invention can 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. MODALITIES

[0106] The invention also provides the following non-limiting embodiments. 1) Isolated FN3 domain that specifically binds to a human CD8A protein comprising the amino acid sequence SEQ ID NO: 35. 2) Isolated FN3 domain from modality 1, wherein the FN3 domain cross-reacts with the monkey-cynomolgus CD8A protein comprising the amino acid sequence of SEQ ID NO: 271. Petition 870260068423, dated 10 / 07 / 2026, page 66 / 201 52 / 165 3) The FN3 domain isolated from modality 2, in which a) The FN3 domain is based on a Tencon sequence of SEQ ID NO: 1; b) the FN3 domain is based on a Tencon27 sequence from SEQ ID NO: 4; and / or c) the isolated FN3 domain of the library comprising the sequence of SEQ IDs NOs: 2, 3, 5, 6, 7 or 8. 4) The isolated FN3 domain of embodiment 3, in which the FN3 domain is conjugated with a second molecule. 5) The isolated FN3 domain of modality 4, in which the second molecule is a detectable label. 6) The isolated FN3 domain of modality 5, in which the detectable labeling is a radioactive isotope, magnetic spheres, metallic spheres, colloidal particles, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, digoxigenin, or hapten. 7) The isolated FN3 domain of modality 3, in which the FN3 domain has a cysteine ​​substitution at residue position 54 corresponding to SEQ IDs 79, 81, 83, 89, 122 and 68. 8) The isolated FN3 domain of modality 3, in which the FN3 domain does not activate CD8+ T cells in vitro, as measured by the enzyme-linked immunospot assay (ELISPOT). Petition 870260068423, dated 10 / 07 / 2026, page 67 / 201 53 / 165 9) The isolated FN3 domain of modality 3, in which the FN3 domain competes for binding to the human CD8A protein with the FN3 domain comprising the amino acid sequence of one of the SEQ ID NOS: 229-234. 10) The isolated FN3 domain of modality 3, in which the FN3 domain binds to human CD8A protein with an affinity (KD) between about 0.02 and about 6.6 nM measured by surface plasmon resonance performed according to the conditions described in Example 3. 11) The isolated FN3 domain of modality 3, wherein the FN3 domain comprises any of the amino acid sequences of SEQ ID NOs: 40-269. 12) The isolated FN3 domain of embodiment 11, which also includes a methionine at the N-terminus of the FN3 domain. 13) The FN3 domain isolated from modality 3, where the FN3 domain is coupled to a fraction that extends the half-life. 14) The isolated FN3 domain of modality 13, in which the half-life extension fraction is a CD8-binding molecule, a polyethylene glycol (PEG), CD8, a CD8 variant, or at least a portion of an Fc region of an immunoglobulin. 15) An isolated polynucleotide that encodes the FN3 domain of modality 3. Petition 870260068423, dated 10 / 07 / 2026, page 68 / 201 54 / 165 16) A vector comprising the polynucleotide of modality 15. 17) An isolated host cell comprising the vector of modality 16. 18) A method for producing an FN3 domain that specifically binds to a human CD8A protein, comprising culturing the isolated host cell of modality 17 under conditions such that the FN3 domain is expressed and purifying the FN3 domain. 19) A diagnostic kit comprising the FN3 domain of modality 3. 20) A diagnostic or capture agent comprising the FN3 domain of modality 3. 21) The diagnostic or capture agent of modality 20, in which the FN3 domain has a cysteine ​​substitution at residue position 54 of SEQ IDs 79, 81, 83, 89, 122 and / or 68. 22) The diagnostic agent of modality 21, in which the substituted cysteine ​​is conjugated with Zr-89 or I-124. 23) A method for detecting CD8-expressing cells in a biological sample comprising treating the biological sample with the diagnostic reagent of modality 20, and evaluating the binding of the biological sample to the FN3 domain of that diagnostic agent. Petition 870260068423, dated 10 / 07 / 2026, p. 69 / 201 55 / 165 24) The method of modality 23, in which the diagnostic agent has a cysteine ​​substitution at residue position 54 of SEQ ID NOs 79, 81, 83, 89, 122 and / or 68 and the substituted cysteine ​​is conjugated to Zr-89 or I-124. EXAMPLES

[0107] The following examples are provided to supplement the previous disclosure and to provide a better understanding of the subject matter described in this document. These examples should not be considered to limit the subject matter described. It is understood that the examples and embodiments described in this document are for illustrative purposes only and that various modifications or alterations thereof will be apparent to those skilled in the art and should be included within and may be made without departing from the true scope of the invention. EXAMPLE 1. BUILDING TENCON LIBRARIES WITH RANDOMIZED LOOPS

[0108] Tencon (SEQ ID NO: 1) is an immunoglobulin-like scaffold, a type III fibronectin (FN3) domain, designed from a consensus sequence of fifteen human tenascin-C FN3 domains (Jacobs et al., Protein Engineering, Design and Selection 25: 107-117, 2012; Pat. No. 8,278,419). The crystal structure of Tencon shows six surface-exposed loops connecting seven beta strands. Petition 870260068423, dated 10 / 07 / 2026, page 70 / 201 56 / 165 These loops, or selected residues within each loop, can be randomized to construct libraries of fibronectin type III (FN3) domains that can be used to screen new molecules that bind to specific targets.

[0109] Tencon: LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSER SYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO 1):

[0110] Several libraries were generated using the tencon scaffolding and various design strategies. In general, the TCL1 and TCL2 libraries produced good folders. The generation of TCL1 and TCL2 libraries is described in detail in Publ. Int. Pat. NoWO2014081944A2. TCL1 Library Construction

[0111] A library designed to randomize only the FG loop of Tencon (TQ1, SEQ ID NO: 1) 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 is produced that incorporates sequences for a Tac promoter, a coding sequence from the Tencon library, a RepA coding sequence, a cis element, and an ori element. After expression in an in vitro transcription / translation system, a Tencon-RepA fusion protein complex is produced. Petition 870260068423, dated 10 / 07 / 2026, page 71 / 201 57 / 165 in cis to the DNA that encodes it. Complexes that bind to a target molecule are then isolated and amplified by polymerase chain reaction (PCR), as described below.

[0112] The construction of the TCL1 library for use with the cis presentation was achieved through successive rounds of PCR is used 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 digestion to produce the entire construct. The TCL1 library was designed to incorporate random amino acids only in the Tencon FG loop, 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 a termination codon in the FG loop. The TCL1 library contains six separate sub-libraries, each with a different randomized FG loop length, from 7 to 12 residuals, in order to further increase diversity.

[0113] TCL1 Library (SEQ ID NO: 2) LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSER SYDLTGLKPGTEYTVSIYGVX7-12PLSAEFTT; in what Petition 870260068423, dated 10 / 07 / 2026, p. 72 / 201 58 / 165 Xi, X2, X3, X4, X5, X6, X7 are any amino acids; and X8, X9, X10, X11, and X12 are any amino acids that are excluded. Construction of the TCL2 Library

[0114] The TCL2 library was constructed in which both BC and FG loops of Tencon were randomized and the amino acid distribution at each position was strictly controlled. Table 2 shows the amino acid distribution at the desired loop positions in the TCL2 library. The designed amino acid distribution had two objectives. First, the library was skewed towards residues that were predicted to be structurally important for Tencon folding and stability based on Tencon crystal structure analysis and / or homology modeling. For example, position 29 was fixed to be only a subset of hydrophobic amino acids, since this residue was buried in the hydrophobic core of the Tencon fold.A second design layer involved polarizing the amino acid distribution relative to that of residues preferentially found in the HCDR3 heavy chain of antibodies, to efficiently produce high-affinity ligands (Birtalan et al., J Mol Biol 377:151828, 2008; Olson et al., Protein Sci 16:476-84, 2007). For this purpose, the “designed distribution” in Table 1 refers to... Petition 870260068423, dated 10 / 07 / 2026, page 73 / 201 59 / 165 corresponds to the following distribution: 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 lacks methionine, cysteine, and STOP codons.

[0115] TCL2 Library (SEQ ID NO: 3) LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVGEAINLT VPGSERSYDLTGLKPGTEYTVSIYGVX9X10X11X12X13SX14X15LSAEFTT; in which X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr orVal; X3 Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr orVal; X4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile,Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr orVal; X5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, lie,Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr orVal; X6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, lie,Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr orVal; X7 is Phe, Ile, Leu, Val or Tyr; Petition 870260068423, of 10 / 07 / 2026, p. 74 / 201 60 / 165 Xg is Asp, Glu or Thr; X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X10 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; Xiié Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X12 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X13 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X14 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and X15 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val. Table 2. Position of Waste WT Distribution in the TCL2 Library Waste * 22 T projected distribution 23 A projected distribution Projected distribution of 50% P 24 P + 25 D projected distribution Petition 870260068423, dated 10 / 07 / 2026, page 75 / 201 61 / 165 26 A Projected distribution of 20% A + 20% G + 27 A Projected distribution 28 F 20% F, 20% IY, 20% L, 20% V, 20% 29 D 33% D, 33% E, , 33% T 75 K Projected distribution 76 G Projected distribution 77 G Projected distribution 78 H Projected distribution 79 R Projected distribution 80 S 100% S 81 N Projected distribution 82 P Projected distribution of 50% P * Waste numbering is based on the Tencon sequence of SEQ ID NO: 1

[0116] Subsequently, these libraries were improved in several ways, including building the libraries on a stabilized Tencon framework (US Pat. No. 8,569,227) that incorporates the E11R / L17A / N46V / E86I substitutions (Tencon27; SEQ ID NO: 4) when compared to the wild-type tencon, as well as altering the randomized positions in the BC and FG loops. Tencon27 is described in Int. Ped. Pat. No. WO2013049275. From this, new libraries designed to randomize only the FG loop of the Tencon were developed. Petition 870260068423, dated 10 / 07 / 2026, p. 76 / 201 62 / 165 (TCL9 library), or a combination of the BC and FG loops (TCL7 library) were generated. These libraries were built for use with the cis display system (Odegrip et al., Proc Natl Acad Sci USA 101: 2806-2810, 2004). Details of this design are shown below: Stabilized Tencon (Tencon27) (SEQ ID NO: 4) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT TCL7 (loops FG and BC randomizados) (SEQ ID NO: 5) LPAPKNLVVSRVTEDSARLSWX1X2X3X4X5X6X7X8X9FDSFLIQYQESEKVGEAI VLTVPGSERSYDLTGLKPGTEYTVSIYGVX10X11X12X13X14X15X16X17X18X19SNPLSA IFTT; em que X1, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y;e X7, X8, X9, X17, X18 e N, P, Q, R, S, T, V, W, Y or included. TCL9 (loop FG randomizado) (SEQ ID NO: 6) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGV X1X2X3X4X5X6X7X8X9 X10X11X12SNPLSAIFTT; X1, X2, X3, X4, X5, X6 e N, P, Q, R, S, T, V, W ou Y;e X8, R, S, T, V, W, Y or others. Petition 870260068423, dated 10 / 07 / 2026, page 77 / 201 63 / 165

[0117] For the construction of the library, DNA fragments encoding randomized BC loops (positions of lengths 6-9) or FG loops (positions of lengths 7-12) were synthesized using Slonomics technology (Sloning Biotechnology GmbH) to control the amino acid distribution of the library and to eliminate stop codons. Two different sets of DNA molecules that randomized either the BC loop or the FG loops were synthesized independently and then combined using PCR to produce the complete library product. Building FG loop libraries (TCL9)

[0118] A set of synthetic DNA molecules consisting of a 5' Tac promoter followed by the complete Tencon gene sequence was produced, with the exception of random codons in the FG loop (SEQ ID NOs: 26-31). For FG loop randomization, all amino acids except cysteine ​​and methionine were encoded in 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. Sublibraries of each length variation were synthesized individually on a 2 pg scale and then amplified by PCR using Sloning-FOR (SEQ ID NO: 9) and Sloning-Rev (SEQ ID NO: 10) oligos.

[0119] Fragment 3' of the library is a sequence Petition 870260068423, dated 10 / 07 / 2026, p. 78 / 201 64 / 165 of constant DNA containing display elements, including a PspOMI restriction site, the coding region of the repA gene, and 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 purified on a gel. To ligate the 5' portion of the library DNA to the 3' DNA containing the repA gene, 2 pmol (~540 ng to 560 ng) of 5' DNA was ligated to an equal molar (~1.25 pg) of 3' repA DNA in the presence of Notl enzyme and PspOMI and T4 ligase at 37°C overnight. The product of the linked library was amplified using 12 PCR cycles with the POP2250 (SEQ ID NO: 11) and DigLigRev (SEQ ID NO: 12) oligos. For each sub-library, the DNA resulting from 12 PCR reactions was combined and purified by Qiagen spin column. The throughput for each TCL9 sub-library ranged from 3234 pg. Building FG / BC Loop Libraries (TCL7)

[0120] The TCL7 library provides a library with randomized Tencon BC and FG loops. In this library, BC loops of 6-9 amino acid lengths were combinatorially mixed with randomized FG loops of 7-12 amino acid lengths. The synthetic fragments of Petition 870260068423, dated 10 / 07 / 2026, page 79 / 201 65 / 165 Tencon BC6, BC7, BC8, and BC9 (SEQ ID NO: 13-16) were engineered to include the Tencon gene encoding for the N-terminal portion of the protein up to and including the residue. VX such that the BC loop is replaced with 6, 7, 8 or randomized amino acids. These fragments were synthesized before the discovery of the L17A, N46V and E83I (CEN5243) mutations, but these mutations were introduced in the molecular biology steps described below. In order to combine this fragment with fragments encoding randomized FG loops, the following steps were taken.

[0121] First, a DNA fragment encoding the Tac promoter and the 5' Tencon sequence up to the nucleotide encoding amino acid A17 (130mer-L17A, SEQ ID N17) was produced by PCR using POP2222ext (SEQ ID N18) and LS1114 (SEQ ID NO: 19) oligos. This was done to include the L17A mutation in the library (CEN5243). Next, DNA fragments encoding Tencon residues R18-V75, including randomized BC loops, were amplified by PCR using BC6, BC7, BC8, or BC9 as templates and LS1115 (SEQ ID N20) and LS1117 (SEQ ID N21) oligos. This PCR step introduced a BsaI site at the 3' end. These DNA fragments were subsequently ligated by overlapping PCR using POP2222ext and LS1117 oligos as primers. The resulting 240 bp PCR product was Petition 870260068423, dated 10 / 07 / 2026, p. 80 / 201 66 / 165 DNA was pooled and purified using the Qiagen PCR purification kit. The purified DNA was digested with BsaI-HF and purified on a gel.

[0122] The fragments encoding the FG loop were amplified by PCR using FG7 (SEQ ID N31), FG8 (SEQ ID N30), FG9 (SEQ ID N29), FG10 (SEQ ID N28), FG11 (SEQ ID NO: 27) and FG12 (SEQ ID NO: 26) as templates with the oligonucleotides SDG10 (SEQ ID NO: 22) and SDG24 (SEQ ID NO: 23) to incorporate a BsaI restriction site and N46V and E86I variations (CEN5243).

[0123] Targeted BC fragments and FG fragments were ligated together in a single step using a 3-way ligation. Four ligation reactions out of 16 possible combinations were established, with each ligation reaction combining two BC loop lengths with two FG loop lengths. Each ligation contained ~300 ng of the total BC fragment and 300 ng of the FG fragment. These 4 ligation sets were then amplified by PCR using POP2222 (SEQ ID NO: 24) and SDG28 (SEQ ID NO: 25) oligos. 7.5 pg of each reaction product were then digested with Not1 and cleaned with a Qiagen PCR purification column. 5.2 pg 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 Petition 870260068423, dated 10 / 07 / 2026, p. 81 / 201 67 / 165 oligos POP2222. EXAMPLE 2: GENERATING TENCON.COM LIBRARIES ALTERNATIVE BONDING SURFACES

[0124] 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 a support protein selected to bind maltose-binding protein (MBP) from a library in which BC, DE, and FG loops were randomized showed it to have a broadly curved interface that fits the active site of MBP (Koide et al., Proc Natl Acad Sci USA 104: 6632-6637, 2007). In contrast, an ankyrin repeat support protein that was selected to bind MBP was found to have a much flatter interaction surface and to bind the outer surface of MBP distant from the active site (Binz et al., Nat Biotechnol 22: 575-582, 2004). These results suggest that the shape of the binding surface of a support molecule (curved vs.The planar structure of the scaffold can dictate which target proteins or specific epitopes on those target proteins can be effectively bound by the scaffold. Published efforts surrounding engineered protein scaffolds containing FN3 domains for protein binding have relied on the projection of adjacent loops for target binding, producing... Petition 870260068423, dated 10 / 07 / 2026, page 82 / 201 68 / 165 thus curved linking surfaces. This approach may limit the number of targets and epitopes accessible by such scaffolding.

[0125] Tencon and other FN3 domains contain two sets of CDR-type loops located on opposite faces of the molecule, the first set formed by loops BC, DE, and FG, and the second set formed by loops AB, CD, and EF. The two sets of loops are separated by the beta strands that form the center of the FN3 structure. If the Tencon image is rotated 90 degrees, an alternative surface can be visualized. This slightly concave surface is formed by loops CD and FG and two antiparallel beta strands, beta strands C and F, and is referred to in this document as the C-CD-F-FG surface. The C-CD-F-FG surface can be used as a template for designing libraries of protein structure interaction surfaces by randomizing a subset of residues that form the surface. The beta strands have a repetitive structure with the side chain of all other residues exposed to the protein surface.Thus, a library can be made up of some or all of the residues exposed to the surface on the beta strips. By choosing the appropriate residues on the beta strips, the inherent stability of the Tencon framework should be minimally compromised, while still providing a surface. Petition 870260068423, dated 10 / 07 / 2026, p. 83 / 201 69 / 165 unique for interaction with other proteins.

[0126] The TCL14 library (SEQ ID NO: 7) was designed to support Tencon27 (SEQ ID NO: 4).

[0127] A full description of the methods used to build this library is described in Publ. Pat. US No. 2013 / 0226834. TCL14 Library (SEQ ID NO: 7): LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIVLTV PGSERSYDLTGLKPGTEYX8VX9IX10GVKGGX11X12SX13PLSAIFTT; in what X1, X2, X3, X4, X5, X6, X10, X11, X12, and

[0128] The two beta strands that form the CCD-F-FG surface in Tencon27 have a total of 9 surface-exposed residues that can be randomized; Strand C: 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 have 7 potential residues: K75, G76, G77, H78, R79, S80, and N81. The selected residuals were chosen for inclusion in the TCL14 design due to the larger theoretical library size if all 22 residuals were randomized.

[0129] Thirteen positions in Tencon were chosen for randomization: L32, Q34 and Q36 in chain C, S38, E39, K40 Petition 870260068423, dated 10 / 07 / 2026, p. 84 / 201 70 / 165 and V41 in the CD loop, T68, S70, and Y72 in the F strand, H78, R79, and N81 in the FG loop. In the C and F strands, S30 and E66 were not randomized as they are slightly beyond the CD and FG loops and do not appear to be as clearly 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 is located at the surface junction. The FG loop had K75, G76, G77, and S80 excluded. Glycines were excluded for the reasons stated 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 loop and was a less attractive candidate for randomization.Although the aforementioned residuals 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 specific target of the selected TCL14 target.

[0130] Following the production of TCL14, 3 additional Tencon libraries of similar design were produced. These two libraries, TCL19, TCL21 and TCL23, are randomized in the same positions as TCL14 (see above), however the amino acid distribution that occurs in these Petition 870260068423, dated 10 / 07 / 2026, page 85 / 201 71 / 165 positions is altered (Table 3). TCL19 and TCL21 were designed to include an equal distribution of 18 natural amino acids at each position (5.55% each), excluding only cysteine ​​and methionine. TCL23 was designed such that each randomized position approximates the amino acid distribution found in the HCDR3 loops of functional antibodies (Birtalan et al., J Mol Biol 377: 1518-1528, 2008), as described in Table 3. As in the TCL21 library, cysteine ​​and methionine were excluded.

[0131] A third additional library was created to expand the potential target linkage surface of the other library of libraries. In this library, TCL24, 4 additional Tencon positions were randomized compared to the TCL14, TCL19, TCL21, and TCL23 libraries. These positions include N46 and T48 of the D strand and S84 and I86 of the G strand. Positions 46, 48, 84, and 86 were chosen in particular because the side strands of these residues are exposed to the surface from the beta D and G chains and are structurally adjacent to random portions of the C and F chains, thus increasing the surface area accessible for binding to target proteins. The amino acid distribution used at each position for TCL24 is identical to that described for TCL19 and TCL21 in Table 3. Petition 870260068423, dated 10 / 07 / 2026, p. 86 / 201 72 / 165

[0132] TCL24 Library (SEQ ID NO: 8) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9 VPGSERSYDLTGLKPGTEYX10VX11IX12GVKGGX13X14SX15PLX16AX17FTT; where X1, X2, X3, X4, X5, X6, X10, X11, Table 3. Frequency of amino acids (%) at each random 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 Petition 870260068423, dated 10 / 07 / 2026, page 87 / 201 73 / 165 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

[0133] The TCL21 library was generated using Colibra (Isogenica) library technology to control amino acid distributions. Fragments of the TCL19, TCL23, and TCL24 genes were generated using Slonomics (Morphosys) technology to control amino acid distributions. PCR was used to amplify each library after initial synthesis followed by gene ligation for RepA so that it could 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 loop libraries. EXAMPLE 3: SELECTION OF TYPE III FIBRONECTIN (FN3) DOMAINS THAT BIND TO CD8A Design and production of human CD8 alpha antigens:

[0134] Two constructs of human CD8 alpha (Swiss Prot P01732) were expressed and purified from HEK cells to produce recombinant protein for the Petition 870260068423, dated 10 / 07 / 2026, page 88 / 201 74 / 165 selection CIS-Display (Table 4). Table 4: CD8A constructs generated for use as antigens Construct SEQ ID NO: Description CD8W7 35 Human CD8 alpha residues 22-167 fused with the Fc fragment of human IgG1 CD8W13 36 Human CD8 alpha residues 22-182 fused with the Fc fragment of human IgG1

[0135] 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 (SEQ ID NO. 4). The CD8 alpha and Fc fragment sequences were linked by a linker containing a signaling marker sequence and polyhistidine (SEQ ID NO. 5).

[0136] Plasmids encoding these proteins were transfected into HEK 293-Expi cells by transient transfection, and culture supernatants were collected by centrifugation at 6000 xg and clarified with a 0.2 micron filter. The supernatants were loaded onto a HiTrap Mabsure Select column (GE Healthcare), and the CD8A proteins were eluted in 0.1 M Na-Acetate pH 3.5 and neutralized by the addition of 2 M Tris at pH 7. Each sample was then dialyzed to PBS pH 7.4 for biotinylation using a biotinylation kit. Petition 870260068423, dated 10 / 07 / 2026, page 89 / 201 75 / 165 No Weigh EZ-LinkSulfo-NHS-LC-Biotin (Thermo Scientific). Library screening

[0137] 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 DNA from the library were incubated with 0.1 mM complete amino acids, 1X 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, IL), 100 mg / mL herring sperm DNA (Promega, Madison, WI), 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 magnetic neutrovidin beads (Thermo Fisher, Rockford, IL) (Rounds 1, 3, and 5) or magnetic streptavidin beads (Promega, Madison, WI) (Rounds 4 and 5), 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 screening rounds were performed to identify molecules of... Petition 870260068423, dated 10 / 07 / 2026, page 90 / 201 76 / 165 scaffold with improved affinities. In summary, the productions 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 aim of these changes was to simultaneously select ligands with a potentially faster transfer rate and a slower transfer rate, resulting in a substantially lower Kd.

[0138] After filtration, selected FN3 domains were amplified by PCR using Tcon6 (SEQ ID NO: 33) and Tcon5shortE8 6I (SEQ ID NO: 34) oligos, subcloned by annealing onto a pET15-LIC, and transformed into BL21-GOLD (DE3) cells (Agilent, Santa Clara, CA) for soluble expression in E. coli using standard molecular biology techniques. Individual clones were collected and grown to saturation in 1 mL of LB with ampicillin in 96 deep-well plates at 37°C. The following day, 25 pL were transferred to 1 mL of fresh LB-Amp medium in 96 deep-well plates and cultured at 37°C for 2 hours. IPTG was added at a final concentration of 1 mM, and protein expression was induced at 30°C for 16 hours. The cells were Petition 870260068423, dated 10 / 07 / 2026, page 91 / 201 77 / 165 cells were collected by centrifugation and subsequently lysed with Bugbuster HT (Merck Chemicals, Gibbstown, NJ) supplemented with a final 0.2 mg / mL of hen egg white lysozyme (Sigma-Aldrich, St. Louis, MO). Cells were collected 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

[0139] Neutravidin-coated plates were blocked for 1 hour in the T20 Initiation Block (Pierce) and then coated with biotinylated CD8W7 or CD8W13 (the same antigen as in panning) or negative control (human Fc) for 1 hour. The plates were rinsed with TBST and the diluted lysate was applied to the plates for 1 hour. After further washes, the wells were treated with HRP-conjugated anti-FN3 domain antibody (PAB25) for 1 hour and then assayed with POD (Roche). FN3 domain molecules with signals at least 10 times above the bottom were selected for further analysis. Small-scale expression and domain purification FN3 cells identified that bind to CD8A

[0140] Isolated clones of single hits Petition 870260068423, dated 10 / 07 / 2026, page 92 / 201 78 / 165 clones identified by biochemical linkage ELISA were combined into a single impact plate for growth in 96-well block plates; clones were grown in 1 mL cultures (LB medium supplemented with kanamycin for selection) at 37°C overnight with shaking. For protein expression in 96-well block plates, 1 mL of TB medium supplemented with kanamycin was inoculated with 50 µL of the overnight culture and grown at 37°C with continuous shaking at 300 rpm until ODgqq = 0.6-1. Once the target OD was reached, protein expression was induced with the addition of 1 mM IPTG; plates were transferred to 30 (300 rpm) for overnight growth. Cultures were centrifuged overnight to collect cells; bacterial pellets were stored at -80°C until ready for use.The pellets were lysed with BugBuster® HT lysis buffer (Novagen EMD Biosciences) and His-labeled centrins were purified from the lysates clarified 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. The purified samples were exchanged in PBS pH 7.4 for analysis using PD plates. MultiTrap™ G-25 (GE Healthcare). Size Exclusion Chromatography Analysis

[0141] Exclusion chromatography was used Petition 870260068423, dated 10 / 07 / 2026, page 93 / 201 79 / 165 size to determine the aggregation state of the anti-CD8 alpha FN3 domain molecules. Aliquots (10 pL) 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 PBS mobile phase pH 7.4. Column elution was monitored by absorbance at 280 nm. Wild-type Tencon was included in each run as a control. Agilent ChemStation software was used to analyze the elution profiles. Only proteins with elution profiles similar to that of the consensus tenascin protein in the same run were considered for further characterization. After filtration, 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 more than 10 times on the substrate and were free of aggregates by SEC (Table 5, SEQ ID No 40228 and 270). Table 5. Summary of FN3 CD8A binding domains identified from ELISA tests. SEQ Binding Binding Binding to Binding to ID aa T cells T cells NO: cells cells of TT cynomolgus cynomolgus ID human human clone kd (1 / s) 2 uM 0.2 uM Petition 870260068423, dated 10 / 07 / 2026, page 94 / 201 80 / 165 2 µM 0.2 µM (MFI) (MFI) (MFI) (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 Petition: 870260068423, on 10 / 07 / 2026, page. 95 / 201 81 / 165 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, Petition 870260068423, de 10 / 07 / 2026, pág. 96 / 201 82 / 165 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, Petition: 870260068423, on 10 / 07 / 2026, page. 97 / 201 83 / 165 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 Petition: 870260068423, on 10 / 07 / 2026, page. 98 / 201 84 / 165 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, Petition 870260068423, de 10 / 07 / 2026, pág. 99 / 201 85 / 165 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, Petition 870260068423, de 10 / 07 / 2026, pág. 100 / 201 86 / 165 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 Screening for T-cell binding from human donors and cynomolgus monkeys

[0142] The binding of 190 ELISA results to primary human and monkey cynomolgus T cells was assessed by flow cytometry. FN3 domain molecules were diluted to 2 μM and 0.2 μM in PBS and incubated with human or monkey cynomolgus CD8+ T cells in a 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 solution (PAB25). After this incubation, the cells were washed twice with PBS and a PE-conjugated secondary antibody and a viability dye were added. Finally, the cells were washed and resuspended in PBS for flow cytometric analysis using a BD Canto Instrument. Cells were blocked in live cells and the mean fluorescence intensity of bound Centirins (PE channel) was calculated using Cytobank software. The results are summarized in Table Petition 870260068423, dated 10 / 07 / 2026, page 101 / 201 87 / 165 5. Analysis of the dissociation rate of anti-human CD8 alpha centrins

[0143] The purified anti-CD8A FN3 domains were subjected to reduced rate analysis using a surface protein plasmon resonance instrument in order to select clones with the slowest dissociation rates for further characterization. Measured dissociation rates ranged from 2.64E-5 to 1.07E-2 sec-1, as shown in Table 5.

[0144] Goat anti-human Fc IgG (Jackson immunoresearch, Cat # 109-005-098) was directly immobilized on a GLC sensor chip at 10 pg / mL, pH 5.0 via amine coupling (pH 5.0) in all 6 ligand channels in the vertical orientation on the chip with a flow rate of 30 pg / min in PBST (PBS, 0.005% Tween). The immobilized GAG-Fc IgG densities averaged 6000 Response Units (RU) with less than 1% variation between the different channels. Internal human CD8A-Fc was captured in the vertical orientation at 3 different ligand densities, 10, 5, 2.5 pg / mL for 5 minutes at a flow rate of 30 pg / min. All FN3 domains were normalized to a concentration of 3 μM and tested for binding in the horizontal orientation. All 6 analyte channels were used for Petition 870260068423, dated 10 / 07 / 2026, page 102 / 201 88 / 165 FN3 domains were used to maximize screening yield. The dissociation phase was monitored for 15 minutes at a flow rate of 100 μL / min using PBST as running buffer. Surface regeneration was achieved by a short pulse of 0.85% phosphoric acid (contact time of 18s at 100 μL / min). Data analyses were performed using Bio-Rad ProteOn Manager software (version 3.1.0.6). Raw data were double-referenced by subtracting the interspot signals (empty chip surface, no immobilized or captured protein) to correct for non-specific FN3 domain binding to the pre-coated GAH-Fc IgG surface, followed by a double correction using empty L6 channel where no hCD8A-Fc was captured. The processed linkage data were locally fitted to a simple 1:1 Langmuir linkage model to extract the dissociation rate for each FN3 domain linkage to the captured hCD8A-Fc. Example 4: Engineering anti-CD8A domains FN3

[0145] A number of mutations were engineered into superior anti-CD8A candidates in order to eliminate post-translational modification risks of oxidation (methionine, or tryptophan), deamination (NS), isomerization (DG), and clipping (DP). Proline residues found in the beta chains were also mutated, since proline has a Petition 870260068423, dated 10 / 07 / 2026, page 103 / 201 89 / 165 potential to destabilize beta chains (Chiba T., et al. J Biol Chem. 2003;278:47016-24). Only residues derived from positions projected by the FN3 domain library were considered for mutation. Variant sequences were chosen to mimic similar chemical properties of the precursor molecule (e.g., tryptophan to tyrosine) or to replace the amino acid at risk of PTM with an amino acid found in other CD8A FN3 domains at that position. A complete list of projected sequences is found in Table 6. The dissociation rate between each mutant and recombinant CD8 alpha was measured by surface plasmon resonance to estimate the relative binding strengths. Table 6. Dissociation rates of CD8A centrin mutants. Mutants are grouped according to the parent molecule. Sample kd (1 / s) Mutations SEQ ID NO: P282DR9P1359_C5 1.47E-04 Parental 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 Petition 870260068423, dated 10 / 07 / 2026, p. 104 / 201 90 / 165 CD8S401 1.27E-02 W38I 265 CD8S400 2.26E-02 W38L 264 CD8S404 3.09E-02 P3 6A 268 P282DR9P1359 _F5 5.78E-05 Parental 79 CD8S371 1.94E-04 W48Y 235 CD8S377 4.00E-04 W81E 241 CD8S374 4.03E-04 W81Y 238 CD8S372 5.71E-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 Parental 89 CD8S390 1.52E-04 W68Y 254 CD8S389 1.84E-04 W68F 253 Petition 870260068423, dated 10 / 07 / 2026, page 105 / 201 91 / 165 CD8S391 3.20E-04 W68H 255 CD8S405 1.14E-03 P48T 269 P282DR9P1359_ F7 3.39E-04 Parental 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

[0146] From the data presented in Table 6 It is evident that a number of mutations that reduce developmental risks maintain dissociation rates similar to those of the parental molecule. The CD8S402 (DP site deletion), CD8S390 (Trp residue deletion), and CD8S403 (Pro removal from the beta strand) mutants resulted in slower dissociation rates than the appropriate parent molecule, indicative of stronger binding. Several other mutations maintain binding similar to the parent molecule and therefore may be preferred over the parent, since these molecules present fewer CMC-related risks during development. EXAMPLE 5: FN3 DOMAIN AFFINITY MEASUREMENTS CONNECTION TO CD8A.

[0147] Nineteen anti-CD8A candidates were selected for full kinetic analysis of binding to Petition 870260068423, dated 10 / 07 / 2026, page 106 / 201 92 / 165 Recombinant human CD8. These candidates were selected from previous positive results (Table 6) using the criteria of 1) strong binding relative to human T cells, 2) strong binding relative to cyno T cells, 3) minimal reduction in cell binding at 0.2 µM compared to 2 µM, 4) free of aggregates via SEC, 5) removal rate less than 2.07E-3 sec-1, 6) sequence diversity relative to sequence families, and 7) relative propensity for sequences with potential developability challenges (oxidation, deamidation, clipping, and hydrophobicity).

[0148] The affinities of the 19 best candidates, later replicated from the 6 main candidates, binding to hCD8A-Fc were measured on a ProteOn XPR36 instrument (BioRad) using GLC sensor chips under conditions similar to those of the Koff screening. The goat anti-human Fc antibody was immobilized directly on the chip by standard amine coupling at 10 pg / ml, pH 5.0 in all 6 ligand channels in the 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 in 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 an empty control channel for the surface of GAH-Fc IgG. Binding was measured by fluxing five different concentrations. Petition 870260068423, dated 10 / 07 / 2026, page 107 / 201 93 / 165 of the anti-CD8A FN3 domains (1 μM diluted in a 3-fold dilution series) were used as analytes simultaneously in a horizontal orientation on the captured hCD8A-Fc surfaces, with a sixth analyte channel containing only PBST running buffer. All interactions were measured at a flow rate of 100 μL / min with association and dissociation times of 4 and 30 minutes, respectively. Ligand surface regeneration was achieved by a short pulse of 0.85% phosphoric acid (18 s contact time at 100 μL / min). Data analyses were performed using Bio-Rad ProteOn Manager software (version 3.1.0.6).The raw data were double-referenced by subtracting the interspot signals (empty chip surface, no immobilized or captured protein) to correct for non-specific binding of the FN3 domain to the pre-coated GAH-Fc IgG surface, followed by a double-reference using the blank buffer 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 fit well to the simple 1:1 Langmuir binding model, implying reagent problems and / or intrinsically complicated binding mechanisms that cannot be considered for use with a simple 1:1 binding mode. Petition 870260068423, dated 10 / 07 / 2026, page 108 / 201 94 / 165 that the GAH-Fc capture of the hCD8A-Fc format is the least disruptive compared to other formats in introducing potential experimental artifacts (such as loss of ligand activity and / or artificial epitopes / heterogeneous ligand population due to amine coupling), the results of the GAH-Fc capture experiments reported in this paper are considered to represent the most reliable data from ProteR SPR, despite the non-conforming 1:1 Langmuir fit observed in many cases. A heterogeneous ligand model was chosen to fit the data assuming two different ligand species, due to heterogeneity in the population of ligand proteins or due to potentially 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 resulting sensogram reflects the sum of two independent reactions with two sets of rate constants, which were reported for each FN3 domain binding. Table 7: Summary of kinetic affinities for the six main candidates for the anti-CD8A FN3 domains. Note: Affinity, Kd = kd / ka. Sample Affinity population Affinity population Petition 870260068423, dated 10 / 07 / 2026, page 109 / 201 95 / 165 (SEQ ID NO:) smallest largest ka (1 / Ms) kd (1 / s) Kd (nM) ka (1 / Ms) kd (1 / s) Kd (nM) P282DR9P1359_F5 (79) 3.48E+04 6.60E- 05 6, 6 3.80E+05 1.42E- 05 0.04 P282DR9P1359_F7 (81) 4.03E+04 3.65E- 04 12 4.04E+05 7.99E- 05 0.5 P282DR9P1359_G7 (83) 6.84E+04 5.51E- 05 2.1 2.76E+05 1.49E- 05 0.05 P282ER9P1360_C8 (89) 3.09E+04 9.52E- 05 4.1 2.18E+05 4.71E- 05 0.2 P283AR9P1362_D6 (122) 5.62E+04 3.12E- 05 0.98 1.55E+05 1.00E- 06 0.03 P282DR9P1359_C5 (68) 1.92E+04 1.27E- 04 6.5 3.00E+05 5.79E- 06 0.02 EXAMPLE 6: TAGING ANTI-CD8A FN3 DOMAINS WITH DFO E 89ZR

[0149] The anti-CD8A FN3 domains were modified to include a single cysteine ​​residue for conjugation of maleimide-containing chelators or PET labeling. Synthetic plasmid DNA encoding clones P282DR9P1359_F5, P282DR9P1359_F7, P282DR9P1359_G7, P282ER9P1360_C8, P283AR9P1362_D6, and P282DR9P1359_C5 with a cysteine ​​mutation at residue E54 was synthesized on DNA2.0 (Table 8). E54 was chosen as the position for the mutation based on previous studies demonstrating the maintenance of Petition 870260068423, dated 10 / 07 / 2026, page 110 / 201 96 / 165 binding affinity, stability, and expression levels for other FN3 domains mutated at this residue (Goldberg S. et al. Protein Engineering Design and Selection, 2016, electronic publication prior to print). Table 8: Molecules of the modified anti-CD8A FN3 domain Original Clone SEQ ID NO Clone with E54C SEQ ID 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

[0150] Anti-CD8A FN3 dominoes modified with a free cysteine ​​were conjugated with Deferoxamine (DFO) to chelate radiometals. 0.5 mL of a 100-500 μM anti-CD8A FN3 domain solution combined with 10 pL of 500 mM TCEP (Sigma, cat. # 646547), gently washed 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 achieve a final concentration of 3.2 M before incubation on ice for 10 minutes and centrifugation at 16,000 xg or higher to pellet the protein. The resulting pellet was resuspended and washed in 1.0 mL of ammonium sulfate. Petition 870260068423, dated 10 / 07 / 2026, page 111 / 201 97 / 165 ammonium 3.2 M supplemented with 100 mM sodium phosphate, pH 7.2 and 1 mM EDTA, before being centrifuged again. After the second centrifugation step, the resulting pellet was dissolved in 100 mM sodium phosphate, pH 7.0, 1 mM EDTA and combined with 10 µL of 50 mM DFO solution to produce a final molar ratio of 5:1 DFO to anti-CD8A. This reaction was allowed to proceed at room temperature for 30 minutes before deactivating with 5.0 microliters of betamercaptoethanol. The excess DFO was finally removed by a variety of methods including a second round of precipitation with ammonium sulfate as described above, passing through a desalting column such as a Zeba 7k column (Pierce Cat # 89889), or by purification with nickel-NTA resin (Qiagen # 30450). Anti-CD8A FN3 DFO-domain conjugates were formulated in 1X PBS for further analysis.

[0151] After conjugation with 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 maintained strong binding to human CD8A after E54 mutation to Cys and conjugation to DFO (Table 9). Table 9. Binding affinity after DFO conjugation Petition 870260068423, dated 10 / 07 / 2026, page 112 / 201 98 / 165 Sample Population with lower affinity Population with higher affinity ka (1 / Ms) kd (1 / s) Kd (nM) ka (1 / Ms) kd (1 / s) Kd (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 < 1 E-05 < 3.4 3.73E+04 < 1 E-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 CELLS T HUMANAS E CYNO.

[0152] A complete dose-response binding curve was generated for the ten to nine selected anti-CD8A FN3 domains. Each candidate was diluted to 20 μM in PBS followed by a series of 1:3 dilutions to generate an 11- or 18-point dose-response curve. Human or cyno CD8+ T cells were incubated with the diluted FN3 domain for 1 hour at 4°C. The cells were washed once with PBS and incubated with an anti-centivin antibody (PAB25) for 1 hour at 4°C. The cells were washed twice with PBS, followed by incubation with a secondary PE, anti-CD3-PacB, anti-CD4-APC, and a viability dye. Finally, the 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 Petition 870260068423, dated 10 / 07 / 2026, page 113 / 201 99 / 165 Centirins bound (PE channel) and the percentage of cells showing positive staining were calculated using Cytobank software. The results were graphically represented using Prism, and EC50 values ​​were calculated using the 4-parameter dose-response variable slope equation.

[0153] An equilibrium cell binding assay based on MesoScale Discovered Cell Affinity Technology (MSD-CAT) was performed to determine the affinity of six leading anti-CD8A candidates for 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 row). Cell viability was checked before binding measurements, and a viability > 85% was desired for a valid analysis. Since these cells were from different donors, in the case of donor-to-donor variations, only cells from the same donors were combined. Each individual anti-CD8A FN3 domain binding was measured in replicates using cells from the same donors. Cell and FN3 domains were incubated overnight at 4°C in a rotor to reach equilibrium.After incubation, the cells were centrifuged together with cell-bound anti-CD8A FN3 domains and the... Petition 870260068423, dated 10 / 07 / 2026, page 114 / 201 100 / 165 free (unbound) anti-CD8A FN3 domains in the supernatants were quantified using MSD assays in which biotinylated recombinant hCD8A-Fc protein was captured at 0.6 pg / mL in assay buffer onto MSD streptavidin plates overnight for ~16 hours at 4°C. After plate locking, the supernatant with free anti-CD8A FN3 domains was added to the plate and incubated for 1 hour, followed by SulfoTag pAb139 (in-house) detection at 1.6 pg / mL. A buffer control without any FN3 domains and hCD8A (plate bottom binding control) in column 1 and a control of the FN3 domain alone without hCD8A (100% free / unbound) in column 12 were included. The mouse anti-hCD8A mAb (mIgG1k, BD Biosciences, cat # 555364, clone RPAT8) was included as a positive control.Tencon27 was included in the initial assay validation as a negative control, and no significant binding was observed; therefore, it was not included in subsequent cell binding due to cell availability. Plates were read immediately on the MSD Sector Imager 6000™ Reader for luminescence levels after the addition of MSD Read Buffer diluting 1:4 of stock in H2O.

[0154] The raw MSD data were exported and analyzed in Prism using a nonlinear function fit. Petition 870260068423, dated 10 / 07 / 2026, page 115 / 201 A variable slope of 101 / 165 was used to derive the Bmax and Hillslope values. Only those with converged Bmax values ​​and slope within the range of -1.5 to -0.5 (ideal -1.0) will be considered for further analysis. The binding data were then normalized using the Bmax values ​​to calculate the normalized % free FN3 domains. A CD8 surface density of 50,000 receptors per cell was used to calculate the receptor concentration. A saturation criterion of <20% free Centirin at the highest CD8 cell concentrations was required to determine affinity using a Solution Affinity Equation for normalized data; for a 1:1 binding model.

[0155] The anti-CD8A FN3 domains bound to primary cells with affinities ranging from 0.167 to 2.81 nM (Table 10). Table 10: Summary of EC50 values ​​for the six main candidates for the anti-CD8A FN3 domain. Clone ID (SEQ ID NO:) EC50 Binding to Human T Cells by Flow Cytometry (nM) EC50 Binding to Cyno T Cells by Flow Cytometry (nM) Affinity for Human T Cells by MSD-CAT (nM) CD8S365 (232) 556.0 123.6 0.167 CD8S366 162.7 69.5 0.123 Petition 870260068423, dated 10 / 07 / 2026, page 116 / 201 102 / 165 (234) CD8S367 (230) 194.5 50.8 0.225 CD8S368 (229) 154.7 70.0 0.459 CD8S369 (233) 124.2 72.3 2.81 CD8S370 (231) 208.7 67.6 0.869 EXAMPLE 8: ACTIVATION OF HUMAN T CELLS Activation Again

[0156] In order to determine if the anti-CD8A domains FN3 activates 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 FN3 domains at 1 or 10 nM in duplicate with human pan-T cells for an average of 4 days. Two independent donors were tested. A plate-bound anti-CD3 antibody was used with a positive control at 2 doses, 0.1 pg / mL and 0.01 pg / mL. PBS was used as a negative control. Cells were then stained with a viability dye and the following antibody panel: CD4-FITC, CD3-PerCP-Cy5,5, CD69-PacB, CD45RABV605, CD25-BV650, CD127-PE, and CD137-PE-Cy7. CD8+ cells Petition 870260068423, dated 10 / 07 / 2026, page 117 / 201 103 / 165 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 replication values ​​were averaged. The results are summarized in Table 11A (donor 022) and 11B (donor 146). For 365, 366, 367, 368, and 370, small changes in T-cell activation markers were observed in only 1 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 that the molecules do not activate T cells de novo at relevant concentrations. Molecule 369 appears to significantly activate CD137 expression in both donors at the highest dose level. Table 11: Median Fluorescence Intensity (MFI) values ​​for various T cell activation markers in CD8+ T cells for Donor 022 (A) and Donor 146 (B) THE Donor Sample (SEQ ID NO:) Anti-CD8A FN3 conc μM Anti-CD3 ug / mL CD45RA MFI CD25 MFI CD69 MFI CD127 MFI CD137 MFI 022 PBS control 0 0 12856 571 223 651 296 022 PBS 0 0.01 13133 707 403 517 343 Petition 870260068423, dated 10 / 07 / 2026, page 118 / 201 104 / 165 control 022 PBS control 0 0.1 11394 1333 1694 158 529 022 CD8S366 (234) 1 0 15054 949 477 425 310 022 CD8S366 (234) 0.01 0 13336 814 230 586 301 022 CD8S368 (229) 1 0 12992 858 698 367 329 022 CD8S368 (229) 0.01 0 15262 677 276 489 306 022 CD8S367 (230) 1 0 15409 796 401 511 297 022 CD8S367 (230) 0.01 0 13666 723 261 502 312 022 CD8S370 (231) 1 0 12946 916 572 376 353 022 CD8S370 (231) 0.01 0 14973 776 353 435 331 022 CD8S365 (232) 1 0 13935 904 5 62 367 328 022 CD8S365 (232) 0.01 0 15156 697 243 504 323 022 CD8S369 (233) 1 0 13661 783 440 441 5122 022 CD8S369 (233) 0.01 0 16513 717 251 596 416 022 TenCon 1 0 14920 702 284 447 334 Donor Sample Anti- Anti- CD45RA CD25 CD69 CD127 CD137 MFI Petition 870260068423, dated 10 / 07 / 2026, page 119 / 201 105 / 165 (SEQ ID NO:) CD8A FN3 conc μΜ CD3 pg / mL MFI MFI MFI MFI 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 (234) 1 0 8531 673 95 1368 589 146 CD8S366 (234) 0.01 0 9414 623 74 1615 559 146 CD8S368 (229) 1 0 8386 691 96 1301 561 146 CD8S368 (229) 0.01 0 9147 628 82 1424 586 146 CD8S367 (230) 1 0 8167 660 95 1322 581 146 CD8S367 (230) 0.01 0 8734 586 77 1479 571 146 CD8S370 (231) (232) 1 0 8344 813 85 1238 586 146 CD8S365 (232) 0.01 0 8460 628 80 1355 605 146 CD8S369 (233) 1 0 8778 681 92 1369 5690 146 CD8S369 (233) 0.01 0 7862 591 74 1498 784 146 TenCon 1 0 7325 609 78 1198 574 Petition 870260068423, dated 10 / 07 / 2026, page 120 / 201 106 / 165 146 TenCon 0.01 0 7764 596 66 1281 530 Activation of pan T cells

[0157] In order to determine if the anti-CD8A domains FN3 can affect T cell activation markers in pan-activated T cells; the anti-CD8A FN3 domains were also evaluated in combination with a low dose of plaque-bound CD3. In this assay, a sub-optimal concentration (0.01 pg / mL) of plaque-bound anti-CD3 was used to activate T cells in the presence of 1 μM or 10 nM of anti-CD8A. After 4 days, the cells were evaluated 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 replication values ​​were averaged. The results are summarized in Tables 12A (donor 022) and 12B (donor 146). Tables 12A and B: Median Fluorescence Intensity (MFI) values ​​for various T cell activation markers in CD8+ T cells for Donor 022 (A) and Donor 146 (B) in the presence of plaque-bound CD3. THE Donor Sample (SEQ ID NO:) Anti-CD8A FN3 Anti-CD3 pg / mL CD45RA MFI CD25 MFI CD69 MFI CD127 MFI CD137 MFI Petition 870260068423, dated 10 / 07 / 2026, page 121 / 201 107 / 165 conc μM 022 PBS control 0 0 12856 571 223 651 296 022 PBS control 0 0.01 13133 707 403 517 343 022 PBS control 0 0.1 11394 1333 1694 158 529 022 CD8S366 (234) 1 0.01 11918 892 1005 369 376 022 CD8S366 (234) 0.01 0.01 13417 1068 848 384 399 022 CD8S368 (229) 1 0.01 11311 1147 1279 260 428 022 CD8S368 (229) 0.01 0.01 13441 760 599 499 348 022 CD8S367 (230) 1 0.01 13271 1135 1127 367 385 022 CD8S367 (230) 0.01 0.01 14521 960 636 483 362 022 CD8S370 (231) 1 0.01 15138 1103 890 407 378 022 CD8S370 (231) 0.01 0.01 14230 875 612 431 355 022 CD8S365 (232) 1 0.01 14395 1112 907 380 407 022 CD8S365 (232) 0.01 0.01 14006 1175 1063 297 430 022 CD8S369 (233) 1 0, 01 13735 877 759 464 5457 022 CD8S369 (233) 0.01 0.01 13864 842 617 450 498 Petition 870260068423, dated 10 / 07 / 2026, page 122 / 201 108 / 165 022 TenCon 1 0.01 14687 791 553 408 358 022 TenCon 0.01 0.01 13090 759 630 464 368 Donor Sample (SEQ ID NO:) Anti-CD8A FN3 conc μM Anti-CD3, pg / mL CD45RA MFI CD25 MFI CD69 MFI CD127 MFI CD137 MFI 146 PBS control 0 0 12856 571 223 651 296 146 PBS control 0 0.01 13133 707 403 517 343 146 PBS control 0 0.1 11394 1333 1694 158 529 146 CD8S366 (234) 1 0.01 6798 876 163 1095 632 146 CD8S366 (234) 0.01 0.01 8589 775 158 1077 637 146 CD8S368 (229) 1 0.01 6576 945 175 1105 662 146 CD8S368 (229) 0.01 0.01 7608 843 200 950 678 146 CD8S367 (230) 1 0.01 6447 897 173 1088 672 146 CD8S367 (230) 0.01 0.01 7899 801 175 1031 655 146 CD8S370 (231) 1 0.01 7327 992 169 1055 687 146 CD8S370 (231) 0.01 0.01 8676 790 183 946 675 146 CD8S365 1 0.01 6624 977 172 1059 670 Petition 870260068423, dated 10 / 07 / 2026, page 123 / 201 109 / 165 (232) 146 CD8S365 (232) 0.01 0.01 7902 843 193 985 659 146 CD8S369 (233) 1 0.01 7660 933 165 1149 7114 146 CD8S369 (233) 0.01 0.01 7892 854 187 989 842 146 TenCon 1 0.01 8352 829 170 1026 658 146 TenCon 0.01 0.01 7627 761 185 1043 673 Cytokine response

[0158] In order to determine whether any of the observed changes in activation markers resulted in alterations in cytokine production, antigen-dependent T cell activation assays were also performed using two anti-CD8A FN3 domains. For one set of assays, CMV-reactive or M1-reactive PBMCs were thawed and incubated overnight at 37°C in 6-well plates. The following day, PBMCs were collected by pipetting, counted, and plated onto Mabtech ELISpot IFNg plates in the presence or absence of 10 pg / mL of peptide. 1 μM of anti-CD8A FN3-DFO conjugate was added to the wells, and the plates were incubated at 37°C for approximately 24 hours undisturbed. Cells were removed, and the plates were washed 5 times with PBS. The supplied detection antibody was added, and the plates were incubated for 2 hours. The plates were again Petition 870260068423, dated 10 / 07 / 2026, page 124 / 201 110 / 165 wells were 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 point counts were generated using the AID EliSpot Software. Results were presented graphically in Prism. Results are summarized in Fig. 1. In this assay, 365-DFO does not increase the number of IFNγ points compared to the medium alone or to the non-CD8A-bound TenCon control in the absence of peptide (Fig. 1A, 1C). Peptide and CD3 are included as positive controls. In the presence of peptide, 365-DFO does not alter the number of IFNγ points compared to the peptide alone or to the non-CD8A-bound peptide (Fig. 1B, 1D). Mean is included as a negative control and CD3 is included as a positive control.These results suggest that centrin does not affect T cell activation.

[0159] To confirm these results in a long-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 µM in the presence or absence of pp65 peptide at 0.25 / ml. The cells were incubated for 6 days. Petition 870260068423, dated 10 / 07 / 2026, page 125 / 201 111 / 165 at 37°C. At each time point, the cells were centrifuged and the supernatant was collected. The samples were stored at -80°C until analyzed. The thawed samples were analyzed for IFN-gamma using an ELISA based on Meso Scale Discovery (MSD). For this assay, a standard curve was prepared according to the manufacturer's instructions. Samples and standards were added to pre-coated 96-well MSD plates. After 2 hours of incubation, the antibody from the detection kit was added. After another 2 hours of incubation, the plates were washed three times followed by the addition of the supplied reading buffer. The plates were read on the MSD Sector Imager 6000 plate reader. The raw MSD data files were analyzed against the generated standard curves using the MSD Discovery Workbench software. The analyzed data were graphed using the Tibco Spotfire program. The results are summarized in Fig. 2. In this assay, 365-DFO does not increase IFNγ secretion in the medium compared to the isolated medium in the absence of peptide (Fig. 2A). The CMV peptide is included as a positive control. In the presence of peptide, 365-DFO also does not alter the amount of IFNγ secretion compared to the peptide alone (Fig. 2B). Medium is included as a negative control. These results suggest that centrin does not affect T cell activation. Petition 870260068423, dated 10 / 07 / 2026, page 126 / 201 112 / 165 EXAMPLE 9: MARKING OF ANTI-CD8A FN3 DOMAINS WITH I124 / I125

[0160] The current method for radioactive labeling CD8S365 with iodine-124 to produce [124I]-IPEM CD8S 365 (Scheme 1) was adapted from procedures in the literature (Bioconjugate Chem. 1991, 2, 435-440; ChemistryOpen 2015, 4, 174-182). Na124l Iodogen-AcOl M of sodium phosphate mMEDTA, pH = 6.5-7 Scheme 1: Synthesis of [124I]-IPEM CD8S 365

[0161] To a 1.5 mL Eppendorf tube was added in order Na124I solution (< 13 μL, <2.5 mCi), AcOH (5 μL to acidify the solution), 1- (4(tributylstanyl)phenethyl)-1H-pyrrole-2,5-dione (75 μL, 1.00 mg / mL in MeCN) and iodogen solution (5 μΣ, 1.00 mg / mL in MeCN). The reaction was allowed to proceed for 5 min at room temperature.

[0162] The crude reaction mixture was diluted with 0.5 mL of 20% EtOH / H2O and purified directly by preparative HPLC, retention time = 14.4 min (Fig. 3). [124I]-IPEM was collected in a 1 drachm vial that was pretreated with Sigma-Cote™ (then washed with 3 mL of 70% EtOH, followed by 3 mL of H2O); total volume collected by preparative HPLC <750 μΣ. Petition 870260068423, dated 10 / 07 / 2026, p. 127 / 201 113 / 165

[0163] An aliquot (~ 5-25 μθί) of the purified fraction was then injected into analytical HPLC (Fig. 4, retention time = 11.7 min).

[0164] The purified [124I]-IPEM was then concentrated under vacuum at room temperature to a volume <100 μΣ.

[0165] Sodium phosphate buffer (1.0 M sodium phosphate, 1 mM EDTA, pH = 6.86) (>25 μΣ) was added to bring the pH to ~6.5-7 (verified by strip). Finally, CD8S 365 (c ~4.57 mg / mL in 100 mM sodium phosphate buffer, 1 mM EDTA, pH = 6.86) was freshly reduced in an amount appropriate to achieve the target specific activity (i.e., if targeting specific activity of 25 mCi / mg and 2.0 mCi of [124I]-IPEM were collected to add 17.5 μΣ of centirin ac ~4.57 mg / mL). The conjugation reaction was allowed to proceed for 60 minutes at room temperature, and the reaction progress was examined to verify that the conversion exceeded 90% using iTLC.

[0166] Purification consisted of diluting the reaction solution with 10% PBS / EtOH (1 mL, pH = 7) by transferring the reaction solution from the 1 drachm flask to a Vivaspin 6 5kDa MWCO centrifugal filter (see appendix for preconditioning). After transfer, the Eppendorf reaction was washed with 10% PBS / EtOH (2 x 1 mL, pH = 7). Petition 870260068423, dated 10 / 07 / 2026, page 128 / 201 114 / 165 and the washes were added to the filter. The crude reaction mixture was centrifuged at 4000 rpm, at 20°C for 30 min. After centrifugation, 500 μL of solution remained and was found to have radiochemical purity (RCP) > 95% by radio TLC (Fig. 5). The purified [124I]-IPEM CD8S 365 was diluted to a volume of 500 μL with PBS / 10% EtOH if the volume was <500 μL and then filtered through a Millex-GV 0.22 μL hydrophilic Durapore membrane (PVDF).

[0167] The radiochemical yield of the protocol is ~ 50% with radiochemical purity >95% RCP by radio TLC). Analytical reversed-phase HPLC was used to determine the protein concentration and specific activity of the final product. The average integration of the peak at a retention time of 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 10X dilution with LAL reagent water. EXAMPLE 10: DETECTION OF CD8 EXPRESSION IN SKYNOLOGY MONKEYS

[0168] Two anti-CD8A FN3 molecules (CD8S365 and Petition 870260068423, dated 10 / 07 / 2026, page 129 / 201 115 / 165 CD8S368) were selected for PET imaging in non-human primates (NHP). Anti-CD8A molecules were radiolabeled with Zr-89 (Zevacor, Somerset, NJ) or I-124 (CPDC, Hamilton, Canada and Zevacor, Somerset, NJ). Approximately 1-2 mCi of radiolabeled anti-CD8A molecules were injected into the saphenous vein of a female NHP (cynomolgus monkey) while anesthetized with isoflurane in oxygen. Each animal was scanned using a microPET scanner. A large-diameter Focus 220 PET scanner (Siemens, Knoxville, TN) was used, with the bed moved to accommodate the entire animal body (from head to lower abdomen). Each examination lasted approximately 1 hour, and examinations were acquired at 15 min, 2 hours, and 24 h after injection. PET images were reconstructed using a 2D maximum likelihood expectation maximization (ML-EM) algorithm, resulting in 3D voxel images measuring 1,898 x 1,898 x 0,796 mm, with dimensions of 128 x 128 x 475 mm. Blood samples were obtained at various time points from the saphenous vein in the leg opposite the injection site, and blood radioactivity was counted using a well counter.

[0169] PET images were analyzed using PMOD v3.7 software (PMOD, Zurich, Switzerland). Regions of interest were manually drawn around organs such as the spleen, kidneys, and liver. Counts were converted into units of percentage of injected dose per gram of Petition 870260068423, dated 10 / 07 / 2026, page 130 / 201 116 / 165 tissue (% ID / g), while blood radioactivity was presented as % ID. A representative PET image is shown in Fig. 9.

[0170] The blood kinetics for each NHP and each anti-CD8A domain molecule FN3 (labeled with Zr-89 or I124) is shown in Table 13 and summarized in Fig. 10. For the same animals and anti-CD8A molecules, the organ biodistributions are shown in Table 14 (units are % ID / g) and summarized in Fig. 11. The Zr-89-labeled molecules exhibited radioisotope residualization 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. Spleen uptake was very similar between the two different molecules and two different radioisotopes for all time points. Table 13. Blood kinetics for each centrin, radiolabeled with 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) 368 124 I- 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 Petition 870260068423, dated 10 / 07 / 2026, p. 131 / 201 117 / 165 2.00 6.12 1.00 24.20 1.13 24.51 1.17 16.01 3.70 3.77 1.50 18.82 2.33 10.92 2.07 12.18 3.33 8.40 3.88 8.20 24.00 1.94 23.03 1.24

[0171] For the same animals and anti-CD8A molecules, organ biodistributions are shown in Table 14 (units are % ID / g) and summarized in Fig. 11. Table 14. Organ absorption for the different centrins, labeled Zr-89 or I-124 (entries are % ID / g). 365 Zr-89 Liver Time Kidney Spleen (h) 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 Time Kidney Spleen Liver (h) 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 Petition 870260068423, dated 10 / 07 / 2026, page 132 / 201 118 / 165 I-124 Time Kidney Spleen Liver (h) 0.25 h 0.104 0.0324 0.0291 2 h 0.065 0.0222 0.0142 24 h Not collected due to technical problem 368 I-124 Time Kidney Spleen Liver (h) 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 CYNOMOLPHIANS MONKEYS

[0172] To test the specificity of anti-CD8A molecules, the same monkeys were treated with a CD8-depleting chemical antibody (mouse CMT807 anti-CD8 human V antibody) to reduce CD8+ T cells prior to imaging. The animals were administered subcutaneously with 10 mg / kg of CD8-depleting antibody 3 days prior to imaging. CD8 depletion was confirmed by measuring the percentage of CD8 T cells in collected blood samples. Petition 870260068423, dated 10 / 07 / 2026, page 133 / 201 119 / 165 of each animal before and after depletion (Fig. 12).

[0173] Approximately 1-2 mCi of radiolabeled FN3 anti-CD8 domain molecule CD8S365 [I-124] was injected into the saphenous vein of a female NHP (cynomolgus monkey) while anesthetized with isoflurane in oxygen. Each animal was scanned on a large-diameter Focus 220 microPET scanner (Siemens, Knoxville, TN), with the bed moved to accommodate the entire body of the animal (head down to lower abdomen). Each examination lasted approximately 1 h and examinations were acquired at 15 min, 2 h and 24 h after injection. PET images were reconstructed using a 2D maximum likelihood expectation maximization (MLEM) algorithm, in 3D voxel images measuring 1.898 x 1.898 x 0.796 mm, dimensions 128 x 128 x 475. Blood samples were obtained at various time points from the saphenous vein in the leg opposite to the injection, and blood radioactivity was counted using a well counter.

[0174] PET images were analyzed using PMOD v3.7 software (PMOD, Zurich, Switzerland). Regions of interest were manually drawn around organs such as the spleen, kidneys, and liver. Counts were converted to units of percentage of injected dose per gram of tissue (% ID / g), while blood radioactivity was presented as % ID. A representative PET image is Petition 870260068423, dated 10 / 07 / 2026, page 134 / 201 120 / 165 shown in Fig. 13 for a depleted animal, showing a complete absence of the spleen signal observed in the non-depleted animal in Fig. 9.

[0175] Blood kinetics for each NHP, both depleted and non-depleted, is shown in Fig. 14, while organ uptakes are shown in Fig. 15. There is little difference in blood kinetics between depleted and non-depleted animals. Spleen uptake at the first time point is similar between depletion and non-depletion, as it is dominated by blood flow. However, at later time points (2 h) spleen uptake in depleted animals is less than half that observed in non-depleted animals, and is essentially at background levels, demonstrating the CD8A specificity of the radiolabeled centrin. EXAMPLE 12: SENSITIVITY AND SPECIFICITY OF PET IMAGING IN CD8-EXPRESSING TUMORS

[0176] In order to determine the smallest number of cells that can be detected with FN3 anti-CD8A domain molecules and PET, a study was conducted in mice using different numbers of CD8 overexpressing cells. Forty 4-5 week old female NOD-scid IL2rynu11 (NSG) mice (JAX Laboratory) were used and were acclimated for 7-10 days. The mice were Petition 870260068423, dated 10 / 07 / 2026, p. 135 / 201 121 / 165 mice were housed in VCI cages under a 12-hour light / dark cycle (lights on at 06:30 h) at a temperature of 19–22 °C. Mice were fed a standard autoclaved laboratory diet and water ad libitum. Mice were tagged on the ear and their tails were tattooed 5–7 days before the start of the study to identify each animal.

[0177] Cell lines overexpressing parental CD8+ HEK-293 and HEK-293-luc were maintained as 2D cultures. Mice were subcutaneously implanted with a total of 106 tumor cells in a 1:1 medium for the Cultrex mixture containing varying proportions of HEK-293-Luc CD8+ expressing cells and parental HEK-293 cells. Once the tumors were palpable, approximately 10-14 days old and 200-300 mm3 in size, human CD8+ cells were visualized using [I-124]CD8-S365.

[0178] The expression of luciferase from HEK-293Luc CD8+ cells was quantified in vivo using bioluminescence imaging on an IVIS Spectrum optical imager (Perkin Elmer). Dynamic optical imaging was performed immediately after injection of 150 mg / kg of D-luciferin to identify the light emission peak.

[0179] Approximately 0.2-0.5 mCi of radiolabeled anti-CD8A FN3 domain molecules were injected into the vein. Petition 870260068423, dated 10 / 07 / 2026, p. 136 / 201 122 / 165 of the tail while anesthetized with isoflurane in oxygen. Each animal was scanned on an Inveon microPET-CT scanner (Siemens, Knoxville, TN) for 20 min of static scanning. Scans were acquired within 2-3 hours after marker injection. PET images were reconstructed using a 2D maximum likelihood expectation maximization (ML-EM) algorithm, in 3D voxel images with a size of 0.776 x 0.776 x 0.796 mm and dimensions of 128 x 128 x 159 mm.

[0180] PET images were analyzed using PMOD v3.7 software (PMOD, Zurich, Switzerland). Regions of interest were manually drawn around the tumor and other organs such as the spleen, kidneys, and liver. Counts were converted to units of percentage of 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 / cm² / steradian.

[0181] Time-activity curves of radioactively labeled anti-CD8A FN3 domain molecules in blood and tumor for HEK293 CD8+ cells and parental cells are shown in Fig. 17 and Fig. 18. There is a significant increase in anti-CD8A FN3 binding in CD8-expressing cells compared to parental cells, while the Petition 870260068423, dated 10 / 07 / 2026, page 137 / 201 123 / 165 blood activity is the same for both. The incorporation of anti-CD8A FN3 by CD8+ HEK293 cells is shown in Fig. 19, as a function of the number of implanted cells. Based on these data, it is estimated that the lowest detection level is approximately 7.5 x 10⁶ cells. Sequence Listing SEQ ID NO: 1 = Original Tencon Sequence LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSER SYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT SEQ ID NO: 2= TCL1 Library LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSER SYDLTGLKPGTEYTVSIYGV(X) 7-12PLSAEFTT; In what X1, X2, X3, X4, X5, X6, X7 are any amino acids; and X8, X9, X10, X11, and X12 are any amino acids that are deleted. SEQ ID NO: 3=TCL2 library LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVGEAINLT VPGSERSYDLTGLKPGTEYTVSIYGVX9X10X11X12X13SX14X15LSAEFTT; in what X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; Petition 870260068423, dated 10 / 07 / 2026, page 138 / 201 124 / 165 Shah Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X7 is Phe, Ile, Leu, Val or Tyr; X8 is Asp, Glu, or Thr; X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X10 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile,Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr orVal; X11 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile,Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr orVal; X12 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile,Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr orVal; X13 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile,Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr orVal; X14 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile,Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; in X15 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile,Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr orVal. Petition 870260068423, of 10 / 07 / 2026, p. 139 / 201 125 / 165 SEQ ID NO: 4= Stabilized Tencon (Tencon 27) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT SEQ ID NO: 5= TCL7 (loops FG and BC) LPAPKNLVVSRVTEDSARLSWX1X2X3X4X5X6X7X8X9FDSFLIQYQESEKVGEAI VLTVPGSERSYDLTGLKPGTEYTVSIYGVX10X11X12X13X14X15X16X17X18X19SNPLSA IFTT; Em que X1, A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y;e X7, N, P, Q, R, S, T, V, W, Y or deleted SEQ ID NO: 6= TCL9 (loop FG) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGV X1X2X3X4X5X6X7X8X9 X10X11X12SNPLSAIFTT; em que X1, X2, X3, X4, X5, X6 e N, P, Q, R, S, T, V, W or Y; e X8, X9, X10, X11 e Q, R, S, T, V, W, Y or delete. SEQ ID NO: 7= biblioteca TCL14 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIVLTV PGSERSYDLTGLKPGTEYX8VX9IX10GVKGGX11X12SX13PLSAIFTT; em que Petition: 870260068423, on 10 / 07 / 2026, page. 140 / 201 126 / 165 Xi, X2, X3, X4, X5, X6, X10, X11, X12 e G, H, I, K, L, N, P, Q, R, S, T, V, W,Y, or M. SEQ ID NO: 8= biblioteca TCL24 Biblioteca TCL24 (SEQ ID NO: 8) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9 VPGSERSYDLTGLKPGTEYX10VX11IX12GVKGGX13X14SX15PLX16AX17FTT; Em que X1, 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 CGGCGGTTAGAACGCCGGCTACAATTAATAC SEQ ID NO: 12 = DigLigRev CATGATTACGCCAAGCTCAGAA SEQ ID NO: 13 = BC9 GTGACACGGCGGTTAGAACGCGGCTAATTAATACATAACCCCATCCCCCTGT TGACAATTAATCATCGGCTCGTATAATGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGGATCTACCATGCTGCCGGCGCGAAAACCTGGTTGTTTCTGAAGTTAC CGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNNNNNTTYGACT CTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTT CCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGGTACCGAATACCGT Petition 870260068423, of 10 / 07 / 2026, p. 141 / 201 127 / 165 TTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC SEQ ID NO: 14 = BC8 GTGACACGGCGGTTAGAACGCGGCTAATTAATACATAACCCCATCCCCCTGT TGACAATTAATCATCGGCTCGTATAATGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGGATCTACCATGCTGCCGGCGCGAAAACCTGGTTGTTTCTGAAGTTAC CGAAGACTCTCTGCGTCTGTCTTGGGNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTT TCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCG GGTTCTGAACGTTCTTACGACCTGACCGGTCTGAACCGGGTACCGATCGATCTACCTAGTCTAGTCTAGTTGACTGAATTCGAATCTGAACGTTCTTACGACCT SEQ ID NO: 15 = BC7 GTGACACGGCGGTTAGAACGCGGCTAATTAATACATAACCCCATCCCCCTGT TGACAATTAATCATCGGCTCGTATAATGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGGATCTACCATGCTGCCGGCGCGAAAACCTGGTTGTTTCTGAAGTTAC CGAAGACTCTCTGCGTCTGTCTTGGGNNNNNNNNNNNNNNNNNNTTYGACTCTTTCC TGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGT TCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGTACCGA SEQ ID NO: 16 = BC6 GTGACACGGCGGTTAGAACGCGGCTAATTAATACATAACCCCATCCCCCTGT TGACAATTAATCATCGGCTCGTATAATGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGGATCTACCATGCTGCCGGCGCGAAAACCTGGTTGTTTCTGAAGTTAC CGAAGACTCTCTGCGTCTGTCTTGGGNNNNNNNNNNNNNNNNTTYGACTCTTTTCCTGA TCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCT GAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACCGTTTCTATCTA Petition 870260068423, of 10 / 07 / 2026, p. 142 / 201 128 / 165 CGGTGTTCTTAGAAGCTTCCCAAAGGC SEQ ID NO: 17 = 130mer-L17A CGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAAT CATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGG ATCTACCATGCTG SEQ ID NO: 18 = POP222ext CGG CGG TTA GAA CGC GGC TAC YOU ARE 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 CATGCGGTCTCTTCCGAAAAAGTTGGTGAAGCGATCGTCCTGACCGTTCCGGGT SEQ ID NO: 23 =SDG24 GGTGGTGAAGATCGCAGACAGCGGGTTAG SEQ ID NO: 24 = POP2222 CGGCGGTTAGAACGCGGCTAC SEQ ID NO: 25 =SDG28 AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCACCGCCGGTGGTGAAGAT CGCAGAC Petição 870260068423, de 10 / 07 / 2026, pág. 143 / 201 129 / 165 SEQ ID NO: 26 = FG12 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGT TGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTAC CGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGA TCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCT GAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTA CGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTG CGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCG GCCGCAACTGATCTTGGC SEQ ID NO: 27 = FG11 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGT TGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTAC CGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGA TCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCT GAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTA CGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGA TCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCC GCAACTGATCTTGGC SEQ ID NO: 28 = FG10 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGT TGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTAC Petição 870260068423, de 10 / 07 / 2026, pág. 144 / 201 130 / 165 CGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGA TCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCT GAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTA CGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCT TCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCA ACTGATCTTGGC SEQ ID NO: 29 = FG9 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGT TGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTAC CGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGA TCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCT GAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTA CGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCA CCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACT GATCTTGGC SEQ ID NO: 30 = FG8 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGT TGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTAC CGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGA TCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCT GAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTA CGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCA Petição 870260068423, de 10 / 07 / 2026, pág. 145 / 201 131 / 165 CCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGAT CTTGGC SEQ ID NO: 31 = FG7 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGT TGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTAC CGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGA TCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCT GAACGTTCTTACGACCTGACCGGTCTGAAACCGGTACCGAATACACCGTTTCTATCTA CGGTGTTNNNNNNNNNNNNNNNNNNNNNNCTAACCCGCTGTCTGCGATCTTCACCACCG GCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTT GGC SEQ ID NO: 32 FG Tencon loop KGGHRSN SEQ ID NO: 33 = Tcon 6 AAGAAGGAGAACCGGTATGCTGCCGGCGCCGAAAC 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 > SEQ ID NO: 35: CD8W7 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQ NKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLP AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAGSGSGSGSDYKDDDPPTPCCPDK ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEWKFNWYVDGVVHNAKTKP Petition 870260068423, of 10 / 07 / 2026, p. 146 / 201 132 / 165 REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > SEQ ID NO: 36: CD8W13 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQ NKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLP AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDGGGGSDYKDDD DKGGGGSHHHHHHDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK > SEQ ID NO: 37: peptídeo de sinal mIgGK Metdtllllwvllllwvpgstg > SEQ ID NO: 38: Fc humano Dkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevk fnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapie ktiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennyk ttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk > SEQ ID NO: 39: linker sequence Ggggsdykddddkggggshhhhhh Table 15. Clone ID SEQ Amino Acid Sequence Petition 870260068423, dated 10 / 07 / 2026, page 147 / 201 133 / 165 ID NO: P282AR9P1356_ A10 40 LPAPKNLVVSRVTEDSARLSWHTATNSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVDYNPTGRVPSSNPLSAIFTT P282AR9P1356_ A4 41 LPAPKNLVVSRVTEDSARLSWVKRPNSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVVDYEGRPRWSNPLSAIFTT P282AR9P1356_ A6 42 LPAPKNLVVSRVTEDSARLSWSKTDSSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVVYIEGNPVFSNPLSAIFTT P282AR9P1356_ B9 43 LPAPKNLVVSRVTEDSARLSWPEGDRPFFDSFLIQY QESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSI YGVDVKWEGNRPVASNPLSAIFTT P282AR9P1356_ D3 44 LPAPKNLVVSRVTEDSARLSWTRHETSFDSFLIQYR ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVVVEYDAAGNPKYSNPLSAIFTT P282AR9P1356_ H1 45 LPAPKNLVVSRVTEDSARLSWIPNPSSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVVFDPVGFPSHSNPLSAIFTT P282AR9P1356_ H6 46 LPAPKNLVVSRVTEDSARLSWRKRANSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVHVEYDQHGRPRWSNPLSAIFTT Petition 870260068423, de 10 / 07 / 2026, pág. 148 / 201 134 / 165 P282BR9P1357_ A9 47 LPAPKNLVVSRVTEDSARLSWKANRTTDLHFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVQYDGQQPLYSNPLSAIFTT P282BR9P1357_ B2 48 LPAPKNLVVSRVTEDSARLSWNPSEDPQRFDSFLIQ YQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVS IYGVDVKWEGNRPVASNPLSAIFTT P282BR9P1357_ C10 49 LPAPKNLVVSRVTEDSARLSWWSNDNRPIFDSFLIQ YQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVS IYGVDVKWEGNRPVASNPLSAIFTT P282BR9P1357_ C4 50 LPAPNNLVVSRVTEDSARLSWPFVSQNKPHFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVKWEGNRPVASNPLSAIFTT P282BR9P1357_ D12 51 LPAPKNLVVSRVTEDSARLSWGQYITAFSFDSFLIQ YQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVS IYGVDVAWFQGKPTWSNPLSAIFTT P282BR9P1357_ D2 52 LPAPKNLVVSRVTEDSARLSWIKDGHPRHFDSFLIQ YQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVS IYGVDVVYDRGQLISSNPLSAIFTT P282BR9P1357_ E5 53 LPAPKNLVVSRVTEDSARLSWWPRKYQRPFDSFLIQ YQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVS IYGVDIEWIGNRPIASNPLSAIFTT P282BR9P1357_ G9 54 LPAPKNLVVSRVTEDSARLSWPIASQIHSPFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV Petition: 870260068423, on 10 / 07 / 2026, page. 149 / 201 135 / 165 SIYGVDVKYDIDSRPISSNPLSAIFTT P282BR9P1357_ H3 55 LPAPKNLVVSRVTEDSARLSWKKREYQDPGFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVKWEGNRPVASNPLSAIFTT P282CR9P1358_ C2 56 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIAYP EWPSNGEAIVLTVPGSERSYDLTGLKPGTEYAVFIW GVKGGAFSNPLSAIFTT P282CR9P1358_ C5 57 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIAYP EWPDSGEAIVLTVPGSERSYDLTGLKPGTEYAVFIW GVKGGPLSHPLSAIFTT P282CR9P1358_D10 58 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLISYP EYPPPGEAIVLTVPGSERSYDLTGLKPGTEYFVIIF GVKGGDTSWPLSAIFTT P282CR9P1358_ F11 59 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIAYP EWPIFEGEAIVLTVPGSERSYDLTGLKPGTEYFVVI YGVKGGEQSSPLSAIFTT P282CR9P1358_ F5 60 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWISYP EWPPDGEAIVLTVPGSERSYDLTGLKPGTEYFVIIW GVKGGETSAPLSAIFTT P282DR9P1359_ A12 61 LPAPKNLVVSRVTEDSARLSWTAPEAAFDSFQIAYP EWPPPREAIVLTVPGSERSYDLTGLKPGTEYFVVIQ GVKGGEISWPLSAIFTT P282DR9P1359_ 62 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIGYP Petition 870260068423, dated 10 / 07 / 2026, pág. 150 / 201 136 / 165 A7 ELEKLGYGEAIVLTVPGSERSYDLTGLKPGTEYWVI IWGVKGGENSWPLSAIFTT P282DR9P1359_ A8 63 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIAYP EWPVQGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY GVKGGELSPPLSAIFTT P282DR9P1359_ B2 64 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIAYT EWPIPYEEAGQEGEAIVLTVPGSERSYDLTGLKPGT EYWVSIYGVKGGPNSQPLSAIFTT P282DR9P1359_ C10 65 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIVYP EWPTDGEAIVLTVPGSERSYDLTGLKPGTEYAVFIW GVKGGNQSWPLSAIFTT P282DR9P1359_ C11 66 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIAYP EFPPPGEAIVLTVPGSERSYDLTGLKPGTEYYVIII GVKGGTDSWPLSAIFTT P282DR9P1359_ C12 67 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYISYP EWPVPGEAIVLTVPGSERSYDLTGLKPGTEYWVVIY GVKGGALSVPLSAIFTT P282DR9P1359_ C5 68 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWITYP EWPDPGGEAIVLTVPGSERSYDLTGLKPGTEYFVVI YGVKGGEIYSPLSAIFTT P282DR9P1359_ D12 69 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIAYP ETATWGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY GVKGGFESAPLSAIFTT Petição 870260068423, de 10 / 07 / 2026, pág. 151 / 201 137 / 165 P282DR9P1359_ E11 70 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYISYP EWPPVGEAIVLTVPGSERSYDLTGLKPGTEYWVIIY GVKGGAISTPLSAIFTT P282DR9P1359_ E2 71 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIFYP EIVTWGEAIVLTVPGSERSYDLTGLKPGTEYWVNIV GVKGGDNSWPLSAIFTT P282DR9P1359_ E3 72 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYP ELPLGGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY GVKGGVESFPLSAIFTT P282DR9P1359_ E5 73 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAISYP EWPVPGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY GVKGGLYSAPLSAIFTT P282DR9P1359_ E6 74 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIAYP EWPVQGEAIVLTVPGSERSYDLTGLKPGTEYFVVIQ GVKGGTPSWPLSAIFTT P282DR9P1359_ E8 75 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYP EWPVIGEAIVLTVPGSERSYDLTGLKPGTEYWVIIQ GVKGGYTSWPLSAIFTT P282DR9P1359_ F11 76 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIFYP ELPIHGEAIVLTVPGSERSYDLTGLKPGTEYWVNIT GVKGGDFSWPLSAIFTT P282DR9P1359_ F2 77 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYP EALHPGYGEAIVLTVPGSERSYDLTGLKPGTEYWVI Petição 870260068423, de 10 / 07 / 2026, pág. 152 / 201 138 / 165 IGGVKGGQKSWPLSAIFTTGGHHHDHH P282DR9P1359_ F3 78 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYITYP EWPVQGEAIVLTVPGSERSYDLTGLKPGTEYWVIIY GVKGGTESEPLSAIFTT P282DR9P1359_ F5 79 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYP EWPPPGEAIVLTVPGSERSYDLTGLKPGTEYFVIIQ GVKGGVESWPLSAIFTT P282DR9P1359_ F6 80 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPTTGEAIVLTVPGSERSYDLTGLKPGTEYFVVIW GVKGGDHSAPLSAIFTT P282DR9P1359_ F7 81 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYP EWPPQGEAIVLTVPGSERSYDLTGLKPGTEYFVVIY GVKGGSYSAPLSAIFTT P282DR9P1359_ G4 82 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIAYP EWPPPGEAIVLTVPGSERSYDLTGLKPGPEYFVVIQ GVKGGDPSFPLSAIFTTGGNHHHHH P282DR9P1359_ G7 83 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAITYI EKEHIEDGEAIVLTVPGSERSYDLTGLKPGTEYWVP IWGVKGGANSWPLSAIFTT P282DR9P1359_ H5 84 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYP EALHPGYGEAIVLTVPGSERSYDLTGLKPGTEYFVV IYGVKGGTNSEPLSAIFTT P282ER9P1360_ 85 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYY Petition 870260068423, de 10 / 07 / 2026, pág. 153 / 201 139 / 165 A9 EPVDSGEAITLPPGSERSYDLTGLKPGTEYWVVIT GVKGGAPSTPLGAIFTT P282ER9P1360_ C1 86 LPAPKNLVVSRVTEDSARLSWTTPDAAFDSFGILYY EPVDSGEAITLPVPGSERSYDLTGLKPGSTEYKWKGIVGITG P282ER9P1360_ C4 87 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGITYY EPNHGGEAISLSVPGSERSYDPTGLKPGTEYWVVIT GVKGGAPSTPLGAIFTT P282ER9P1360_ C6 88 LSAPKNLVVSRVTEDSARVDSFGYWTY EPVDSGEAITLPPGSERSYDLTGLKPGTEYWVVIT GVKGGAPSTPLGAIFTT P282ER9P1360_ C8 89 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYY EPVDSGEAITLPVPGSERSYDLTGLKPGTEYWKVGVGTTWGTTW P282ER9P1360_ D11 90 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYY EPVDSGEAITLPVPGSERSYDLTGLKPGTEYFVIIV GVKGGYPSIPLGAAFTT P282ER9P1360_ E4 91 LPAPKNLVVSRVDSARVDSARVDSFGYWWTY EPVDSGEAITLPVLGSERSYDLTGLKPGTEYWVVIT GVKGGAPSTPLGAIFTT P282ER9P1360_ F11 92 LPAPKNLVVSRVTEDSARLSWIAPDAAFDSSFSIAYV EAELVGEAIQLVVPGSERSYDLTGLKPGSYDLTGLKPGTEYWVGVGG Petition 870260068423, of 10 / 07 / 2026, p. 154 / 201 140 / 165 P282ER9P1360_ G10 93 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIWYV EQHPFGEAIPLFVPGSERSYDLTGLKPGTEYTVGIR GVKGGNFSTPLIAHFTT P282ER9P1360_ G7 94 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYY EPVDSGEAITLPVPGSERSYDLTGLKPGTEYWVIT GVKGGAPSTPLGAILTT P282ER9P1360_ H10 95 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYPP EWPFAGEAIGLPVPGSERSYDLTGLKPGTEYFVVIY GVKGGELSEPLTAQFTT P282ER9P1360_ H2 96 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSSFSIAYV EAELVGEAIQLVVPGSERSYDLTGLKPGTEVGVGFTG P282ER9P1360_ H3 97 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSSIAYV EAELVGEAIQLVVPGSERSYDLTGLKPGTEYWVVIL GVKGGNPSNPLGASFTT P282FR9P1361_ A3 98 LPAPKNLVVSRVTEDS EYGYPGEAIVLTVPGSERSYDLTGLKPGTEYDVAIV GVKGGNRSYPLSAIFTT P282FR9P1361_ A5 99 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYY EPVDSGEAITLPVPGSERSYDLTGLKPGTEYKVGVTTVGTFGTFGTT P282FR9P1361_ C7 100 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIWYH EYGGDGEAIVLTVPGSERSYDLTGLKPGTEYDVAIW Petition 870260068423, of 10 / 07 / 2026, p. 155 / 201 141 / 165 GVKGGDVSYPLSAIFTT P282FR9P1361_ D3 101 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIWYA EYGYPGEAIVLTVPGSERSYDLTGLNPGTEYDVAIS GVKGGPRSYPLSAIFTT P282FR9P1361_ E12 102 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSLGITYW ESPYAGEAIVLTVPGSERSYDLTGLKPGTEYGVFIL GVKGGYPSAPLSAIFTT P282FR9P1361_ F1 103 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIWYA EYGYSGEAIVLTVPGSERSYDLTGLKPGTEYDVAIW GVKGGVRSYPLSAIFTT P282FR9P1361_ F11 104 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIWYR EYGGSGEAIVLTVPGSERSYDLTGLKPGTEYDVAIW GVKGGVRSYPLSAIFTT P282FR9P1361_ F2 105 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIWYA EYGYPGEAIVLTVPGSERSYDLTGLKPGTEYDVAIS GIKGGPRSYPLSAIFTT P282FR9P1361_ F3 106 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIWYA EYGYPGEAIVLTVPGSERSYDLTGLKPGTEYDVAIS GAKGGPRSYPLSAIFTT P282FR9P1361_ F7 107 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIWYR EYATGEAIVLTVPGSERSYDLTGLKPGTEYDVVITG VKGGYPSYPLSAIFTT P282FR9P1361_ 108 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGITYW Petição 870260068423, de 10 / 07 / 2026, pág. 156 / 201 142 / 165 G9 ESPYAGEAIVLTVPGSERSYDLTGLKPGTEYGVFIL GVKGGYPSAPLSAIFTT P282FR9P1361_ H4 109 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIWYA EYGYSGEAIVLTVPGSERSYDLTGLKPGTEYDVAIY GVKGGSPSYPLSAIFTT P282FR9P1361_ H5 110 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIWYA EYGYPGEAIVLTVPGSERSYDLTGLKPGTEYDVAIS GVKGGPRSYPLSAIFTT P283AR9P1362_ A3 111 LPAPKNLVVSRVTEDSARLSWKRIDSPFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ A4 112 LPAPKNLVVSRVTEDSARLSWIGHDSGFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ B10 113 LPAPKNLVVSRVTEDSARLSWKRRWDSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVEWFNGLPHHSNPLSAIFTT P283AR9P1362_ B2 114 LPAPKNLVVSRVTEDSARLSWAKHPNSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVVVNELNNPLFSNPLSAIFTT P283AR9P1362_ B8 115 LPAPKNLVVSRVTEDSARLSWWTSPLPFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT Petition: 870260068423, on 10 / 07 / 2026, page. 157 / 201 143 / 165 P283AR9P1362_ C12 116 LPAPKNLVVSRVTEDSARLSWAKNLHSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ C6 117 LPAPKNLVVSRVTEDSARLSWYPSDPPFDFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVPNYHSRRSYYYSNPLSAIFTT P283AR9P1362_ C7 118 LPAPKNLVVSRVTEDSARLSWVKRATSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVRYNEGQPIWSNPLSAIFTT P283AR9P1362_ D2 119 LPAPKNLVVSRVTEDSARLSWQRPKSGFDFDLQY QESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSI YGVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ D3 120 LPAPKNLVVSRVTEDSARLSWPVESNAFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVEYDQHGRPRWSNPLSAIFTT P283AR9P1362_ D4 121 LPAPKNLVVSRVTEDSARLSWVREHDSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ D6 122 LPAPKNLVVSRVTEDSARLSWAKRPGAFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ D7 123 LPAPKNLVVSRVTEDSARLSWVKRATSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY Petition 870260068423, de 10 / 07 / 2026, pág. 158 / 201 144 / 165 GVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ E9 124 LPAPKNLVVSRVTEDSARLSWVPSPWGFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ F12 125 LPAPKNLVVSRVTEDSARLSWARNITSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ F2 126 LPAPKNLVVSRVTEDSARLSWRKKDHPFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ F8 127 LPAPKNLVVSRVTEDSARLSWGYYHGHFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKWEGNRPVASNPLSAIFTT P283AR9P1362_ G11 128 LPAPKNLVVSRVTEDSARLSWRKEATSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT P283AR9P1362_ G3 129 LPAPKNLVVSRVTEDSARLSWVKRATSFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVDVKWEGNRPVASNPLSAIFTT P283AR9P1362_H11 130 LPAPKNLVVSRVTEDSARLSWPKIQGQHFDSFLIQY QESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSI YGVDVKYDIDSRPISSNPLSAIFTT P283BR9P1363_ 131 LPAPKNLVVSRVTEDSARLSWQRADDILPYFDSFLI Petition 870260068423, dated 10 / 07 / 2026, p. 159 / 201 145 / 165 A10 QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVKYDIDSRPISSNPLSAIFTT P283BR9P1363_ A8 132 LPAPKNLVVSRVTEDSARLSWVRSDTARFFDSFLIQ YQESEKVGEAIVLTVPGSERSYDYLTTVSYD IYGVDVKYDIDSRPISSNPLSAIFTT P283BR9P1363_ B2 133 LPAPKNLVVSRVTEDSARLSWASTVDPHPRFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVKYDIDSRPISSNPLSAIFTT P136349 B13634 LPAPKNLVVSRVTEDSARLSWQRHSDAHPLFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVKWEGNRPVASNPLSAIFTT P283BR9P1363_ C4 135 LPAPKNLVVSRVTEDSARLSWPIVFLIFLINTQH YQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVS IYGVDVQYTATGQPERSNPLSAIFTT P283BR9P1363_ C8 136 LPAPKNLVVSRVTEDSARLSWAKTSDLHPLFDSFLI QYQESEKVGEAIVLTVPGSERSYDYLTTVGYLTV SIYGVDVKWEGNRPVASNPLSAIFTT P283BR9P1363_ D11 137 LPAPKNLVVSRVTEDSARLSWNKKHDGQPTFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVVEGSYPASSIFPT P28336363 138 LPAPKNLVVSRVTEDSARLSWIKSETSQPAFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVKWEGNRPVASNPLSAIFTT Petition 870260068423, of 10 / 07 / 2026, p. 160 / 201 146 / 165 P283BR9P1363_ E6 139 LPAPKNLVVSRVTEDSARLSWYARKFISPFDSFLIQ YQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVS IYGVDVKWEGNRPVASNPLSAIFTT P283BR9P1363_ F2 140 LPAPKNLVVSRVTEDSARLSWYRPDNRAGAFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVKYDIDSRPISSNPLSAIFTT P283BR9P1363_ F4 141 LPAPKNLVVSRVTEDSARLSWERIVQTPHFDFLIQ YQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVS IYGVDVKWEGNRPVASNPLSAIFTT P283BR9P1363_ F6 142 LPAPKNLVVSRVTEDSARLSWPEEAVTATSFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVKWEGNRPVASNPLSAIFTT P283BR9P1363_ G2 143 LPAPKNLVVSRVTEDSARLSWPKNQTNRHFDSFLIQ YQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVS IYGVDVKWEGNRPVASNPLSAIFTT P283BR9P1363_ G5 144 LPAPKNLVVSRVTEDSARLSWYRATTPAPHDFSLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTV SIYGVDVKWEGNRPVASNPLSAIFTT P283BR9P1363_ G7 145 LPAPKNLVVSRVTEDSARLSWSAKKFPRHFDSFLIQ YQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVS IYGVDVKWEGNRPVASNPLSAIFTT P283DR9P1364_ A4 146 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIAYP EWPVQGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY Petition 870260068423, de 10 / 07 / 2026, pág. 161 / 201 147 / 165 GVKGGDWSEPLSAIFTT P283DR9P1364_ A7 147 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIAYP EWPVRGDAIVLTVPGSERSYDLTGLKPGTEYWVIIQ GVKGGTDSFPLSAIFTT P283DR9P1364_ B1 148 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIAYP EIPLGGEAIVLTVPGSERSYDLTGLKPGTEYFVVIY GVKGGLLSSPLSAIFTT P283DR9P1364_ B11 149 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYISYP EWEQLGEAIVLTVPGSERSYDLTGLKPGTEYFVVIY GVKGGALSAPLSAIFTT P283DR9P1364_ B4 150 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAISYP EWPPPGEAIVLTVPGSERSYDLTGLKPGTEYWVIIL GVKGGDQSWPLSAIFTT P283DR9P1364_ C10 151 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYP EWPKDGEAIVLTVPGSERSYDLTGLKPGTEYAVFIW GVKGGVYSNPLSAIFTT P283DR9P1364_ D11 152 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPPKGEAIVLTVPGSERSYDLTGLKPGTEYFVVIY GVKGGIHSAPLSAIFTT P283DR9P1364_ D8 153 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYP ETPIQGEAIVLTVPGSERSYDLTGLKPGTEYFVIIH GVKGGITSFPLSAIFTT P283DR9P1364_ 154 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGISYP Petition 870260068423, de 10 / 07 / 2026, pág. 162 / 201 148 / 165 D9 EWPPLGEAIVLTVPGSERSYDLTGLKPGTEYWVIIF GVKGGERSWPLSAIFTT P283DR9P1364_ E3 155 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIAYP ELPIGGEAIVLTVPGSERSYDLTGLKPGTEYFVIIR GVKGGTLSPPLSAIFTT P283DR9P1364_ E5 156 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWYSYP EWPVPGEAIVLTVPGSERSYDLTGLKPGTEYWVIIQ GVKGGKLSWPLSAIFTT P283DR9P1364_ E7 157 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYP EWPVRGEAIVLTVPGSERSYDLTGLKPGTEYWVIIY GVKGGDRSNPLSAIFTT P283DR9P1364_ E8 158 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYP EWPVHGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY GVKGGVLSEPLSAIFTT P283DR9P1364_ E9 159 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPTKGEAIVLTVPGSERSYDLTGLKPGTEYFVVIN GVKGGWRSFPLSAIFTT P283DR9P1364_ F2 160 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIAYP EWPVPGEAIVLTVPGSERSYDLTGLKPGTEYFVIIQ GVKGGFGSFPLSAIFTT P283DR9P1364_ F6 161 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIAYP EREQDKWGEAIVLTVPGSERSYDLTGLKPGTEYWVI IQGVKGGRPSTPSAILTT Petition 870260068423, de 10 / 07 / 2026, pág. 163 / 201 149 / 165 P283DR9P1364_ F8 162 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYP EWPPGEAIVLTVPGSERSYDLTGLKPGTEYFVIIYG VKGGWTSPPLSAIFTT P283DR9P1364_ G10 163 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYP EWPGSGEAIVLTVPGSERSYDLTGLKPGTEYFVVIF GVKGGSQSWPLSAIFTT P283DR9P1364_ G9 164 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIWYP EWPVGGEAIVLTVPGSERSYDLTGLKPGTEYWVNIS GVKGGEYSFPLSAIFTT P283DR9P1364_ H1 165 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQISYP EWPVHGEAIVLTVPGSERSYDLTGLKPGTEYWVIIW GVKGGRQSWPLSAIFTT P283DR9P1364_ H11 166 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP ELPLGGEAIVLTVPGSERSYDLTGLKPGTEYFVIIW GVKGGDRSEPLSAIFTT P283DR9P1364_ H6 167 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIAYP ETPVRGEAIVLTVPGSERSYDLTGLKPGTEYFVIII GVKGGQESFPLSAIFTT P283DR9P1364_ H9 168 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSISYI EYPEIPGGEAIVLTVPGSERSYDLTGLKPGTEYWVP IWGVKGGIQSWPLSAIFTT P283ER9P1365_ A1 169 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIAYV EWWHRGEAISLPVPGSERSYDLTGLKPGTEYNVIIT Petition 870260068423, dated 10 / 07 / 2026, page 164 / 201 150 / 165 GVKGGIPSHPLGAIFTT P283ER9P1365_ A7 170 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYW ESEVYGEAIALPVPGSERSYDLTGLKPGTEYQVSII GVKGGVYSQPLAAIFTT P283ER9_P1365 B1361 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIGYA EPVVTGEAISLSVPGSERSYDLTGLKPGTEYWVVII GVKGGINSYPLGAIFTT P283ER9P1365_ C1 172 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYW ESEVYGEAIALPVTGSERSYDLTGLKPGTEYQVSII GVKGGVYSQPLAAIFTT P283ER9P1365_ E2 173 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYR ESEFRGEAIALPVTGSERSYDLT GLKGGVTGVKGVKF P283ER9P1365_ F4 174 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYR ESEFRGEAIALPVPGSERSYDLTGLKPGTKYSVIII GVKGGEFSQPLGAIFTT P283ER9P1365_ G1 175 LPAPKNLVVSRVDSW ESEFRGEAIALSVPGSERSYDLTGLKPGTKYRVIII GVKGGEFSQPLGAIFTT P283ER9P1365_ G3 176 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGISYY EWAPNGEAIQLSVPGSERSYDLTGLVIKPGTEYKHIPGGIFGGIFGITHT P283ER9P1365_ 177 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYR Petition 870260068423, of 10 / 07 / 2026, p. 165 / 201 151 / 165 H3 ESEFRGEAIALPVPGSERSYDLTGLKPGTKYRVIII GVKGGEFSQPLSAIFTT P283FR9P1366_ A1 178 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWITYP EWPVPGEAIVLTVPGSERSYDLTGLKPGTEYAVFIW GVKGGDASEPLSAIFTT P283FR9P1366_ A5 179 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIAYP EWPTRGEAIVLTVPGSERSYDLTGLKPGTEYFVVIY GVKGGSPSPPLSAIFTT P283FR9P1366_ A9 180 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYG EYPGPGEAIVLTVPGSERSYDLTGLKPGTEYWVPIW GVKGGELSEPLSAIFTT P283FR9P1366_ B7 181 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWITYP EWPVNGEAIVLTVPGSERSYDLTGLKPGTEYWVVIW GVKGGVESPPLSAIFTT P283FR9P1366_ C2 182 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKISYP EWPPEGEAIVLTVPGSERSYDLTGLKPGTEYAVFIW CVKGGEHSWPLSAIFTT P283FR9P1366_ C3 183 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIAYP EWPDGGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY GVKGGILSPPLSAIFTT P283FR9P1366_ C4 184 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYP EWPVRGEAIVLTVPGSERSYDLTGLKPGTEYWVIII GVKGGEDSWPLSAIFTT Petição 870260068423, de 10 / 07 / 2026, pág. 166 / 201 152 / 165 P283FR9P1366_ C6 185 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYP EWPVYGEAIVLTVPGSERSYDLTGLKPGTEYFVVIY GVKGGNYSDPLSAIFTT P283FR9P1366_ D12 186 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPLGGEAIVLTVPGSERSYDLTGLKPGTEYWVIIL GVKGGDQSWPLSAIFTT P283FR9P1366_ D6 187 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIFYP ELVFPGEAIVLTVPGSERSYDLTGLKPGTEYWVNIS GVKGGEHSWPLSAIFTT P283FR9P1366_ D7 188 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYP ELPVKGEAIVLTVPGSERSYDLTGLKPGTEYFVVIW GVKGGTYSGPLSAIFTT P283FR9P1366_ D8 189 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIAYP EIPIAGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY GVKGGDWSDPLSAIFTT P283FR9P1366_ E11 190 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPVPGEAIVLTVPGSERSYDLTGLKPGTEYWVIIK GVKGGNISWPLSAIFTT P283FR9P1366_ F5 191 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIGYP EWPIKGEAIVLTVPGSERSYDLTGLKPGTEYWVIIW GVKGGDRSEPLSAIFTT P283FR9P1366_ F8 192 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYP EIAKWGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY Petição 870260068423, de 10 / 07 / 2026, pág. 167 / 201 153 / 165 GVKGGVHSFPLSAIFTT P283FR9P1366_ F9 193 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIFYP ELPIAGEAIVLTVPGSERSYDLTGLKPGTEYWVNIS GVKGGYESWPLSAIFTT P283FR9P1366 GLKGGYESWPLSAIFTT P283FR9P1366 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSYYP ELPVEGEAIVLTVPGSERSYDLTGLKPGTEYWVIIW GVKGGATSEPLSAIFTT P283FR9P1366_ G5 195 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYP EYPALGEAIVLTVPGSERSYDLTGLKPGTEYFVIII GVKGGDESFPLSAIFTT P283FR9P1366_ G8 196 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP ELPIGGEAIVLTVPGSERSYDLT GLKPGTEYF P283FR9P1366_ H10 197 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYP EWPPEGEAIVLTVPGSERSYDLTGLKPGTEYFVVIY GVKGGHLSDPLSAIFTT P283FR9P1366_ H11 198 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIQYL ETAPDGEAIVLTVPGSERSYDLTGLKPGTEYYVWIP GVKGGAFSPLSAIFTT P283FR9P1366_ H3 199 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYP EWPIKGEAIVLTVPGSERSYDLTGLKPGTEYWVVIY GVKGGVFSEPLSAIFTT P283FR9P1366_ 200 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIYIEN Petition 870260068423, of 10 / 07 / 2026, p. 168 / 201 154 / 165 H5 KVNGEAIVLTVPGSERSYDLTGLKPGTEYHVTIGGV KGGTESNTLSAIFTT P283FR9P1366_ H6 201 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPVTGEAIVLTVPGSERSYDLTGLKPGTEYWVIIF GVKGGERSWPLSAIFTT P283FR9P1366_ H7 202 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYP EYPALGEAIVLTVPGSERSYDLTGLKPGTEYFVIIA GVKGGIQSWPLSAIFTT P283FR9P1366_ H8 203 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYISYP EWPGSGEAIVLTVPGSERSYDLTGLKPGTEYAVFIW CVKGGWLSDPLSAIFTT P283FR9P1366_ H9 204 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIAYP EWPVNGEAIVLTVPGSERSYDLTGLKPGTEYWVVIW GVKGGVNSYPLSAIFTT P283GR7P1367_ A11 205 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPTDGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY GVKGGSYSEPLSAIFTT P283GR7P1367_ B4 206 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSILYY ELPPSGEAIVLTVPGSERSYDLTGLKPGTEYTVSIF GVKGGDNSFPLSAIFTT P283GR7P1367_ B7 207 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPTDGEAIVLTVPGSERSYDLTGLKPGTEYFVVIY GVKGGHWSYPLSAIFTT Petição 870260068423, de 10 / 07 / 2026, pág. 169 / 201 155 / 165 P283GR7P1367_ B9 208 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIWYH EYHPRGEAIVLTVPSSERSYDLTGLKPGTEYDVVIS GVKGGHWSYPLSAIFTT P283GR7P1367_ C9 209 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIGYP EWPLGGEAIVLTVPGSERSYDLTGLKPGTEYWVIIY GVKGGEYSDPLSAIFTT P283GR7P1367_ E5 210 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIWYH EYHPRGEAIVLTVPGSERSYDLTGLKPGTEYDVVIS GVKGGHWSYPLSAIFTT P283GR7P1367_ F5 211 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPTDGEAIVLTVPGSERSYDLTGLKPGTEYFVIIY GVKGGALSRPLSAIFTT P283GR7P1367_ G8 212 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIAYP EYVWGGEATSLGEAIVLTVPGSERSYDLTGLKPGTE YFVVITGVKGGLGSYPLSAIFTT P283GR7P1367_ H2 213 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPTDGEAIVLTVPGSERSYDLTGLKPGTEYFVVIY GVKGGGRSYPLSAIFTT P283GR7P1367_ H8 214 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSINYW EEDPAGEAIVLTVPGSERSYDLTGLKPGTEYRVLIG GVKGGHGSLPLSAIFTT P283GR7P1367_ H9 215 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYP EWPTDGEAIVLTVPGSERSYDLTGLKPGTEYFVVIY Petition 870260068423, dated 10 / 07 / 2026, page 170 / 201 156 / 165 GVKGGGRSAPLSAIFTT P283HR7P1368_ A10 216 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIFYL EPFPRGEAIPLEVPGSERSYDLTGLKPGTEYSVDIR GVKGGDHSDPLWAYFTT P283HR7P1368_ B12 217 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIGYV EFTRAGEAISLSVPGSERSYDLTGLKPGTEYHVVII GVKGGEPSHPLGAPFTT P283HR7P1368_ C3 218 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIGYA EPAVTGEAISLSVPGSKRSYDLTGLKPGTEYWVVII GVKGGINSYPLGASFTT P283HR7P1368_ D1 219 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGISYY EWAPNGEAIQLSVPGSERSYDLTGLKPGTEYHVVII GVKGGEPSHPLGAPFTT P283HR7P1368_ D2 220 LPAPKNLVVSRVTEDSARLSWTAPDAAFNSFGIGYA EPAVTGEAISLSVPGSERSYDLTGLKPGTEYWVVII GVKGGINSYPLGASFTT P283HR7P1368_ D4 221 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIWCV EPIPEGEAIPLFVPGSERSYDLTGLKPGTEYRVGIR GVKGGTFSSPLAAPFTT P283HR7P1368_ F10 222 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYR ESEFRGEAIALPVPGSERSYDLTGLKPGTKYRVIII GVKGGEFSQPLGAIFTT P283HR7P1368_ 223 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIGYI Petição 870260068423, de 10 / 07 / 2026, pág. 171 / 201 157 / 165 F6 EWVHRGEAISLHVPGSERSYDLTGLKPGTEYVVAIV GVKGGEPSTPLGAPFTT P283HR7P1368_ G1 224 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLITYW EIEPEGEAIFLGVPGSERSYDLTGLKPGTEYRVQIN GVKGGTISYPLFAGFTT P283HR7P1368_ G10 225 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIAYV EWWHRGEAISLPVPGSERSYDLTGLKPGTEYWVTIL GVKGGIISTPLGASFTT P283HR7P1368_ G11 226 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIGYA EPAVTGEAISLSVPGSERSYDLTGLKPGTEYWVVII GVKGGINSYPLGASFTT P283HR7P1368_ H1 227 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIAYI ETARWGEAISLTVPGSERSYDLTGLKPGTEYNVVII GVKGGTPSHPLGAPFTT P283HR7P1368_ H8 228 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGITYL DPRNGEAISLNVPGSERSYDLTGLKPGTEYWVVIIG VKGGINSYPLGASFTT CD8S368 229 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIAYP EWPPPGEAIVLTVPGSCRSYDLTGLKPGTEYFVIIQ GVKGGVESWPLSAIFTT CD8S367 230 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYP EWPPQGEAIVLTVPGSCRSYDLTGLKPGTEYFVVIY Petition 870260068423, dated 10 / 07 / 2026, page 172 / 201 158 / 165 GVKGGSYSAPLSAIFTT CD8S370 231 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAITYI EKEHIEDGEAIVLTVPGSCRSYDLTGLKPGTEYWVP IWGVKGGANSWPLSAIFTT CD8S365 232 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGILYY EPVDSGEAITLPVPGSCRSYDLTGLKPGTEYWVVIT GVKGGAPSTPLGTIFTT CD8S369 233 LPAPKNLVVSRVTEDSARLSWAKRPGAFDSFLIQYQ ESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVSIY GVDVKYDIDSRPISSNPLSAIFTT CD8S366 234 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWITYP EWPDPGGEAIVLTVPGSCRSYDLTGLKPGTEYFVVI YGVKGGEIYSPLSAIFTT Clone SEQ ID NO: Main Sequence CD8S3 71 235 P282DR9P1359_F5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFQIAYPEYPPPGEAIVLT VPGSERSYDLTGLKPGTEYFVIIQ GVKGGVESWPLSAIFTT Petition 870260068423, dated 10 / 07 / 2026, page 173 / 201 159 / 165 CD8S3 72 236 P282DR9P1359_F5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFQIAYPELPPPGEAIVLT VPGSERSYDLTGLKPGTEYFVIIQ GVKGGVESWPLSAIFTT CD8S3 73 237 P282DR9P1359_F5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFQIAYPEIPPPGEAIVLT VPGSERSYDLTGLKPGTEYFVIIQ GVKGGVESWPLSAIFTT CD8S3 74 238 P282DR9P1359_F5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFQIAYPEWPPPGEAIVLT VPGSERSYDLTGLKPGTEYFVIIQ GVKGGVESYPLSAIFTT CD8S3 75 239 P282DR9P1359_F5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFQIAYPEWPPPGEAIVLT VPGSERSYDLTGLKPGTEYFVIIQ GVKGGVESLPLSAIFTT CD8S3 76 240 P282DR9P1359_F5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFQIAYPEWPPPGEAIVLT VPGSERSYDLTGLKPGTEYFVIIQ GVKGGVESSPLSAIFTT CD8S3 77 241 P282DR9P1359_F5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFQIAYPEWPPPGEAIVLT VPGSERSYDLTGLKPGTEYFVIIQ Petition 870260068423, dated 10 / 07 / 2026, page 174 / 201 160 / 165 GVKGGVESEPLSAIFTT CD8S3 78 242 P282DR9P1359 _G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIEEGEAIV LTVPGSERSYDLTGLKPGTEYWVP IWGVKGGANSWPLSAIFTT CD8S3 79 243 P282DR9P1359 _G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIESGEAIV LTVPGSERSYDLTGLKPGTEYWVP IWGVKGGANSWPLSAIFTT CD8S3 80 244 P282DR9P1359 _G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIEDGEAIV LTVPGSERSYDLTGLKPGTEYYVP IWGVKGGANSWPLSAIFTT CD8S3 81 245 P282DR9P1359 _G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIEDGEAIV LTVPGSERSYDLTGLKPGTEYFVP IWGVKGGANSWPLSAIFTT CD8S3 82 246 P282DR9P1359 _G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIEDGEAIV LTVPGSERSYDLTGLKPGTEYSVP IWGVKGGANSWPLSAIFTT CD8S3 83 247 P282DR9P1359 _G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIEDGEAIV Petition 870260068423, de 10 / 07 / 2026, pág. 175 / 201 161 / 165 LTVPGSERSYDLTGLKPGTEYWVP IYGVKGGANSWPLSAIFTT CD8S3 84 248 P282DR9P1359_G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIEDGEAIV LTVPGSERSYDLTGLKPGTEYWVP IFGVKGGANSWPLSAIFTT CD8S3 85 249 P282DR9P1359_G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIEDGEAIV LTVPGSERSYDLTGLKPGTEYWVP ISGVKGGANSWPLSAIFTT CD8S3 86 250 P282DR9P1359_G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIEDGEAIV LTVPGSERSYDLTGLKPGTEYWVP IWGVKGANSYPLSAIFT CD8S3 87,251 P282DR9P1359_G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIEDGEAIV LTVPGSERSYDLTGLKPGTEYWVP IWGVKGGANSEPLSAIFTT CD8S3 88 252 P282DR9P1359_G7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAITYIEKEHIEDGEAIV LTVPGSERSYDLTGLKPGTEYWVP IWGVKGGAQSWPLSAIFTT CD8S3 253 P282ER9P1360_C8 LPAPKNLVVSRVTEDSARLSWTAP Petition 870260068423, de 10 / 07 / 2026, pág. 176 / 201 162 / 165 89 DAAFDSFGILYYEPVDSGEAITLP VPGSERSYDLTGLKPGTEYFVVIT GVKGGAPSTPLGTIFTT CD8S3 90 254 P282ER9P1360_C8 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFGILYYEPVDSGEAITLP VPGSERSYDLTGLKPGTEYYVVIT GVKGGAPSTPLGTIFTT CD8S3 91 255 P282ER9P1360_C8 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFGILYYEPVDSGEAITLP VPGSERSYDLTGLKPGTEYHVVIT GVKGGAPSTPLGTIFTT CD8S3 92 256 P282DR9P1359_F7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAIAYPEYPPQGEAIVLT VPGSERSYDLTGLKPGTEYFVVIY GVKGGSYSAPLSAIFTT CD8S3 93 257 P282DR9P1359_F7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAIAYPELPPQGEAIVLT VPGSERSYDLTGLKPGTEYFVVIY GVKGGSYSAPLSAIFTT CD8S3 94 258 P282DR9P1359_F7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAIAYPEIPPQGEAIVLT VPGSERSYDLTGLKPGTEYFVVIY GVKGGSYSAPLSAIFTT Petition 870260068423, dated 10 / 07 / 2026, page 177 / 201 163 / 165 CD8S3 95 259 P282DR9P1359_F7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAIAYPEHPPQGEAIVLT VPGSERSYDLTGLKPGTEYFVVIY GVKGGSYSAPLSAIFTT CD8S3 96 260 P282DR9P1359_C5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFYITYPEWPDPGGEAIVL TVPGSERSYDLTGLKPGTEYFVVI YGVKGGEIYSPLSAIFTT CD8S3 97 261 P282DR9P1359_C5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFQITYPEWPDPGGEAIVL TVPGSERSYDLTGLKPGTEYFVVI YGVKGGEIYSPLSAIFTT CD8S3 98 262 P282DR9P1359_C5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFSITYPEWPDPGGEAIVL TVPGSERSYDLTGLKPGTEYFVVI YGVKGGEIYSPLSAIFTT CD8S3 99 263 P282DR9P1359_C5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFWITYPEYPDPGGEAIVL TVPGSERSYDLTGLKPGTEYFVVI YGVKGGEIYSPLSAIFTT CD8S4 00 264 P282DR9P1359_C5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFWITYPELPDPGGEAIVL TVPGSERSYDLTGLKPGTEYFVVI Petition 870260068423, dated 10 / 07 / 2026, page 178 / 201 164 / 165 YGVKGGEIYSPLSAIFTT CD8S4 01 265 P282DR9P1359_C5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFWITYPEIPDPGGEAIVL TVPGSERSYDLTGLKPGTEYFVVI YGVKGGEIYSPLSAIFTT CD8S4 02 266 P282DR9P1359_C5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFWITYPEWPPPGGEAIVL TVPGSERSYDLTGLKPGTEYFVVI YGVKGGEIYSPLSAIFTT CD8S4 03 267 P282DR9P1359_F7 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFAIAYAEWPPQGEAIVLT VPGSERSYDLTGLKPGTEYFVVIY GVKGGSYSAPLSAIFT CD8S4 04 268 P282DR9P1359_C5 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFWITYAEWPDPGGEAIVL TVPGSERSYDLTGLKPGTEYFVVI YGVKGGEIYSPLSAIFTT CD8S4 05 269 P282ER9P1360_C8 LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFGILYYEPVDSGEAITLT VPGSERSYDLTGLKPGTEYWVVIT GVKGGAPSTPLGTIFTT SEQ ID NO: 270 Tencon25 LPAPKNLVVSEVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSER Petition 870260068423, de 10 / 07 / 2026, pág. 179 / 201 165 / 165 SYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT SEQ ID NO: 271 Cyno CD8alpha MRNQAPGRPKGATSPPPLPTGSRAPPVAPELRAEPRPGERVMAPPVTALLLPLV LLLHAARPNQFRVSPLGRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGTAARPTFLLY LSQNKPKAAEGLDTQRFSGKRLGDTFVLTLRDFRQENEGYYFCSALSNSIMYFSHFVPV FLPAKPTTTPARPPTPAPTTASQPLSLRPEACRPAAGGSVNTRGLDFACDIYIWAPLA GACGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGGKPSLSDRYV Petition 870260068423, dated 10 / 07 / 2026, pages 180 / 201

Claims

1 / 2 CLAIMS 1. Isolated FN3 domain for CD8A binding that specifically binds to a human CD8A protein characterized by comprising an amino acid sequence of any of the SEQ ID NO: 229, 230, 231, 232, 233 or 234.

2. Isolated FN3 domain binding to CD8A, according to claim 1, characterized in that the FN3 domain binding to CD8A is conjugated to a second molecule.

3. Isolated FN3 domain binding to CD8A, according to claim 2, characterized in that the second molecule is a detectable marker.

4. Isolated FN3 domain binding to CD8A, according to claim 3, characterized in that the detectable marker is a radioactive isotope, magnetic spheres, metallic spheres, colloidal particles, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, digoxigenin or hapten.

5. Isolated FN3 domain for binding to CD8A, according to claim 1, characterized by further comprising a methionine at the N-terminal of the FN3 domain for binding to CD8A.

6. Isolated FN3 domain for CD8A binding, according to claim 1, characterized in that the FN3 domain for CD8A binding is coupled to a half-life extending portion.

7. Isolated FN3 domain for CD8A binding, according to Petition 870260068423, dated 10 / 07 / 2026, page 198 / 201 2 / 2 claim 6, characterized in that the half-life-extending portion is albumin, an albumin-binding molecule, a polyethylene glycol (PEG) or an Fc region of an immunoglobulin.

8. Diagnostic kit characterized by comprising the FN3 domain for binding to CD8A as defined in claim 1.

9. A trapping agent characterized by comprising the FN3 domain for binding to CD8A as defined in claim 1.

10. Method for detecting cells expressing CD8 in a biological sample, characterized by comprising treating the biological sample with the capture agent as defined in claim 9, and detecting the binding of the biological sample to the CD8A-binding FN3 domain of that capture agent. Petition 870260068423, dated 10 / 07 / 2026, pp. 199 / 201

Images