WO2025101471A1

WO2025101471A1 - Anti-cd27 antibody cell-based biological potency assay

Info

Publication number: WO2025101471A1
Application number: PCT/US2024/054492
Authority: WO
Inventors: Susan J. ABBONDANZO; Dhanvanthri S. Deevi; Kevin GURNEY; Asra MIRZA; Amy H. M. TOU; Aarron Willingham; Junming Yie
Original assignee: Merck Sharp and Dohme LLC
Current assignee: Merck Sharp and Dohme LLC
Priority date: 2023-11-09
Filing date: 2024-11-05
Publication date: 2025-05-15
Anticipated expiration: 2026-05-09

Abstract

This disclosure relates generally to a cell-based functional assay to determine the potency of anti-CD27 antibodies.

Description

25857 ANTI-CD27 ANTIBODY CELL-BASED BIOLOGICAL POTENCY ASSAY CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The application claims the benefit of priority to U.S. Provisional Application No. 63/597,534, filed November 9, 2023, the contents of each of which are incorporated herein by reference in their entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0002] The sequence listing of the present application is submitted electronically via Patent Center with a file name “25857-WO-PCT_SL.xml”, creation date of September 9, 2024, and a size of 10,116 bytes. This sequence listing submitted via Patent Center is part of the specification and is herein incorporated by reference in its entirety. FIELD [0003] This disclosure relates generally to a cell-based potency assay to measure the biological activity of anti-CD27 antibodies. BACKGROUND OF THE INVENTION [0004] CD27 is a co-stimulatory member of the Tumor Necrosis Factor Receptor (TNFR) superfamily, which was identified as a membrane molecule on human T cells (van Lier et al., 1987, J Immunol 139:1589-96). According to current evidence, CD27 has a single ligand, CD70, which is also a TNF (Tumor Necrosis Factor) family member (Goodwin et al., 1993, Cell 73:447-56). CD27 and CD70 have been identified and cloned in the mouse system (Gravestein et al., 1993, Eur J Immunol 23:943-50; Tesselaar et al., J Immunol 159:4959-65). CD27 is constitutively expressed on naïve, activated and memory T cells, Natural Killer (NK) T cells, memory B cells, and a subset of NK cells that are potentially activated by CD70 expressed on sister immune cells (Baars et al., 1995, J Immunol 154:17-25; Hamann et al., 1997, J Exp Med 186:1407-18). CD27 signaling plays an important role in both generation and maintenance of cytotoxic T lymphocyte responses. [0005] CD27 co-stimulation during antigen presentation promotes T cell activation, clonal expansion, differentiation into effector cells, and memory programming. These effects appear to be especially relevant in the context of low affinity CD8 T cells, and may potentiate development of a broader T cell repertoire. CD27 also plays an important role in survival of activated T cells after they traffic to non-lymphoid organs. [0006] An anti-CD27 antibody acts as an inhibitor of the co-stimulatory receptor CD27 and a cancer immunotherapeutic with the potential to combine with immune checkpoint inhibitors (including anti-PD1 antibodies such as pembrolizumab), therapeutic cancer vaccines, or therapies that induce immunogenic cell death to increase benefit to patients with various tumor types. Given that anti-CD27 antibody signaling enhances priming of cytotoxic T lymphocyte responses even in the absence of primed antigen presenting cells, the use of an anti-CD27 antibody as a monotherapy or in combination therapy with immune checkpoint inhibitors (including anti- programmed cell death protein 1 (PD-1) antibodies such as pembrolizumab), therapeutic cancer vaccines, or therapies that induce immunogenic cell death to increase benefit to patients with various tumor types represents an attractive immunotherapy approach for cancers with minimal T cell infiltration and/or inadequate immunological responses. [0007] Furthermore, it is desirable to establish a robust cell-based assay to determine the potency of anti-CD27 antibody drug products for pre-clinical and clinical use. SUMMARY OF THE INVENTION [0008] The present disclosure encompasses an insight that methods for determining the potency of anti-CD27 antibodies can be improved by clustering a plurality of the anti-CD27 antibodies. In some embodiments, the method comprises: a) providing a reporter cell line wherein the reporter cell line is a T cell line, comprising one or more nucleic acid molecules encoding a reporter molecule operably linked to a promoter responsive to CD27 signaling and encoding CD27; b) clustering a plurality of anti-CD27 antibodies; c) incubating the anti-CD27 antibodies with the reporter cell line, to allow binding of an antigen binding domain of the anti-CD27 antibodies to the CD27 on the reporter cell line resulting in the expression of the reporter molecule; and d) measuring the activity of the reporter molecule to determine the potency of the anti-CD27 antibodies. [0009] The summary of the technology described herein is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims. [0010] In one aspect, provided are methods of determining the potency of anti-CD27 antibodies to produce an immune response comprising: a) providing a reporter cell line wherein the reporter cell line is a T cell line comprising one or more nucleic acid molecules encoding a reporter molecule operably linked to a promoter responsive to CD27 signaling and encoding CD27; b) clustering a plurality of anti-CD27 antibodies; c) incubating the anti-CD27 antibodies with the reporter cell line, to allow binding of an antigen binding domain of the anti-CD27 antibodies to the CD27 on the reporter cell line resulting in the expression of the reporter molecule; and d) measuring the activity of the reporter molecule to determine the potency of the anti-CD27 antibodies. [0011] In some embodiments, the step b) comprises providing an accessory cell line expressing a Fcγ receptor. In some embodiments, the Fcγ receptor is FcγRIIB. [0012] In some embodiments, the methods further comprise co-culturing the reporter cell line and the accessory cell line. In some embodiments, the reporter cell line and the accessory cell line are pre-mixed before the co-culturing step. In some embodiments, the reporter cell line and the accessory cell line are sequentially added to an assay plate comprising a plurality of wells. [0013] In some embodiments, the methods comprise incubating the anti-CD27 antibodies with the co-cultured reporter cell line and accessory cell line, to allow Fc regions of the anti-CD27 antibodies to bind to the Fcγ receptor on the accessory cell line inducing CD27 clustering on the surface of the reporter cell line. In some embodiments, the accessory cell line is CD70 negative. In some embodiments, the accessory cell line is a K562 cell line. [0014] In some embodiments, the reporter cell line and the accessory cell line are co-cultured at a ratio of about 1:5 to about 5:1. In some embodiments, the reporter cell line and the accessory cell line are pre-mixed in the ratio of 1:1 before co-culture. [0015] In some embodiments, the co-culture of the reporter cell line and the accessory cell line is incubated with the anti-CD27 antibodies provided in a series of dilutions. [0016] In some embodiments, the co-culture of the reporter cell line and the accessory cell line is incubated with the anti-CD27 antibodies for at least 2 hours. In some embodiments, the co- culture of the reporter cell line and the accessory cell line is incubated with the anti-CD27 antibodies for 2 to 6 hours. In some embodiments, the co-culture of the reporter cell line and the accessory cell line is incubated with the anti-CD27 antibodies for about 5 hours. [0017] In some embodiments, cell seeding density of the reporter cell line is from 10,000 to 500,000 cells/well. In some embodiments, cell seeding density of the reporter cell line is from 50,000 to 200,000 cells/well. In some embodiments, cell seeding density of the accessory cell line is from 10,000 to 500,000 cells/well. In some embodiments, cell seeding density of the accessory cell line is from 50,000 to 200,000 cells/well. [0018] In some embodiments, the step b) comprises providing anti-CD27 antibodies immobilized by an antibody-binding protein. In some embodiments, the antibody-binding protein is Protein A. [0019] In some embodiments, the methods further comprise incubating the immobilized anti- CD27 antibodies with the reporter cell line, to allow binding of an antigen binding domain of the 25857 anti-CD27 antibodies to the CD27 on the reporter cell line resulting in the expression of the reporter molecule. [0020] In some embodiments, the anti-CD27 antibodies are immobilized by the antibody- binding protein on a plate. [0021] In some embodiments, the reporter cell line is a Jurkat T cell line. [0022] In some embodiments, the promoter responsive to CD27 signaling is an NFκB promoter, an NFAT promoter, or an AP-1 promoter. In some embodiments, the promoter responsive to CD27 signaling is an NFκB promoter. [0023] In some embodiments, the reporter molecule is a luciferase, a fluorescent protein, an alkaline phosphatase, a beta lactamase, or a beta galactosidase. In some embodiments, the reporter molecule is a firefly luciferase, a Renilla luciferase, or a nanoluciferase. In some embodiments, the reporter molecule is nanoluciferase. [0024] In some embodiments, the anti-CD27 antibodies comprise (a) three heavy chain complementarity determining regions (HC-CDRs), wherein HC-CDR1 is SEQ ID NO: 1, HC- CDR2 is SEQ ID NO: 2, and HC-CDR3 is SEQ ID NO: 3; and (b) three light chain complementarity determining regions (LC-CDRs), wherein LC-CDR1 is SEQ ID NO: 4, LC- CDR2 is SEQ ID NO: 5, and LC-CDR3 is SEQ ID NO: 6. [0025] In some embodiments, the anti-CD27 antibodies comprise a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 9. [0026] In some embodiments, the anti-CD27 antibodies comprise a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 8 and the light chain comprises SEQ ID NO: 10. [0027] In one aspect, provided are methods for determining any degradation of anti-CD27 antibodies from a first time period to a second time period, comprising: (i) performing a measurement of the potency of the anti-CD27 antibodies; (ii) performing a second measurement of the potency of the anti-CD27 antibodies according to the method of any one of claims 1-32 at a second later time period; and (iii) comparing the potency from the initial measurement and the potency from the second measurement; wherein a reduction in the measurement of potency from the first time period to the second later time period is indicative of a degradation of the anti- CD27 antibodies. [0028] In one aspect, provided are kits for determining the potency of anti-CD27 antibodies, comprising a first cell line which is a human T cell line comprising one or more nucleic acid molecules encoding a reporter molecule operably linked to a promoter responsive to CD27 signaling and encoding CD27, a second cell line that is FcγRIIb positive and CD70 negative, media that includes a substrate for the nucleic acid molecule encoding the reporter molecule of the first cell line, and anti-CD27 antibodies. BRIEF DESCRIPTION OF THE FIGURES [0029] FIG.1A shows the expression of CD27 on engineered Jurkat cells by florescence- activated cell-sorting (FACS) binding assay. [0030] FIG.1B shows the expression of CD70 and FcγRIIb on JY cells. FcγRIIb positive human B cell line JY was tested for presence of CD70 by flow cytometry. JY cells showed significant expression of CD70, which cannot be used in a co-culture system as CD70 is ligand/co-receptor for CD27. [0031] FIG.1C shows the expression of FcγRIIb on human myelogenous leukemia cell line K562. K562 is negative for CD70 but expresses FcγRIIb on cell surface. [0032] FIG.1D shows that co-culturing K562 cell line with human T lymphocyte cell line Jurkat with over expression of CD27 and NF-kB driven Nanoluciferase (“Jurkat-CD27-Nanoluc cell line”) significantly increased boserolimab-induced luciferase reporter gene activation as compared to Jurkat-CD27-Nanoluc cell line alone. The enhanced luciferase reporter gene activity is believed to be due to a clustering effect from interaction of the Fc region of boserolimab and FcγRIIb on K562 cells, as illustrated in the FIG.1E and FIG.1F. [0033] FIG.1E illustrates signal transduction of CD27 receptor in Jurkat-CD27-Nanoluc cells with boserolimab followed by nuclear factor kappa B (NFκB)-mediated nanoluciferase expression. [0034] FIG.1F illustrates enhanced luciferase expression through CD27- NFκB axis with receptor clustering mechanism found to be mediated by boserolimab binding to FcγRIIb on K562 cells and CD27 on Jurkat cells. [0035] FIG.2A shows another assay performance improvement believed to be due to a clustering effect from immobilization. Immobilizing boserolimab by Protein A significantly increased boserolimab-induced luciferase reporter gene activation as compared to Jurkat-CD27- Nanoluc cell line alone. Half of a 96-well cell culture plate was coated with Protein A whereas the other half of the plate was not. Immobilizing boserolimab by anchoring the Fc part to Protein A led to CD27 clustering and increased luciferase expression with Jurkat-CD27-Nanoluc cell line. In contrast, plates with no Protein A showed base level luciferase expression. [0036] FIG.2B illustrates Protein A-mediated receptor clustering induced by boserolimab and ultimately increased luciferase expression in the cells that gives a signal-to-noise ratio. [0037] FIG.3A illustrates Jurkat-CD27-Nanoluc Stable cell line selection. Jurkat cells stably expressing CD27 were transfected with NFκB-Nanoluciferase reporter by Lipofectamine 2000. The bar graph represents luminescence read out (RLU) or fold change from cherry-picked single cell clones in response to tumor necrosis factor alpha (TNFα) at 10, 100 and 1000 ng/mL. TNFα is a strong activator of NFκB promoter, thus it was used to assess luciferase reporter activity. The top 12 clones with strong TNFα-induced activation were expanded for further screening. [0038] FIG.3B shows the boserolimab dose response of the top 12 clones from the initial TNFα screening tested in the presence of K562 cells. The curves represent raw luminescence signal (top graph) or fold activation (bottom graph) from 12 clones. The top 5 clones were expanded to retest. [0039] FIG.3C shows the boserolimab dose response of the top 5 clones retested in the presence of K562 cells. The final clone 10G11 was chosen based on the performance and cell growth. [0040] FIGs.4A and 4B show assay optimization with adding two cell lines sequentially vs pre-mixing. We found that pre-mixing Jurkat-CD27-Nanoluc cell line 10G11 with K562 instead of adding them sequentially improved assay performance. Shown is the dose response curve of reference and material various relative potency (RP): 35%, 71%, 141% and control at 100% of reference. [0041] FIGs.5A-5D show assay optimization with cell seeding density with 1:1 ratio of 10G11 and K562 cells.100,000 cells/well of 10G11 with same number of K562 seem to perform the best with low assay variation. [0042] FIG.6 shows assay optimization with different 10G11 cell seeding density from 50,000 to 200,000 cells/well and fixed number of K562 cells at 100,000 cells/well. They all show a well- formed dose-response curve.10G11 cells at 100,000 cells/well appear to give a slightly better performance. [0043] FIGs.7A-7D show assay optimization with a time course of various incubation times. All incubation times show well-formed dose-response curve with good accuracy. [0044] FIGs.8A-8B show assay optimization for cell passage. Assay performance was maintained over prolonged cell passage. Cells showed similar performance at passage 15 and 30. [0045] FIGs.9A-9C show a qualification study of the optimized CD27 functional cell-based assay. FIG.9A shows relative potency values by each potency level. FIG.9B. shows the percent Relative Bias estimates (red circle) with its 90% confidence intervals. FIG.9C shows the linearity assessment of relative potency vs. target potency in Ln scale. [0046] FIG.10A shows the results of a CD27 cell-based reporter assay when co-culturing Jurkat-CD27-NFκB-Nanoluc cells and K562 cells. [0047] FIG.10B shows the results of a CD27 cell-based reporter assay when culturing Jurkat- CD27-NFκB-Nanoluc cells only. [0048] FIG.11A shows the results of a CD27 cell-based reporter assay when K562 cells were pre-incubated with anti-CD32 Fab for blocking FCγRIIb receptors on K562. [0049] FIG.11B shows the results of a CD27 cell-based reporter assay –when K562 cells were pre-incubated with mouse IgG1 Fab as negative control. [0050] FIGs.12A and 12B show a Complement Dependent Cytotoxicity (CDC) Assay, and an Antibody Dependent Cellular Cytotoxicity (ADCC) Surrogate Assay using Jurkat-CD16-NFAT- Luc Reporter Cells, respectively. [0051] FIG.13 shows clone selection in a cell based functional assay. DETAILED DESCRIPTION OF THE DISCLOSURE [0052] The present disclosure provides methods to determine the potency of antibodies using a reporter assay system. The present disclosure extends an insight that an assay for determining the potency of antibodies can be improved by clustering the antibodies. [0053] In one aspect, provided are methods for determining the potency of antibodies (e.g., anti-CD27 antibodies) to produce an immune response, that include steps of a) providing a reporter cell line wherein the reporter cell line is a T cell line comprising one or more nucleic acid molecules encoding a reporter molecule operably linked to a promoter responsive to CD27 signaling and encoding CD27; b) clustering a plurality of an anti-CD27 antibody; c) incubating the anti-CD27 antibodies with the reporter cell line, to allow binding of an antigen binding domain of the anti-CD27 antibodies to the CD27 on the reporter cell line resulting in the expression of the reporter molecule; and d) measuring the activity of the reporter molecule to determine the potency of the anti-CD27 antibodies. Definitions [0054] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group. [0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art. [0056] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. [0057] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides). [0058] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and/or values. [0059] As used herein, the term “comprising” may include the embodiments “consisting of” and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as “consisting of” and “consisting essentially of” the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds. [0060] “Stably transfected” refers to the foreign gene being part of the host genome and is therefore replicated. This is typically initiated by transiently transfecting a cell with the foreign gene but through a process of careful selection and amplification, and stable clones are generated. [0061] The term “antibody,” “immunoglobulin,” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically encompasses, for example, individual monoclonal antibodies (including neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibodies, e.g., sc-Fv; multispecific antibodies formed from antibody fragments. [0062] A “Fab fragment” is comprised of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” can be the product of papain cleavage of an antibody. An “Fc” region contains two heavy chain fragments comprising the CH3 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. [0063] A “Fab’ fragment” contains one light chain and a portion or fragment of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and C H2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab’ fragments to form a F(ab’) 2 molecule. A “F(ab’)2 fragment” contains two light chains and two heavy chains containing a portion 20 of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab’) 2 fragment thus is composed of two Fab’ fragments that are held together by a disulfide bond between the two heavy chains. An “F(ab’)2 fragment” can be the product of pepsin cleavage of an antibody. The “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions. [0064] A “coding sequence” or a sequence “encoding” an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in production of the product. [0065] A “promoter” or “promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. [0066] A nucleic acid or polynucleotide is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. [0067] The terms “express” and “expression” refer to allowing or causing the information in a gene, RNA or DNA sequence to become manifest; for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene. For example, a DNA sequence is expressed in or by a cell to form an “expression product” such as an RNA (e.g., mRNA) or a protein. The expression product itself may also be said to be “expressed” by the cell. [0068] The term “luminescent substrate” refers to a substrate that luminesces upon catalysis by a reporter molecule, e.g., luciferin. For example, the reporter molecule may be a luminescent enzyme or luminescent reporter enzyme that catalyzes a reaction with a luminescent substrate, 25857 e.g., luciferase or nanoluciferase. Various luciferase genes encoding luciferase are commercially available for use in the invention, including luciferase genes from fireflies, sea pansy (Renilla reniformis), ostracods (Cypridina hilgendorfii), and copepods (Gaussia princeps), and NanoLuc®, a modified 19 kilodalton (kDa) luciferase derived from deep sea shrimp (Oplophorus gracilirostris) that can be purchased from Promega Corp. [0069] “Read-out signal” refers to a signal produced from the reporter gene protein expression. The signal can be emitted by the protein or reaction of the protein with a substrate. [0070] “Receptor clustering” refers to the spatial clustering of receptors on a cell surface, e.g., CD27 receptor on a cell surface. Receptor clustering may increase the sensitivity of the antibody binding to the receptor (illustrated in FIG.1F). [0071] The terms “culture” and “cell culture” are used interchangeably and refer to a cell population that is maintained in a medium under conditions suitable to survival and/or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms also refer to the combination comprising the cell population and the medium in which the population is maintained. Suitable culture conditions for mammalian cells are known in the art. See e.g., Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). [0072] The term “co-culture” as used here refers to culturing two or more types of cells together in the same cell culture system with some degree of direct or indirect interaction between the cells. [0073] The term “cell culture medium,” or “culture medium,” refers to any nutrient solution used for growing or maintaining cells (e.g., mammalian cells) and which generally provides at least one or more components from the following: essential and nonessential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for at least minimal growth and/or survival. The solution may also contain components that enhance growth and/or survival above the minimal rate, including hormones and growth factors. [0074] “Assay media” refers to a solution comprising a nutrient(s) for cells such as glucose, vitamins, amino acids, or a combination thereof and serum, and optionally antibiotics and a buffer. In some embodiments, the assay media of the invention includes the test substance at various dilutions to be tested. [0075] As used here, the term “passage” also referred to as “subculturing” is the process of harvesting cells from a culture and transferring them to a new culture to enable further propagation of the cell line. The term “passage number” refers to the number of times the cell culture has been subcultured. 25857 Reporter Cell Line [0076] The methods provided herein include a reporter cell line, also referred to as a first cell line or a responding cell line. In some embodiments, the reporter cell line is a T cell line expressing CD27. In some embodiments, the reporter cell is a human T cell line. In some embodiments, the reporter cell line is the Jurkat T cell line that is stably transfected with CD27. In some embodiments, the CD27 gene is introduced into a cell line using a vector. In some embodiments, the CD27 gene is introduced into a cell line using vectors such as plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and/or viral vectors (e.g., retroviruses and lentiviruses). In some embodiments, the CD27 is introduced into a cell line using a virus vector such as a lentivirus vector. [0077] In some embodiments, a reporter cell line is stably transfected with a reporter gene. In some embodiments, the reporter gene of the reporter cell line expresses a reporter molecule upon activation with an anti-CD27 antibody. In some embodiments, the reporter gene expresses a reporter molecule in the presence of a promoter. In some embodiments, the promoter is responsive to activation by the binding of CD27 antibody to the CD27 receptor. In some embodiments, the reporter gene comprises a luciferase gene coding sequence. In some embodiments, the luciferase gene coding sequence is operably linked and/or driven by a NFκB promoter that expresses the reporter molecule luciferase in response to binding of the anti-CD27 antibody to CD27 receptor. In some embodiments, the reporter gene comprises a nanoluciferase gene. In some embodiments, the nanoluciferase gene is operably linked and/or driven by a NFκB promoter that expresses the reporter molecule nanoluciferase in response to binding of the anti- CD27 antibody to CD27 receptor. [0078] In some embodiments, a reporter gene is a gene encoding a reporter molecule that is detectable by fluorescence, luminescence, color change, enzyme assay, or histochemistry. For example, a fluorescent reporter protein encoded by a reporter gene may be a fluorescent protein that fluoresces when exposed to a certain wavelength of light (e.g., GFP). A reporter protein may be a reporter enzyme that catalyzes a reaction with a substrate to produce an observable change in that substrate. Enzymes such as luciferase (e.g., substrate luciferin) or β-lactamase (e.g., substrate CCF4) can cause luminescence or allow fluorescence on substrate cleavage. Enzymes such as β-galactosidase (e.g., substrate X-gal (5-bromo-4-chloro- 3-indolyl-P-D- galactopyranoside)) and secreted alkaline phosphatase (e.g., substrate PNPP (p-Nitrophenyl Phosphate, Disodium Salt)) can result in a visualizable precipitate upon substrate cleavage. 25857 [0079] In some embodiments, the signal is fluorescence or luminescence. In some embodiments, the reporter molecule is NanoLuc that luminesces in the presence of the luminescent substrate NanoGlo (Promega). Clustering [0080] In some embodiments, the method provided herein includes clustering a plurality of anti-CD27 antibodies. In some embodiments, clustering a plurality of anti-CD27 antibodies refers to the spatial clustering of anti-CD27 antibodies. In some embodiments, the methods comprise providing an accessory cell that clusters a plurality of anti-CD27 antibodies. In some embodiments, the methods comprise immobilizing anti-CD27 antibodies on a plate. [0081] In some embodiments, an accessory cell contributes to CD27 receptor clustering on a reporter cell line. The accessory cell line is also referred to as a secondary cell or an Fc gamma Receptor (FcγR)-expressing cell. FcγR family members, e.g., FcγRIIB can contribute to the immune cell receptor clustering mediated by Fc fragment of antibody engagement while maintaining binding of F(ab)s to target (Stewart et al. Journal for ImmunoTherapy of Cancer 2014, 2:29). [0082] In some embodiments, the accessory cell line is negative for CD70, which is a natural CD27 ligand. Absence of CD70 in the accessory cell line enhances the activity of the anti-CD27 antibody to be tested. In some embodiments, the accessory cell line is K562, a human myelogenous leukemia cell line. [0083] In some embodiments, anti-CD27 antibody is immobilized by a superantigen. The superantigen anchors the Fc portion of the antibody leading to CD27 clustering in the reporter cell line (illustrated in FIG.2B). In some embodiments, the superantigen is Protein A. Anti-CD27 Antibody [0084] In some embodiments, the test substance is an anti-CD27 antibody that binds the CD27 receptor. In some embodiments, the anti-CD27 antibody is hCD27.15, the sequence of which is disclosed in WO2012/004367, or 1F5, the sequence of which is disclosed in US20110274685, which are incorporated herein by reference. In some embodiments, an anti-CD27 antibody comprising the light chain amino acid sequence of SEQ ID NO: 10 and comprising the heavy chain amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-CD27 antibody is boserolimab. In some embodiments, the anti-CD27 antibodies comprise (a) three heavy chain complementarity determining regions (HC-CDRs), wherein HC-CDR1 is the amino acid sequence of SEQ ID NO: 1, HC-CDR2 is the amino acid sequence of SEQ ID NO: 2, and HC- 25857 CDR3 is the amino acid sequence of SEQ ID NO: 3; and (b) three light chain complementarity determining regions (LC-CDRs), wherein LC-CDR1 is the amino acid sequence of SEQ ID NO: 4, LC-CDR2 is the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 is the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD27 antibodies comprise a heavy chain variable region (VH) of SEQ ID NO: 7 and a light chain variable region (VL) of SEQ ID NO: 9. In some embodiments, the anti-CD27 antibodies comprise a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 8 and the light chain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD27 antibody is boserolimab, comprising two light chains comprising the amino acid sequence of SEQ ID NO: 10 and comprising two heavy chains comprising the amino acid sequence of SEQ ID NO: 8. Table 1. The amino acid sequences of boserolimab S_{EQ ID NO: Amino acid sequence} H - CDR1 1 NYGMN Q A K T D S

[0085] Antibodies and fragments thereof that bind to the same epitope as any of the anti-CD27 antibodies or antigen-binding fragments thereof of the present invention also may be used as part of the present invention. In some embodiments, an antibody or antigen-binding fragment thereof that binds CD27 and has VL domains and VH domains with at least 99% 98%, 97%, 96%, 95%, 25857 90%, 85%, 80% or 75% sequence identity to one or more of the VL domains or VH domains described herein and exhibits specific binding to CD27 may also be utilized in the methods and uses described herein. In another embodiment, the binding antibody or antigen-binding fragment thereof of the present invention comprises VL and VH domains (with and without signal sequence) having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acid substitutions, and exhibits specific binding to CD27. Assay [0086] In some embodiments, the methods provided herein includes clustering a plurality of anti-CD27 antibodies using an accessory cell. In some embodiments, the methods comprise co- culturing a reporter cell line and accessory cell line in the present of an anti-CD27 antibody. [0087] In some embodiments, the reporter cell line and the accessory cell line are pre-mixed before co-culturing. In some embodiments, the reporter cell line and the accessory cell line are sequentially added to an assay plate comprising a plurality of wells. In some embodiments, the reporter cell line is added to the assay plate first. In some embodiments, the accessory cell line is added to the assay plate first. [0088] In some embodiments, the reporter cell line and the accessory cell line are co-cultured at a ratio of about 1:10 to about 10:1. In some embodiments, the reporter cell line and the accessory cell line are co-cultured at a ratio of about 1:10 to about 5:1. In some embodiments, the reporter cell line and the accessory cell line are co-cultured at a ratio of about 1:10 to about 1:1. In some embodiments, the reporter cell line and the accessory cell line are co-cultured at a ratio of about 1:5 to about 10:1. In some embodiments, the reporter cell line and the accessory cell line are co- cultured at a ratio of about 1:1 to about 10:1. In some embodiments, the reporter cell line and the accessory cell line are co-cultured at a ratio of about 1:5 to about 5:1. In some embodiments, the reporter cell line and the accessory cell line are co-cultured at a ratio of about 1:5 to about 1:1. In some embodiments, the reporter cell line and the accessory cell line are co-cultured at a ratio of about 1:1 to about 5:1. In some embodiments, the reporter cell line and the accessory cell line are co-cultured at a ratio of about 1:1 to about 1:1. [0089] In some embodiments, the reporter cells are at a passage of about 2 to about 50 at the time of determining the potency. In some embodiments, the reporter cells are at a passage of about 15 to about 30 at the time of determining the potency. [0090] In some embodiments, the accessory cells are at a passage of about 2 to about 50 at the time of determining the potency. In some embodiments, the accessory cells are at a passage of about 15 to about 30 at the time of determining the potency. 25857 [0091] In some embodiments, the reporter cells are seeded in a multi-well plate. In some embodiments, the multiple well plate has 4 to 100 wells. In some embodiments, the multiple well plate has 6 wells, 12 wells, 24 wells, 48 wells, or 96 wells. In some embodiments, the multiple well plate has 6 wells, 12 wells, 24 wells, 48 wells, or 96 wells. In some embodiments, the multiple well plate has growth area of about 0.1 cm² to about 10 cm². In some embodiments, the multiple well plate has approximate growth area of 9.5 cm², 3.8 cm², 1.9cm², 0.95 cm², or 0.32cm². The growth area of the multiple well plate may vary with different commercial manufacturers. [0092] In some embodiments, the reporter cell line is seeded at a density of about 25,000 to about 500,000 cells/well. In some embodiments, the reporter cell line is seeded at a density of about 50,000 to about 200,000 cells/well. In some embodiments, the reporter cell line is seeded at a density of about 50,000 to about 150,000 cells/well. In some embodiments, the reporter cell line is seeded at a density of about 100,000 to about 200,000 cells/well. [0093] In some embodiments, the accessory cell line is at a density of about 25,000 to about 500,000 cells/well. In some embodiments, the accessory cell line is at a density of about 50,000 to about 200,000 cells/well. In some embodiments, the accessory cell line is at a density of about 50,000 to about 150,000 cells/well. In some embodiments, the accessory cell line is seeded at a density of about 100,000 cells/well. [0094] In some embodiments, the reporter cell line and the accessory cell line are seeded at a density of about 100,000 cells/well in a 96 well plate. [0095] In some embodiments, time of incubation of the cells with the test substance is from about 2 hours to about 10 hours. In some embodiments, time of incubation of the cells with the test substance is about 4 hours to about 5 hours. GENERAL METHODS [0096] Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 19892^nd Edition, 20013^rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol.217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol.1), cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol.3), and bioinformatics (Vol.4). 25857 [0097] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol.1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol.2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol.3, John Wiley and Sons, Inc., NY, NY, pp.16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp.45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp.384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol.1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York). [0098] Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp.139-243; Carpenter, et al. (2000) J. Immunol.165:6205; He, et al. (1998) J. Immunol.160:1029; Tang et al. (1999) J. Biol. Chem.274:27371-27378; Baca et al. (1997) J. Biol. Chem.272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol.224:487-499; U.S. Pat. No.6,329,511). [0099] Having described different embodiments of the invention herein with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims. EXAMPLES [0100] The following examples are meant to be illustrative and should not be construed as further limiting. 25857 Definitions for Examples CD27 Cluster of differentiation antigen 27, T Cell-surface Immunoreceptor BSC Biological Safety Cabinet BSL Bio Safety Level DMSO Dimethyl Sulfoxide FBS-HI Fetal Bovine Serum, Heat Inactivated SOP Standard Operating Procedure RPMI Roswell Park Memorial Institute PEN/STREP Penicillin/Streptomycin [0101] Reagents used in the examples described herein are listed in Table 2, below. Equivalent reagents, and in-lab prepared reagents can also be used. Table 2. Reagents (those listed or equivalent, unless otherwise noted) Identification Manufacturer Cat. Number

Preparation of In-Lab Reagents FBS-HI Solution [0102] FBS-HI was thawed at 5±3 °C, mixed well by gently swirling, and aliquoted in single- use aliquots in 50-mL centrifuge tubes. The aliquots were stored at -20±5 °C. ^ One day prior to use, the needed number of aliquots were pulled from the freezer and thawed at 5±3 °C or in a water bath set to 37 °C before use. 25857 Penicillin-Streptomycin Solution [0103] Penicillin-Streptomycin solution was thawed at 5±3 °C, mixed well by swirling, and aliquoted in single-use aliquots of 6 mL in 15-mL centrifuge tubes. The aliquots were stored at - 20±5 °C. ^ One day prior to use, the needed number of aliquots were pulled from the freezer and thawed at 5±3 ̊C or in a water bath set to 37 °C before use. Assay Media Preparation [0104] The ingredients listed in Table 3 were mixed to prepare “Assay Media”. The mixed solution was filtered through a 0.2 micron Polyethersulfone (PES) filtering unit and stored at 5±3 °C. Assay Media was pre-warmed in a water bath set to 37 °C or put it a BSC (e.g., Nuaire LabGard, Nu-440-600) 30 minutes before each use. Volumes were scaled up proportionally. Table 3. Assay Media Ingredients Reagents Volume (mL) Final Concentration RPMI1640 m di m 445 89% Example 1

: s ab s ng e ce nes 1.1 Jurkat CD27 expression cell line [0105] Parental Jurkat, Clone E6-1 cells were purchased from ATCC^®. A lentiviral construct containing nucleic acid encoding human CD27 (pCD810-Neo-CD27) was transduced into human T lymphocyte cell line Jurkat (“Jurkat CD27 cell line”), and a stable pool was generated with geneticin selection. [0106] The Jurkat-CD27-NFκB-Nanoluc cell line was generated by transfecting Jurkat-CD27 cells with the plasmid pNL3.2.NFκB-RE[NlucP/NFκB-RE/Hygro] vector (Promega) using Lipofectamine 2000. A stable pool was made under geneticin and hygromycin selection. [0107] The Jurkat-CD27-NFkB-Nanoluc stable pool underwent limiting dilution to generate single clones which were further screened by testing with TNF- α at 1000, 100 and 10 ng/ml for Nanoluciferase activity. The top 12 high responding clones were further tested in a functional assay (12 single clones at 100,000 cells per well were co-cultured with 100,000 K562 cells per well) and ranked according to the assay performance criteria. The top 5 clones were retested. A 4-PL dose response curve is shown in FIG.13. The final clone 10G11 was chosen due to superior 25857 response and cell growth. This clone was expanded for cell banking and utilized for further assay development and pre-qualification of the cell-based functional assay. [0108] The Jurkat CD27 cell line was maintained in culture medium containing 500 mg/mL of G418 (Gibco Cat#10131-027), 100 U/mL of Penicillin-Streptomycin (Gibco Cat# 15140-122), and 10% Fetal Bovine Serum (HyClone Cat#SH30088.03) in RPMI1640 basal media (Gibco Cat# 22-400-089). 1.2 K562 [0109] The human myelogenous leukemia cell line K562 cells (ATCC Accession No. CRL- 3344) were maintained in culture medium containing 100 U/mL of Penicillin-Streptomycin (Gibco Cat# 15140-122) and 10% Fetal Bovine Serum (HyClone Cat#SH30088.03) in RPMI1640 basal media (Gibco Cat# 22-400-089) (“K562 Complete Growth Medium”). 1.3 Jurkat-CD27-Nanoluc [0110] The human T lymphocyte cell line Jurkat with expression of CD27 and NF-kB driven Nanoluciferase (“Jurkat-CD27-Nanoluc cell line”) was generated as explained in Example 4. The Jurkat-CD27-Nanoluc cell line was maintained in culture medium containing 500 mg/mL of G418 (Gibco Cat#10131-027), 500 mg/mL Hygromycin (Invitrogen Cat#10687-010), 100 U/mL of Penicillin-Streptomycin (Gibco Cat# 15140-122), and 10% Fetal Bovine Serum (HyClone Cat#SH30088.03) in RPMI1640 basal media (Gibco Cat# 22-400-089) (“Jurkat-CD27-Nanoluc Complete Growth Medium”). 1.4 Jurkat-CD27- NLuc cells and K562 cells Recovery and Routine Passaging / Maintenance Recovery of Cells [0111] Before starting the procedure, Recovery Medium (10% FBS and 100U/ml Penicillin- Streptomycin in RPMI1640) was pre-warmed in a water bath at 37±2 ºC. The Recovery Medium is the same as the complete medium for each cell line without selection. [0112] The Jurkat-CD27-Nanoluc complete media is composed of RPMI 1640 supplemented with 10% fetal bovine serum (heat-inactivated), 100 U/mL Penicillin-Streptomycin, 500 μg/mL Hygromycin B, and 500 μg/mL Geneticin. The K562 complete growth medium is composed of RPMI 1640 supplemented with 10% fetal bovine serum (heat-inactivated) and 100 U/mL Penicillin-Streptomycin. Day 1 a. One cryovial of frozen cells was removed from a vapor-phase liquid nitrogen storage and placed in a 37 °C water bath until the cells were just thawed (approximately 1-2 minutes). b. After thawing, the vials were surface-sterilized with a sterilizing wipe (e.g., Fisher Catalog# 18-318-007) and placed inside a BSC. 25857 c. The thawed cells were gently transferred with a pipette from the vial and the suspension was slowly dispensed into 10 mL of pre-warmed Recovery Medium in a 15-mL centrifuge tube. The cell suspension was centrifuged at 150 x g at room temperature for 5 minutes and the centrifugation was stopped with a low brake so as not to disturb the cell pellet. d. The supernatant was carefully removed, making sure not to disturb the cell pellet and gently cells were resuspended in 5 mL of pre-warmed Recovery Medium. The cell suspension was gently mixed by pipetting it up and down 3-4 times with a serological pipette. e. The cell suspension was transferred into a T75 tissue culture flask, and an additional 15 mL of Recovery Medium was added. The cell culture was then incubated in the 37°C + 5% CO2 humidified tissue culture incubator until day 3. Day 3 a. The tissue culture flask was removed from the incubator and the cells were observed under an inverted light microscope to confirm normal morphology and detect signs of contamination. b. 1mL of the cell suspension was removed to count the cells and determine cell density and viability of the cells. c. The cells were then passaged and maintained as appropriate with the viable cell density (VCD). Routine Passaging and Maintenance of Jurkat-CD27-Nanoluc cells and K562 cells [0113] Passage +1 began in this step when the cells were passaged in complete growth media and transferred to a new flask for the first time after initial recovery. a. After the cells were accommodated into the complete growth media routine passage was performed in T175 (175 cm² cell culture flask with filter cap, Nunc, Cat. No.178883, for 50-60 mL), or T75 (75 cm² cell culture flask with filter cap, Nunc, Cat. No.156499 for 20-30 mL) cell culture flasks. b. The cell culture flask was removed from the incubator and the cells were observed under an inverted light microscope to confirm normal morphology and detect signs of contamination. c. 1 mL of the cell suspension was removed to count the cells and determine cell density and viability using an automated cell counter (Beckman Coulter, Vi-Cell XR). Cells can be manually counted using a light-inverted microscope and hemacytometer in the alternative. d. Examples of seeding densities for routine maintenance are provided. Densities may be adjusted as needed, but it is advisable to not allow the cells grow to a density greater than 2.0 x 10⁶ cells/mL. 25857 Table 4. Suggested Cell Seeding Densities for Routine Passaging # of Days till Seeding Density Cell Line Medium to Use the Passage or * ng g

sensitive to change of pH due to long culture. However, if the cells have to be split after 4 days in culture, then at least one more round of normal passaging at 48-hour interval is needed before they can be used for potency assay. Freezing and Banking of cells a. Cell cultures described above were expanded as necessary for the desired number of vials. b. Cell freezing medium (Life Technologies, Cat. No.12648-010) was kept on ice until needed. c. Cells were transferred from multiple culture flasks to 50 mL centrifuge tubes and centrifuged at 200 x g at room temperature for 5-7 minutes and the centrifugation was stopped with a low brake. d. Medium was removed and the cells were resuspended in ice cold freezing medium at the desired concentration (e.g., 5x10⁶/mL). The cell suspension was aliquoted into cryovials at a desired volume (e.g., 1 mL) _{e. The cells were frozen overnight in a suitable cell freezing container like CoolCell} Container in a freezer unit set to -70 ± 10 °C. The cells were transferred to a vapor phase of liquid nitrogen drawer for storage after 24 to 72 hours. 25857 Example 2: CD27 expression on Jurkat-CD27-NFkB/NLuc cells [0114] The expression levels of CD27 on the cell surface of Jurkat-CD27 cells was determined by flow cytometry. Cells at log phase of growth were collected from the culture flask, and pelleted by centrifugation at 1000 rpm for 5 minutes in a tabletop centrifuge (Beckman GS-6) using swing out bucket rotor (GH-3.8). The resulting cell pellet was gently re-suspended into 5 mL of FACS buffer (phosphate-buffered saline (PBS) with 0.5% bovine serum albumin (BSA) and 0.05% sodium azide) and kept on ice. Cells were diluted with FACS buffer (200 mL cells + 800 mL FACS buffer), and cell viability and count were measured using a Vi-Cell XR instrument. Cell count was diluted to a final concentration of 100,000 cells per 200 mL. Cells were distributed to four BD Falcon FACS tubes (Ref#352093): 1) Cells alone, 2) Secondary antibody alone, 3) Human IgG1 Isotype control (10 mg/mL; see below), 4) boserolimab (10 mg/mL). To tube 3) Human IgG1 isotype was added to a final concentration of 10mg/mL (Biolegend Ultra-LEAF purified human IgG1 isotype control clone ET901, Cat#403102, 1 mg/mL). To tube 4) boserolimab was added to a final concentration of 10mg/mL (51 mg/mL). After one hour of incubation on ice, cells were washed thrice by centrifugation with 800 mL of FACS buffer. To tube 1) 200 mL of FACS buffer was added. To tubes 2), 3) and 4) Secondary antibody (R-Phycoerythrin conjugated goat anti-human IgG, Jackson Research Labs, Code#109- 116-170) at 1:200 dilution with FACS buffer in a volume of 200 mL was added and incubated on ice for additional hour. (Secondary antibody: R-Phycoerythrin conjugated goat anti-human IgG, Jackson Research Labs, Code#109-116-170). After one hour of incubation on ice, cells were washed thrice by centrifugation with 800 mL of FACS buffer and re-suspended in 200 mL same buffer. Samples were run on BD FACS Canto II. The data were analyzed using FlowJo 10.1 software. Results are shown in FIG.1A. Example 3: Cell surface expression of CD70 and FcγRIIb on JY cells and K562 cells [0115] The JY cells and K562 cells were collected from the culture flask, and pelleted by centrifugation at 1000 rpm for 5 minutes in a table-top centrifuge (Beckman GS-6) using swing out bucket rotor (GH-3.8). The cell pellet was gently re-suspended into 5 mL of FAS buffer (PBS with 0.5% BSA and 0.05% Sodium azide) and kept on ice. Cells were diluted with PBS with 0.5% BSA (200 mL cells + 800 mL PBS-Serum), and cell viability and count were measured using Vi-Cell XR instrument. Cell count was adjusted to contain 200,000 cells per 160 mL.160 mL of the solution having cells were distributed to two BD Falcon FACS tubes (Ref#352093) labelled as: Cells alone, Anti-CD70-FITC + anti-FcγRIIb (APC) (FITC Mouse Anti-Human CD70 Clone Ki-24, BD Biosciences Cat#555834, APC Mouse Anti-Human CD32 (FcγRIIb) 25857 Clone FLI8.26, BD Biosciences Cat#559769).20 mL of each of the pre-diluted antibodies were added to tube#2 and 40 mL of FACS buffer to tube#1. After one hour of incubation on ice, cells are washed thrice by centrifugation with 800 mL of FACS buffer and re-suspended in 200 mL same buffer. Samples were run on BD FACS Canto II. The data was analyzed using FlowJo 10.1 software. Results are shown in FIG.1B and FIG.1C. Example 4: Generation of the Jurkat-CD27-Nanoluc stable cell line [0116] The Jurkat cells expressing CD27 were transfected with the plasmid pNL3.2.NFκB- RE[NlucP/NFκB-RE/Hygro] vector (Promega) using Lipofectamine 2000 (Invitrogen Cat# 11- 668-500). Cells were washed with PBS, counted and re-suspended in OPTI-MEM medium (Invitrogen Cat# 31-985-088) at 4x10⁵ cell per mL and plated 2 mL per well into a 6 well plate. As per the transfection protocol from ThermoFisher, for each of the well in a 6 well plate, 4 mg of plasmid DNA was complexed with 10 mL of Lipofectamine 2000 per well in 0.25 mL of OPTI-MEM for 20 minutes. A master mix of DNA-Lipofectamine 2000 complex was prepared for 3 wells. To each well 0.25 mL of DNA complex was added dropwise with gentle swirling of plate. The plate was incubated for 24 hours in a CO2incubator. Cells were washed by centrifugation and switched to selection medium containing 800 mg/mL of G418 (Gibco Cat#10131-027), 500 µg Hygromycin (Invitrogen Cat#10687-010), 1X of Penicillin- Streptomycin (Gibco Cat# 15140-122), and 10% Fetal Bovine Serum (HyClone Cat#SH30088.03) in RPMI1640 basal media (Gibco Cat# 22-400-089). Fresh selection medium was changed every three days for two weeks. The surviving cells were pooled and culture was expanded to a T75 flask in 3 week time. The functional activity of NFκB driven Nanoluciferase reporter was tested using recombinant human TNFα on the stable pool of cells. Once the activity was confirmed, the cells were expanded and a batch of cells were frozen. The rest of the cells were diluted to obtain 0.2 cells per well of 96 well plate in a volume of 100 mL and seeded on to 12 plates to obtain single cell clones. The limiting dilutions culture resulted in picking up 120 clones that started proliferation, and that were narrowed down to 40 of the best growing clones that were transferred to 24 well plates.40 clones were further shortlisted by testing with TNFα at 1000, 100 and 10 ng/mL for Nanoluciferase activity. The top 12 high responding clones were further tested at 100,000 cells per well co-cultured with 100,000 K562 cells per well with boserolimab and dose response curves were generated. The 5 best clones that showed good dose response curves in a 4-PL curve were selected for retesting. Results are shown in FIGs.3A-C. Final clone 10G11 was chosen due to superior response and cell growth. This clone was expanded and utilized for further assay development. 25857 Example 5: CD27 Cell-based reporter assay optimization [0117] The cell-based functional assay was established utilizing Jurkat-CD27-Nanoluc Clone#10G11 with NFκB promoter driven nanoluciferase reporter gene and CD27 over- expression, hereinafter called as 10G11 in a co-culture assay with K562 cells. Boserolimab bound to CD27 receptor on T cells and induced NFκB activation for cytokine secretion response. In this assay the nanoluciferase expression was considered as NFκB activation signal.10G11 cells were cultured in RPMI 1640 Medium (RPMI 1640 Medium, GlutaMAX™, HEPES) containing 10% Heat Inactivated Fetal Bovine Serum, 100 U/mL Penicillin Streptomycin, 500 µg/mL Hygromycin B, and 500 µg/mL Geneticin. K562 cells were cultured in RPMI 1640 Medium (RPMI 1640 Medium, GlutaMAX™, HEPES) containing 10% Heat Inactivated Fetal Bovine Serum and 100 U/mL Penicillin Streptomycin. For the assay, 10-150 mL of both cell lines were harvested and centrifuged at 300xg at room temperature for 5 minutes to pellet cells. The supernatant was removed and cells were resuspended in RPMI 1640 Medium (RPMI 1640 Medium, GlutaMAX™, HEPES) containing 10% Heat Inactivated Fetal Bovine Serum, and 100 U/mL Penicillin Streptomycin. The cells were counted, and plates were seeded at a volume of 40 µL/well with a concentration of 1.0 x 10⁵ cells/well in a 96-well tissue culture plate.20 µL/well of boserolimab dilutions made in the medium was added and incubated for 5 hours in CO2 incubator. The nanoluciferase was tested with Nano-Glo reagent from Promega and read on SpectraMax^® instrument. Data were analyzed by SoftMax^® Pro software. [0118] Pre-mixing Jurkat-CD27-Nanoluc cell line 10G11 with K562 (instead of adding them sequentially) improved assay performance. FIGs.4A and 4B show the dose response curves of reference and material relative potency of 35%, 71%, 141% and control at 100% of reference. The pre-mixing step lowered assay variation while simplifying assay procedure, since pre-mixing allows larger volume of pre-mix to be added to the reaction instead of two smaller volumes, which simplifies assay procedure and reduces variation introduced by sample dispersion. The precision was also improved.20 mg/mL of boserolimab with 8-point 5-fold dilutions was used, and the incubation time for the study was 5 hours. [0119] Cell seeding density was studied with a 1:1 ratio of 10G11 and K562 cells.100,000 cells/well of 10G11 with the same number of K562 seemed to perform the best with low assay variation.20 mg/mL of boserolimab with 8-point 5-fold dilutions was used, and the incubation time for the study was 5 hours. Results are shown in FIGs.5A-5D. [0120] Cell seeding density was studied with different 10G11 cell seeding density from 50,000 to 200,000 cells/well and fixed number of K562 cells at 100K cells/well. They all show a well- formed dose-response curve.10G11 cells at 100K seem to give slightly better performance.20 mg/mL of boserolimab with 8-point 5-fold dilutions was used, and the incubation time for the study was 5 hours. Results are shown in FIG.6. [0121] A time course study using 3 hours, 4 hours, 5 hours, and 6 hours incubation times was performed. All incubation times show well-formed dose-response curves with good accuracy.20 mg/mL of boserolimab with 8-point 5-fold dilutions was used. Results are shown in FIGs.7A- 7D. [0122] The assay was tested over different passage numbers for Clone 10G11. Assay performance was maintained over prolonged cell passage. Cells showed similar performance at passage 15 and 30.20 mg/mL of boserolimab was used at 1:4 dilutions. The 10G11 cells were pre-mixed with K562 cells for the assay and incubated with boserolimab for 5 hours. Results are shown in FIG.8A for high passage (P30) and FIG.8B for low passage (P15). Example 6: Alternative assay performance improvement by clustering effect via immobilization. [0123] Immobilizing boserolimab by Protein A significantly increased boserolimab-induced luciferase reporter gene activation as compared with Jurkat-CD27-Nanoluc cell line alone (See FIG.2B). Half of a 96-well cell culture plate was coated with Protein A whereas the other half plate was not. Immobilizing boserolimab by anchoring the Fc part to Protein A led to CD27 clustering and increased luciferase expression with the Jurkat-CD27-Nanoluc cell line. In contrast, plates with no Protein A showed base level luciferase expression. [0124] A cartoon drawing (FIG.2B) illustrates Protein A-mediated receptor clustering of boserolimab and the ultimate increased luciferase expression in the cells that gives a signal-to- noise ratio. Results are shown in FIG.2A. Example 7: CD27 cell-based reporter assay Assay Procedure [0125] On the day of the experiment, the Assay Media was warmed in a water bath set to 37ºC. Concurrently, needed reagents were pulled (e.g., reference standard, control, test samples) from cold storage and allowed to equilibrate to room temperature on the bench top or in BSC. Preparation of Standards, Assay Control, and Test Sample Dilutions [0126] Each dilution was thoroughly mixed by vortexing during preparation. For each of the assay plates, boserolimab dilutions were performed in singleton and tested in duplicate (i.e., each assay dilution is loaded into 2 wells) on each plate. All prepared dilutions of all assay and articles were discarded after completion of the assay using appropriate safety procedures. Dilution of Boserolimab Reference Standard, Assay Control, or Test Sample(s) in Assay Media [0127] Boserolimab Reference standard was diluted in Assay Media to 40 μg/mL in 1.5 or 2mL dilution tubes (see Table 5). Per each assay plate, separate (independent) dilutions of Reference Standard were prepared. [0128] Boserolimab Assay Control was diluted in Assay Media to 40 µg/mL in 1.5 or 2mL dilution tubes (see Table 5). Per each assay plate, separate (independent) dilutions of Assay Control were prepared. [0129] Boserolimab Test Sample(s) were diluted in Assay Media to 40 µg/mL in 1.5 or 2mL dilution tubes (see Table 5). Per each assay plate, separate (independent) dilutions of Test Sample(s) were prepared. Table 5. Example dilution scheme (if Reference Standard, Assay Control, or Diluted Test Sample concentration is 51.0 mg/mL). Starting Volume of Diluted Ste Concentration Test Sample, Assay Volume of Media Final Concentration

Dilution plates and assay plates [0130] Dilution plates were set up according to the sample dilution Table 6.

25857 Table 6. Sample Serial Dilution Table Volume of Diluted Final 2X Concentration Test S tion # of boserolimab Volume o ample, Concentration of Dilu f Assay Assay Control or boserolimab in

polystyrene tissue culture treated plates and labelling them to match the respective dilution plates numbering unless otherwise indicated. Preparation of Jurkat-CD27- NanoLuc cells and K562 cells for Assay [0132] 45-50 mL of Jurkat-CD27-NanoLuc cell suspension were harvested in a 50-mL centrifuge tube.45-50 mL of K562 cell suspension were harvested in a 50-mL centrifuge tube. One flask is typically sufficient for 3~4 assay plates. The tubes with harvested cells were centrifuged at 300xg at room temperature for 5 minutes and the centrifugation was stopped with low brake to pellet cells. [0133] Once centrifugation stopped, the supernatants was aspirated carefully from the centrifuged tubes while making sure not to touch the pellet.8-10 mL of assay media was added to each tube and the pellets were gently resuspended with a serological pipette by pipetting slowly up and down approximately 4-5 times until all cells are re-suspended with no visible clumps. Cell density was determined using automated cell counter like Vi-Cell instrument (Beckman Coulter, Vi-Cell XR) using 1:10 dilution of cells suspension. (Example: 100 µL of cell suspension plus 900 µL of assay medium). The cell density of Jurkat-CD27-NanoLuc and K562 used in the assay was 4 x 10⁶ cells/mL. 25857 Preparation of Cell Assay Mixture [0134] Based on the results from cell counting and resuspension after centrifugation, the final cell suspension volume for the number of the plates in the assay was calculated. Table 7. Dilution for final cell culture volume Number of Target Cell Seeding Prepare the minimum volume of each of Target Cell Seeding plates in the Density for Jurkat- Jurkat-CD27-NanoLuc and K562 cell CD27-NanoLuc cells Density for K562 )

^^^^^^^^^^^^ ^^^^ ^^^^^^^^ ^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^ ^^^^^^ ^^^^^^^^^^^^^^ ^^^^^^^^ ^^^^^^^^^^^^^^ (Equation 1)

^^^^^^^^^^^^ ^^^^ ^^^^^^^^^^ ^^^^^^^^^^ ^^^^^^ ൌ_{^^^^^^^^^^^^^^ ^^^^^^^^^^^^ ^^^^^^ െ ^^^^^^^^^^^^ ^^^^ ^^^^^^^^ ^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^ ^^^^^^} (Equation 2) [0136] For example, when viable cell density is 8 x 10⁶ cells/mL, 9 mL (for 3 plates) of Jurkat- CD27-NanoLuc cells at 4 x 10⁶ cells/mL should be prepared for final assay cells suspension. Then it should be mixed [9 mL x (4 x10⁶/8 x10⁶)] = 4.5 mL of the re-suspended cells with [9 mL - 4.5 mL] = 4.5 mL of assay media. [0137] The required assay media was added in a 50-mL centrifuge tube, and then the required volume of re-suspended Jurkat-CD27-NanoLuc and K562 cells was added into the same 50-mL centrifuge tube. The combined cell suspension was gently mixed by gently pipetting up and down approximately 4-5 times using a serological pipette. The combined cell suspension of Jurkat- CD27-NanoLuc and K562 cells was poured carefully into a sterile 50 mL reagent reservoir, and mixed up and down with a pipette 1-2 times. [0138] Using a multi-channel pipette, 50 mL of the combined cell suspension was added to each well of the assay plates as quickly as possible, and dispense against the wall of the well. The plates were gently rocked for uniform distribution of cell suspension to the bottom of the plate. 25857 The plates were visually inspected to ensure all liquid settled to the bottom of the plate. Where needed, the plates were gently tapped without causing splashing. [0139] 50 µL from all wells of the dilution plate(s) was transferred, with a manual multi- channel pipette, to the corresponding wells of the respective assay plate(s), making sure pipette tips were changed between row-to-row or column-to-column transfers in all plates. [0140] After adding Protein A dilutions, the plates were gently tapped without causing splashing or the plate(s) were put on a plate shaker set to 300 rpm for 2 minutes to mix contents in each well of assay plate(s). [0141] The assay plates were placed in a humidified incubator set at 37 ºC and 5% CO2 for 4.5± 0.5 hours. Nano-GLO^® Reagent was removed from the freezer and left on a bench to equilibrate to room temperature. Detection of Assay Plates with Nano-Glo^® Reagent [0142] The assay plates were removed from the incubator and allowed to equilibrate to room temperature for 5 to 10 minutes. Room temperature equilibrated Nano-Glo^® Reagent was prepared for use in the assay following the manufacturer’s instructions. Nano-Glo^® Luciferase Assay Substrate was taken out from -20^oC freezer and mixed with Nano-Glo^® Luciferase Assay Buffer at 1:50 ratio using a dark polypropylene tube to protect the substrate from light. Freshly re-constituted Nano-Glo^® mixture was used. The prepared reagent mixture was poured into a reagent reservoir. [0143] 100 μL of Nano-Glo^® substrate solution was added to all wells of the assay plate by dispensing against the wall of the well, and not touching the 100 μL content already in the wells. [0144] The plates were placed on a bench top shaker set to 300 rpm and gently shaken for 10- 15 minutes at room temperature, with a covering to protect from light with black lids. Reading the Plates and Finishing the Assay [0145] After 10-15 minutes mixing on shaker, the first assay plate was removed from the shaker. Once logged on to SpectraMax^® M5e or M5 plate reader software using unique login ID and password, the drawer of SpectraMax^® reader was opened. The first cell plate was put on the plate nest. “Plate 1” icon followed by “Start” button in protocol template were clicked on. After reading was complete, the plate was removed from the plate nest and the next plate was put on. “Plate 2” icon followed by “Start” button in the template were clicked on. The reading was carried on until all assay plates were read. 25857 Example 8: Data Analysis [0146] Raw data containing counts of relative light units (RLU) was captured using non-GXP SoftMax^® Pro software. Data files were uploaded into a data repository at the end of the experiment. Calculations [0147] All calculations, including assessment of comparison to acceptance criteria were performed and recorded within the SoftMax^® Pro data file. [0148] The mean RLU were plotted as a function of boserolimab concentration to generate sigmoidal dose-response curves. The data were fit to the four-parameter logistic (4PL) equation: ^_{^ ൌ ^^ ^ ^^^ െ ^^^} _{^^ ^ ^^^} ^^ (Equation 3)

where y is the mean luminescence upper asymptote, A is the lower asymptote, x is the boserolimab concentration (ng/mL), C is the inflection point or EC₅₀ (ng/mL), and B is the slope parameter of the 4PL curve fit. [0149] Initially, SoftMax^® template fit the raw data for each individual test article (e.g., Reference, Control, and Samples) independently of each other. This is called unrestricted fit, from which parameters A, B, C, and D for each individual dose-response curve were calculated and recorded. [0150] Determined A and D parameters were used to calculate the relative % difference of the upper asymptotes (%D Difference) and the relative % difference of the lower asymptotes (%A difference) between Reference and Test samples (or Control) using the following formulas: ห_{^^^^^^^^^^^ െ ^^^^^^^^^^^ ห} _{^^^^^^^ ^^^௧^^^^} 4)

5) 25857 [0151] Determined B (Slope) parameters were used in the calculation of Slopes Ratio using the following equation: ^^ ^_{^^^^^^^^^^^^^^^^^^^ ൌ ^^^^^^^^^} ^^_{^^^^^^^^^^^^௧^^^^} (Equation 6) [0152] A and D

Standard were used to calculate the ratio of the asymptotes as follows: ^^ ^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^ _^^^^^^^^^ _^^^^^^^^^ୀ ^^_^^^^^^^^^ (Equation 7)

Sum of Squared Errors (SSE) [0153] SSE evaluates goodness of fit statistics and measures the total deviation of the observed response values (experimental) from the fitted data (predicted). SSE can be calculated by Equation 8. ୬ S_{SE ൌ ^^y ଶ} _{୧ െ f୧^} (Equation 8)

where n is the number of observations (equals to 8 as per the total number of concentration points), yi is the observed data value and fi is the predicted value from the fit. [0154] SSE in this method is determined based on normalized data to account for possible differences in absolute signal intensity between different instruments. Table 8. SSE calculations using normalized values. Determinant Explanation Example SoftMax^® Equation or ,

25857 [0155] After the unrestricted analysis, the raw data were fit to a restricted 4PL fit, where the Reference curve was analyzed as paired with each test sample (or control) on the plate, (i.e., Reference vs. Sample 1, or Reference vs. Sample 2, or Reference vs. Sample 3, or Reference vs. Assay Control). Both curves in the pair were fit to the same parameter A, the same parameter D, and the same parameter B, allowing only parameter C to fluctuate for the best fit. This is also called Parallel Line Analysis (PLA). PLA results were presented for each pair in a separate graph in the SoftMax^® file. [0156] By fitting a pair of curves using PLA analysis, the potency of the test sample or control was calculated and reported by the SoftMax^® Pro software, using the following equation: C Relative Potency ^%^ _{ୖ^^^୰^୬ୡ^} _{ୗୟ୫୮୪^ ^୭୰ େ୭୬^୰୭୪^} ൌ C _{ൈ 100} _{ୗୟ୫୮୪^ ^୭୰ େ୭୬^୰୭୪^}

(Equation 9) where C is the EC50 parameter calculated from the PLA fit of the two curves. [0157] Reportable Potency result was calculated as geometric mean (GeoMean) of three valid individual relative potency values using the following equation: G_{eoMean ^%^ ൌ 10ெ^^^ ^^^^ ^^ ^^௧^^^௬^} (Equation 10) where Mean (Log10 potency) is the average of decimal logarithm values of three individual relative potency values. [0158] Variability of the Reportable Potency was calculated as geometric standard deviation (%GSD) of three relative potency values using the following equation: G_{SD ^%^ ൌ ൫10ௌ௧^^௩^^^^^^ ^^௧^^^௬^ െ 1൯ ൈ 100}

(Equation 11) where St.Dev.(Log10 potency) is standard deviation of decimal logarithm values of three individual relative potency values. Valid Test Acceptance Criteria [0159] Acceptance criteria in the method was based on method development and pre- qualification data 25857 Table 9. Assay Acceptance Criteria (per each of the three assay plates). Assay Article ID Assay Acceptance Parameter Acceptance Criterion Asymptotes ratio ≥ 3.0 f

at plate and repeated. Table 10. Sample Acceptance Criteria (per each of the three assay plates) Test Article ID Sample Acceptance Parameter Acceptance Criterion f

[0161] If any of the sample acceptance criteria failed for a particular sample, that sample was rejected, and the test was repeated. During the repeat, positions of the samples on repeat plate were the same as on the original plate. [0162] If there are multiple and systemic failures of Test Acceptance Criteria (e.g., > 3 subsequent fails of the assay and >3 subsequent fails of a particular sample), consult with Quality to assess path forward. Acceptance Criteria for Validity of Geometrical Mean Relative Potency (Reportable Result) [0163] The %GSD of the three valid relative potency values was ≤ 30%. [0164] If %GSD > 30%, all the data was technically evaluated, and results were compared between all three valid plates. If, upon evaluation, atypical results were identified (e.g., sigmoidal shape of the curves is non-conforming to the typically obtained curves, or >3 hot wells in the media-only wells, etc.), those plate(s) were invalidated and repeated while maintaining the same 25857 plate number(s) and sample positions as in the original plate(s). If no atypical behavior was identified in any of the plates, the sample results produced on all three plates were invalidated and the assay was repeated while maintaining the same sample positions as in the original plates. Example 9. Statistical Analysis [0165] The pre-qualification study was performed by 2 analysts testing 4 plates per day over the course of 3 days, using the range of 35-200% target potency levels. The samples tested were at targeted relative potency values of 35%, 50%, 71%, 100%, 141% and 200%. Table 11 provides a summary of the results of this pre-qualification. Statistical Analysis [0166] The qualification study consisted of 24 plates executed by two analysts over 3 days with 4 plates done per day. Up to 4 samples were tested on one plate together with the Reference Standard and Cell Control. The samples tested had targeted relative potency values of 35%, 50%, 71%, 100%, 141% and 200%, prepared by dilution of Reference Standard. Each analyst generated n=6 values at each potency concentration giving a total of n=12 per potency level. The 100% potency concentration included n=18 values for each analyst per day, giving a total of n=36. The data for each assay plate from the qualification study were documented. All statistical analyses of the data were done using JMP® version 14.1 software. [0167] An assessment on the %Relative Accuracy, Precision, and Dilutional Linearity was done on the relative potency values from the 24 plates. Relative Accuracy was determined by measuring the geometric mean (GM) of the relative potencies of all samples divided by its target potency at each level. Dilutional Linearity was determined by the linear regression of relative potency values versus target potency levels on the log scale. Intermediate Precision was assessed for each potency level as well as combined over all potency levels by doing a variance component analysis of between-run and within-run variability, where run is defined as an analyst testing 4 plates per day. Results Plot of Data used in pre-qualification results [0168] A pre-qualification data which passed all system suitability criteria for the two analysts testing four plates per day over three days at each potency level is shown in FIG.9A. 25857 Table 11. Summary of Pre-Qualification results. Qualification Parameter Qualification Results Relative Accuracy The %relative bias ranges from -2.2% to -5.6%. y

[0169] Accuracy was based on the percent relative bias of each target potency level according to the formula % Relative Bias=100*(Geomean/Target-1) (Equation 12) [0170] The Geomean of the relative potency values was calculated as the antilog of the average log relative potency values. The results of the Geomean, %Relative Bias and its 90% confidence intervals at each target potency levels from the 24 plates are given in Table 12. The largest %Relative Bias was -5.6 % at the 35% and 71% potency levels with the 90% confidence interval of (-13.6%,3.3%) and (-12.1%, 1.4%), respectively. Table 12. Accuracy/Relative Bias for different target potency levels Target mean Lower 90% Upper 90 Lower 90% Upper 90% Potency N Geo % % Relative Relative Relative

Dilutional Linearity (Relative Accuracy) [0171] Linearity was also assessed through graphical representation of the natural log relative potency versus natural log target potency with accompanying regression line, which is shown in FIG.9C. The overall coefficient of determination (R²) was determined as 0.944 with a slope of 1.023 in the log scale. It confirms that the assay is linear within the range of 35% to 200%. [0172] The assays’ ability to generate proportional results can be achieved through the calculation of proportional bias, which is related to the slope (b) from the regression of log relative potency on log target potency. The formula is given in Equation 2. 25857 %_{Proportional Bias (Pbias) =100^ ^2^ ^ 1 ^1 ^} (Equation 13) [0173] The result of the %Proportional Trend Bias for Linearity over the range of 35% to 200% was 1.6% and given in Table 13. This implies that the estimated 2-fold increase in observed potency is about 1.6% more than expected for a perfectly linear assay. This is not unusual performance for a bioassay. The bias is reported per 2-fold dilution since the 2-fold is a common scale. Table 13. Summary statistics for assessing linearity Intercept Slope Proportional Trend Lower 95% CI on Upper 95% CI on Bias (%) PBias PBias

[0174] Intermediate precision considers the overall variability from different analysts, days and plates. A variance component analysis was performed on the natural log transformed relative potencies to estimate between- and within-run variation per potency level. The variance estimates (s²= s²between-run+ s² within-run;S=√s²) were converted back to the original units and expressed in terms of % RSD using the following formula given in Equation 14. %_{RSD =100^} e^s 2 ^ 1 _{; %GSD=100^^^^} ^{^} _{െ 1^} (Equation 14) [0175] The precision estimates for each target potency level estimated are summarized in Table 14 in terms of %RSD and %GSD. In Table 14, under “Random Effect” column, “Run” means the variability between runs, “Residual” is the variability within runs or “Repeatability”, and “Total” is the Intermediate precision. The intermediate precision %RSD ranges from 11.0% to 17.4%. The estimate of ‘run’ variability is 0 at all potency levels which indicates that the contribution of its variability to the total intermediate precision is negligible compared to within- run variability. The average %RSD over all potency levels is 14.0%. The 95% confidence intervals on %RSD are also provided for information purposes. Table 14. Variability Estimates at each target potency level Target Random Effect %RSD 95% Lower RSD 95% Upper RSD %GSD

25857 Target Potency (%) Random Effect %RSD 95% Lower RSD 95% Upper RSD %GSD 50 R n 00 00 00 00

[0176] The variance estimates of Run and within Run (Residual) from the pre-qualification study was used to determine a testing format to predict assay variability for various numbers of runs and plates per run. The estimates of run and within-Run variability at each target potency were used in calculating the reportable values and predicted %RSD using Equation 15. The results are given in Table 15. ^_{^^^^^^^^^^^^^ %^^^^^^ ൌ ^}^ ఙ మ మ ^ _ೃೠ^ ఙ_ೈ^^ ^^ _{^ ା ^^^షೃೠ^} _{^⋅ ^} _{^^^^ ^}

(Equation 15) where, k is number of runs and n is number of plates. Table 15. Format Variability Target # Run Residual Total %RSD (1 %RSD %RSD %RSD )

25857 141 2 0 0.0129 0.0129 8.0 5.7 4.6 4.0 200 2 0 0.0211 0.0211 10.3 7.3 5.9 5.1 35 3 0 00297 00297 100 70 58 50

runs with either 1, 2, 3 or 4 plates per run. The current testing scheme of doing 4 plates per run would give a highest %RSD of the reportable value of 8.6% at 35% potency level. Example 10. Heat stressed stability samples [0178] Boserolimab was staged for 6 months at 40 ^oC for an accelerated stability study. Samples were pulled at the initial time point and at 2, 3, and 6-month time points. The relative potency of these samples was measured by CD27 cell-based reporter assay as described in Example 7 followed by data analysis using SoftMax Pro software as described in Example 8. [0179] The assay results indicate that these samples lost potency due to boserolimab degradation and post-translational modifications. Fragmentation was shown by an increase in low molecular weight species (LMWS) through non-reducing sodium dodecyl sulfate (SDS) capillary electrophoresis (CE-SDS NR) from 3.3% to 26%. Aggregation was observed by an increase in high molecular weight (HMW) species through ultra-performance size exclusion chromatography (UP-SEC) from 0.3% to 1.8%, and an increase in acidic variants through ion- exchange chromatography (IEX) from 14.3% to 81.9%. [0180] The CD27 cell-based assay was more stable compared to the binding ELISA. The relative potency of the CD27 cell-based assay dropped to 20% in CBA, while the ELISA’s relative potency decreased to 73% at the 40°C 6-month time point. This information can be found in Table 16. [0181] A key difference between these two assays is that the cell-based assay relies on a fully intact antibody for full activity, while the ELISA only measures the binding of the Fab portion of boserolimab to recombinant CD27 protein. 25857 Table 16. Correlation of boserolimab potency loss with other stability data changes [0182] When t assay, a stepwise

decrease in potency was observed in the curve shape as the samples were held for longer periods at 40°C (FIG.10A). However, when the same samples were added only to the Jurkat-CD27- NFκB-NanoLuc T cells without the K562 cells, the assay did not indicate any stability, and there was no significant difference in potency between the 40°C samples (FIG.10B). It was also observed that the single cell culture assay window was smaller compared to the co-culture assay window. [0183] These side-by-side experimental results demonstrated that the CD27 receptor clustering effect induced by co-culturing K562 cells with Jurkat-CD27-NFκB-NanoLuc cells was vital for CD27-mediated Jurkat T cell activation. In conclusion, the CD27 cell-based reporter assay requires an intact or fully functional boserolimab for full activation of Jurkat-CD27-NFκB- NanoLuc cells, as suggested by the loss of potency due to deamidation of key residues (resulting in an increase in acidic variants) and fragmentation (resulting in an increase in low molecular weight species) observed in the 40°C stability samples. Additionally, these structural changes leading to a loss of potency were only observed when the K562 cells were cultured with Jurkat- CD27-NFκB-NanoLuc cells. [0184] To investigate the critical factor in K562 cells that contributes to the boserolimab structure-function activity, the hypothesis was proposed that the Fc gamma receptor IIb protein (CD32b) expressed on K562 cells plays a significant role. To test this hypothesis, K562 cells were pre-incubated with either 10 μg/mL anti-CD32 Fab or mouse IgG1 Fab (used as a negative control) for a period of 30-60 minutes prior to co-culturing them with Jurkat-CD27-NFκB- NanoLuc cells in the CD27 cell-based reporter assay. [0185] The results of the assay demonstrated that blocking FcγRIIb receptors on K562 cells with 10 µg/mL anti-CD32 Fab disrupts the cross-linking between the target and APC cells. This interference led to a smaller assay window and rendered the assay ineffective in indicating 25857 stability. These results align with the findings obtained from the CD27 cell-based reporter assay of single cell culture (FIG.11A). [0186] When K562 cells were pre-incubated with 10 μg/mL mouse IgG1 Fab simultaneously as a negative control for 30-60 minutes before co-culturing with Jurkat-CD27-NFκB-Nanoluc cells in the CD27 cell-based reporter assay, no disruption of boserolimab clustering CD27 receptors was observed (FIG.11B). The assay results indicated a higher assay window and a decrease in potency of the samples, which correlated with the degree of change in the Fc region of boserolimab due to fragmentation or deamidation (acidic variants). [0187] Other potential mechanisms for the activity of boserolimab that rely on the Fc portion of the antibody include complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). In vivo, boserolimab binding to CD27 positive cells could activate complement proteins in the serum, leading to cell death (CDC), or recruit NK cells to induce cell death (ADCC) of boserolimab-coated cells. [0188] To model these activities in vitro, boserolimab was added to Jurkat-CD27 T cells and incubated either with serum complement to assess complement-mediated killing of target cells (CDC assay) or with Jurkat-CD16-NFAT-Luc cells to evaluate activation of the CD16 reporter cell (a surrogate assay for NK cell-mediated ADCC assay). Similar to the findings in the CD27 cell-based reporter assay, an increasing loss of CDC (FIG.12A) and ADCC (FIG.12B) activity were observed with the escalating heat treatment of the stability samples at 40°C. This indicates that the stressed conditions, including deamidation and fragmentation, also impair these effector function activities. [0189] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. [0190] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

25857 WHAT IS CLAIMED IS: 1. A method of determining the potency of anti-CD27 antibodies to produce an immune response comprising: a) providing a reporter cell line wherein the reporter cell line is a T cell line comprising one or more nucleic acid molecules encoding a reporter molecule operably linked to a promoter responsive to CD27 signaling and encoding CD27; b) clustering a plurality of anti-CD27 antibodies; c) incubating the anti-CD27 antibodies with the reporter cell line, to allow binding of an antigen binding domain of the anti-CD27 antibodies to the CD27 on the reporter cell line resulting in the expression of the reporter molecule; and d) measuring the activity of the reporter molecule to determine the potency of the anti-CD27 antibodies. 2. The method of claim 1, wherein the step b) comprises providing an accessory cell line expressing a Fcγ receptor. 3. The method of claim 2, wherein the Fcγ receptor is FcγRIIB. 4. The method of any one of claims 2-3, further comprising co-culturing the reporter cell line and the accessory cell line. 5. The method of claim 4, wherein the reporter cell line and the accessory cell line are pre-mixed before the co-culturing step. 6. The method of claim 4, wherein the reporter cell line and the accessory cell line are sequentially added to an assay plate comprising a plurality of wells. 7. The method of any one of claims 2-6, further comprising incubating the anti- CD27 antibodies with the co-cultured reporter cell line and accessory cell line, to allow Fc regions of the anti-CD27 antibodies to bind to the Fcγ receptor on the accessory cell line inducing CD27 clustering on the surface of the reporter cell line. 25857 8. The method of any one of claims 2-7, wherein the accessory cell line is CD70 negative. 9. The method of any one of claims 2-8, wherein the accessory cell line is a K562 cell line. 10. The method of any one of claims 2-9, wherein the reporter cell line and the accessory cell line are co-cultured at a ratio of about 1:5 to about 5:1. 11. The method of claim 10, wherein the reporter cell line and the accessory cell line are pre-mixed in the ratio of 1:1 before co-culture. 12. The method of any one of claims 2-11, wherein the co-culture of the reporter cell line and the accessory cell line is incubated with the anti-CD27 antibodies provided in a series of dilutions. 13. The method of any one of claims 2-12, wherein the co-culture of the reporter cell line and the accessory cell line is incubated with the anti-CD27 antibodies for at least 2 hours. 14. The method of any one of claims 2-13, wherein the co-culture of the reporter cell line and the accessory cell line is incubated with the anti-CD27 antibodies for 2 to 6 hours. 15. The method of any one of claims 2-14, wherein the co-culture of the reporter cell line and the accessory cell line is incubated with the anti-CD27 antibodies for about 5 hours. 16. The method of any one of claims 6-15, wherein cell seeding density of the reporter cell line is from 10,000 to 500,000 cells/well. 17. The method of claim 16, wherein cell seeding density of the reporter cell line is from 50,000 to 200,000 cells/well. 18. The method of any one of claims 6-17, wherein cell seeding density of the accessory cell line is from 10,000 to 500,000 cells/well. 25857 19. The method of claim 18, wherein cell seeding density of the accessory cell line is from 50,000 to 200,000 cells/well. 20. The method of claim 1, wherein step b) comprises providing anti-CD27 antibodies immobilized by an antibody-binding protein. 21. The method of claim 20, wherein the antibody-binding protein is Protein A. 22. The method of any one of claims 20-21, further comprising incubating the immobilized anti-CD27 antibodies with the reporter cell line, to allow binding of an antigen binding domain of the anti-CD27 antibodies to the CD27 on the reporter cell line resulting in the expression of the reporter molecule. 23. The method of any one of claims 20-22, wherein the anti-CD27 antibodies are immobilized by the antibody-binding protein on a plate. 24. The method of any one of claims 1-23, wherein the reporter cell line is a Jurkat T cell line. 25. The method of any one of claims 1-24, wherein the promoter responsive to CD27 signaling is an NFκB promoter, an NFAT promoter, or an AP-1 promoter. 26. The method of any one of claims 1-25, wherein the promoter responsive to CD27 signaling is an NFκB promoter. 27. The method of any one of claims 1-26, wherein the reporter molecule is a luciferase, a fluorescent protein, an alkaline phosphatase, a beta lactamase, or a beta galactosidase. 28. The method of any one of claims 1-27, wherein the reporter molecule is a firefly luciferase, a Renilla luciferase, or a nanoluciferase. 29. The method of any one of claims 1-28, wherein the reporter molecule is nanoluciferase. 25857 30. The method of any one of claims 1-29 wherein the anti-CD27 antibodies comprise: (a) three heavy chain complementarity determining regions (HC-CDRs), wherein HC-CDR1 is SEQ ID NO: 1, HC-CDR2 is SEQ ID NO: 2, and HC-CDR3 is SEQ ID NO: 3; and (b) three light chain complementarity determining regions (LC-CDRs), wherein LC-CDR1 is SEQ ID NO: 4, LC-CDR2 is SEQ ID NO: 5, and LC-CDR3 is SEQ ID NO: 6. 31. The method of any one of claims 1-29, wherein the anti-CD27 antibodies comprise a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 9. 32. The method of any one of claims 1-29, wherein the anti-CD27 antibodies comprise a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 8 and the light chain comprises SEQ ID NO: 10. 33. A method for determining any degradation of anti-CD27 antibodies from a first time period to a second time period, comprising: (i) performing a measurement of the potency of the anti-CD27 antibodies according to the method of any one of claims 1-32 at a first time period; (ii) performing a second measurement of the potency of the anti-CD27 antibodies according to the method of any one of claims 1-32 at a second later time period; and (iii) comparing the potency from the initial measurement and the potency from the second measurement; wherein a reduction in the measurement of potency from the first time period to the second later time period is indicative of a degradation of the anti-CD27 antibodies. 34. A kit for determining the potency of anti-CD27 antibodies, comprising a first cell line which is a human T cell line comprising one or more nucleic acid molecules encoding a reporter molecule operably linked to a promoter responsive to CD27 signaling and encoding CD27, a second cell line that is FcγRIIb positive and CD70 negative, media that includes a 25857 substrate for the nucleic acid molecule encoding the reporter molecule of the first cell line, and anti-CD27 antibodies.