Genetically engineered multi-component systems for the identification and characterization of T cell receptors and T cell antigens

Abstract

Providing a systematic approach to developing effective vaccines against common and emerging pathogens. [Solution] The multi-component system comprises a genetically engineered antigen-presenting cell (eAPC) (component A) and a gene donor vector (component C) for delivery of one or more ORFs encoding analyte antigen-presenting complexes (aAPXs) and / or analyte antigenic molecules (aAMs), where component A a. lacks endogenous surface expression of at least one family of aAPXs and / or aAMs, b. contains at least two genomic acceptor sites for recombinase-mediated exchange (RMCE) (components B and D), each for integration of at least one ORF encoding at least one aAPX and / or aAM, and component C matches component B, and is designed to deliver c. a single ORF encoding at least one aAPX and / or aAM or d. two or more ORFs encoding at least one aAPX and / or aAM.

Description

Technical Field

[0001] Field of the Invention The present invention relates to the construction, assembly, and use of a multi-component system composed of at least three components: a genetically engineered antigen-presenting cell (eAPC), a genetically engineered genomic acceptor site, and a matching gene donor vector. The present invention is used for the rapid and high-throughput generation of stable derivative cells presenting various forms of antigenic molecules for the identification and characterization of antigen and cognate TCR sequences.

Background Art

[0002] Introduction to the Invention The immune surveillance mechanism by T lymphocytes (T cells) is a central function in the adaptive immunity of all jawed vertebrates. The immune surveillance mechanism by T cells is achieved through the rich functional diversity across T cell subtypes, which contribute to the elimination of pathogen infections and tumor cells, organize the adaptive immune response to symbiotic non-self factors such as invading pathogens, commensal microorganisms, and molecular components of food, and further maintain self-tolerance. To respond to various foreign and self factors, T cells must be able to specifically detect the molecular components of these foreign and self factors. Thus, T cells must be able to detect the majority of self and non-self molecules encountered by an individual with sufficient specificity to initiate an effective response against pathogenic organisms and diseased self while avoiding the initiation of such a response against healthy self. The highly complex nature of this task becomes apparent when considering the virtually infinite diversity of both foreign and self molecules, and pathogenic organisms are under evolutionary pressure to escape detection by T cells.

[0003] T cell receptor (TCR) T cells are mainly defined by the expression of the T cell receptor (TCR). The TCR is a component of T cells that plays a role in interacting with and sensing the targets of T cell adaptive immunity. Generally speaking, the TCR is composed of a heterodimeric protein complex presented on the cell surface. Each of the two TCR chains belongs to the immunoglobulin superfamily (IgSF) domain, and is composed of two extracellular domains, a variable (V) region and a constant (C) region that form an antiparallel β-sheet. These are tethered to the cell membrane by a type I transmembrane domain adjacent to a short cytoplasmic tail. The quality of T cells to adapt to and detect various molecular components results from variations in the TCR chains that occur during T cell development. This variation occurs by somatic recombination in a manner similar to antibody production in B cells.

[0004] TCR Chain Diversity The T cell pool consists of subpopulations with heterogeneous functions and phenotypes. However, T cells can be broadly classified into αβ or γδ according to the somatic rearranged TCR isoforms expressed on their surface. There are two TCR chain pair isoforms, namely the pair of TCRα (TRA) and TCRβ (TRB), and the pair of TCR gamma (TRG) and TCR delta (TRD). T cells expressing the TRA:TRB pair are called αβ T cells, and T cells expressing the TRG:TRD pair are often called γδ T cells. Both αβ and γδ forms of TCR are responsible for recognizing various ligands, namely "antigens", and each T cell generates new αβ or γδ receptor chains during T cell maturation. These new TCR chain pairs achieve diversity of recognition by generating receptor sequence diversity in a process called somatic V(D)J recombination, and after somatic V(D)J recombination, each T cell expresses a copy of a single TCR that has been rearranged separately. In the TRA and TRG gene loci, several separate variable (V) and functional (J) gene segments are available for recombination and are juxtaposed to the constant (C) gene segments, so it is called VJ recombination. Recombination at the TRB and TRD gene loci further includes diversity (D) gene segments, so it is called VDJ recombination. Each recombined TCR has the ability of unique ligand specificity, which is determined by the structure of the ligand-binding site formed by the α and β chains in the case of αβ T cells, or the γ and δ chains in the case of γδ T cells. The structural diversity of TCRs is mainly limited to three short hairpin loops of each chain, called complementarity-determining regions (CDRs). The three CDRs are contributed by each chain of the receptor chain pair, and these six CDR loops together exist at the distal end of the TCR extracellular domain away from the membrane to form the antigen-binding site. Sequence diversity in each TCR chain is achieved in two forms. First, the random selection of gene segments for recombination results in basic sequence diversity. For example, TRB recombination occurs among 47 unique V, 2 unique D, and 13 unique J germline gene segments. Generally, the V gene segment contributes to both the CDR1 and CDR2 loops and is thus encoded in the germline. The second form for generating sequence diversity occurs within the hypervariable CDR3 loop, which results from the random deletion of templated nucleotides and the addition of non-templated nucleotides at the junctions between the recombined V, (D), and J gene segments.

[0005] TCR:CD3 complex Mature αβ and γδ TCR chain pairs are presented as a complex on the cell surface together with several accessory CD3 subunits called ε, γ, δ, and ζ. These subunits associate with the αβ or γδ TCR as three dimers ( εγ, εδ, ζζ). This TCR:CD3 complex forms a unit for initiating a cellular signaling response upon the binding of the αβ or γδ TCR to cognate antigen. The CD3 accessory subunits bound as the TCR:CD3 complex contribute to a signaling motif called the immunoreceptor activation tyrosine motif (ITAM). CD3ε, CD3γ, and CD3δ each contribute to a single ITAM, while the CD3ζ homodimer contains three ITAMs. The three CD3 dimers ( εγ, εδ, ζζ) assembled with the TCR thus contribute to ten ITAMs. Upon TCR ligation with cognate antigen, phosphorylation of tandem tyrosine residues creates paired docking sites for proteins containing Src homology 2 (SH2) domains, such as the important 70 kDa ζ-chain-associated protein (ZAP-70). Recruitment of such proteins initiates the formation of the TCR:CD3 signaling complex, which ultimately is responsible for T cell activation and differentiation.

[0006] αβ T cells αβ T cells are generally more abundant in humans than the corresponding γδ T cells. Many αβ T cells interact with peptide antigens presented by the HLA complex on the cell surface. Peptide-HLA (pHLA)-recognizing T cells were the first to be described and are the best characterized. Rarer forms of αβ T cells have also been described. Mucosal-associated invariant T (MAIT) cells are thought to have relatively limited α and β chain diversity and recognize bacterial metabolites rather than protein fragments. Invariant natural killer T cells (iNK T cells) and germline-encoded mycolyl-reactive T cells (GEM T cells) are restricted to the recognition of glycolipids cross-presented by non-HLA molecules. Most iNK T cells are thought to interact with glycolipids presented by CD1d, while GEM T cells interact with glycolipids presented by CD1b. Yet another form of T cells is thought to interact with glycolipids associated with CD1a and CD1c, but such cells have not yet been characterized in detail.

[0007] conventional αβ T cells An important feature of most αβ T cells is the recognition of peptide antigens associated with HLA molecules. These are often referred to as "conventional" αβ T cells. Within an individual, self-HLA molecules present peptides from self and foreign proteins to T cells, providing an essential basis for adaptive immunity against malignant lesions and foreign pathogens, symbiotic organisms, food, and for adaptive tolerance towards self. The HLA locus encoding the HLA proteins is the most gene-dense and polymorphic region of the human genome, with over 12,000 alleles described in humans. The high degree of polymorphism at the HLA locus ensures diversity in peptide antigen presentation between individuals, which is important for immunity at the population level.

[0008] HLA Class I and II There are two forms of classical HLA complexes, namely HLA Class I (HLA I) and HLA Class II (HLA II). There are three classical HLA I genes, namely HLA-A, HLA-B, and HLA-C. These genes encode transmembrane α chains that associate with the invariant β2-microglobulin (β2M) chain. The HLA I α chain consists of three domains of the immunoglobulin fold type, namely α1, α2, and α3. The α3 domain is near the membrane and is mostly invariant, while the α1 and α2 domains together form an antigen-binding cleft that is polymorphic and far from the membrane. There are six classical HLA II genes, namely HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1. These genes encode paired DP, DQ, and DR heterodimer HLA complexes that contain α and β chains. Each chain has two major structural domains of the immunoglobulin fold type, where the α2 and β2 domains contain modules that are mostly invariant and near the membrane, similar to those of the HLA I α3 domain. The HLA II α2 and β2 domains together form an antigen-binding cleft that is far from the membrane and is a highly polymorphic region. The antigen-binding grooves of HLAI and HLAII contain two anti-parallel α helices on a platform of eight anti-parallel β sheets. In this groove, peptide antigens bind and are presented in an extended conformation. The peptide-contact residues in HLAI and HLAII are sites of most sequence polymorphisms, which constitute the molecular basis of the diverse peptide repertoires presented by different HLA alleles. Peptides contact the antigen-binding groove extensively, and as a result, each HLA allele imposes distinct sequence constraints and preferences on the presented peptides. A given peptide thus binds only to a limited number of HLAs, and reciprocally, each allele accepts only a specific fraction of the peptide collection from a given protein. The set of HLAI and HLAII alleles present in an individual is called the HLA haplotype. Polymorphisms of the HLAI and HLAII genes and co-dominant expression of inherited alleles drive a very large diversity of HLA haplotypes across the human population, which poses a major obstacle to the standardization of the analysis of these HLA-antigen-TCR interactions when combined with the vast sequence diversity of the αβ TCR.

[0009] αβ TCR Binding to HLAI and HLAII The αβ TCR recognizes peptides as part of a mixed pHLA binding interface formed by residues of both HLA and peptide antigens (altered self). The HLAI complex is presented on the surface of almost all nucleated cells and is generally thought to present peptides derived from endogenous proteins. T cells can thus examine the endogenous cellular proteome of HLAI-presenting cells by sampling the pHLAI complexes of interacting cells. Since binding of HLAI requires expression of the TCR co-receptor CD8 by the interacting T cell, HLAI sampling is CD8 +It is restricted to αβ T cells. In contrast, surface expression of the HLAII complex is mainly restricted to professional APCs and is generally thought to present peptides derived from proteins that are exogenous to the presenting cell. Interaction T cells can thus examine the proteome of the extracellular microenvironment in which the presenting cell resides. Since binding of HLAII requires expression of the TCR co-receptor CD4 by the interacting T cells, HLAII sampling is CD4 + It is restricted to αβ T cells.

[0010] Thymic selection of αβ TCR The role of the above αβ TCR is the detection of the pHLA complex, and TCR-presenting T cells can enhance responses that are closely related to the role of that T cell in established immunity. The αβ TCR repertoire that occurs in an individual should be the reason for the vast and unpredictable diversity of all foreign antigens that are likely to be encountered before actual diversity occurs in relation to a particular haplotype. This result is achieved against the background that a very diverse and large number of αβ TCRs occur in a somewhat randomized manner with the ability to recognize pHLA complexes that are not specified other than being specifically taught to avoid strong interactions with self-pHLA. This is carefully regulated during T cell maturation in a process called thymic selection. During the first step of T cell maturation in the thymus, T cells with αβ TCRs that cannot interact with self pHLA complexes with sufficient affinity are deprived of survival signals and eliminated. This step, called positive selection, ensures that surviving T cells have a TCR repertoire that can potentially recognize at least foreign or altered peptides presented in association with the correct HLA. Subsequently, αβ TCRs that interact strongly with self pHLA and thus have the ability to drive autoimmunity are actively removed by the process of negative selection. This combination of positive and negative selection results in T cells having αβ TCRs with low affinity for peripheral self pHLA. This establishes an αβ T cell repertoire that is self-restricted but not autoreactive. This highly individualized nature of T cell development with respect to HLA haplotypes highlights the difficulty in standardized analysis of αβ TCR-antigen-HLA interactions. Furthermore, this forms the basis for both graft rejection and graft-versus-host disease, as well as the general principle that αβ TCRs identified in one individual can have completely different effects in a second individual, which has clear implications for TCR-based and T cell-based therapeutic and diagnostic strategies as they appear in actual clinical practice.

[0011] unconventional αβ T cells The non-HLA restricted or "unconventional" forms of αβ T cells have very different molecular antigen targets. These unconventional αβ T cells do not bind to classical HLA complexes, but instead bind to conserved HLA-like proteins such as the CD1 family or MR1. The CD1 family includes four forms (CD1a, b, c, and d) that are involved in antigen cross-presentation. These cell surface complexes have an α-chain that is very similar to HLA-I, which forms a heterodimer with β2M. Small hydrophobic pockets presented on the surface of the α-chain far from the membrane form binding sites for lipid-based antigens derived from pathogens. Innate like NK T cells (iNK T cells) are the best understood example of lipid / CD1 family recognition, and GEM T cells represent another prominent example. "Type I" iNK T cells are known to strongly interact with the lipid α-GalCer associated with CD1d. These iNK T cells exhibit a very limited TCR diversity with a fixed TCR α-chain (Vα10 / Jα18) and a limited number of β-chains (with restricted vβ usage), which are analogous to natural pathogen-associated molecular pattern (PAMPS) recognition receptors such as Toll-like and Nod-like receptors. In contrast, "Type II" NK T cells exhibit a more diverse TCR repertoire and are thought to have more diverse forms of CD1d-lipid complex binding. GEM T cells recognize mycobacterial-derived glycolipids presented by CD1b, but the molecular details of antigen presentation by CD1a, b, and c and their T cell recognition are only just beginning to be understood. MAIT cells predominantly express an invariant TCR α-chain (TRAV1-2 ligated to TRAJ33, TRAJ20, or TRAJ12), which can pair with a range of TCR β-chains. Instead of peptides or lipids, the MAIT TCR can bind to pathogen-derived folate and riboflavin-based metabolites presented by the HLA-I-like molecule MR1. The limited but significant diversity observed in the TCRs of MAIT TCRs is thought to allow for the recognition of diverse but correlated metabolites associated with conserved MR1. It is not well understood how non-classical HLA-restricted αβ T cell TCRs are selected in the thymus during maturation. However, the basic processes of negative and positive selection outlined above would seem to apply, and there is some evidence suggesting that this occurs in specialized niches within the thymus.

[0012] γδ T cells In contrast to the detailed mechanistic understanding of αβ TCR development and pHLA binding, relatively little is known about the relationship between antigen targets and their γδ T cell counterparts. This is in part because of their relatively low abundance in the circulating T cell compartment. However, γδ T cells are widely thought not to be strictly HLA-restricted and, like antibodies, are thought to recognize surface antigens more freely. Additionally, more recently, γδ T cells have been recognized to be able to dominate the resident T cell compartment of epithelial tissues, which are the main sites of interaction of the immune system with foreign antigens. Furthermore, various mechanisms for γδ T cell tumor immunosurveillance and other forms of surveillance of the dysregulated self have begun to be elucidated in the literature. The specific antigen targets of both natural-like and adaptive γδ T cells are still mostly undefined, but the tissue distribution and rapid recognition of PAMPs suggest a fundamental role for γδ T cells in both the early response to foreign antigens and the early life when the adaptive immune system is still maturing. The diverse functions of γδ T cells are thought to be based on the use of different VγVδ gene segments and can be broadly understood in two main categories in which γδ T cells, mainly together with invariant TCRs, mediate natural-like recognition of PAMPs very early during infection. In addition to PAMPs, these types of γδ T cells are thought to recognize self-molecules, including phosphoantigens, which can give very early signs of cellular stress, infection, and perhaps tumorigenesis. The recognition of PAMPs and such so-called danger associated molecular patterns (DAMPs) as well as a number of tissue-restricted natural-like γδ T cells strongly suggests that these cells are well-suited to respond rapidly to antigenic loads without the need for prior activation, homing, and clonal expansion.

[0013] The second form of γδ T cells is actually more adaptable and is thought to have a highly diverse γδ TCR repertoire and the ability to circulate through the periphery and access lymphoid tissues directly. Such antigen-specific γδ T cells have been described for common human pathogens such as CMV and are thought to form memory responses. However, γδ T cells show only relatively limited clonal expansion after activation, and little data are available regarding the degree of TCR diversity and specific responses of γδ T cells in the peripheral circulation or tissues. Furthermore, γδ TCRs do not interact with pHLA complexes and thus are generally thought not to bind peptide antigens in this context, although only a few antigen targets of γδ T cells have been characterized and the underlying molecular framework is poorly understood.

[0014] Due to the low frequency of peripheral γδ T cells and the difficulty of studying tissue-resident T cells in humans, our knowledge of how this important and diverse type of T cell participates in adaptive immune responses has been limited. This emerging area of research requires more reliable techniques to capture and characterize rare γδ T cells, isolate their TCR pairs, and identify their cognate antigens.

[0015] Antigens and Antigen-Presenting Cells In the context of T cells and their relationship with the TCR, an antigen can be defined as any molecule that can bind to the TCR and initiate signal transduction within the T cell. The best-characterized T cell antigens are peptides presented in the context of HLA I and HLA II complexes and recognized by conventional αβ T cells. However, in recent years, it has become clear that unconventional αβ T cells and γδ T cells can bind a wide range of biomolecules as antigens, such as lipids, lipopeptides, glycopeptides, glycolipids, as well as a series of metabolites and catabolites. Furthermore, it has been shown that γδ T cells can directly bind to fully folded proteins in an antibody-like manner. Thus, the view that T cell antigens are mainly restricted to peptides presented by HLA has expanded over the past 20 years to include almost all biomolecules. With this concept in mind, it is appropriate to define what can be considered an antigen-presenting cell (APC). Antigens and Antigen-Presenting Cells

[0016] As defined in the previous section, HLA I and HLA II have non-overlapping expression profiles across cell types. It is widely accepted that almost all nucleated cells present HLA I complexes on their cell surface and thus have the ability to present peptide antigens for T cell sampling. In contrast, HLA II has a restricted expression profile and is expressed only on the surface of cells with specialized roles in antigen presentation, including dendritic cells (DCs), macrophages, and B cells, at least under steady-state conditions. These specialized cell types are often referred to as professional APCs. For the purposes of this document, the term "APC" is used to denote any nucleated cell capable of presenting an antigen for sampling by αβ or γδ T cells. Such antigens are not limited to those presented as "cargo" in specific antigen-presenting complexes, such as HLA and HLA-like molecules, but can include any cell surface-presented moiety capable of binding to αβ or γδ TCR-bearing cells.

[0017] Therapeutic Use of TCR Adoptive transfer of primary T cells was first tested in a clinical setting in the early 1990s, using T cells expanded ex vivo against viral antigens to confer viral immunity to immunocompromised patients. A similar approach using primary T cells expanded ex vivo against specific cancer antigens was tested soon after for the treatment of malignancies. A limitation in these early approaches that continues to be an issue today is the lack of understanding of the nature and diversity of T cells, which is at odds with the need to finely optimize the composition in therapeutic products. Currently, the use of primary T cells expanded ex vivo has been largely abandoned in the pharmaceutical industry, except for a handful of initiatives using primary T cells with specificity for viral antigens.

[0018] In recent years, the ability to stably introduce genetic material into primary human cells has led to an increase in the types of experimental gene-edited T cell therapeutics. Such therapeutic cell products aim to harness the power of T cell responses and redirect T cell specificity towards disease-associated antigen targets, such as antigens expressed only by malignant cells. These rely mainly on transferring chimeric antigen receptors (CARs) into recipient T cells rather than actual TCR chain pairs. A CAR is a signaling receptor element, such as the ζ chain of the CD3 complex, grafted with a targeting moiety (often a single-chain antibody element targeting a surface-expressed protein of malignant cells) to generate a synthetic chimeric receptor that mimics CD3-TCR function. These so-called CAR T cell (CAR-T) products have shown variable degrees of success in clinical trials to date, and despite their potential, it is not straightforward to interpret beyond tumors with unique molecular targets, such as B cell malignancies. Instead, there has been growing interest in transferring full-length TCR chain pair ORFs into T cells. Such TCR-engineered T cell therapeutics are currently limited by difficult manufacturing processes and, like CAR-T products, the lack of identified antigen targets and targeting constructs. To date, the use of αβ TCRs for the recognition of peptide antigens presented by HLAI on malignant cells has been the focus, and the fundamental difficulty with this approach is the requirement for antigens specific to malignant cells.

[0019] Since TCR-pHLA interactions are of relatively low affinity, natural TCRs do not appear to be optimal for engineered TCR T cell therapies. Several approaches have been devised for in vitro affinity maturation of TCRs in the same way as single-chain antibody affinity maturation. These TCR affinity maturation approaches also generally use a single-chain format that fuses the V region of one chain to the V region of another chain to create a single polypeptide construct. Such single polypeptides can then be used in phage or yeast display systems adapted from the antibody engineering workflow and can undergo several rounds of selection based on target binding. In such single-chain TCR approaches, there are two inherent limitations in obtaining functional TCR chain pairs. First, the selection is based on binding affinity to the target. However, it has been well documented that TCR affinity does not always correlate with the strength or potency of TCR signaling output. Second, selection of single-chain constructs based on affinity is not always interpreted as equivalent affinity once they are reconstituted as full-length receptors.

[0020] In the context of therapy, there are further important limitations in affinity matured TCR pairs. Namely, considering that their sequences have been altered, the resulting constructs by definition are no longer subject to thymic selection, where TCRs that strongly react with self-antigens are deleted from the repertoire. Thus, these modified TCRs have an inherent risk of being autoreactive, and it is very difficult to eliminate this risk in vitro using current methods. For the same reason, any selected or engineered TCRs for therapeutic use need to be individualized. If TCRs are artificially engineered or native TCRs are used across individuals, cross-reactivity needs to be eliminated based on the HLA haplotype and presented peptide repertoire of each specific individual in order to avoid potentially devastating autoimmunity. This is because thymic selection occurs in the context of all available HLA molecules that are specific only to that given individual. The potential for such cross-reactivity is unknown. However, the ability of our TCR repertoire to recognize pHLA complexes of other individuals of the same species as foreign is a fundamental property of adaptive immunity and supports graft rejection and graft-versus-host disease. Recent clinical trials using mature TCR chain pairs against the cancer-specific melanoma-associated antigen (MAGE) have shed light on the potential problems of bypassing thymic selection. When autologous T cells with mature TCRs were infused back into two cancer patients, these patients rapidly developed fatal heart disease. Subsequent studies revealed that the MAGE-specific matured TCRs were cross-reactive with peptides presented by HLA-I from the cardiac protein titin. This strongly suggests that cross-reactivity is a distinct possibility in the therapeutic use of TCRs.

[0021] Another means of achieving the use of TCRs for therapeutic purposes is to use them as affinity reagents in much the same way as antibody therapeutic substances. Single-chain TCR molecules have been tested for delivering conjugate drug substances to specific populations of HLA-antigen-expressing cells. Such an approach is generally considered to be safer than CAR-T or TCR-engineered T cell therapies because the administration of the drug substance can simply be withdrawn. However, due to the potential for cross-reactivity and unpredictable off-target effects, there are still potential limitations in this context.

[0022] Detection of TCR repertoire in clinical diagnosis In a related perspective, there is increasing interest in using the detection of the amount of specific TCR sequences for clinical diagnostic purposes. Especially with the emergence of deep sequencing methods, it is possible to grasp the complete TCR diversity for the entire and specific matching αβ pairs within an individual. This could potentially serve as a means of diagnosing specific conditions and disease states by simply detecting the amount of expanded T cell clones as a surrogate readout for establishing the immune response to disease-related antigens in patients. However, such an inclusive approach is currently limited to very strong immune responses with established clinical time points and suffers from the underlying difficulty of identifying the specific antigen targets of any particular TCR identified by sequencing.

[0023] Use of T cell antigens for therapy and diagnosis The fundamental strength of harnessing the adaptive immune response can be rephrased as the central technical difficulty that the exquisite specificity of TCR-antigen interactions requires detailed knowledge of antigens specifically associated with each pathogen, cancer cell, or autoimmune disease. Furthermore, since each antigen can be presented by a specific antigen-presenting complex or its allele, antigen discovery must be performed for each relevant HLA gene and allele. For some infectious diseases such as HIV, influenza, and CMV that are associated with a strong adaptive immune response and generally exhibit a conserved epitope response hierarchy, the most important epitopes have been mapped in association with several common HLAs. Similarly, in the fields of cancer, allergy, and autoimmunity, there has been an increasing number of systematic attempts to map relevant T cell antigens. However, these are difficult procedures, and the effort to systematically represent T cell antigens associated with different clinical situations is hampered by the absence of an efficient, robust, rapid, and adaptable protocol. In particular, cancer cells are a difficult and important aspect. This is because most of the peptides presented on the surface of malignant cells are self-antigens or very similar to self-antigens. Thus, thymic selection will eliminate TCRs that can strongly recognize these peptides. At the same time, tumors develop to evade immune recognition. This means that potent immune responses against established tumors are relatively rare and it is difficult to predict or discover the targets. However, these responses do exist and, importantly, are usually associated with better outcomes. The targets of such responses, tumor-associated antigens (TAAs), in most cases have distinguishable features from self, are overexpressed during cancer development, are absent in the cell type at this stage of development, or are derived from proteins specifically altered by genetic mutations or post-translational modifications such as phosphorylation.

[0024] When available, knowledge of such epitopes allows investigation of T cell responses relevant for fundamental discoveries, diagnostic purposes, and, for example, testing vaccine efficacy. Importantly, these offer both highly specific targets for T cell tolerance induction in allergy and autoimmunity and, critically, targets for specific immunotherapy and valuable targets against malignant cells. Malignant lesions are particularly valuable targets as promising candidates for cell immunotherapy, and progress in T cell manipulation is slowed by the lack of confirmed target TAAs beyond the few cases where specific markers for cancer types happen to be available. In view of the potential of cell therapy and the lack of confirmed targets, the identification of promising TCR antigens remains one of the most pressing obstacles to TCR-based immunotherapy, particularly in efforts to treat cancer.

[0025] Technical aspects of TCR and T cell antigen analysis Overall, the development of TCR-based therapies is still in its infancy and has had limited success. Although potentially enormous, diagnostic approaches have rarely been implemented in comparative clinical studies aimed at evaluating a patient's disease state or response to therapy. Technologies that are not well developed for the stable capture of native TCR chain pairs and the systematic analysis of TCR-antigen interactions in the functional relationships of cell-cell communication at high throughput are the main obstacles to the development of TCR-based therapies and diagnostics.

[0026] Deep sequencing approaches have improved our understanding of T cell receptor diversity in health and disease. However, these approaches have generally focused on short segments spanning the CDR3 region of the TCRβ chain. Most studies have ignored the contribution of the TCRα chain, and few have attempted to analyze the antigen specificity of paired αβ chains and TCRs determined to be of interest. Recent workflows using single cell encapsulation and genetic barcoding have enabled the pairing of native TCRαβ or γδ chain pairs and the analysis of full-length sequences, but such workflows are still at the experimental stage.

[0027] Isolated TCR chain pairs can be analyzed for antigen specificity in either biophysical or functional formats. Biophysical analysis requires the recombinant production of both the TCR and the analyte antigen in soluble form. In the case of HLA-restricted TCRs, this would thus require the production of all individual TCRs and cognate pHLA complexes. This is technically very difficult, slow, and low throughput. Furthermore, such analysis provides only interaction affinity, which has low relevance to functional characteristics in a predictable manner. Until recently, detailed functional analysis of isolated TCR sequences in relation to cells has been limited by the cumbersome protocols of transfection of the analyte TCR chain pair into primary T cells or immortalized T cell lines and detection of cell responses by traditional flow cytometry analysis of cell activation or detection of factors secreted from transfected cells upon antigen loading. In a recent publication by Guo et al., rapid cloning, expression, and functional characterization of paired TCR chains from single cells were reported (Molecular Therapy - Methods and clinical development (2016) 3:15054). In this study, the analyte human αβ TCR pair was expressed in a reporter cell line lacking αβ TCR expression, which included a green fluorescent protein (GFP) reporter system linked to the Nur77 promoter that is activated upon TCR stimulation. This system is still inefficient due to the lack of standardized TCR integration into the reporter cell line genome and does not provide a systematic method for antigen loading of cells by APC elements.

[0028] Similar to the workflow for identification of TCRs against known T cell antigens, de novo discovery of novel T cell antigens in health and disease is still very difficult. Most approaches are still essentially biophysical and aim to test candidate antigens in immunization protocols or generate them by identification of cognate TCRs as described above. There is little or no standardization in the field of T cell antigen discovery, which is mostly limited to academic research.

[0029] With the growing interest in TCRs and their homologs for both therapeutic and diagnostic uses, and the emergence of means to capture a significant number of native TCRαβ and γδ chain pairs, there is still a lack of reliable, high-throughput, standardized techniques for the systematic analysis of TCR-antigen interactions. Importantly, there is a lack of a standardized system for the functional analysis of TCR chain pairs in the native context of cell-cell communication where TCRs and antigens are both presented by live cells. Furthermore, there is a lack of a system that can achieve TCR candidate selection and / or affinity / functional maturation of TCR chain pairs in relevant contexts of cell-cell communication.

[0030] Therapeutic uses of T cell antigens With the rapid expansion of knowledge in T cell biology, there has been a growing interest in the use of T cell antigens in therapeutic agents. This has mainly taken the form of several types of immune strategies. Most prominently, the use of next-generation sequencing approaches allows for the identification of mutagenized sequences in tumor cells. Such sequences may represent T cell antigens that are potentially unique to cancer cells and thus may be immunogens for personalized vaccines against the sequenced tumors. However, for many of the genetic mutations that can be observed, there are no high-throughput formats for analyzing these potential T cell antigens for their ability to be presented by the patient HLA repertoire or whether these antigens are immunogenic. At present, predictions of mutant peptide binding are performed computationally for a very small number of HLA alleles. These prediction models generally provide information on whether a given peptide sequence binds to HLA and do not generally predict the potential immunogenicity of the bound antigen. Furthermore, such computer models are not reliable for antigens that do not present the canonical "anchoring" residues for the HLA allele against which the antigen is being analyzed. Summary of the Invention Problems to be Solved by the Invention

[0031] For example, tolerance therapies for allergies and autoimmune syndromes, as well as other immune approaches including prophylactic vaccination against pathogens, required detailed knowledge of T cell antigens. In the case of the latter prophylactic vaccines, there is still a surprisingly poor knowledge of T cell antigens derived from pathogens common to all HLA alleles, with knowledge only of a handful of HLA alleles. There is a need for a systematic approach to expand this knowledge to develop vaccines effective against common and emerging pathogens.

Means for Solving the Problems

[0032] The present invention addresses the above need. Specifically, the present invention relates to the construction, assembly, and use of a multi-component system composed of at least three components: a genetically engineered antigen-presenting cell (eAPC), a genetically engineered genomic acceptor site, and a matching gene donor vector. The present invention is used for the rapid and high-throughput generation of stable derivative cells presenting antigenic molecules in various forms for the identification and characterization of antigens and cognate TCR sequences. Specifically, the eAPC is genetically engineered by genome editing so as not to have cell surface presentation of human leukocyte antigen (HLA) molecules, HLA-like molecules, and other forms of antigen-presenting molecules and antigenic molecules. In addition, the eAPC as part of the multi-component system includes a genomic acceptor site for the insertion of an antigen-presenting molecule encoding an open reading frame (ORF) and, if necessary, the insertion of an analyte antigen encoded genetically. The system further comprises a gene donor vector designed to target the genomic acceptor site of the APC so as to rapidly deliver an ORF encoding an analyte antigen molecule and / or an antigen-presenting complex. This multi-component system may be used as an analytical system in clinical immunodiagnosis. Furthermore, the present invention relates to the use of the multi-component system for identifying and characterizing T cell antigens and cognate TCRs for the manufacture of immunotherapeutic and immunodiagnostic agents.

[0033] The present invention enables a highly standardized system for the assembly of various analyte eAPC forms in a systematic manner. This standardization and systematization are achieved by highly defined and controllable genomic integration of antigen presentation complexes and antigenic molecule ORFs using a matched donor vector / genomic acceptor site subsystem. This controllable and predictable system brings significant efficiency to the process of generating eAPC populations, reducing the cycle time and cost of this process. Previous systems relied on random integration using uninduced genomic integration and / or viral approaches. Furthermore, the system design is, in part, to ensure a controllable copy number, usually a single copy, of the integrated ORF and enable strict control of the achievable expression levels of the integrated ORF products. More importantly, the ability to integrate ORFs in single copy from a vector pool enables so-called "shotgun integration" (each cell into which the donor vector is integrated can receive only a single ORF from a library of vectors that may encode diverse ORF populations). This enables conversion of a library of ORFs encoded by donor vectors into a library of eAPCs that express a single desired analyte ORF for each eAPC clone subpopulation (essentially a cell-based array system similar to bacteriophage or yeast display systems). This array system can facilitate the identification of unknown analyte antigen sequences within a sequence library based on the reactivity of TCR or other affinity reagents when presented by eAPCs, and subsequent recovery of the carrier eAPCs of the unknown "reactive" sequences. Furthermore, shotgun integration enables efficient production of each analyte antigen within target cells. Compared to transient transfection of a large pool of analyte antigen sequences (resulting in only a slight level of transcripts available for any given analyte antigen), each cell in the eAPC library generated by shotgun integration reliably expresses a single analyte for surface presentation by eAPCs, thus facilitating the identification of analyte antigens by various means.

[0034] The present invention relates to the provision of a genetically engineered multi-component system, the components of which are used in the production of one or more analyte eAPCs. These analyte eAPCs are then combined with one or more analyte TCRs (collectively, the eAPC:TCR system, eAPC:T) to obtain one or more outputs. Here, the analyte TCR can be provided as a soluble or immobilized reagent and can be presented on the surface of a cell or presented by a non-cell-based particle (NCBP). The eAPC presents a candidate analyte antigen to the analyte TCR.

[0035] The minimal form of the multi-component system comprises a first component (referred to as component A) as an eAPC that includes a second component as a genomic acceptor site, and a third component is a gene donor vector (referred to as component C) (Figure 1). The eAPC is the basic component of the multi-component system to which all other components of the system relate. Thus, the eAPC is either natural or contains an engineered feature that renders it suitable for use in creating a population of analyte eAPCs.

[0036] With respect to the present invention, the eAPC (component A) i. lacks the endogenous surface expression of at least one family of antigen presentation complexes (aAPX) and / or analyte antigenic molecules (aAM), ii. contains at least one genomic acceptor site (referred to as component B), wherein i) may be obtained by selection of a naturally occurring cell population lacking the expression of aAPX and / or aAM, or may be engineered to lack such expression, and ii) may be synthetic or may be introduced by specific or non-specific genomic integration.

[0037] The selection of candidate eAPC cells lacking the desired aAPX and / or aAM expression from a naturally occurring cell population can be achieved by methods well known in the art. This can be accomplished directly by staining target cells with an affinity reagent specific for the desired aAPX and / or aAM that is lacking from the eAPC, and selecting cells lacking target aAPX and / or aAM expression. Manipulating cells to lack aAPX and / or aAM expression can be achieved by non-targeted or targeted means. Non-targeted mutagenesis of cells can be achieved by providing the cells with a chemical, radiological, or other mutagen, and then selecting cells lacking target aAPX and / or aAM expression. Targeted mutagenesis of genomic loci can be achieved by different means including, but not limited to: i. Zinc finger nucleases, ii. CRISPR / Cas9-mediated targeting, iii. Site-directed mutagenesis by synthetic transcriptional activator-like effector nucleases (TALEN) wherein the site-specific nuclease induces site-specific DNA repair error mutagenesis at the target locus, and thereafter, mutant cells are obtained by selection of cells lacking target aAPX and / or aAM expression. Component A (eAPC) may optionally include additional T cell co-stimulatory receptors. This feature enables robust or varying forms of communication between the analyte eAPC and the analyte TCR-presenting cell (analyte TC), and tunable communication is appropriate for the identification or characterization of specific analyte TCRs and / or analyte antigens. With respect to the present invention, various forms of CD28 ligation at the analyte TC can be facilitated by including one or more of CD80, CD86, and / or additional B7 family proteins.

[0038] ​Component A (eAPC) may further include, as necessary, the introduction of cell surface adhesion molecule components or the removal of endogenous cell surface adhesion molecules, to promote, respectively, the binding of eAPC to the analyte TC and the formation of immunological synapses, or to avoid strong binding and the formation of harmful cell clustering within the eAPC:T combination system. Adhesion molecules that can be introduced as additional ORFs into Component A or removed genetically from Component A can be selected from the integrin family of adhesion proteins. eAPC may, as necessary, have the ability to process antigens and load them as cargo onto aAPX by a native processing and loading mechanism (referred to as aAPX:aAM). An eAPC having the ability to process antigens and load them as cargo onto aAPX by a native processing and loading mechanism may also have the ability to process and load a cargo molecule (CM) that is endogenous to the eAPC or the culture system (including eAPC) (the aAPX loaded with CM is called the aAPX:CM complex) (Figure 17).

[0039] The second component of the minimal multi-component system is a gene donor vector (Component C) used for the integration of at least one ORF encoding at least one aAPX and / or aAM (Figure 1). Component C is a gene donor vector that is coupled to the genomic acceptor site of Component B contained within the genome of eAPC (Component A). Component C is designed for the integration of one or more ORFs encoding aAPX and / or aAM, encoded by the gene donor vector, into the genomic acceptor site (Component B). Here, this integration results in the expression of aAPX and / or aAM by the target eAPC. For the present invention, the paired gene donor vector and genomic acceptor site are also referred to as an integration couple. In an amplified form multi-component system, component A (eAPC) may further comprise a second genomic acceptor site (referred to as component D) coupled to a second gene donor vector (referred to as component E; this is also added to the system) (Figure 2). The multi-component system may further comprise one or more additional integration couples.

[0040] A multi-component system comprising eAPC and one or two integration couples is of the derivative eAPC form: i.eAPC-p ii.eAPC-a iii.eAPC-pa and is used for the production of, wherein each gene donor vector may comprise one or more ORFs encoding one or more aAPX and / or aAM, for incorporating the ORF into the coupled genomic acceptor site, i) expresses at least one aAPX, ii) expresses at least one aAM, and iii) expresses at least one aAPX and at least one aAM (Figure 3). The gene donor vector and the genomic acceptor site function as an integration couple subsystem of the multi-component system. The gene donor vector must first be combined with a target ORF, where the basic donor vector comes to encode the target ORF. The assembled and ready donor vector is then introduced into the target eAPC to exchange the target ORF with the genomic acceptor site, such that the target ORF is incorporated into the coupled acceptor site of the target cell (Figure 4).

[0041] A multi-component system comprising gene donor vector components C and / or E may be combined with at least one ORF encoding at least one aAPX and / or aAM such that components C' and / or E' are obtained. Here, the combination is defined as the ligation of genetic material in the correct orientation into the correct coding frame of the gene donor vector. The combination of one or more ORFs into gene donor vectors C and / or E is performed using a unique library of ORFs, i. Separate reactions for obtaining separate libraries of C' and / or E' vectors encoding multiple ORFs ii. A single reaction for obtaining a pooled library of C' and / or E' vectors encoding multiple ORFs This may be performed multiple times as. Here, the separate libraries may be combined with component A multiple times such that a separate library of eAPCs with unique ORFs encoding unique aAPX and / or aAM is obtained, and the pooled libraries may be combined with component A as a single event such that a pooled library of eAPCs each having unique ORFs encoding unique aAPX and / or aAM is obtained. Efficient integration of one or more ORFs with a predictable copy number into the genomic acceptor site is highly advantageous for the operation of standardized eAPCs where an analyte eAPC population can be rapidly prepared and characterized. Thus, the genomic acceptor site and the coupled donor vector are important for the function of eAPCs. Furthermore, it is highly desirable to have eAPCs such that components B and D are isolated from each other and donor vector component C is not incorporated by component B and vice versa. In addition, it is also desirable that component B and / or component D are suitable for a method of making eAPCs where the introduction of a defined single aAPX- and / or aAM-containing construct is rapid and repeatable and the likelihood of correct integration and delivery of only a single analyte is high.

[0042] The genomic acceptor site is: i. A synthetic construct designed for recombinase-mediated cassette exchange (RMCE) ii. A synthetic construct designed for site-specific homologous recombination iii. A native genomic site for site-specific homologous recombination and can be selected from, where i) is preferred. The RMCE method may employ selected heterospecific sites specific for individual recombinase enzymes such that each of components B and D has an isolated specificity. Component B and / or component D, which are genomic acceptor sites, are the following genetic elements: i. Heterospecific recombinase sites ii. homology arm iii. eukaryotic promoter iv. eukaryotic conditional regulatory element v. eukaryotic terminator vi. selection marker vii. splice acceptor site viii. splice donor site ix. non-protein-coding gene x. insulator xi. mobile gene element xii. meganuclease recognition site xiii. internal ribosome entry site (IRES) xiv. viral self-cleaving peptide element xv. Kozak consensus sequence comprises at least one of

[0043] The preferred genomic acceptor site is composed of two different arrangements using the following selected elements from the previously described list of elements. The first arrangement is for accepting, by RMCE integration, a single ORF encoding one or more aAPX and / or aAM and / or a selection marker for integration, where the arrangement is 5'-[A] [B] [C] [D] [E] [F]-3' where A) is an element iii) constitutive or inducible eukaryotic promoter, B) is an element i) heterospecific recombinase site 1, C) is an element xv) Kozak consensus sequence, D) is an element vi) FACS and / or MACS compatible coding protein marker, E) is an element i) heterospecific recombinase site 2, F) is an element v) eukaryotic terminator.

[0044] The second arrangement is for accepting, by RMCE integration, two ORFs encoding one or more aAPX and / or aAM and / or a selectable marker for integration, where the arrangement is: 5'-[A] [B] [C] [D] [E] [F] [G] [H] [I]-3' where A) is element iii) a constitutive or inducible eukaryotic promoter, B) is element i) a heterospecific recombinase site 1, C) is element xv) a Kozak consensus sequence, D) is element vi) a FACS and / or MACS compatible encoded protein marker 1, E) is element v) a eukaryotic bidirectional terminator, F) is element vi) a FACS and / or MACS compatible encoded protein marker 2, G) is element xv) a Kozak consensus sequence, H) is element i) a heterospecific recombinase site 2, I) is element iii) a constitutive or inducible eukaryotic promoter, Furthermore, in this second arrangement, elements F, G and I are encoded in the antisense direction.

[0045] Component C and / or E are the following gene elements: i. A heterospecific recombinase site ii. A homology arm iii. A eukaryotic promoter iv. A eukaryotic conditional regulatory element v. A eukaryotic terminator vi. A selectable marker vii. A splice acceptor site viii. A splice donor site ix. A non-protein coding gene x. An insulator xi. Mobile genetic element xii. Meganuclease recognition site xiii. Internal ribosome entry site (IRES) xiv. Viral self-cleaving f element xv. Kozak consensus sequence xvi. Selection marker for integration xvii. Antibiotic resistance cassette xviii. Bacterial origin of replication xix. Yeast origin of replication xx. Cloning site is composed of at least one of

[0046] In a preferred embodiment of the gene donor vector, component C and / or component E is composed of two different, possible arrangements using the following selected elements from the previously described list of elements. The first arrangement is for the delivery of a single ORF encoding one or more aAPX and / or aAM and / or a selection marker for integration by RMCE integration, where the arrangement is 5'-[A] [B] [C] [D] [E]-3' where A) is heterospecific recombinase site 1, B) is Kozak consensus sequence, C) is a cloning site for a single ORF encoding one or more aAPX and / or aAM and / or an integration selection marker, D) is heterospecific recombinase site 2, E) is an antibiotic resistance cassette and a bacterial origin of replication in a non-specific orientation, Furthermore, element viii and / or xiv may be used to link multiple aAPX and / or aAM and / or element xvi together.

[0047] The second arrangement is for the delivery of two ORFs encoding one or more aAPX and / or aAM and / or a selectable marker for integration by RMCE, where the arrangement is: 5'-[A] [B] [C] [D] [E] [F]-3' where A) is element i) a heterospecific recombinase site 1, B) is element xv) a Kozak consensus sequence, C) is a cloning site for the introduction of two or more ORFs encoding one or more aAPX and / or aAM and / or element xvi) a selectable marker for integration and having a eukaryotic terminator, D) is element xv) a Kozak consensus sequence (antisense orientation), E) is element i) a heterospecific recombinase site 2, F) is element xvii) an antibiotic resistance cassette and element xviii) a bacterial origin of replication in a non-specific orientation, Furthermore, element viii and / or xiv may be used to ligate together multiple aAPX and / or aAM and / or element xvi within each ORF.

[0048] Production of analyte eAPC using a multi-component system The above multi-component system may be used in multiple ways during its operation to create distinct forms of analyte eAPC or a library thereof that serve to present analyte aAPX, aAM, aAPX:aAM, and aAPX:CM to an analyte TCR within an eAPC:T combination system (see Figure 27). A multi-component system comprising a single integration couple may be used to produce eAPC-p in one step from component A by providing a component C' combined with an ORF of aAPX. This aAPX is integrated into site B and component B' is produced. The resulting cell line expresses the provided aAPX and the aAPX is presented on the cell surface (Figure 5). A multi-component system comprising two integration couples may be used to produce eAPC-p from component A in one step by providing component C' combined with the ORF of aAPX. This aAPX is integrated into site B and component B' is produced. The resulting cell line expresses the provided aAPX, and the aAPX is presented on the cell surface. The second integration couple D / E remains unmodified and can be used in downstream integration steps (Figure 6). A multi-component system comprising a single integration couple may be used to produce eAPC-a from component A in one step by providing component C' combined with the ORF of aAM. This aAM is integrated into site B and component B' is produced. The resulting cell line expresses the provided aAM, and the aAM is presented on the cell surface or retained intracellularly (Figure 7). A multi-component system comprising two integration couples may be used to produce eAPC-a from component A in one step by providing component C' combined with the ORF of aAM. This aAM is integrated into site B and component B' is produced. The resulting cell line expresses the provided aAM, and the aAM is presented on the cell surface or retained intracellularly. The second integration couple D / E remains unmodified and can be used in downstream integration steps (Figure 8).

[0049] A multi-component system comprising a single integration couple may be used to produce eAPC-pa from component A in one step by providing component C' combined with two ORFs, one encoding aAPX and the other encoding aAM. Both aAPX and aAM are integrated into site B and component B' is produced. The resulting cell line expresses the provided aAPX and aAM, and may present aAPX:aAM on the cell surface (Figure 9). A multi-component system comprising two integration couples may be used to produce eAPC-pa from component A in one step by providing component C' combined with two ORFs, one encoding aAPX and the other encoding aAM. Both aAPX and aAM are integrated into site B to produce component B'. The resulting cell line may express the provided aAPX and aAM and present aAPX:aAM on the cell surface. The second integration couple D / E remains unmodified and may be used in downstream integration steps (Figure 10). A multi-component system comprising two integration couples may be used to produce eAPC-pa from component A in one step by providing component C' and E' each combined with one ORF encoding either aAPX or aAM. Both aAPX and aAM are integrated into site B or D to produce components B' and D'. The resulting cell line may express the provided aAPX and aAM and present aAPX:aAM on the cell surface (Figure 11). A multi-component system comprising two integration couples may be used to produce eAPC-pa from component A in two steps by first providing component C' combined with the ORF encoding aAPX. This aAPX is integrated into site B to produce component B'. The resulting cell line expresses the provided aAPX, which is presented on the cell surface (eAPC-p intermediate). The second integration couple D / E remains unmodified. In the second step, a donor vector provides E' combined with the ORF encoding aAM. This aAM is integrated into site E to produce component E'. The resulting cell line may express the provided aAM, which is processed in aAPX and loaded as a cargo to form an aAPX:aAM complex on the cell surface (Figure 12). A multi-component system comprising two integrated couples may be used to produce eAPC-pa from component A in two steps by first providing component C' combined with an ORF encoding aAM. This aAM is integrated at site B and component B' is produced. The resulting cell line expresses the provided aAM (eAPC-a intermediate). The second integrated couple D / E remains unmodified. In the second step, a donor vector is provided with E' combined with an ORF encoding aAPX. This aAPX is integrated at site E and component E' is produced. The resulting cell line expresses the provided aAPX, which is presented on the cell surface. The aAM integrated in the first step may be processed in aAPX and loaded as a payload to form an aAPX:aAM complex on the cell surface (Figure 13).

[0050] In the above examples of producing analyte eAPC-p, eAPC-a and eAPC-pa populations from eAPC, the multi-component system is used to produce distinct populations of analyte eAPC expressing defined aAPX and / or aAM by providing known aAPX and aAM candidates in a defined manner. This method may be repeated multiple times to construct a library of eAPC-p, eAPC-a and eAPC-pa provided to the eAPC:T combination system during operation of the system. An alternative approach is to employ a pooled library of candidate aAPX and / or aAM ORFs combined with gene donor vectors and integrate these in a single reaction to obtain a pooled library of analyte eAPC-p, eAPC-a or eAPC-pa expressing multiple aAPX, aAM and / or aAPX:aAM This method of converting a vector pool into a pool of eAPC-p, -a and / or -pa is called shotgun integration. This is particularly useful when analyzing a large library of candidate aAM against a fixed aAPX or vice versa. A multi-component system comprising two integration couples may be used to produce eAPC-pa from component A in two steps by first providing component C' combined with an ORF encoding aAPX. This aAPX is integrated into site B and component B' is produced. The resulting cell line expresses the provided aAPX on the cell surface (eAPC-p intermediate). The second integration couple D / E remains unmodified. In the second step, a library of multiple E' is provided, where the library of donor vectors comprises a pool of vectors each combined with a single ORF encoding aAM. Each aAM is integrated into site E in a single cell and component E' is produced. The resulting cell pool comprises a population of cells, where each cell has a single random aAM ORF from the original vector pool integrated therein. The aAM integrated in the second step may be processed and loaded as a cargo in the aAPX integrated in the first step to form an aAPX:aAM complex on the cell surface (Figure 14).

[0051] A multi-component system comprising two integration couples may be used to produce eAPC-pa from component A in two steps by first providing component C' combined with an ORF encoding aAM. This aAM is integrated into site B and component B' is produced. The resulting cell line expresses the provided aAM (eAPC-a intermediate). The second integration couple D / E remains unmodified. In the second step, a library of multiple E' is provided, where the library of donor vectors comprises a pool of vectors each combined with a single ORF encoding aAPX. Each aAPX is integrated into site E within a single cell and component E' is produced. The resulting cell pool comprises a population of cells, where each cell has a single random aAPX ORF from the original vector pool integrated therein. The aAM integrated in the first step may be processed and loaded as a cargo in the aAPX integrated in the second step to form an aAPX:aAM complex on the cell surface (Figure 15). A multi-component system comprising two integrated couples may be used to produce eAPC-pa from component A in one step by providing components C' and E' each combined with a library of ORFs encoding either a library of aAPX or a library of aAM. Both aAPX and aAM are integrated at site B or D, and B' and D' are produced. The resulting cell pool contains a population of cells, where each cell has a single random aAPX ORF and a single random aAM ORF from the original vector pool integrated therein. Within each cell in the pooled library, the integrated aAM may be processed and loaded as a cargo onto the aAPX integrated in the same cell to form an aAPX:aAM complex on the cell surface. This pooled library may contain all possible combinations of aAPX:aAM from the provided set of aAPX and aAM (Figure 16). In the above shotgun integration method for providing a pooled library of eAPC-pa, the robustness of the system relies on a single-copy genomic acceptor site. This ensures that only a single analyte is introduced into each cell via the integration couple. This single-copy genomic acceptor site is one of the optional flanks of eAPCS. Because multiple copies of the same genomic acceptor site may be beneficial in providing integration steps that can be obtained in the produced eAPC with multiple "alleles" from the provided library of vectors.

[0052] With respect to the present invention, aAPX is the following: i. One or more members of HLA class I ii. One or more members of HLA class II iii. One or more non-HLA antigen-presenting complexes may be selected from one of. With respect to the present invention, aAPX is the following: i. A polypeptide or a complex of polypeptides provided as an analyte antigen ii. A peptide derived from a polypeptide provided as an analyte antigen iii. A peptide provided as an analyte antigen iv. A metabolite provided as an analyte antigen v. A polypeptide or a complex of polypeptides translated from an analyte antigenic molecule ORF vi. A peptide derived from a polypeptide translated from an analyte antigenic molecule ORF vii. A peptide derived from a change in the Component A proteome viii. A polypeptide derived from the Component A proteome ix. A metabolite derived from a change in the Component A metabolome may be selected from one of the following.

[0053] Contact between the analyte eAPC and the analyte TC The present invention relates to the provision of a genetically engineered multi-component system. One or more analyte eAPC populations are produced using Component A and one or more Component C' and / or one or more Component E'. These analyte eAPCs are then combined with one or more analyte TCRs to construct an eAPC:TCR analysis system (eAPC:T) and obtain one or more outputs (Figure 27). Here, the eAPC provides the analyte antigen, and the analyte TCR is as follows: i. A TCR molecule and / or ii. A molecule having an affinity for the analyte antigen may be represented by, and the analyte TCR is, within the eAPC:T system, as follows: i. An analyte TCR presenting cell (TC) and / or ii. A soluble or immobilized affinity reactant and / or iii. A non-cell-based particle (NCBP) may be presented in different manners to the eAPC represented as, where i) The analyte TCR-presenting cell (TC) is considered to be any TC capable of presenting the analyte TCR to the eAPC; ii) The affinity reagent is considered to be any reagent made as an analyte for exploring TCR binding and / or stimulation on the cell surface of the eAPC in the APC:T system. This reagent can be an analyte TCR multimer reagent (e.g., TCR “tetramer”) used to stain the eAPC. In this regard, the affinity reagent can be an antibody or the like; iii) The non-cell-based particle (NCBP) acts in a manner similar to the affinity reagent insofar as the particle is the analyte TCR or other substance for which binding of the analyte antigen on the eAPC surface is to be evaluated within the eAPC:T system. However, the NCBP can also be considered a larger substance capable of further carrying genetic or other information that acts directly or indirectly as an identifier of the presented analyte TCR or other conjugate. A representative example of the NCBP is a bacteriophage in a phage display scenario, where the phage can display an antibody fragment-antigen binding (FAB). The positively labeled eAPC can be recovered with the phage and sequenced to identify the FAB specific for the analyte antigen on the eAPC surface.

[0054] Furthermore, the presentation of the analyte TCR by cells can be any of the following: i. Primary T cells ii. Recombinant T cells iii. Genetically engineered TCR-presenting cells iv. Genetically engineered cells presenting molecules having an affinity for the analyte antigen (collectively referred to as analyte TCs). The eAPC:T analysis system consists of one or more analyte eAPC populations and analyte TCRs (Figure 27). The analyte eAPC population is produced using a multi-component system as described above (Figures 3 - 16). The eAPC:T system is provided in a form that allows physical contact between the analyte eAPC and the analyte TCR, enabling complex formation between the analyte antigen presented by one or more analyte eAPCs and the analyte TCR.

[0055] An analyte antigen is any substance to which an analyte TCR can presumably bind in the eAPC:T system: i. aAPX (analyte antigen presenting complex) and / or ii. aAM (analyte antigenic molecule) and / or iii. aAPX:aAM (analyte antigen presenting complex presenting an analyte antigenic molecule) and / or iv. CM (non-analyte cargo molecule) and / or v. aAPX:CM (analyte antigen presenting complex presenting a cargo molecule) wherein aAPX is a complex capable of presenting an aAM, aAM is any molecule that can be directly recognized by a TCR or recognized by a TCR when loaded onto an aAPX, aAPX:aAM is an aAPX loaded with an aAM, CM is a cargo molecule that can be loaded onto an aAPX but is not an analyte and thus can be derived from an analyte antigen presenting cell (APC) or the assay system itself, and aAPX:CM is an aAPX loaded with CM.

[0056] For the purposes of the present invention, the eAPC:T system is: i. input of a single analyte eAPC or ii. input of a library of pooled analyte eAPCs and consists of the following: iii. input of a single analyte TC, or iv. input of a single analyte affinity reagent, or v. input of a single analyte NCBP, or vi. input of a pooled library of analyte TCs, or vii. input of a pooled library of analyte affinity reagents, or viii. input of a pooled library of analyte NCBPs in combination with one of the following.

[0057] Contact in a buffer system The contact between the analyte eAPC and the analyte TC is carried out in a permissive cell culture system or buffered medium, where the system contains a medium that permits the function of both the analyte eAPC and the analyte TC cells, the analyte affinity reagent or the analyte NCBP. The contact between the soluble analyte TCR, the immobilized analyte TCR and / or the analyte NCBP and the analyte eAPC is carried out in a permissive buffered system, where the system contains a buffered medium that permits the function of both the analyte TCR and the analyte eAPC cells.

[0058] Labeling of eAPC with an affinity reagent or NCBP The analyte eAPC obtained from the multi-component system can be used for the characterization of the analyte antigen presented by the eAPC. This characterization can be carried out in such a manner that the analyte eAPC is labeled by contacting it with an immobilized or soluble affinity reagent or NCBP (Figure 24). The labeling of eAPC can be detected by direct observation of the label by methods such as flow cytometry, microscopy, spectroscopy or luminometry, or by capture with an immobilized affinity reagent or NCBP for the identification of the analyte antigen.

[0059] Definition of signal response The analyte eAPC obtained from the multi-component system is used for the characterization of the signal response of the analyte eAPC to the analyte TCR, where this signal response can be either binary or varying gradually, and can be measured as being specific to the eAPC (Figure 21) and / or, if included, specific to the analyte TC (Figure 20). This signal can be detected by methods such as flow cytometry, microscopy, spectroscopy or luminometry or other methods known to those skilled in the art.

[0060] General method - Selection of eAPC A method of selecting one or more analyte eAPCs from an eAPC:T combination system or a library of analyte eAPCs to obtain one or more analyte eAPCs, wherein the expressed analyte antigen binds to one or more analyte TCRs: i. Combining one or more analyte eAPCs with one or more analyte TCRs to effect contact between the analyte antigen and the analyte TCR, and at least one of the following: ii. Measuring the formation of complexes, if any, between one or more analyte antigens and one or more analyte TCRs, and / or iii. Measuring the signal from a labeled analyte TCR, and / or iv. Measuring the signal response, if any, of the analyte eAPC induced by complex formation between one or more analyte antigens and one or more analyte TCRs, and / or v. Measuring the signal response, if any, of the analyte TC induced by complex formation between one or more analyte antigens and one or more analyte TCRs, and vi. Selection of one or more analyte eAPCs based on steps ii, iii, iv, and / or v, wherein the selection is made by positive and / or negative measurements and wherein i, iv, and vi or i, v, and vi include preferred configurations.

[0061] General method - Selection of analyte TCRs A method of selecting one or more analyte TCRs from an input analyte TCR or a library of analyte TCRs to obtain one or more analyte TCRs, wherein the expressed analyte antigen binds to one or more analyte TCRs: i. Combining one or more analyte APCs with one or more analyte TCRs to effect contact between the analyte antigen presented by the analyte APC and one or more analyte TCRs, and ii. If any, measuring the formation of complexes between one or more analyte antigens and one or more analyte TCRs, and / or iii. Measuring the signal from the labeled analyte TCR, and / or iv. If any, measuring the signal response in one or more analyte TCs induced by complex formation between an analyte TCR and an analyte antigen, and / or v. If any, measuring the signal response by an analyte APC induced by complex formation between one or more analyte TCRs and one or more analyte antigens, vi. Selection of one or more analyte APCs from steps ii, iii, iv and / or v, wherein the selection is made by positive and / or negative measurements comprising, wherein i, iv and v include preferred configurations.

[0062] General methods for signal response Methods for selecting an analyte eAPC and / or an analyte TC and / or an affinity reagent and / or an NCBP from an eAPC:T combination system based on the reported signal response are: i. Determining the native signal transduction response, and / or ii. Determining the synthetic signal transduction response (when the eAPC includes such a response circuit and / or when the analyte TC includes an equivalent synthetic reporter circuit) comprising. Induced native or synthetic signal responses specific to the APC and / or the analyte TC are as follows: i. Secreted biomolecules ii. Secreted chemical substances iii. Intracellular biomolecules iv. Intracellular chemical substances v. Surface-expressed biomolecules vi. The cytotoxic effect of the analyte TC on the analyte eAPC vii. The paracrine effect of analyte TC on analyte eAPC, such that the signal response is induced in the analyte APC and determined by detecting any increase or decrease in any of i to v viii. Proliferation of analyte TC ix. The immunological synapse between analyte TC and analyte eAPC is measured by detecting one or more increases or decreases of, wherein the detected signal response is compared to the non-induced signal response state specific to analyte eAPC and / or analyte TC prior to the assembly of the eAPC:T combination system and / or the combination systems assembled in parallel, wherein analyte eAPC and / or analyte TC can present a control analyte antigen and / or analyte TCR species and / or soluble analyte antigen known not to induce a signal response within the eAPC:T combination system used.

[0063] Method of selection by labeling and / or signal response A method for selecting analyte eAPC and / or analyte affinity reagent and / or analyte NCBP from an eAPC:T combination system comprises: i. Determining the labeling of eAPC by an affinity reagent or NCBP including ii. Determining the native signal transduction response, and / or iii. Determining the synthetic signal transduction response (if the eAPC includes such a response circuit) may also be included, wherein selecting eAPC and / or affinity reagent and / or NCBP by detecting the labeling of eAPC may include detecting the surface labeling of eAPC by an affinity reagent and / or NCBP by including a detectable label in the affinity reagent and / or NCBP. This detectable label may be fluorescence, luminescence, spectroscopy, chemical, radiochemical or an affinity moiety. Thus, this selection of eAPC may be performed based on FACS, MACS or equivalent high-throughput screening and selection methods.

[0064] Summary In the eAPC:T combination system, the measurement of signal responses in one or more analyte eAPCs or one or more analyte TCs, or the labeling of eAPCs that can be mediated by complex formation between an analyte antigen and an analyte TCR (Figure 27, step iv) is important for the selection of the primary system output (Figure 27, step v), where the primary system output is a single cell or cell pool, and / or a single affinity reagent or affinity reagent pool, and / or a single NCBP or NCBP pool. Here, the selection of cells or reagents can be based on the presence or absence of signal responses reported in either and / or both of the contacting analyte eAPCs or analyte TC cells, or by the measurable labeling of eAPCs with affinity reagents or NCBPs.

[0065] Obtaining the primary system output from the eAPC:T system The present invention relates to the provision of a genetically engineered multi-component system. Using component A and one or more component C's and / or one or more component E's, one or more analyte eAPC populations are produced. These analytes are then combined with one or more analyte TCRs by an eAPC:T system to obtain one or more outputs. The analyte TCRs are provided as soluble or immobilized reagents, presented on the cell surface, or presented by cell-free based particles (NCBPs). The presentation of analyte TCRs by cells can be in any of the following forms: i. Primary T cells ii. Recombinant T cells iii. Genetically engineered TCR-presenting cells iv. Genetically engineered cells presenting molecules having affinity for the analyte antigen (Collectively referred to as analyte TC above) can be in any of these forms.

[0066] This system is composed of the selection of one or more analyte eAPC populations and analyte TCRs (Figure 27). The analyte eAPC population is produced using the multi-component system as described above (Figures 3 - 16). The eAPC:T system is provided in a format that allows physical contact between the analyte eAPC and the analyte TCR, where this contact allows complex formation between the analyte antigen presented by one or more analyte eAPCs and the analyte TCR, where the analyte antigen is one of the following: i. aAPX and / or ii. aAM and / or iii. aAPX:aAM and / or iv. CM and / or v. aAPX:CM wherein the analyte TCR is presented by the analyte TC, or by any of a soluble or immobilized analyte affinity reagent, or by the analyte NCBP for possible binding to the analyte antigen presented by the analyte eAPC, and complex formation can lead to stabilization of the complex, leading to labeling of the eAPC and / or induction of signal transduction within the analyte eAPC, and the labeling of the eAPC and / or induction of signal transduction within the analyte eAPC and / or the analyte TC can be reported and measured.

[0067] The manner of reporting the induced signal response and / or labeling of the eAPC is as described above, and it is these reported responses and / or labels that need to be measured upon acquisition of the primary output of the multi-component system constructed in the eAPC:T system. The primary output from the eAPC:T system is the selected cell population and / or the selected affinity reagent or the selected NCBP, where the selection is based on: i. a measurable label of the eAPC by the affinity reagent or NCBP, and / or ii. a signal response detected in the eAPC, and / or iii. the absence of a detected signal response in the eAPC, and / or iv. a signal response detected in the analyte TC, and / or Lack of signal response detected in analyte TC Performed based on this, where the primary output can be represented as a single cell or cell pool and / or one or more eAPC-binding affinity reagents or NCBPs (affinity reagents or NCBPs bound to eAPC).

[0068] From the eAPC:T combination system, an analyte affinity reagent, NCBP, or analyte TC and / or analyte eAPC can be selected based on the response in the contacting cells. That is, analyte TC can be selected based on the reported response or lack thereof in the contacting analyte eAPC. Conversely, analyte eAPC can be selected based on the reported response or lack thereof in the contacting analyte TCR, or when the analyte TC is an analyte affinity reagent or NCBP, the analyte affinity reagent or NCBP can be selected from the eAPC response.

[0069] The eAPC and / or analyte TC output from the system is the selected cell, where the selection is made based on the presence or absence of the reported signal response in either the analyte TC or eAPC. These cells may contain one or more eAPCs and / or one or more analyte TCs. Here, the selected cells may contain a single cell, a cell pool of the same identity, or a cell pool of different identities (Figure 27 step v). The primary analyte affinity reagent or NCBP output from the system is the selected cell with or without the bound affinity reagent or NCBP, where the selection is made based on the presence or absence of the signal response labeled or reported by the analyte eAPC. Here, the selected affinity reagent or NCBP may contain a single affinity reagent or NCBP, a pool of affinity reagents or NCBPs of the same identity, or a pool of affinity reagents or NCBPs of different identities (Figure 27 step v).

[0070] Output from the binary composition The signal reported in analyte eAPC and / or analyte TC in the eAPC:T combination system may be used to select an analyte cell population to provide a primary output. With respect to the present invention, the primary output of analyte eAPC can be achieved by selecting a desired analyte eAPC population labeled with analyte TCR from a binary system when the eAPC:T combination system is of a binary composition of one or more analyte eAPCs and analyte TCR (e.g., Figure 24). The primary output of an analyte affinity reagent or NCBP can be achieved by selecting a desired analyte eAPC population labeled with the analyte affinity reagent or analyte NCBP from a binary system when the eAPC:T combination system is of a binary composition of one or more analyte eAPCs and an analyte affinity reagent or analyte NCBP (e.g., Figure 24). The primary output of eAPC and / or analyte TC type can be achieved by selecting a desired analyte APC and / or analyte TC population from the combined culture system when the eAPC:T combination system is of the nature of a fixed analyte eAPC and a pooled library analyte TC (e.g., Figure 22) or when the eAPC:T combination system is of the nature of a fixed analyte TC and a pooled analyte eAPC library (e.g., Figure 23). The primary output of analyte eAPC can be achieved by selecting a desired analyte eAPC population from the combined culture system when the eAPC:T combination system is of the nature of a fixed analyte TCR and a pooled library analyte eAPC (e.g., Figure 24) or when the eAPC:T combination system is of the nature of a fixed eAPC and a pooled soluble analyte affinity reagent or NCBP library. The primary output of the analyte affinity reagent or analyte NCBP can be achieved by selecting the desired analyte affinity reagent or analyte NCBP population from the combined culture system when the eAPC:T combination system is of the nature of a fixed soluble analyte affinity reagent or analyte NCBP and a pooled library analyte eAPC (e.g., Figure 24), or when the eAPC:T combination system is of the nature of a fixed eAPC and a pooled analyte affinity reagent or analyte NCBP library.

[0071] Modes of obtaining the output There are several different modes by which the primary output can be obtained, where each mode involves a cell sorting step. Sorting may be achieved by fluorescence-activated cell sorting (FACS) and / or magnetic-activated cell sorting (MACS) and / or different affinity-activated cell sorting methods. The eAPC and / or analyte TC cells and / or eAPC-binding affinity reagent or NCBP as the primary output may obtain single cells by single cell sorting and / or obtain cell pools by cell pool sorting. The eAPC and / or analyte TC cells as the primary output may obtain single cells by single cell sorting and, if necessary, then proliferate the single cells to obtain a monoclonal pool of the selected eAPC or analyte TC cells. The eAPC and / or analyte TC cells as the primary output may also obtain cell pools by cell pool sorting and, if necessary, then proliferate the cell pools to obtain a pool of the selected eAPC and / or TC cells.

[0072] Obtaining the terminal system output from the eAPC:T system Following the above method of obtaining the primary output which is the analyte eAPC and / or analyte TC and / or analyte NCBP selected based on the measured signal response or stable complex formation, the terminal output from the eAPC:T system can be obtained by further processing of the selected eAPC and / or analyte TC and / or NCBP primary output (Figure 27, step vi). The terminal output from the multi-component system is presented by analyte APC or analyte TC or analyte affinity reagent or analyte NCBP, and is obtained as the primary output from the multi-component system by selection from the eAPC:T combination system. i.aAPX and / or ii.aAM and / or iii.aAPX:aAM and / or iv.CM and / or v.aAPX:CM and / or vi.TCR is the identity of.

[0073] In the eAPC:T system, it is frequent that the analyte molecules presented by analyte eAPC and analyte TC are encoded by genes. For example, when NCBP is displayed by bacteriophage, it is also the case when analyte NCBP has an identity encoded by a gene. Therefore, in order to identify the analyte molecules presented by analyte eAPC or analyte TC or analyte NCBP, gene sequencing of the prepared analyte eAPC, TC and NCBP may be performed. The selected primary output obtains the gene sequence for the genome or transcriptome of the selected and / or expanded cells: i.aAPX and / or ii.aAM and / or iii.aAPX:aAM iv.CM and / or v.aAPX:CM and / or vi.analyte TCR may be processed to determine the identity of, where the resulting identity represents the terminal output of the eAPC:T system. NCBP having a gene component may obtain the gene sequence for the genome or transcriptome of the selected NCBP and be processed to determine the identity of the analyte TCR, where the resulting identity represents the terminal output of the eAPC:T system.

[0074] The eAPC may obtain gene sequences for component B' and / or component D' of the sorted and / or expanded TC cells and be processed to determine the identity of the analyte antigen, where the resulting identity of the analyte antigen represents the end output of the eAPC:T system. The analyte TC may obtain gene sequences for the genome or transcriptome of the sorted and / or expanded TC cells and be processed to determine the identity of the analyte TCR, where the resulting identity of the TCR represents the end output of the eAPC:T system.

[0075] Gene sequencing can be achieved in various ways, from various sources of genetic material, with or without specific processing. Prior to the sequencing step i. extracting genomic DNA, and / or ii. extracting RNA transcripts of component B' and / or D', and / or iii. amplifying the DNA and / or RNA transcripts of component B' and / or D' by PCR and / or RT-PCR may be performed. The sequencing step may be destructive to the eAPC or TC, NCBp, or a pool thereof obtained as the primary output of the multi-component system.

[0076] If it is desirable to obtain the primary output from an eAPC:T system where the sequencing step is destructive to the primary output eAPC, an output eAPC equivalent to the analyte eAPC may be created using the sequence information obtained as the end output of the multi-component system. In the context of the analyte molecule encoded by a gene, the end output of the eAPC:T system may be obtained by obtaining sequence information from component B' and / or D' and / or the cell genome and / or transcriptome. However, in some embodiments, the antigen information is not encoded by a gene. Antigens that are post-translationally modified, antigens provided to the eAPC:T combinatorial system by non-genetic means, antigens emerging from the induced or modified state of the analyte eAPC proteome or metabolite, and CMs specific to the eAPC:T system may not be rationally identified by gene sequencing means.

[0077] In important cases of aAMs that can be provided to the eAPC:T system by non-genetic means, there are two different ways in which the provided aAM can be presented by the APC as an aAPX:aAM complex. In the first scenario, the aAM can bind directly to aAPX and is provided in a form that forms the aAPX:aAM complex on the cell surface (Figure 18). An example of such an aAM is a peptide antigen for the HLA complex. In the second scenario, the aAM is taken up by the analyte eAPC, loaded as a cargo in aAPX, and provided in a form that is processed to form the aAPX:aAM complex on the cell surface (Figure 19). A method for selecting and identifying an aAM cargo or a CM cargo, wherein the cargo is a metabolite and / or peptide loaded into the aAPX of an eAPC obtained by being selected as the primary output of a multi-component system, i. isolating the aAPX:aAM or aAPX:CM or the loaded aM or the loaded CM, ii. identifying the loaded cargo, wherein the identified loaded cargo (CM or aAM) is the end output of the multi-component system.

[0078] Generally, there are two modes by which a cargo molecule can be identified from a selected APC. First, forced release of the cargo from aAPX:aAM or aAPX:CM results in isolation of aAM or CM that can then be used for subsequent identification (Figure 25). This example is the acid wash of an eAPC that liberates peptide aAM from the HLA complex. Second, capture of aAPX:aAM or aAPX:CM (e.g., by complex dissociation and immunoaffinity isolation methods) results in isolation of the aAPX:aAM or aAPX:CM complex, and aAM or CM can be identified (Figure 26). Methods for identifying isolated aAM and / or CM directly or from isolated aAPX:aAM or aAPX:CM complexes are: i. Mass spectrometry ii. Peptide sequencing analysis and can include, where the aAM and / or CM identities included are the end output of a multi-component system.

[0079] Determination of the affinity of an analyte TCR for an analyte antigen using an eAPC:T system Following the above-described method for obtaining a primary output, where the primary output is an analyte eAPC cell selected based on a measured signal response, the eAPC primary output can be subjected to affinity analysis to determine the affinity of the analyte antigen for cognate analyte TCR, where the analyte antigen is one of: i. aAPX and / or ii. aAM and / or iii. aAPX:aAM and / or iv. CM and / or v. aAPX:CM and where the analyte TCR is provided as a soluble affinity reagent or presented by an analyte TC or analyte NCBP, and the affinity of the analyte antigen is determined by the following method: i. Labeling a selected analyte eAPC with analyte TCR at a range of concentrations ii. Performing FACS analysis on the stained analyte eAPC of step a iii. Determining the intensity of the fluorescent label of the analyte eAPC for the analyte TCR within a predetermined concentration range; iv. Calculating the affinity of the analyte antigen for the analyte TCR is determined by.

[0080] Regarding the present invention, the affinity of the analyte antigen may be determined by a method described previously (which may also include a labeled reference), and the affinity is calculated using the ratio of the fluorescence intensity of the analyte antigen to the fluorescence intensity of the reference, where the labeled reference is: i. An analyte eAPC labeled with an affinity reagent for one analyte antigen; ii. An analyte eTPC-t labeled with an affinity reagent for one or more CD3 proteins iii. A cell or particle presenting an analyte antigen as a labeled reference is selected from. Description of the Drawings The present invention will be further described in the following non-limiting drawings.

Brief Description of the Drawings

[0081]

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Example

[0082] Materials and Methods Electroporation of ARH-77 Cells Per reaction, 4×10 6 cells were electroporated using Glutamax-I (Life Technologies) in 500 μl RPMI 1640 with Gene Pulser XCELL TM (Bio-Rad) at the following settings: square wave 285 V, pulse length 12.5 ms, 2 pulses at 1-second intervals. The DNA concentrations used for Cas9 plasmid V1.A.8 were 10 μg / ml and 7.5 μg / ml for the gRNAs targeting the integration sites (V2.I.10 and V2.J.1 for integration at the HLA endogenous locus, V2.J.6 for the target AAVS1 site) (Table 3). The integration vector was electroporated at a concentration of 7.5 μg / ul. For HDR integration at the HLA locus, HLA class I V1.C.6 and V1.C.9 plasmids were used. For HDR integration at the AAVS1 locus, HLA class I V1.F.8 and V1.F.10 and HLA class II V1.I.5 and V1.I.7 were used. Variants of the pp65 ORF were integrated into pre-generated HLA monoallelic cell lines. Plasmids V1.G.9 and V1.H.1 containing pp65 in a form linked to the GFP marker were used for this purpose. To generate an ARH-77 HLA-null cell line with one RMCE site, plasmids with heterospecific recombinase sites adjacent to the marker were used. V4.B.2 was used for RFP and V4.B.3 for BFP. The same plasmids were co-electroporated to generate stable cell lines containing two RMCE sites. Monoallelic HLA cell lines were also generated using RMCE, where vector V4.D.2 was electroporated into cells containing one RMCE site. After electroporation, the cells were incubated for 2 days in culture medium RPMI 1640 containing Glutamax-I + 10% FBS (37 °C, 5% CO 2 ) and then analyzed.

[0083] Transfection of HEK293 cells One day before transfection, the cells were seeded at 1.2 - 1.4×10 6Cells were seeded at a density of cells / 60 mm dish in 90% DMEM + 2 mM L-glutamine + 10% HI-FBS (Life Technologies). The next day, 65% confluent cells were transfected with a total of 5 μg of DNA and jetPEI® (Polyplus transfection reagent, Life Technologies) (N / P ratio 6). Transfection was performed after changing the medium. The stock solutions of DNA and jetPEI® were diluted in sterile 1 M NaCl and 150 mM NaCl, respectively. The final volume of each solution was set to 50% of the total mixing volume. Then, the PEI solution was added to the diluted DNA, and the mixture was incubated at room temperature for 15 minutes. Finally, the DNA / PEI mixture was carefully added to the 60 mm dish, taking care not to disrupt the cell film. Cells were incubated at (37 °C, 5% CO 2 , 95% relative humidity) for 48 hours, and then GFP expression analysis was performed. Due to the deletion of the HLA class I gene, cells were transfected with a DNA vector (V1.A.8) encoding 0.42 μg of Cas9_GFP, gRNAs targeting HLA-A, B, and C (V2.A.1, V2.A.7, and V2.B.3, respectively), and an empty vector (V1.C.2). For the integration of the RMCE site at the AAVS1 locus, cells were transfected with 0.5 μg of V1.A.8; 0.625 μg of gRNA V2.J.6 and 0.75 μg of a plasmid encoding two markers flanked by the RMCE site (V4.B.2 for RFP and V4.B.3 for BFP), and the empty vector V1.C.2 was used to fill 5 μg of DNA.

[0084] Selection of polyclonal GFP-expressing cells Cells transfected by electroporation or transfection with Cas9-P2A-GFP (V1.A.8) or a plasmid encoding the GFP selection marker (V1.A.4) were sorted for transient GFP expression using a FACSJAzz TM cell sorter (BD Biosciences). HEK 293 cells were TrypLE TMCollected using Express Trypsin (ThermoFisher Scientific), resuspended in an appropriate volume of DPBS 1× (Life Technologies), and then cell sorting was performed in DMEM 1× medium containing 20% HI-FBS and Anti 100X (Life Technologies). ARH-77 cells were washed and resuspended in an appropriate volume of DPBS, and then sorting was performed in RPMI 1640 containing Glutamax-I, 20% HI-FBS, and Anti 100X (Life Technologies).

[0085] Sorting of polyclonal and monoclonal cells with stable expression of the component of interest To obtain a population of cells constitutively expressing the integrated protein or marker, cells were sorted 7 - 15 days after the first GFP+ selection. For cells predicted to express surface proteins, sorting was performed after antibody staining. For the HLA class I gene, PE-Cy TM 5 mouse anti-human HLA-ABC antibody (BD Biosciences) was used. Staining for HLA-DR and HLA-DP was performed using Alexa Fluor® 647 mouse anti-human HLA-DR, DP, DQ (BD Biosciences). For HEK 293-derived cell lines, cells were collected using TrypLE TM Collected using Express Trypsin (ThermoFisher Scientific), washed in an appropriate volume of DPBS 1× (Life Technologies), and then cell sorting was performed in DMEM 1× medium containing 20% HI-FBS and Anti 100X (Life Technologies). ARH-77-derived cell lines were washed in an appropriate volume of DPBS, and then sorting was performed in RPMI 1640 containing Glutamax-I, 20% HI-FBS, and Anti 100X (Life Technologies).

[0086]

Table 1

[0087] For HLA knockout or integration, cell selection was performed based on loss or gain of HLA expression, respectively. Cells integrated with the RMCE site were sorted based on the expression of BFP and RFP markers, and HLA monoclonal clones integrated with the pp65 variant were sorted for GFP expression (Table 4). Monoclonal selection of cells expressing the gene of interest was performed in 96-well plates containing 200 μl of growth medium. One to two plates were sorted per sample. Immediately afterwards, polyclonal selection of the remaining cells was performed in FACS tubes using a two-way sorting setting on the cell sorter Influx TM (BD Biosciences).

[0088] Phenotypic screening of monoclonal populations A sample of 20,000 cells from the expanded monoclonal population was transferred to a microtiter plate for analysis. The cells were resuspended in 250 μl of 1× DPBS (Life Technologies) and analyzed on a LRSFortessa TM (BD Biosciences). BFP and RFP expression were detected using PMTs for BV421 and the PE-Texas Red fluorophore, respectively. For proteins with surface expression, the cells were first incubated with PE-Cy TMStaining was performed using 5 mouse anti-human HLA-ABC antibody (BD Biosciences) or Alexa Fluor® 647 mouse anti-human HLA-DR, DP, DQ (BD Biosciences). The staining solution was prepared using the recommended antibody volume diluted in 100 μl of staining buffer (DPBS + 2% FBS). Cells were incubated at 4 °C for 1 hour and then washed twice with 500 μl of staining buffer before analysis. The selected monoclonal clones were maintained in normal growth medium. HEK239 cells were grown in DMEM + 2 mM L-glutamine + 10% HI-FBS (Life Technologies), and ARH-7 cells were grown in RPMI 1640 containing Glutamax-I + 10% HI-FBS. The cell confluence was monitored daily until it reached 10 - 12×10 6 . DNA was extracted from 5×10 6 cells using the QIAamp DNA Minikit (Qiagen). The remaining cells were further grown and cryopreserved at a density of 3×10 6 cells / ml in 70% growth medium + 20% HI-FBS + 10% DMSO.

[0089]

Table 2

[0090] Verification of integration into the correct genomic location Monoclonal antibodies with the desired phenotypic characteristics were screened, evaluated at the molecular level, and this was carried out by PCR using Q5® Hot Start High-Fidelity DNA Polymerase (NBE) in a 20 μl reagent using the components and volumes recommended by the manufacturer. Primers 9.C.4 and 9.D.6 were used to determine whether the HLA I ORF was integrated into the HLA locus; correct right homologous arm recombination was indicated by a 1 kb amplicon (Table 5). For HLA integration at the AAVS1 locus, four sets of primers were used: 9.C.3 and 9.C.8 to evaluate correct left homologous arm recombination (1.1 kb), 9.C.4 and 9.D.1 to evaluate right homologous arm recombination (660 bp), 1.C.5 and 9.C.5 to amplify the CMV promoter (810 bp) of the internal construct, and 1.C.2 and 9.C.10 (380 bp) to obtain an amplicon for the SV40 pA terminator of the internal construct. Evaluation of RMCE site integration in HEK293 and ARH-77 HLA-null cell lines was carried out using primer sets 2 and 4. To confirm HLA class I deletion in HEK293 cells, specific HLA primers were used as follows: 4.A.3 and 4.A.4 targeting HLA-A, 4.A.7 and 4.B.1 for HLA-B, and 4.B.5 and 8.A.1 for HLA-C. First, a PCR Master Mix was prepared using all components (Q5® reaction buffer, dNTPs, Hot-Start Q5® DNA Polymerase, primer Fwd and Rev, 100 ng of DNA template and H2O). The PCR reaction was performed using a C1000 Touch TM Thermal Cycler (Bio-Rad). PCR products were electrophoresed in a 1% agarose gel in 1× TAE buffer using a PowerPac Basic (Bio-Rad), stained with 10,000-fold diluted sybersafe, and analyzed using a Fusion SL (Vilber Lourmat).

[0091]

Table 3

[0092] Identification of gene copy number The DNA of the selected monoclonal antibody was analyzed using primers specific for the gene of interest that target the gene and a probe that recognizes the fragment of the integrated gene and extends to the homologous arm. For HLA class I integration at the HLA locus, primers 4.I.9 and 9.C were used to amplify the gene of interest, and 8.B.2 conjugated with FAM was used as a probe. For the construct integrated at the AAVS1 locus, primers 9.D.6 and 9.D.7 and probe 9.J.2 (also conjugated with FAM) were used. In all cases, the reference gene (TRAC) was simultaneously screened, and the chromosomal copy number was determined using primers 10.A.9 and 10.A.10 and the fluorescent probe 10.B.6 conjugated with HEX. From the integration copy number, for the reference gene (TRAC), ARH-77 cells were considered diploid and HEK293 cells were considered triploid. Prior to digital droplet PCR, the DNA was digested with MfeI (NEB) to separate tandem integrations. The reaction setup and cycle conditions were according to the protocol of QX200 Droplet Reader and Droplet Generator and C1000 Touch TM deep-well Thermal cycler (Bio-Rad) using ddPCR TM Supermix for Probes (No dUTP) (Bio-Rad). Data was acquired using QuantaSoft TM software, using Ch1 for the detection of FAM and Ch2 for HEX. TM

[0093]

Table 4

[0094]

Table 5

[0095] HLA-A in the eAPC cell line * Flp-mediated integration of the 02:01 sequence eAPC cells were electroporated with a vector encoding Flp, DNA encoding a marker for tracking delivery (a vector encoding GFP), and a vector containing HLA-A * 02:01. HLA-A * The 02:01 sequence also encoded a linker and a 3×Myc-tag at the 3'-end. The electroporation conditions used were 258 V, 12.5 ms, 2 pulses, and 1 pulse interval. The ratio of each integration vector to the Flp-vector was 1:3. Cells electroporated with only the GFP-vector and non-electroporated cells were used as controls, respectively, to set the gate for GFP selection 2 days later. The next day (2 days after electroporation), the cells were analyzed and sorted based on GFP expression. The cells were sorted using a BD Influx cell sorter. Three days after electroporation, sorting based on GFP expression was performed to enrich the electroporated cells. Seven to eight days after electroporation, the cells were harvested and surface stained for HLA-ABC expression. BFP+ve RFP-ve HLA+ve cells were sorted into single cells to make them monoclonal. To genotype the cells, a PCR reaction was performed using 100 ng of DNA as a template to verify whether integration occurred at the integration site where integration was expected. A forward primer targeting the integration cassette (Pan_HLA_GT_F1 (Insert SEQ ID NO)) and a reverse primer targeting just outside the integration site (SV40pA_GT_R1 Insert SEQ ID NO) were used, and the PCR products were electrophoresed on a 1% agarose gel.

[0096] Flp-mediated integration of the HCMV ORF sequence in the eAPC-p cell line eAPC-p cells were electroporated with a vector encoding Flp, DNA encoding a marker for tracking delivery (a vector encoding GFP), and a vector containing the HCMV pp28, pp52, or pp65 aAM-ORF. The HCMV-ORF sequence also encoded a linker and a 3×Myc-tag at the 3' end. The electroporation conditions used were 258 V, 12.5 ms, 2 pulses, and a 1-pulse interval. The ratio of each integration vector to the Flp-vector was 1:3. To set the gate for GFP sorting 2 days later, cells electroporated with only the GFP-vector and non-electroporated cells were used as controls, respectively. The next day (2 days after electroporation), the cells were analyzed and sorted based on GFP expression. The cells were sorted using a BD Influx cell sorter.

[0097] Flp-mediated shotgun integration of three HCMV ORF sequences in the eAPC-p cell line eAPC-p cells were electroporated with a vector encoding Flp, DNA encoding a marker for tracking delivery (a vector encoding GFP), and a vector containing the HCMV pp28, pp52, or pp65 aAM-ORF. The HCMV-ORF sequence encoded a linker and a 3×Myc-tag also at the 3' end. The electroporation conditions used were 258 V, 12.5 ms, 2 pulses, and a 1-pulse interval. For shotgun integration, the vectors containing the HCMV-ORF were pooled at a ratio of 1:1:1 and the mixture was electroporated into eAPC-p cells. The resulting eAPC-pa cells were polyclonal. Individual monoclonal cells were sorted and genetically characterized to demonstrate that the polyclonal consisted of cells containing all three HCMV-ORFs.

[0098] PCR reaction to evaluate RMCE-integration of HCMV ORF into component D Primers used to evaluate the integration of HCMV ORFs were annealed to the linker (Forward Primer 10.D.1 (Insert SEQ ID)) and the EF1aplha promoter (Reverse Primer 15.H.4). The expected sizes were 0.8 kb for pp28, 1.5 kb for pp52, and 1.9 kb for pp65.

[0099]

Table 6

[0100]

Table 7

[0101] PCR products were electrophoresed on a 1% agarose gel in 1×TAE buffer using PowerPac Basic (Bio-Rad), stained with 10,000-fold diluted sybersafe, and analyzed using Fusion SL (Vilber Lourmat).

[0102] Proliferation of antigen-specific CD8+ cells Peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors known to have CD8+ T cells specific for CMV-A.0201-NLVP using Ficoll Paque Plus (GE Healthcare). Cells were stained with surface antibodies against CD markers. The specific T cell population was sorted using a BD Influx cell sorter, pelleted, and resuspended at 200,000 cells / ml in OSG medium + 10% HS.

[0103] Pulse of eAP with peptide multimers HLA-A * 02:01 eAPCs were pulsed with 1 μM peptide (NLVPMVATV (SEQ ID NO: 3) or in complete OSG medium + 10% HS) for 4 hours. Cells were washed three times in phosphate-buffered saline (PBS) and resuspended at 100,000 cells / ml in OSG medium + 10% human serum (HS).

[0104] Co - culture of antigen - specific CD8+ and eAPC CD8+ cells were co - cultured with eAPC in a 96 - well polystyrene (wp) round - bottom plate at a 1:1 ratio, i.e., 5000 cells of each cell type in a 100 μL culture volume, a total of 10000 cells / well. As a result of restimulation on the 9th day of culture, 150 μL of the culture was maintained and restimulated with 5000 fresh pulsed eAPC cells at a volume of 50 μL per well. In a parallel experiment, CD8+ T cells were co - cultured with an eAPC - pa cell line stably expressing pp65 in 96 wp round - bottom at a 1:1 ratio, 5000 cells of each cell type in a 100 μL culture volume, a total of 10000 cells / well. Unpulsed HLA - null and eAPC - pa cells were included as controls. No restimulation was performed.

[0105] Phenotyping Phenotyping was performed on the 14th day. Five replicates and 20 - well pools were phenotyped per condition. Cells were stained separately with 100 - fold diluted DCM (Zombie NIR), followed by staining with 50 - fold diluted multimers (Table 4) for 10 minutes, and then surface markers (Table 4) were added at 25 μL / sample for 30 - 60 minutes. Cells were resuspended in staining buffer (PBS + 2% FBS), data was acquired on an LSRFortessa, and analyzed with FlowJo.

[0106] Proliferation of antigen - specific CD4+ cells Peripheral blood mononuclear cells (PBMCs) were obtained from healthy blood donors known to have CD4+ T cells specific for INFL - DRB1 * 01:01 - PKYV and isolated using Ficoll Paque Plus (GE Healthcare). Cells were stained with surface antibodies against CD markers. Specific T - cell populations were sorted using a BD Influx cell sorter, pelleted, and resuspended in OSG medium + 10% HS at 100000 or 400000 cells / ml.

[0107] Pulsing of eAPC with Peptide Multimers HLA DRB1 * 6 eAPC was pulsed for 2 hours with 1 μM peptide (PKYVKQNTLKLAT (SEQ ID NO: 1) or in complete OSG medium + 10% HS). Cells were washed three times in phosphate-buffered saline (PBS) and resuspended at 5000 cells / ml in OSG medium + 10% human serum (HS).

[0108] Co-culture of Antigen-Specific CD4+ and eAPC CD4+ cells were co-cultured with eAPC in 96wp round bottom at 250 eAPC and 5,000 to 20,000 CD4+ cells in a 100 μL culture volume. Cultures were maintained in OSG + 10% HS. On day 1, 100 U / mL IL-2 was administered to some cultures. Cultures not receiving IL-2 were given medium. IL-2 was added to cultures cultured for 14 days on day 7.

[0109] Phenotyping Phenotyping was performed on day 14. Five replicates and 20-well pools were phenotyped per condition. Cells were stained. Cells were stained separately with 100-fold diluted DCM (Zombie NIR), followed by staining with 50-fold diluted multimer (Table 4) for 10 minutes, and then surface markers (Table 4) were added at 25 μL / sample for 30 - 60 minutes. Cells were resuspended in staining buffer (PBS + 2% FBS), data was acquired on an LSR Fortessa, and analyzed with FlowJo.

[0110] Cytotoxicity Assay Pulsing of eAPC with Peptide 2×10 6 Cells were pulsed overnight with 1 μM NLVPMVATV (SEQ ID NO: 3) peptide in 2 ml of complete Roswell Park Memorial Institute (RPMI) medium, harvested, washed three times with PBS, and resuspended in complete RPMI.

[0111] Generation of Antigen-Specific CD8+ T Cells The antigen-specific CD8+ T cells were derived from PBMC of healthy donors. The cells were stained with surface antibodies against CD markers. The specific T cell population was sorted using a BD Influx cell sorter, counted, and stored in liquid nitrogen. One day before the experiment, the cells were thawed and rested overnight in complete OSG medium. The cells were counted and resuspended in complete OSG medium.

[0112] Co-culture of eAPC and antigen-specific CD8+ cells Peptide-pulsed eAPC and non-pulsed eAPC were co-cultured with cytotoxic CD8+ T cells. 10,000 eAPC per well were seeded in 96-well plates (in 50 μl of complete RPMI). The eAPC were co-cultured with CD8+ T cells at increasing ratios. The ratios tested were eAPC alone 1:0 (eAPC:CD8+), 1:1 (eAPC:CD8+), 1:8 (eAPC:CD8+) in a total volume of 100 μl. The cells were co-cultured for 4 - 5 hours.

[0113] Staining The cells were transferred from the wells to microtubes (1 well → 1 microtube), and 400 μl of RPMI was added per tube. The cells were centrifuged at 400 g for 3 minutes, the supernatant was removed, and the cell pellet was resuspended in 25 μl of staining mix or RPMI (unstained control) (staining mix: Annexin V BV711 + CD80 APC + CD8 APC-H7) and incubated at 450 rpm for 20 minutes at RT. Staining was terminated by adding 400 μl of RPMI per tube, followed by centrifugation and removal of the supernatant. The staining is described in Table 4. The cell pellet was resuspended in 150 μl of RPMI containing 1 μg / ml propidium iodide (stained samples) or 150 μl of RPMI (unstained samples), and the samples were transferred to 96-well plates for data acquisition by Fortessa.

[0114] Metal affinity chromatography The peptides used in the pulse experiment were purchased from Genscript Biotech. APD-2: NLVPMVATV (SEQ ID NO: 3) pp65 is a wild-type peptide and is restricted to binding to HLA-A * 02:01, and APD-21: NLGPMAAGV (SEQ ID NO: 4) pp65 is a triple mutant peptide of ADP-2 NLVPMVATV (SEQ ID NO: 3) pp65 with V3G, T8G, V6A mutations. APD-11: VYALPLKML (SEQ ID NO: 5) is a wild-type peptide restricted to binding to HLA-A * 24:02. Cells were cultured in RPMI supplemented with 10% FBS at 37 °C and 5% CO 2 On the day of the experiment, the cells were collected, washed twice in warm PBS 1×, and re-seeded at 2×10 6 cells / ml and pulsed with 1 μM peptide for 2 hours. The pulsed cells were collected, washed twice with ice-cold PBS, and lysed in ice-cold lysis buffer (150 mM sodium chloride (NaCl), 50 mM Tris pH 8, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 5 mM imidazole, 0.2 mM iodoacetamide, and 1× Halt protease inhibitor cocktail (Thermo Scientific)), vigorously stirred, and incubated at 4 °C for 20 minutes. The clarified lysate was applied to HisPur TMIt was mixed with nickel-nitriloacetic acid (Ni-NTA) resin (Thermo Scientific) and rotated at 4 °C for 2 hours. After removing the unbound fraction of the lysate, the resin was washed twice with a high-salt buffer (250 mM NaCl, 50 mM Tris pH 8, 25 mM imidazole) and twice with a low-salt buffer (50 mM NaCl, 50 mM Tris pH 8). The washed beads were collected in the low-salt wash buffer and transferred to a spin column (Thermo Scientific). The bound fraction was eluted with 10% acetic acid and ultrafiltered through a 3 kD Nanosep Omega column (Pall). The peptide fraction was subjected to liquid extraction by mixing it 1:1 with water-saturated ethyl acetate and stirring vigorously enough to remove the organic phase. Subsequently, solid-phase extraction was performed with a stage tip assembled using a two-layer Empore styrene divinylbenzene-reversed-phase sulfonate (SDB-RPS) matrix 47 mm disk (3M). The SDB-RPS membrane was activated with acetonitrile and equilibrated with SDB wash buffer (0.2% TFA, milli-Q, pH < 2), after which the sample was loaded. Then, after washing the SDB membrane twice, the adsorbed peptide fraction was eluted with elution buffer (80% acetonitrile (ACN), 1% NH 3 4, milli-Q, pH > 10). The sample was transferred to an HPLC glass vial, dried under reduced pressure, stored at -20 °C, and then subjected to LC-MS / MS analysis.

[0115] Mass spectrometry The peptide was resuspended in 10 μl of Solvent A (3% CAN, 0.1% formic acid (FA), MQ) and then subjected to LC-MS / MS analysis. Each sample was analyzed on a Q Exactive HF (Thermo Fisher, Germany) connected to a Dionex nano-UHPLC system (Thermo Fisher Scientific) by injecting 8 μl from each sample vial. The UHPLC was equipped with a trap column (Acclaim PepMap 100, 75 μm × 2 cm, nanoviper, C18, 3 μm, 100 Å; Thermo Fisher Scientific) and an analytical column (PepMap RSLC C18, 2 μm, 100 Å, 50 μm × 50 cm; Thermo Fisher Scientific) heated to 50 °C. The mobile phase buffer for nLC separation consisted of Solvent A and Solvent B (95% CAN, 0.1% FA, MQ). The peptide was eluted during a 30-minute gradient and sprayed directly into the mass spectrometer. The flow rate was set at 400 nL / min and the LC gradient was as follows: 2 - 5% Solvent B within 5 minutes, 5 - 40% Solvent B within 30 minutes, 40 - 47% Solvent B within 5 minutes, 47 - 100% Solvent B within 5 minutes, 100% Solvent B for 8 minutes, and 2% Solvent B for 5 minutes. Nano-spray was achieved with an applied voltage of 1.8 kV. The mass spectrometer was programmed in data-dependent acquisition (DDA) mode (top 10 peaks) to perform a Fourier transform survey scan from 400 - 1600 m / z (resolution 60,000 at 200 m / z), AGC target 1e6, and maximum injection time 250 ms. MS2 scans were acquired for the 10 most abundant MS1 ions with a charge state of 1 - 7 using HCD fragmentation and a quadrupole isolation window of 1.2 m / z for 30 seconds of dynamic exclusion.

[0116] Data analysis The raw MS files were searched against a peptide fasta file containing the peptides used in the experiment and supplemented with a list of common LC-MS / MS contaminants using MaxQuant (version 1.5.6.5). The digestion specificity was set to non-specific, the peptide variable modification was set to allow oxidation (M), the initial search tolerance was set to 20 ppm, and the FDR was set to 1%.

[0117] Example Example 1: Deletion of the APX gene family by targeted mutagenesis In this example, how targeted mutagenesis of the genes encoding the antigen presentation complex (APX) family was achieved to generate the first trait of the genetically engineered antigen-presenting cell (eAPC) is described. The trait is a deletion of surface expression of at least one member of the APX family. In this example, the targeted APX consisted of three members of the major HLA class I family, HLA-A, HLA-B, and HLA-C, in the HEK293 cell line. HEK293 cells were derived from human embryonic kidney cells that exhibit endogenous surface expression of HLA-ABC. Cytogenetic analysis demonstrated that this cell line had a karyotype close to triploid, and thus HEK293 cells encode three alleles of each HLA-A, HLA-B, and HLA-C gene. Targeted mutagenesis of the HLA-A, HLA-B, and HLA-C genes was performed using a genetically engineered CRISPR / Cas9 system in which Cas9 nuclease activity was targeted to the HLA-A, HLA-B, and HLA-C loci by synthetic guide RNAs (gRNAs). Four to five unique gRNAs were designed to target conserved nucleotide sequences for each HLA gene locus, and the targeted sites were directed towards the start point of the gene coding sequence. This was because this was more likely to generate null alleles. The efficiency of the gRNAs that induced mutations at the targeted loci was determined, and the most efficient gRNAs were for HLA-A, HLA-B, and HLA-C null (HLA-ABC) null)It was selected to generate HEK293 cell lines.

[0118] Plasmids encoding optimal gRNAs targeting the HLA-A, HLA-B, and HLA-C loci were transfected into HEK293 cells together with the plasmid encoding Cas9-P2A-GFP as described in the "Methods". Cells positive for Cas9-P2A-GFP plasmid uptake were FAC sorted based on GFP fluorescence 2 days after transfection (Figure 28a). GFP-sorted cells were further grown for more than 5 days to allow sufficient time for gene editing events to occur and, in the case of deleterious mutations, sufficient time to lose expression of the remaining endogenous HLAI proteins. After this growth period, cells were stained with a pan-HLA-ABC antibody, and cells with reduced HLA-ABC expression on the surface were identified (Figure 28b). Absence of pan-HLA-ABC antibody staining suggested that each HLA-A, HLA-B, and HLA-C allele had mutated. Individual HLA-ABC negative cells were sorted and grown into monoclonal populations.

[0119] HLA-ABC null Monoclonality was confirmed by the absence of surface expression of HLA-ABC. A subset of the monoclonal clones was shown to lack surface expression of HLA-ABC. Three examples of the monoclonal clones ACL-414, ACL-415, and ACL-416 are shown in Figure 29. Further genetic characterization of monoclonal clones lacking HLAI surface expression was performed by determining that the cell lines have genetic mutations underlying all alleles of the HLA-A, HLA-B, and HLA-C genes (Figure 30). Genetic characterization was performed by PCR using primers spanning the gRNA genomic target sites for detection of amplicon size changes and / or primers used as templates for sequencing. Figure 30 shows HLA-ABC containing gene deletions in alleles of the HLA-A, HLA-B, and HLA-C genes detected by short PCR amplicons compared to the amplicon sizes of the parental cell line (e.g., ACL-414). null Shows the selection of monoclonal clones. In conclusion, the genetically modified HEK293 cell lines (including ACL-414, ACL-415 and ACL-416) were demonstrated to lack surface expression of HLA-ABC and thus had the first trait of genetically engineered antigen-presenting cells (eAPCs).

[0120] Example 2: Preparation of eAPCs Containing Component B In this example, a method for stably integrating Component B into the monoclonal strain ACL-414 to generate the second trait of eAPCs is described. The second trait included at least one genomic acceptor site for the integration of at least one ORF, where the genomic acceptor site was a synthetic construct designed for recombinase-mediated cassette exchange (RMCE). null In this example, the genomic integration site (Component B) was composed of selected genetic elements. Two unique heterospecific recombinase sites, FRT and F3, which flanked the ORF encoding the selectable marker blue fluorescent protein (BFP). An EF1a promoter was encoded on the 5' side of the FRT site and an SV40 polyadenylation signal terminator was encoded on the 3' side of the F3 site. The advantage of having non-coding cis-regulatory elements located outside the heterospecific recombinase sites is that they are not required in the matched gene donor vector (Component C). Thus, after cellular delivery of the gene donor vector, transient expression of the encoded ORF is not observed. This has made the selection of successful RMCE more reliable such that cellular expression of the ORF from the gene donor vector likely occurs only after correct integration into Component B, because the appropriate cis-regulatory elements are included (see Example 6).

[0121] To facilitate stable genomic integration of Component B into the genomic safe harbor locus (AAVS1), a plasmid was constructed in which the DNA element of Component B was flanked by AAVS1 left and right homology arms. Each arm was composed of a >500 bp sequence homologous to the AAVS1 genomic locus. Stable integration of Component B was achieved by the process of homology-directed recombination (HDR) at the genomic safe harbor locus AAVS1. The ACL-414 cell line was transfected with a plasmid encoding an optimal gRNA targeting the AAVS1 locus, a plasmid encoding Cas9-P2A-GFP, and a plasmid encoding the Component B gene element flanked by AAVS1 left and right homology arms. Cells positive for Cas9-P2A-GFP plasmid uptake were FAC sorted based on GFP fluorescence 2 days after transfection (Figure 31a). The GFP-sorted cells were further grown for more than 7 days to allow sufficient time for HDR to occur and for the transient expression of the selection marker BFP to be lost. After this growth period, the cells were analyzed by a FACS instrument, and individual BFP-positive cells were sorted and grown into monoclonal populations (Figure 31c).

[0122] Individual monoclonal clones were selected as eAPCs for single integration of component B into the desired AAVS1 genomic locus based on maintained BFP expression. Cell lines ACL-469 and ACL-470 were monoclonal clones in which BFP expression was maintained (Figures 32a and b). Genetic characterization was performed on DNA extracted from monoclonal ACL-469 and ACL-470, demonstrating that component B was integrated into their genomes and that component B was integrated into the AAVS1 site (Figure 33). Confirmation of genomic integration was determined by detection of a PCR amplicon of the expected size using primers specific for component B (Figure 33a). Confirmation that component B was integrated into the AAVS1 site was determined by detection of a PCR amplicon of the expected size using primers designed against the AAVS1 genomic sequence distal to the region encoded by the homology arms and primers unique to the SV40 pA terminator encoded by component B (Figure 33b). The copy number of component B was determined by digital droplet PCR, where the number of component B and reference gene DNA molecules was measured and a ratio was calculated (Table 1). Monoclonal ACL-469 and ACL-470 contained component B molecules and reference gene molecules in a ratio of 1 to 3. Considering that the parental HEK293 cell line has a karyotype close to triploid, this demonstrates single integration of component B into the ACL-469 and ACL-470 cell lines. In conclusion, the genetically modified ACL-469 and ACL-470 cell lines are HLA-ABC null and contain a single copy of a synthetic genomic acceptor site designed for RMCE, thus demonstrating the generation of eAPCs with a single synthetic integration acceptor site.

[0123] Example 3: Generation of eAPCs Containing Component B and Component D In this example, component B and component D were introduced into HLA-ABC nullA method for stably integrating into the monoclonal strain ACL-414 to generate a second trait of eAPC is described. The second trait includes two genomic acceptor sites for the integration of at least one ORF, where the genomic acceptor sites are synthetic constructs designed for recombinase-mediated cassette exchange (RMCE). This example uses the same method and components as described in Example 2, except that a second genomic acceptor site (Component D) is added. The Component D gene element is composed of two unique heterospecific recombinase sites F14 and F15 that are different from Component B. These sites flanked an ORF encoding the selectable marker red fluorescent protein (RFP). An EF1a promoter was encoded on the 5' side of the F14 site, and an SV40 polyadenylation signal terminator was encoded on the 3' side of the F15 site. As in Example 2, the Component D gene element was flanked by AAVS1 left and right homology arms, each composed of >500 bp sequences homologous to the AAVS1 genomic locus. Component B and Component D were integrated into AAVS1 as described in Example 2, except for the addition of the plasmid encoding the Component D element to the transfection mix. Cells positive for Cas9-P2A-GFP plasmid uptake were FAC sorted based on GFP fluorescence 2 days after transfection (Figure 31a). The GFP-sorted cells were further grown for more than 7 days, after which the cells were analyzed with a FACS instrument, and individual BFP and RFP positive cells were sorted and grown to form monoclonal populations (Figure 31b).

[0124] Individual monoclonal strains were selected as eAPCs for single integration of component B and single integration of component D into different AAVS1 alleles based on maintained BFP and RFP expression. The cell line ACL-472 was a representative monoclonal with maintained BFP and RFP expression (Figure 32c). As described in Example 2, genetic characterization was performed on DNA extracted from the monoclonal ACL-472, demonstrating that components B and D were integrated into their genomes and that both components were integrated into the AAVS1 site (Figure 33). The copy numbers of both components B and D were determined by digital droplet PCR, where the numbers of component B, D, and reference gene DNA molecules were measured and ratios were calculated. Monoclonal ACL-472 contained component B and D molecules and reference gene molecules in a ratio of 2 to 3 (Table 2). Considering that the parental HEK293 cell line has a karyotype close to triploid, this demonstrates single integration of component B and single integration of component D into the ACL-472 cell line. In conclusion, the genetically modified ACL-472 cell line is HLA-ABC null and contains a single copy of a synthetic genomic acceptor site designed for RMCE, components B and D, demonstrating the generation of an eAPC with two unique synthetic integration acceptor sites.

[0125] Example 4: eAPC-p constructed in one step using one integration couple, where component C' encodes a single HLAI ORF In this example, a method for constructing an eAPC-p in which the genomic acceptor site (component B) is a native genomic site and the gene donor vector (component C') contains a single ORF encoding one analyte antigen presentation complex (aAPX) in one step using one integration couple is described. In this example, the eAPC is a genetically modified ARH-77 cell line (referred to as ACL-128), where two families of APX major HLA class I family and HLA class II were mutated. The basic cell line ARH-77 is a B-lymphoblast derived from plasmacytic leukemia that exhibits strong HLA-A, B, C and HLA-DR, DP, DQ cell surface expression. Cytogenetic analysis has demonstrated that the basic ARH-77 cell line has a karyotype close to diploid, but also shows a deletion of chromosome 6p21, which is the region encoding the HLA locus. DNA sequencing of the ARH-77 locus confirmed that ARH-77 encodes only a single allele of the HLA-A, HLA-B and HLA-C as well as the HLA-DRA, HLA-DRB, HLA-DQA, HLA-DQB, HLA-DPA and HLA-DPB gene families. HLA-ABC null and HLA-DR,DP,DQ null The cell line ACL-128 was generated by CRISPR / cas9 targeted mutagenesis with gRNAs targeting the HLA-A, HLA-B and HLA-C as well as the HLA-DRA, HLA-DRB, HLA-DQA, HLA-DQB, HLA-DPA and HLA-DPB gene families using the method described in Example 1. Surface labeling with pan-anti-HLA-ABC or pan-anti-HLA-DR,DP,DQ confirmed that ACL-128 lacks surface expression of both APX families (Figure 34b and 35 and Figure 37b, respectively).

[0126] In this example, the genomic acceptor site component B is the native AAVS1 genomic site, and targeted integration was achieved by HDR. The gene donor vector (component C) was matched to component B by encoding AAVS1 left and right homology arms, each composed of >500bp sequences homologous to the AAVS1 genomic locus. Between the AAVS1 left and right homology arms, the plasmid encoded a CMV promoter and an SV40 terminator. The aAPX of interest was cloned between the promoter and the terminator to generate component C'. In this example, component C' is one aAPX, HLA-A *24:02 or HLA-B * -07:02, each comprising a single ORF encoding (component C' HLA-A*24:02 and component C' HLA-B*-07:02 respectively). The method of constructing eAPC-p was by HDR-mediated integration of component C' into component B to produce component B'. The cell line ACL-128 was electroporated with a plasmid encoding an optimal gRNA targeting the AAVS1 locus, Cas9-P2A-GFP, and component C'. Cells positive for Cas9-P2A-GFP plasmid uptake were FAC sorted two days after electroporation based on GFP fluorescence (Figure 34a). The GFP-sorted cells were further grown for more than 7 days to allow sufficient time for HDR to occur and for transient expression of aAPX to be lost. After this growth period, the cells were stained with a pan-HLA-ABC antibody and cells that acquired expression of the analyte HLA on the surface were identified (Figure 34b). The presence of pan-HLA-ABC antibody staining indicated that the analyte HLA ORF encoded by component C' was integrated into the genome. Individual HLA-ABC positive stained cells were sorted and grown to form a monoclonal population of eAPC-p.

[0127] Individual monoclonal strains were selected as eAPC-p based on maintained analyte HLA surface expression and integration of the analyte ORF into the genomic acceptor site (production of component B'). The cell lines ACL-321 and ACL-331 are HLA-A * 24:02 or HLA-B *-07:02 analyte HLA surface expression was maintained in a representative monoclonal antibody (Figure 35). Gene characterization was performed on DNA extracted from the selected monoclonal antibodies ACL-321, ACL-327, ACL-331, and ACL-332, demonstrating that their genomes incorporated component C', that this incorporation occurred at the AAVS1 genomic acceptor site, and that component B' was generated (Figure 36). Confirmation of genomic integration was determined by detection of a PCR amplicon of the expected size using primers specific for component C' (Figure 36a). The presence of component B' was confirmed by detection of a PCR amplicon of the expected size using primers designed against the AAVS1 genomic sequence distal to the region encoded by the homology arms and primers unique to the SV40 pA terminator linked to the analyte HLA ORF (Figure 36b). As a result, aAPX HLA-A * 24:02 or HLA-B * Generation of gene-modified ACL-321 and ACL-331 cell lines containing copies of the -07:02 ORF within genomic acceptor site component B' resulted in the finding that the analyte aAPX alone is the major HLA class I member expressed on the cell surface. Thus, this demonstrated the generation of two defined eAPC-p cell lines using a multi-component system.

[0128] Example 5: eAPC-p encoding an HLAII ORF with paired component C' constructed in one step using one integration couple In this example, a method for constructing eAPC-p in one step using one integration couple is described. Here, the genomic acceptor site component B is the native genomic site, and the gene donor vector (component C') comprises a single ORF encoding two aAPX chains. This example utilized eAPC, ACL-128, and component B, all as defined in Example 4. However, component C' was HLA-DRA * linked by a viral self-cleaving peptide element to the HLA-DRB1 *01:01 allele, or HLA-DPB1 * HLA-DPA1 linked by a viral self-cleaving peptide element to the 04:01 allele * contained a single ORF encoding the 01:03 allele (each, Component C' HLA-DRA*01:01 / HLA-DRB1*01:01 and Component C' HLA-DPA1*01:03 / HLA-DPB1*04:01 so called). The viral self-cleaving peptide element, when transcribed, encoded a peptide sequence that resulted in self-cleavage of the synthesized peptide, yielding two polypeptides that defined each HLA chain.

[0129] The method of constructing eAPC-p was as described in Example 4, except that identification of cells that acquired expression of the analyte HLA on the surface was performed by cell surface labeling with pan - anti-HLA-DR, DP, DQ antibodies (Figure 37). The presence of pan - anti-HLA-DR, DP, DQ antibody staining indicated that the analyte HLA ORF encoded by Component C' was integrated into the genome. Individual HLA-DR, DP, DQ positive stained cells were sorted and grown into a monoclonal population of eAPC-p. As described in Example 4, individual monoclonal strains were selected as eAPC-p based on maintained analyte HLA surface expression and integration of the analyte ORF into the genomic acceptor site (generation of Component B'). Cell lines ACL-341 and ACL-350 were representative monoclonals in which surface expression of the analyte HLA of HLA-DRA * 01:01 / HLA-DRB1 * 01:01 or HLA-DPA1 * 01:03 / HLA-DPB1 * 04:01 was maintained (Figure 38). In conclusion, respectively aAPX HLA-DRA * 01:01 / HLA-DRB1 * 01:01 or HLA-DPA1 * 01:03 / HLA-DPB1 *The generation of the genetically modified ACL-341 and ACL-350 cell lines containing a copy of the ORF within genomic acceptor site component B' resulted in the finding that only the analyte aAPX is the major HLA class II member expressed on the cell surface. Thus, this demonstrated the generation of two defined eAPC-p cell lines using a multi-component system.

[0130] Example 6: eAPC-p in which component B is a synthetic construct, constructed in one step using one integration couple In this example, a method for constructing eAPC-p in one step using one integration couple is described. Here, the genomic acceptor site component B is a synthetic construct designed for the RMCE genomic site, and the gene donor vector (component C') contained a single ORF encoding one aAPX. In this example, the genomic integration site (component B) was composed of selected gene elements. Two unique heterospecific recombinase sites, FRT and F3, which flanked an ORF encoding the selectable marker blue fluorescent protein (BFP). An EF1a promoter was encoded 5' of the FRT site and an SV40 polyadenylation signal terminator was encoded 3' of the F3 site. The gene elements of component B were integrated into the cell line ACL-128 by electroporation using the same plasmid as described in Example 2. As described in Example 2, individual monoclonal strains were selected based on maintained BFP expression and genetically characterized to contain a single integration of component B at the desired AAVS1 genomic location (Figure 39a). The resulting eAPC cell line ACL-385 was HLA-ABC null and HLA-DR, DP, DQ null and contained a single copy of the synthetic genomic acceptor site component B designed for RMCE.

[0131] The gene donor vector (Component C) was matched to Component B such that Component C encodes the same heterospecific recombinase sites FRT and F3. The aAPX ORF of interest (optionally encoding a Kozak sequence immediately prior to the start codon) was cloned between the two heterospecific recombinase sites to generate Component C'. In this example, Component C' contained a single ORF encoding one aAPX HLA-A * 02:01 (referred to as Component C' FRT:HLA-A*02:01:F3 ). eAPC-p was generated by RMCE via electroporation of the cell line ACL-385 using a plasmid encoding the Tyr-recombinase Flp and Component C' FRT:HLA-A*02:01:F3 . Four to ten days after electroporation, individual cells that were positive for HLAI surface expression and negative / reduced for the fluorescent protein marker BFP encoded by the Component B selectable marker were sorted. More grown individual monoclonal strains were selected based on maintained HLAI allele expression and loss of BFP fluorescence (indicating that the expected RMCE had occurred). To identify this monoclonal, both phenotypic and genetic tests were performed. First, all monoclonal cell lines were screened for lack of cell surface HLA-ABC expression and BFP fluorescence (Figure 39). Genomic DNA was extracted from the cell lines, e.g., ACL-421 and ACL-422, and the integration of Component C' into Component B resulting in Component B' was confirmed by detection of a PCR product specific to Component B' (Figure 40). In conclusion, the generation of the genetically modified ACL-421 and ACL-422 cell lines each containing a copy of the aAPX HLA-A * 02:01 ORF in the synthetic genomic acceptor site Component B' resulted in the result that only the analyte aAPX is the major HLA class I member expressed on the cell surface. Thus, this demonstrated the generation of two defined eAPC-p cell lines using a multi-component system.

[0132] Example 7: eAPC-pa Constructed in Two Steps Using Two Integration Couples In this example, a method for constructing eAPC-pa in two steps is described. Step 1 Genomic acceptor site Component B was a native genomic site, and the gene donor vector (Component C') comprised a single ORF encoding one aAPX. Step 2 The genomic acceptor site (Component D) was a second native genomic site, and the gene donor vector Component E' comprised a single ORF encoding one analyte antigen molecule (aAM). In this example, eAPC was ACL-128, the genomic acceptor site Component B was the mutant HLA-A allele genomic site (HLA-A null referred to as), and Step 1 in which targeted integration was achieved by HDR was performed. Component C was composed of >500bp sequences homologous to the HLA-A null genomic locus each, and the gene donor vector (Component C) was matched to Component B by encoding the HLA-A null left and right homology arms. Between the HLA-A null left and right homology arms, the plasmid encoded a CMV promoter and an SV40 terminator. The aAPX of interest was cloned between the promoter and the terminator to generate Component C'. In this example, Component C' comprised a single ORF encoding one aAPX HLA-A * 02:01 or HLA-B * -35:01 (referred to as Component C' HLA-A*02:01 and Component C' HLA-B*-35:01 respectively). The integration of Component C' into Component B and the selection of monoclonal eAPC-p cell lines were the same as in Example 4, except that a gRNA targeting the HLA-A null genomic locus was used to promote the HDR integration of Component C' into Component B. Monoclonal eAPC-p ACL-191 and ACL-286 expressed HLA-A * 02:01 or HLA-B * -35:01 on the cell surface (Figure 41a).

[0133] In this example, the genomic acceptor site (Component D) was the native AAVS1 genomic site, and Step 2 of achieving targeted integration by HDR was performed. Component E was matched to Component D by encoding AAVS1 left and right homology arms each composed of a >500 bp sequence homologous to the AAVS1 genomic locus. Between the AAVS1 left and right homology arms, the plasmid encoded a CMV promoter and an SV40 terminator. The aAM of interest was cloned between the promoter and the terminator to generate Component E'. In this example, Component E' contained a single ORF encoding a selection marker GFP linked to the aAM ORF encoding hCMV-pp65 (referred to as Component E' GFP:2A:pp63 ). The viral self-cleaving peptide elment encoded a peptide sequence that, when transcribed, resulted in self-cleavage of the synthesized peptide, yielding two polypeptides, GFP and the intracellular hCMV-pp65 protein. The incorporation of Component E' into Component D was as in Example 4. Individual monoclonal strains ACL-391 and ACL-395 were selected as eAPC-pa based on maintained selection marker GFP expression (Figure 41b). As a conclusion, genetically modified ACL-391 and ACL-395 cell lines were generated that contained copies of the aAPX HLA-A * 02:01 or HLA-B * -35:01 ORF in the genomic acceptor site Component B' and the aAM ORF pp65 in the genomic acceptor site Component D'. These genetic modifications resulted in the aAPX being the only major HLA class I member expressed on the cell surface, and the aAM also being expressed on the cell surface. Therefore, this demonstrated the generation of two defined eAPC-pa cell lines using a multi-component system.

[0134] Example 8: eACP-p constructed in one step, where Component C' encodes a single HLAI ORF In this example, we describe the one-step conversion of eAPC to eAPC-p by a single integration coupling event for incorporating a single HLA I ORF encoding an analyte antigen-presenting complex (aAPX). Here, the eAPC contains two synthetic genomic acceptor site components B and D designed for RMCE-based genomic integration. The generated eAPC-p has one genomic acceptor site (component B') occupied by the HLA I ORF, while the remaining component D is available for additional integration coupling events (Figure 6). This example utilized the eAPC (ACL-402) prepared in Example 3, which contains components B and D. Here, component B contains two unique heterospecific recombinase sites F14 and F15 that flank an ORF encoding the selectable marker red fluorescent protein (RFP). An EF1a promoter was encoded on the 5'-side of the F14 site, and an SV40 polyadenylation signal terminator was encoded on the 3'-side of the F15 site. Component D contains two unique heterospecific recombinase sites FRT and F3 that flank an ORF encoding the selectable marker blue fluorescent protein (BFP). An EF1a promoter was encoded on the 5'-side of the FRT site, and an SV40 polyadenylation signal terminator was encoded on the 3'-side of the F15 site. This example uses a component C gene donor vector that contains heterospecific recombinase sites F14 and F15 and thus matches component B. Two independent components C' were prepared from component C. Here, one vector (V4.H.5) contains a Kozak sequence, start codon, and an aAPX ORF encoding HLA-A * 02:01 between the F14 / F15 sites, and the second vector (V4.H.6) contains a Kozak sequence, start codon, and an aAPX ORF encoding HLA-A * 24:02 between the F14 / F15 sites.

[0135] eAPC (ACL-402) was combined by electroporation independently with a vector encoding the expression of RMCE recombinase enzyme (Flp, V4.1.8) and each component C' of either V4.H.5 or V4.H.6. The cells were cultured for 4 - 10 days. Then, the cells were selected and sorted based on the loss of the selection marker RFP for integration and the acquisition of HLAI on the cell surface. Then, the more grown individual monoclonal strains were characterized, verified, and selected based on the acquisition of HLAI surface expression and the loss of RFP fluorescence (indicating that the expected conversion to B' of component B occurred). The selected eAPC-p monoclonal ACL-900 (V4.H.5, HLA-A * 02:01) and ACL-963 (V4.H.6, HLA-A * 24:02) were negative for RFP compared to the parental ACL-402 cell line and maintained HLAI surface expression (Figure 43a). Furthermore, both monoclonal strains retained the expression of the selection marker for BFP integration, indicating that component D is not coupled to the component B integration coupling event and is isolated. To further characterize the eAPC-p monoclonal, genomic DNA was extracted from the cells, and confirmation of the integration coupling (resulting in component B') between component C' and component B was performed by detection of a PCR product specific for component B' (Figure 43b, Table 5 lists the primers used for genotyping). Primers targeting the region adjacent to the genomic acceptor site (primer ID 8.B.3) and primers targeting the region within the integration coupling event (primer ID 15.H.2) were designed. Amplification occurred only in the case of specific integration, and no products were generated from control recombination (ACL-3) or off-target recombination. In summary, this example demonstrates two specific examples of the conversion of eAPC to eAPC-p using a multi-component system. Here, two different aAPXs are delivered individually (component C'), and after being integrated into a single genomic acceptor site (component B) by the RMCE genomic integration method, a limited library containing two separate eAPC-p is created. Furthermore, a second genomic acceptor site (component D) is isolated and shown to be unaffected by the component B / component C' integration couple.

[0136] Example 9: eAPC-pa constructed in one step from eAPC-p, where component D' encodes a single analyte antigen molecule (aAM) ORF This example describes a method for constructing multiple eAPC-pa in parallel from the parental eAPC-p (described in Example 8). Here, the genomic acceptor site (component D) is targeted for integration by a primed gene donor vector (component E' comprising a single ORF encoding aAM). In this example, the parental eAPC-p strain used was ACL-900 expressing a single aAPX (HLA-A * 02:01) integrated into component B' (described in Example 8). The eAPC-p component D remained open and contained two unique heterospecific recombinase sites FRT and F3 flanking an ORF encoding the selectable marker blue fluorescent protein (BFP). An EF1a promoter was encoded 5' of the FRT site and an SV40 polyadenylation signal terminator was encoded 3' of the F15 site. In this example, the gene donor vector component E was used, which contained two heterospecific recombinase sites F14 and F15 and thus matched component D. In this example, component E was further primed with one aAM ORF of interest selected from HCMVpp28 (V9.E.6), HCMVpp52 (V9.E.7) or HCMVpp65 (V9.E.8) (each encoding a c-myc tag at the C-terminus). Furthermore, each component E' further contained a Kozak sequence and a start codon immediately 5' of the aAM ORF. Thus, a small separate library of component E' containing three vectors was created.

[0137] eAPC-p (ACL-900, Example 8) was independently combined with a vector encoding the expression of each component E' of RMCE recombinase enzyme (Flp, V4.1.8) and V9.E.6, V9.E.7 or V9.E.8 by electroporation. To cause integration coupling, after incubating the cells for 4 - 10 days, individual eAPC-pa were selected, and single cells (monoclonal) were selected based on the decreased signal of the integration selection marker BFP encoded by component D (Figure 44a). Subsequently, the more grown individual monoclonal eAPC-pa, ACL-1219 (pp28), ACL-1227 (pp52) and ACL-1233 (pp65) were characterized, confirmed, and selected based on the loss of BFP expression and the maintained surface expression of HLAI (which indicates that the expected conversion of component D to D' occurred) (aAPX to component B') (Figure 44b). Furthermore, the maintained surface expression of aAPX indicated that component B' was not affected by the integration coupling event between component D and component E' and was isolated from this event. To further characterize the selected eAPC-pa monoclonal, genomic DNA was extracted, and confirmation of the integration coupling (resulting in component D') between component E' and component D was performed by detection of polymerase chain reaction (PCR) amplicon products specific to component D'. Figure 44c shows two monoclonal of each of the three eAPC-pa. Here, for aAM ORF pp28 (0.8 kb), pp52 (1.5 kb) and pp65 (1.9 kb), amplicon products of the expected size were observed, further confirming that the expected integration event occurred. In summary, this example demonstrates three specific examples of the conversion of eAPC-p to eAPC-pa using a multi-component system. Here, three different aAMs were delivered individually (component E'), and after being integrated into a single genomic acceptor site (component D) by the RMCE genomic integration method, a small library of three separate eAPC-pa with three different aAM ORFs was generated. Furthermore, the loaded second genomic acceptor site (component B') was isolated and shown not to be affected by the component D / component E' integration coupling.

[0138] Example 10: Shotgun integration of multiple analyte antigen molecule ORFs into eAPC-p for generating a pooled eAPC-p library in a single step In this example, a method for generating a pooled eAPC-p library by integrating a pool of primed component E vectors (component E') that collectively encode multiple aAM ORFs (HCMVpp28, HCMVpp52, and HCMVpp65) into the parental eAPC-p (described in Example 8) in a single step is described. Here, each cell has a single random analyte antigen ORF from the original vector pool integrated by component D', such that each eAPC-pa expresses a single random aAM, but the pooled library of eAPC-pa represents, as a population, all of the aAM ORFs encoded by the original pooled vector library. This method of generating a pool of eAPC-pa each expressing a single random ORF from the vector pool is called shotgun integration. In this example, the parental eAPC-p strain used was ACL-905, which expresses aAPX (HLA-A * 02:01) on the cell surface (construction of the cell line is described in Example 8), and components D and E' were as described in Example 9. In this example, the individual component E' vectors V9.E.6, V9.E.7, and V9.E.8 from Example 9, each containing an aAM ORF encoding HCMVpp28, HCMVpp52, and HCMVpp65, respectively, were mixed together at a molar ratio of 1:1:1 to create a vector pool. eAPC-p (ACL-905) was combined with the vector pool and a vector encoding the expression of RMCE recombinase enzyme (Flp, V4.1.8) by electroporation. After incubating the cells for 4 - 10 days, the cells were bulk sorted based on the decreased signal of the selection marker BFP for integration encoded by component D (Figure 45a), and a pooled cell population ACL-1050 was generated (Figure 45b).

[0139] To confirm that the eAPC-pa pool ACL-1050 is composed of a mixture of eAPC-pa, each encoding one of HCMV pp28, HCMV pp52, or HCMV pp65 in component D', individual cells were sorted from the polyclonal population into single cells, and 12 cells for gene characterization were randomly selected. Amplification of component D' was performed using primers across each aAM (Table 5, Figure 45c). Figure 45c shows the amplicons generated for the 12 cells along with the control. Here, for all 12 cells, a single amplicon product was observed that matched the expected size for one of aAM ORF pp28 (0.8 kb), pp52 (1.5 kb), and pp65 (1.9 kb). Furthermore, each aAM ORF was identified at least once. This indicates that the eAPC-pa pool is composed of a mixture of eAPC-pa in which each eAPC-pa in the pool has incorporated a single random aAM ORF from the pool of the original three vectors. In conclusion, this example demonstrates the use of a multi-component system for the single-step conversion of eAPC-p into a pooled library of eAPC-pa by combining eAPC-p with a pooled library of three vectors (component E') encoding three different analyte antigen molecules and utilizing an RMCE-based shotgun integration approach. Furthermore, this example proves that each eAPC-pa in the generated eAPC-pa pool has incorporated a single random aAM ORF from the original vector pool by an integration coupling event between component D and component E' and that all three aAM ORFs are represented within the generated pooled eAPC-p library.

[0140] Example 11: Demonstration of two eAPC:T systems for antigen-specific proliferation of primary CD8 cells induced by eAPC-pa This example describes the composition and use of two different eAPC:T systems. Here, the first system consists of eAPC-p, exogenously provided aAM (which gives rise to aAPX:aAM presented by eAPC-p), and analyte primary T cells (analyte TC), and the second system consists of eAPC-pa presenting aAPX:aAM and analyte primary T cells (analyte TC). Using the eAPC:T system, analyte TC bearing a TCR capable of responding to the analyte antigen (aAPX:aAM) was identified and selected by detecting the proliferation and growth of the analyte TC. In this example, the induced proliferation of antigen-specific CD8+ T cells from the CD8+ T cell population was monitored. Here, aAPX is HLA-A * 02:01 (HLA class I), and aAM is the peptide NLVPMVATV. In the system composed of eAPC-p, aAM is provided exogenously, and in the system composed of eAPC-pa, aAM is naturally processed from the incorporated analyte antigen ORF (HCMVpp65). The cell lines used were eAPC-p (ACL-191) and eAPC-pa (ACL-390) described in Examples 8 and 9, respectively. Analyte TC CD8+ T cells were isolated from healthy blood donors known to have CD8+ T cells specific for the NLVPMVATV peptide as described in "Materials and Methods". In the first eAPC:T system, as described in "Materials and Methods", eAPC-p(ACL-191) was pulsed with exogenous NLVPMVATV (SEQ ID NO: 3) peptide at a peptide concentration of 1 μM for 4 hours. The eAPC:T system was then assembled by combining the pulsed eAPC-p cells with the analyte TC (bulk-sorted CD8+ T cells) and co-culturing them under standard conditions. After 9 days of co-culture, the cells were analyzed by specific staining with CMV-A.0201-NLVP tetramer (aAPX:aAM as a soluble reagent) for cells that formed a cooperative complex between the analyte antigen and the analyte TCR, and the proliferation of antigen-specific T cells was detected by flow cytometry. It was compared with an eAPC:T system containing non-pulsed ACL-191 cells (without aAM) or pulsed HLA-null ACL-128 (without aAPX) cells or non-pulsed HLA-null ACL-128 cells (without aAPX:aAM). Significantly increased proliferation of the analyte TC (CD8+ T cells) confirmed by HLA-A * 02:01-NLVP tetramer staining was observed only in the eAPC:T system containing eAPC-p cells pulsed with NLVPMVATV (Fig. 46a).

[0141] The second eAPC:T system was assembled by combining eAPC-pa(ACL-390) cells with the analyte TC (bulk-sorted CD8+ T cells) and co-culturing them under standard conditions (see Materials and Methods). As in the case of using the first system, the co-cultured cells were collected and analyzed by specific staining with CMV-A.0201-NLVP tetramer (aAPX:aAM as a soluble reagent) for cells induced by the analyte antigen and the analyte TCR. Fig. 46b shows pp65 ORF (aAM) and aAPX (HLA-A *Demonstrate the antigen - specific proliferation of primary CD8+ T cells co - cultured with eAPC - pa(ACL - 390) cells that stably express <02:01> and thus present aAPX:aAM. Compare with two other eAPC:T systems including eAPC - p(ACL - 191) cells with and without stable expression of the pp65 ORF and HLA - null ACL - 128 cells. The proliferation of CD8+ T cells specific for aAPX:aAM presented by eAPC - pa was identified by CMV - A.0201 - NLVP tetramer staining only in the eAPC:T system containing eAPC - pa(ACL - 390). In summary, this example demonstrates the use of eAPC - p and eAPC - pa cells in an engineered eAPC - T system that can selectively expand analyte TCs (CD8+ T cells) for the identification and selection of analyte TCs having an analyte TCR that enables T - cell stimulation by the presented analyte antigen (aAPX:aAM). Further, the two eAPC:T systems demonstrate the use of different forms of aAPX:aAM, where in one system, aAM is provided exogenously and in the second system, aAM is provided by processing by natural cellular machinery from the expressed integrated analyte antigen ORF of eAPC - pa.

[0142] Example 12: Demonstration of an eAPC:T system for antigen - specific proliferation of primary CD4 cells induced by eAPC - pa This example describes the engineering and use of an eAPC:T system. Here, the system is composed of eAPC - p, exogenously provided aAM (resulting in aAPX:aAM presented by eAPC - p), and analyte primary T cells (analyte TCs). Using the eAPC:T system, analyte TCs and analyte TCRs having a TCR that enables a response to the analyte antigen (aAPX:aAM) were identified and selected by detecting the proliferation and growth of specific analyte TCs. In this example, a specific aAPX:AM (aAPX is HLA - DRB1 *Induced proliferation of antigen - specific CD4+ T cells from a CD4+ T cell population by 01:01 (HLA class II), where aAM is the peptide PKYVKQNTLKLAT (SEQ ID NO: 1), was provided exogenously. The cell line used was eAPC - p(ACL - 341) constructed in a manner similar to that described in Examples 8 and 9. Analyte TC CD4+ T cells were isolated from healthy blood donors known to have CD4+ T cells specific for the PKYVKQNTLKLAT peptide as described in the "Materials and Methods" section.

[0143] In this example, eAPC - p(ACL - 341) was pulsed for 2 hours with exogenous PKYVKQNTLKLAT (SEQ ID NO: 1) peptide at a peptide concentration of 1 μM as described in the "Materials and Methods" section. The eAPC:T system was constituted by co - culturing the pulsed eAPC - p cells with the analyte TC (bulk - sorted CD4+ T cells) under standard conditions. After 7 days of co - culture, the cells were analyzed. For cells induced by the presented aAPX:aAM, INFL - DRB1 * was analyzed by specific staining with 01:01 - PKYV tetramer (aAPX:aAM as a soluble reagent), and the proliferation of antigen - specific T cells was detected by flow cytometry. It was compared with an eAPC:T system containing non - pulsed ACL - 341 cells (aAPX:CM). A significant proliferation of analyte TC (CD4+ T cells) confirmed by CMV - A.0201 - NLVP tetramer staining to be specific for the PKYVKQNTLKLAT (SEQ ID NO: 1) peptide was observed only in the eAPC:T system containing eAPC - p cells pulsed with PKYVKQNTLKLAT (Figure 47). In conclusion, this example demonstrates the use of HLA class II - based eAPC - p incorporated into an eAPC:T system that can selectively expand analyte TC (CD4+ T cells) for the identification and selection of analyte TC having an analyte TCR that forms a cooperative complex with the presented analyte antigen (aAPX:aAM).

[0144] Example 13: Demonstration of eAPC:T for antigen-specific cytotoxicity induced by eAPC-pa in co-cultured primary CD8 cells This example describes the construction and use of two different eAPC:T systems. Here, the first system consists of eAPC-p, exogenously provided aAM (resulting in aAPX:aAM presented by eAPC-p), and analyte primary T cells (analyte TC), and the second system consists of eAPC-pa presenting aAPX:aAM and analyte primary T cells (analyte TC). The eAPC:T system was used to confirm the specificity of analyte TC for the presented analyte antigen (aAPX:aAM) by detecting the cytotoxic effect of analyte TC on eAPC-p or -pa. In this example, the cytotoxic effect of antigen-specific CD8+ T cells derived from the CD8+ T cell population and forming a cooperative complex between the analyte TCR and aAPX:AM is demonstrated. Here, aAPX is HLA-A * 02:01 (HLA class I), and aAM is the peptide NLVPMVATV (SEQ ID NO: 3). In the system composed of eAPC-p, aAM is provided exogenously, and in the system composed of eAPC-pa, aAM is naturally processed from the incorporated analyte antigen ORF (HCMVpp65). The cell lines used were eAPC-p (ACL-191) and eAPC-pa (ACL-390) described in Examples 8 and 9, respectively. Analyte TC CD8+ T cells were isolated from healthy blood donors known to have CD8+ T cells specific for the NLVPMVATV peptide as described in "Materials and Methods". In the first eAPC:T system, as described in "Materials and Methods", eAPC-p(ACL-191) was pulsed with exogenous NLVPMVATV peptide at a peptide concentration of 1 μM for 2 hours. The eAPC:T system was then assembled by combining the pulsed eAPC-p cells with the analyte TC (bulk-sorted CD8+ T cells) and co-culturing them under standard conditions. The co-cultured cells were analyzed by evaluating the killing of eAPC-p cells by annexin V and PI staining and flow cytometry for the cooperative complex of the analyte antigen and the analyte TCR. It was compared with an eAPC:T system containing pulsed HLA-null ACL-128 cells (without aAPX) or non-pulsed HLA-null ACL-128 cells (without aAPX:aAM). A significant cytotoxic effect by the analyte TC (CD8+ T cells) was confirmed only in the eAPC:T system containing eAPC-p cells pulsed with NLVPMVATV (Figure 48a).

[0145] The second eAPC:T system was assembled by combining eAPC-pa(ACL-390) cells with the analyte TC (bulk-sorted CD8+ T cells) and co-culturing them under standard conditions. As in the case of using the first system, the co-cultured cells were collected and analyzed by evaluating the killing of eAPC-pa cells by annexin V and PI staining and flow cytometry for the cooperative complex of the analyte antigen and the analyte TCR. Figure 48b demonstrates the antigen-specific cytotoxic effect of primary CD8+ T cells co-cultured with eAPC-pa(ACL-390) cells that have stable expression of pp65 ORF (aAM, component D') and aAPX (HLA-A * 02:01, component B') and thus present aAPX:aAM. It was compared with two other eAPC:T systems containing eAPC-p(ACL-191) cells without stable expression of pp65 ORF (without aAM) and HLA-null ACL-128 cells (without aAPX:aAM). The antigen-specific cytotoxic effect of CD8+ T cells specific for aAPX:aAM presented by eAPC-pa was observed only in the eAPC:T system containing eAPC-pa(ACL-390). In conclusion, this example demonstrates the use of eAPC-p and eAPC-pa cells in an engineered eAPC-T system that can selectively induce cytotoxicity by analyte TCs (CD8+ T cells) for the identification and selection of analyte TCs having an analyte TCR that forms a cooperative complex with the presented analyte antigen (aAPX:aAM). Further, the two eAPC:T systems demonstrate the use of different forms of aAPX:aAM, where in one system, aAM is provided exogenously and in the second system, aAM is provided by processing by native cellular machinery from the expressed integrated analyte antigen ORF of eAPC-pa.

[0146] Example 14: Identification of aAM Loaded on eAPC-p by Mass Spectrometry This example describes the use of eAPC-p administered with an exogenous analyte antigen molecule (aAM). Here, the aAPX:aAM complex is then captured by metal affinity chromatography and the aAM load is identified by mass spectrometry. This identifies that aAPX:aAM contains aAM, i.e., HLA-restricted presentation of antigenic peptides. This example uses the eAPC-p cell line of Example 8. Here, eAPC-p has aAPX incorporated into component B' (ACL-900 (HLA-A * 02:01) and ACL-963 (HLA-A * 24:02)). The aAPX ORF also encodes a 6× histidine tag at the C-terminus for capture by metal affinity chromatography. eAPC-p was combined with exogenous aAM and pulsed with a peptide at a concentration of 1 μM for 2 hours. Here, four separate pulses were performed with one of the following aAMs as the peptide: NLVPMVATV (APD-2, SEQ ID NO: 3), NLGPMAAGV (APD-21, SEQ ID NO: 4) or VYALPLKML (APD-11, SEQ ID NO: 5) or no peptide. After the pulse, eAPC-p was harvested and lysed as described in "Materials and Methods", and then aAPX:aAM was captured by metal affinity chromatography. After capture, the peptides (aAM and CM) were isolated from aAPX, washed with acid and filtered. The peptide fraction was then subjected to liquid extraction and removal of the organic phase, followed by solid phase extraction and subjected to mass spectrometry for identification of the peptide fraction.

[0147] Figure 49 shows a table summarizing the mass spectrometry results of different combinations of eAPC-p / aAM pulses. The results show that the peptides NLVPMVATV and VYALPLKML bind to aAPX HLA-A * 02:01 and aAPX HLA-A * 24:01, respectively, to form a complex, while all other combinations of aAPX:aAM do not form a detectable aAPX:aM complex. These results are consistent with the known peptide-HLA binding affinities of the three peptides. In conclusion, this example demonstrates that eAPC-p can be used for the selective binding of aAM and aAPX by capture of aAPX:aAM and for the subsequent release and enrichment of aAM for identification by mass spectrometry. Thus, this demonstrates that eAPC-p can be used to determine the HLA-restricted presentation of analyte antigenic molecules.

[0148] SEQUENCE LISTING <110> Genovie AB <120> An Engineered Multi-component System for Identification and Characterisation of T-cell receptors and T-cell antigens <130> P018243PCT1 <160> 72 <170> BiSSAP 1.3 <210> 1 <223> Analyte Antigenic Molecule <210> 2 <223> Analyte Antigenic Molecule <210> 3 <223> Analyte Antigenic Molecule, APD-2 <210> 4 <223> Analyte Antigenic Molecule, APD-21 <210> 5 <223> Analyte Antigenic Molecule, APD-11 <210> 6 <223> V1.A.4 pcDNA3.1_GFP <210> 7 <223> SpCas9-2A-GFP Vector V1.A.8 <210> 8 <223> pMA-SV40pA vector V1.C.2 <210> 9 <223> HLA-A 02:01 6xHis + Exon2 / 3-HA-L+R vector V1.C.6 <210> 10 <223> HLA-B 35:01 6xHis + Exon2 / 3-HA-L+R vector V1.C.9 <210> 11 <223> AAVS1-S_A24_6xH vector V1.F.8 <210> 12 <223> AAVS1-L_B07_6xH vector V1.F.10 <210> 13 <223> AAVS1-l_GFP_HCMVpp65_WT vector V1.G.10 <210> 14 <223> AAVS1-l_GFP_HCMVpp65 ANET vector V1.G.9 <210> 15 <223> AAVS1-l_GFP_HCMVpp65 AIN vector V1.H.1 <210> 16 <223> AAVS1_DRA_Flag-DRB1_6xHis vector V1.I.5 <210> 17 <223> AAVS1_DPA1_Flag-DPB1_6xHis vector V1.I.7 <210> 18 <223> HLA-A-sg-sp-opti1 vector V2.A.1 <210> 19 <223> HLA-B-sg-sp-3 vector V2.A.7 <210> 20 <223> HLA-C-sg-sp-4 vector V2.B.3 <210> 21 <223> HLA-A-ex2-3_sg-sp-opti_1 vector V2.I.10 <210> 22 <223> HLA-A-ex2-3_sg-sp-opti_2 vector V2.J.1 <210> 23 <223> AAVSI_sg-sp-opti_3 vector V2.J.6 <210> 24 <223> AAVS_Efla-intron_F14_RFPnls_F15 vector V4.B.2 <210> 25 <223> AAVS_Efla-intron_FRT_BFPnls_F3 vector V4.B.3 <210> 26 <223> pMA_FRT_HLA-A*02:01-6xHis_F3 vector V4.D.2 <210> 27 <223> pMA_F14_HLA-A*02:01-6xHis_F15 vector V4.H.5 <210> 28 <223> pMA_F14_HLA-A*24:02-6xHis_F15 vector V4.H.6 <210> 29 <223> pMA_F14_HLA-B*07:02-6xHis_F15 vector V4.H.7 <210> 30 <223> pMA_F14_HLA-B*35:01-6xHis_F15 vector V4.H.8 <210> 31 <223> CMVpro_FLP_Sv40pA_V2 vector V4.1.8 <210> 32 <223> FRT_HCMVpp28-3xMYC_F3 vector V9.E.6 <210> 33 <223> FRT_HCMVpp52-3xMYC_F3 vector V9.E.7 <210> 34 <223> FRT_HCMVpp52-3xMYC_F3 vector V9.E.8 <210> 35 <223> pMA-sv40_OE_F1 primer 1.C.2 <210> 36 <223> pMA-sv40_OE_R1 primer 1.C.3 <210> 37 <223> HLA-A-GT-Rg3 primer 4.A.3 1 <210> 38 <223> HLA-A-GT-Fg2 primer 4.A.4 <210> 39 <223> HLA-B-GT-Fg2 primer 4.A.7 <210> 40 <223> HLA-B-GT-Rg2 primer 4.B.1 <210> 41 <223> HLA-C-GT-Fg2 primer 4.B.5 <210> 42 <223> HLA-A-02_GT_Rg4 primer 4.I.9 <210> 43 <223> HLA-A-Exon3_HA-RE-BglII_F1 primer 6.I.9 <210> 44 <223> HLA-C-04-GT-Rg1 primer 8.A.1 <210> 45 <223> CMV-pA-HLA-Ex3_Probe_F1 primer 8.B.2 <210> 46 <223> CMV-pro_GT_R1 primer 9.C.3 <210> 47 <223> sv40pA_GT_F1 primer 9.C.4 <210> 48 <223> AAVS1_GT_F1 primer 9.C.5 <210> 49 <223> AAVS1_GT_F3 primer 9.C.7 <210> 50 <223> AAVS1_GT_F4 primer 9.C.8 <210> 51 <223> AAVS1_GT_R2 primer 9.C.10 <210> 52 <223> AAVS1_GT_R3 primer 9.D.1 <210> 53 <223> AAVS1_GT_R4 primer 9.D.2 <210> 54 <223> HLA-A-intron4_GT_R1 primer 9.D.6 <210> 55 <223> sv40pA-GT primer 9.D.7 <210> 56 <223> sv40pA-AAVS1-probe-FAM-F1 primer 9.J.2 <210> 57 <223> TRAC_TCRA-ex1_R1 primer 10.A.9 <210> 58 <223> TRAC_TCRA-promoter_F1 primer 10.A.10 <210> 59 <223> TRAC_probe (HEX) primer 10.B.6 <210> 60 <223> Pan-HLA_GT_F1 primer 8.B.3 <210> 61 <223> SV40pA_GT_R1 primer 15.H.2 <210> 62 <223> 3xMyc_OE_R1 primer 10.C.4 <210> 63 <223> CtermCysLink_OE_R1 primer 10.D.1 <210> 64 <223> Ef1a_intron_GT_F2 primer 15.H.4 <210> 65 <223> HCMVpp65_GT_F2ddPCR primer / probe 21.I.1 <210> 66 <223> HCMVpp28_GT_F1 ddPCR primer / probe 21.I.2 <210> 67 <223> HCMVpp52_GT_F1 ddPCR primer / probe 21.I.3 <210> 68 <223> Myc-Tag_GT_R1 ddPCR primer / probe 20.H.10 <210> 69 <223> Linker-Myc_Probe_Fam ddPCR primer / probe 20.H.9 <210> 70 <223> TRAC-TCRA-ex1-F1 ddPCR primer / probe 10.A.9 <210> 71 <223> TRAC-TCRA-ex1-F1 ddPCR primer / probe <210> 72 <223> TRAC-probe (HEX) ddPCR primer / probe

[0149] List of Abbreviations aAPX analyte antigen presentation complex aAM analyte antigenic molecule APC antigen-presenting cell APX antigen presentation complex BFP blue fluorescent protein CAR-T CAR T cell CM payload molecule CRISPR clustered regularly interspaced short palindromic repeats gRNA Cas9 guide RNA

[0150] CAR chimeric antigen receptor CDR complementarity-determining region C region constant region CMV cytomegalovirus DAMPS danger-associated molecular pattern DC dendritic cell DNA deoxyribonucleic acid D region diversity region eAPC genetically engineered antigen-presenting cell eAPC-p genetically engineered antigen-presenting cell presenting analyte antigen presentation complex

[0151] eAPC-pa genetically engineered antigen-presenting cell presenting analyte antigen presentation complex and analyte antigenic molecule eAPC-a genetically engineered antigen-presenting cell expressing analyte antigenic molecule eAPC:T Analyte eAPC is an eAPC:TCR system in which the analyte eAPC is combined with the analyte TCR FACS Fluorescence-activated cell sorting GEM T cell Germline-encoded mycobacterium-reactive T cell GFP Green fluorescent protein HLAI HLA class I HLAII HLA class II HDR Homology-directed recombination HLA Human leukocyte antigen IgSF Immunoglobulin superfamily

[0152] IRES Internal ribosome entry site iNK T cell Invariant natural killer T cell J region Joining region MACS Magnetic-activated cell sorting MAGE Melanoma-associated antigen MAIT Mucosa-associated invariant T NCBP Non-cell-based particle ORF Open reading frame PAMPS Pathogen-associated molecular pattern PCR Polymerase chain reaction

[0153] RMCE Recombinase-mediated cassette exchange RFP Red fluorescent protein DNA Ribonucleic acid SH2 Src homology 2 T cell T lymphocyte TC Cell presenting a TCR or TCR-mimicking affinity reagent TCR T cell receptor TRA TCRα TRB TCRβ TRD TCRδ

[0154] TCRsp TCR surface protein complexed with CD3 TALEN Transcription activator-like effector nuclease TRG TRCγ TAA Tumor-associated antigen V region Variable region β2M β2-microglobulin ZAP-70 70 kDa ζ-chain-associated protein

[0155] Definition Pair of complementary TCR chains: Two TCR chains whose translated proteins can form TCRsp on the surface of TCR-presenting cells. Affinity: The kinetic or equilibrium parameter of the interaction between two or more molecules or proteins Affinity reagent: Any reagent designed to have specific affinity for an analyte. Often used for affinity for HLA-antigen complexes. Allele: Variant form of a given gene. AM: Analyte antigenic molecule. Generally, a protein expressed by cells from genomic DNA and / or specific transgene sequences, but can also be a metabolite. AM is expressed in cells and fragments can then be presented as cargo by APX or by itself on the cell surface. Whether as cargo or not, AM can then be a target for T cell receptor-bearing cells or related affinity reagents. Amplicon: A DNA or RNA fragment that is a source and / or product of artificial amplification using various methods including PCR.

[0156] Analyte: The substance of interest to be identified and / or measured and / or queried in a combinatorial system. Analyte TC: Analyte cells presenting analyte TCR on their surface, which can be primary T cells, recombinant T cells or genetically engineered TCR-presenting cells. Analyte TCR: TCRsp or TCR-mimicking affinity reagent provided in the form of a soluble reagent, immobilized reagent, presented by NCBP or presented on the cell surface. Antigen: Any molecule that can be bound by a TCR, which can bring about a signal transmitted within a T cell and is often presented by an antigen-presenting complex. Analyte antigen: Collectively, any substance that presents an antigen for analytical determination, represented by the eAPC:T cell system. Antibody: An affinity molecule expressed by specialized cells of the immune system called B cells, containing two chains. B cells express a very large and diverse repertoire of antibodies that generally do not bind to self-proteins but can bind to and neutralize pathogens or toxins threatening the host. Natural or artificially genetically engineered antibodies are often used as affinity reagents. APC: Antigen-presenting cell. A cell that bears AM, APX, APX on its cell surface. APX: Antigen-presenting complex. A protein expressed and presented on the cell surface by a nucleated cell from a gene / ORF encoding genomic DNA and / or a specific introduced gene sequence. APX presents a cargo that is a peptide or other metabolite molecule. C region: Constant region. One of the gene segments used in the assembly of the T cell receptor. The c region is a distinct segment that defines general functions in the immune system rather than driving TCR diversity.

[0157] Cargo loading apparatus: A set of cellular proteins that generate cargo molecules from proteins or other presented molecules found in cells and load them onto APX. CDR: Complementary determining region. Short sequences at the antigen-facing ends of TCRs and antibodies that perform most of the target-binding function. Each antibody and TCR contains six CDRs, which are generally the most variable parts of the molecule that enable the detection of a large number of diverse target molecules. CM: Cargo. A peptide or metabolite presented by an antigen-presenting complex, such as HLA I or HLA II. CM can be expressed by a cell natively from genomic DNA, introduced into the culture medium, or expressed from a specifically introduced gene sequence. Copy number: The total number of copies of a defined sequence encoded within the genome of a cell. Cytogenetic: Genetics related to the structure and function of chromosomes, i.e., determination of the cell's karyotype. Cytotoxicity / Cytotoxicity: The process by which T cells release factors that directly and specifically damage target cells. D region: Diversity region. One of the gene segments used in the assembly of the T cell receptor. Each has many different variations of these regions, and thus each individual can arm themselves with T cells having a very large variety of different TCRs. DNA: Deoxyribonucleic acid. The chemical name of the molecule that forms the genetic material encoding genes and proteins. eAPC:TCR system: eTPC:T system in which the analyte eAPC is combined with the analyte TCR so that primary and terminal outputs can be obtained. Endogenous: Substances originating within the cell.

[0158] Genetically engineered cells: Modified cells whose genome has been genetically engineered by genetic modification. Eukaryotic conditional regulatory element: A DNA sequence that can affect the activity of a promoter and can be induced or suppressed under defined conditions Eukaryotic promoter: A DNA sequence encoding an RNA polymerase binding site and a response element. The sequence of the promoter region controls the binding of RNA polymerase and transcription factors, and thus the promoter plays a major role in determining where and when the gene of interest is expressed. Eukaryotic terminator / signal terminator: A DNA sequence recognized by a protein factor that binds to RNA polymerase II and induces the transcription termination process. This also encodes the polyA signal. FACS / Flow cytometry: Fluorescence-activated cell sorting. An analytical technique that can analyze individual cells for the expression of specific cell surface and intracellular markers. Cell sorting, a variant of this technique, allows cells with a defined set of markers to be recovered for further analysis. Family of APX: A set of several similar genes encoding functionally related proteins that make up the antigen presentation complex. Fluorescent (protein) marker: A molecule that has specific quenching and emission characteristics and can be detected by microscopy, FACS, and related techniques. Gene donor vector: A gene-based vector for delivering genetic material to a genomic recipient site.

[0159] Genomic recipient site: A site within the genome for targeted integration of donor genetic material encoded within a gene donor vector. Heterospecific recombinase site: A DNA sequence recognized by a recombinase enzyme to facilitate crossover of two DNA molecules. HLA I: Human leukocyte antigen class I. A gene expressed in all nucleated cells in humans. The expressed HLA I is transported to the cell surface where it presents short fragments of internal proteins, peptides, as cargo to the T cell receptor. Thus, it presents fragments of endogenous proteins that may be indicative of ongoing infection. HLA I can additionally present peptides generated from proteins added to the culture medium, expressed from transgene elements, or taken up by the cell as cargo. The HLA class I genes are polymorphic, meaning that different individuals may have variations in the same gene that lead to variations in presentation. Related to HLA class II. HLA II: Human leukocyte antigen class II. A gene expressed in certain cells (e.g., dendritic cells) that coordinate and assist the adaptive immune response in humans. It is related to HLA class I. HLA class II proteins are transported to the cell surface where they present short fragments of foreign proteins, peptides, as cargo to the T cell receptor. Thus, it presents fragments of endogenous proteins that may be indicative of an ongoing infection. In addition, HLA II can present peptides generated from proteins added to the culture medium, expressed from transgene elements, or taken up by the cell as cargo. The HLA class II genes are polymorphic, meaning that different individuals may have variations in the same gene that lead to variations in presentation.

[0160] Homologous arm: A DNA extension that has nearly the same sequence identity as a complementary homologous arm and thus promotes the exchange of two DNA molecules by homology-directed repair, a cellular process. Immune surveillance: The process by which the immune system detects infections, neoplastic lesions, or other potentially pathogenic changes and is thereby activated. Insulator: A DNA sequence that prevents a gene from being affected by the activation or repression of neighboring genes. Insulators also prevent the spread of heterochromatin from silenced genes to genes that are actively transcribed. Integration: The physical ligation of a DNA sequence into the chromosome of a cell. Integration couple: A pair of an integration vector and a genomic recipient site Internal ribosome entry site (IRES): A DNA sequence that encodes an RNA element that, when transcribed, allows for the initiation of translation in a cap-independent manner. J region: Joining region. One of the gene segments used in the assembly of the T cell receptor. Each individual has multiple different variations of these regions, allowing each individual to be armed with T cells that have a very large variety of different TCRs. Karyotype: The chromosomal constitution of a cell. Kozak sequence: A short sequence required for the efficient initiation of translation

[0161] Major HLA class I: A family of APXs consisting of the genes HLA-A, HLA-B, and HLA-C. Matched: When two components encode gene elements that direct and limit the interaction between complementary components. Meganuclease recognition site: A DNA sequence recognized by an endodeoxyribonuclease commonly called a meganuclease Metabolite: A molecule produced or modified by a cell's metabolic pathways Mobile genetic element: A DNA sequence that permits the integration of DNA having the activity of a transposase enzyme. Monoclonal cell line: A defined group of cells generated from a single progenitor cell by repeated cell replication. Native: A substance that occurs naturally in a cell. Non-coding gene: A protein non-coding DNA sequence that is transcribed into a functional non-coding RNA molecule. ORF: Open reading frame. An extension of genetic material that encodes a translation frame for the synthesis of a protein (polypeptide) by ribosomes. Paracrine: Signaling by a soluble factor that acts directly on neighboring cells.

[0162] PCR: Polymerase chain reaction in which a specific target DNA molecule is amplified exponentially Peptide: A short stretch of amino acids 6 - 30 amino acids in length. Phenotypic analysis: Analysis of the observable characteristics of a cell. Polymorphism: The existence of different forms in individuals of the same species due to the presence of different alleles of the same gene. Polypeptide: A protein consisting of a stretch of peptides that forms a three-dimensional structure. Primary output: eAPC cells, analyte TC cells, NCBP, or other analyte TCR forms that can induce and / or determine the terminal output. Primer: A short DNA sequence that enables specific recognition of a target DNA sequence, for example during PCR. Promoter: A regulatory DNA element for the controlled initiation of gene expression. Selectable marker: A DNA sequence that confers a trait suitable for an artificial selection method. Shotgun integration: A process in which a library of vectors is introduced into a cell population, by which only a single copy of any given vector insert can be integrated into the genome of each single cell. Used when referring to the integration of pooled vectors into a given cell population via an integration couple.

[0163] Splice acceptor site: A DNA sequence at the 3' end of intron AM, APX CM, or an affinity reagent for interaction with a cell having TCRsp on its surface or a TCRsp-based reagent. Splice donor site: A DNA sequence at the 5' end of an intron. Synthetic: A substance artificially produced or introduced into a cell. T cell: A T lymphocyte. A leukocyte that expresses a T cell receptor on its surface. Selected by the immune system to have the ability to recognize infection and malignant diseases and reject grafts from most members of the same species without reacting with self. TCR: T cell receptor. An affinity molecule expressed by a subset of lymphocytes called T lymphocytes. TCR-mimicking affinity reagent: A protein or molecule that can interact with and bind to an analyte antigen in a manner mimicking a natural TCRsp. TCRsp: A complementary TCR chain pair that complexes with CD3 and is expressed as a surface protein, or a complementary TCR chain pair expressed as a protein presented by a protein or NCBP in the form of a soluble reagent, an immobilized reagent. Terminal output: Analyte antigen and TCR sequences in the form of AM, APX, APX:CM, APX:AM, TCRsp, or a TCR-mimicking affinity reagent.

[0164] TRA: The locus encoding the TCRα. One of four different loci encoding genes capable of forming VDJ-recombined TCR chains. The translated TCRα chain protein typically pairs with the translated TCRβ chain protein to form an α / β TCRsp. TRB: The locus encoding the TCRβ. One of four different loci encoding genes capable of forming VDJ-recombined TCR chains. The translated TCRβ chain protein typically pairs with the TCRα chain protein to form an α / β TCRsp. TRD: The locus encoding the TCRδ. One of four different loci encoding genes capable of forming VDJ-recombined TCR chains. The translated TCRδ chain protein typically pairs with the translated TCRγ chain protein to form a γ / δ TCRsp. TRG: The locus encoding the TCRγ. One of four different loci encoding genes capable of forming VDJ-recombined TCR chains. The translated TCRγ chain protein typically pairs with the transcribed TCRδ chain protein to form a γ / δ TCRsp. V region: Variable region. One of the gene segments used in the assembly of the T cell receptor. Each has many different variations of these regions, such that each individual can arm T cells with a very large variety of different TCRs.

[0165] The present invention is further described in the following items: Item 1. A multi-component system in which the first component is a genetically engineered antigen-presenting cell (eAPC) (referred to as component A), and the second component is a gene donor vector (referred to as component C) for delivering one or more ORFs encoding an analyte antigen-presenting complex (aAPX) and / or an analyte antigenic molecule (aAM). 2. Component A lacks the endogenous surface expression of at least one family of a.aAPX and / or aAM, b. containing at least one genomic integration site (referred to as component B) for the integration of at least one ORF encoding at least aAPX and / or aAM The multi-component system according to item 1. 3. Component C matches component B, and component C a. a single ORF encoding at least one aAPX and / or aAM, and / or b. two or more ORFs encoding at least one aAPX and / or aAM is designed to deliver, the ORF can be stably integrated into the B genomic acceptor site, and a and / or b may or may not encode a selection marker for integration, the multi-component system according to item 1 or 2. 4. Comprising a gene donor vector (referred to as component C) for delivering eAPC (referred to as component A) and one or more ORFs encoding aAPX and / or aAM, and component A a. lacks the endogenous surface expression of at least one family of aAPX and / or aAM, b. contains at least one genomic integration site (referred to as component B) for the integration of at least one ORF encoding at least one aAPX and / or aAM, Component C matches component B, and component C c. a single ORF encoding at least one aAPX and / or aAM, or d. two or more ORFs encoding at least one aAPX and / or aAM is designed to deliver, the ORF can be stably integrated into the B genomic acceptor site, and aAPX and / or aAM are expressed, and c and / or d may or may not encode a selection marker for integration, the multi-component system according to any one of items 1 to 3.

[0166] 5. The multi-component system according to any one of items 1 to 4, wherein component A comprises a further component (referred to as component D) which is a genomic integration site for the integration of one or more ORFs encoding at least one aAPX and / or aAM. 6. A further component (referred to as component E) is a gene vector that matches D, and component E a. a single ORF encoding at least one aAPX and / or aAM, or b. two or more ORFs encoding at least one aAPX and / or aAM is designed to deliver, wherein the ORF can be stably integrated into the D genomic acceptor site, and a and / or b may or may not encode a selection marker for integration, such that aAPX and / or aAM are expressed. The multi-component system according to item 5. 7. The multi-component system according to any one of items 1 to 6, wherein one or more additional genomic acceptor sites and matching gene donor vectors are added as additional components of the system. 8. Comprising genomic acceptor site B and / or D, and genomic acceptor site B and / or D a. a synthetic construct designed for recombinase-mediated cassette exchange (RMCE) b. a synthetic construct designed for site-specific homologous recombination c. a native genomic site for site-specific homologous recombination selected from. The multi-component system according to any one of items 1 to 7. 9. The multi-component system according to any one of items 1 to 8, wherein component A expresses a T cell co-stimulatory receptor.

[0167] 10. The multi-component system according to item 9, wherein component A expresses the T cell co-stimulatory receptors CD80 and / or CD83 and / or CD86. 11. When provided with genetic material encoding one or more ORFs encoding at least one or two or more aAPXs such that aAPX is expressed on the cell surface, the multi-component system according to any one of items 1 to 10, wherein component A can load a cargo molecule (CM) (referred to as aAPX:CM). 12. The multi-component system according to item 11, wherein aAPX can load CM by a natural processing and loading mechanism. 13. The multi-component system according to item 11 or 12, wherein aAPX can load aAM as CM (referred to as aAPX:aAM). 14. aAPX may be any of the following a. One or more members of HLA class I b. One or more members of HLA class II c. One or more non-HLA antigen presentation complexes d. Or a combination of a, b and / or c The multi-component system according to any one of items 1 to 13.

[0168] 15. aAM may be a. A polypeptide or a complex of polypeptides provided as an analyte antigen b. A peptide derived from a polypeptide provided as an analyte antigen c. A peptide provided as an analyte antigen d. A metabolite provided as an analyte antigen e. A polypeptide or a complex of polypeptides translated from an analyte antigenic molecule ORF f. A peptide derived from a polypeptide translated from an analyte antigenic molecule ORF g. A peptide derived from a modification of the component A proteome h. A polypeptide derived from a modification of the component A proteome i. A metabolite derived from a modification of the component A metabolome And / or a combination thereof, the multi-component system according to any one of items 1 to 14. 16. Comprising component B and / or D, wherein component B and / or D comprises at least one of the following genetic elements a. Heterospecific recombinase site b. Homology arm c. Eukaryotic promoter d. Eukaryotic conditional regulatory element e. Eukaryotic terminator f. Selection marker g. Splice acceptor site h. Splice donor site i. Non-protein-coding gene j. Insulator k. Mobile genetic element l. Meganuclease recognition site m. Internal ribosome entry site (IRES) n. Viral self-cleaving peptide element o. Kozak consensus sequence The multi-component system according to any one of items 1 to 15.

[0169] 17. Comprising component C and / or E, wherein component C and / or E comprises at least one of the following genetic elements a. Heterospecific recombinase site b. Homology arm c. Eukaryotic promoter d. Eukaryotic conditional regulatory element e. Eukaryotic terminator f. Selection marker g. Selection marker for integration h. Splice acceptor site i. Splice donor site j. Non-protein-coding gene k. Insulator l. Mobile genetic element m. Meganuclease recognition site n. Internal ribosome entry site (IRES) o. Viral self-cleaving peptide element p. Antibiotic resistance cassette q. Bacterial replication origin r. Yeast replication origin s. Cloning site t. Kozak consensus sequence The multi-component system according to any one of items 1 to 16, comprising at least one of the following: 18. Comprising component B and / or D, wherein component B and / or D is for RMCE integration of a single ORF, as follows: Note: a. Eukaryotic promoter b. Pair of heterospecific recombinase sites c. Kozak consensus sequence d. Selection marker e. Eukaryotic terminator The multi-component system according to any one of items 1 to 17, comprising the following:

[0170] 19. Comprising component B and / or D, wherein component B and / or D is for RMCE integration of two or more ORFs, and the following gene elements: a. Eukaryotic promoter b. Pair of heterospecific recombinase sites c. Two or more Kozak consensus sequences d. Selection marker e. Eukaryotic terminator f. Second eukaryotic promoter g. Second selection marker h. Second eukaryotic terminator The multi-component system according to any one of items 1 to 18, comprising the following: 20. Component C and / or E is present and is for RMCE integration of a single ORF, and the following gene elements: a. Pair of heterospecific recombinase sites b. Kozak consensus sequence c. Antibiotic resistance cassette d. Bacterial replication origin e. Cloning site for introduction of a single ORF encoding one or more aAPX and / or aAM and / or selection marker for integration The multi-component system according to any one of items 1 to 19.

[0171] 21. Component C and / or E is present and is for RMCE integration of two or more ORFs, as follows: a. A pair of heterospecific recombinase sites b. Two or more Kozak consensus sequences c. An antibiotic resistance cassette d. A bacterial or yeast replication origin e. A cloning site for introducing two or more ORFs encoding one or more aAPX and / or aAM and / or a selection marker for integration, together with a eukaryotic terminator The multi-component system according to any one of items 1 to 20, comprising the above. 22. The multi-component system according to any one of items 1 to 21, wherein component C and / or E is combined with at least one ORF encoding at least one aAPX and / or aAM to obtain component C' and / or E'. 23. The multi-component system according to item 22, wherein the combination is performed multiple times so as to obtain a library of component C' and / or E'. 24. One or more of component C' and / or E' are combined with component A to incorporate one or more aAPX ORFs encoded by component C' and / or E' into component B and / or D so that eAPC-p expresses aAPX on the cell surface to obtain a cell (referred to as eAPC-p), where component B and / or D become component B' and / or D'. The multi-component system according to item 22 or 23. 25. One or more of component C' and / or E' are combined with component A to incorporate one or more aAM ORFs encoded by component C' and / or E' into component B and / or D so that eAPC-a expresses aAM on the cell surface or intracellularly to obtain a cell (eAPC-a), where component B and / or D become component B' and / or D'. The multi-component system according to item 22 or 23.

[0172] 26. One or more components C' and / or E' are combined with component A to incorporate into component B and / or D one or more aAPX ORFs and / or one or more aAMs encoded by component C' and / or E' so that eAPC-pa expresses aAPX and aAM and / or aAPX:aAM, thereby obtaining cells (eAPC-pa), where component B and / or D become component B' and / or D', the multi-component system according to item 22 or 23. 27. One or more components C' or E' are combined with eAPC-p to incorporate into component B or D one or more aAM ORFs encoded by component C' or E' so that eAPC-pa expresses aAPX and aAM and / or aAPX:aAM, thereby obtaining cells (eAPC-pa), where component B or D become component B' or D', the multi-component system according to item 24. 28. One or more components C' or E' are combined with eAPC-a to incorporate into component B or D one or more aAPX ORFs encoded by component C' or E' so that eAPC-pa expresses aAPX and aAM and / or aAPX:aAM, thereby obtaining cells (eAPC-pa), where component B or D become component B' or D', the multi-component system according to item 25. 29. a. Combining component A with at least one component C' and / or E', where one or more components C' and / or E' encode one or more aAPXs, and combining with an integration factor, and the following: b. Selecting for loss of a genomic acceptor site selection marker c. Selecting for acquisition of surface expression of one or more aAPXs d. Selecting for acquisition of one or more integration selection markers The method for producing eAPC-p as defined in item 24, comprising at least one of the above.

[0173] 30. The method according to item 29, including b, c and d. 31.1 or two or more components C' and / or E' encode a single aAPX in step a of item 29, the method according to item 29 or 30. 32. The method according to item 31, which is performed multiple times to obtain a discrete and defined library of eAPC-p, and each step a of item 29 is performed using a unique aAPX so that a unique eAPC-p is obtained. 33.1 or two or more components C' and / or E' encode a mixed pool of two or more unique aAPXs in step a of item 29 to obtain a library, where the library comprises a mixed population of eAPC-p that each express a single aAPX from the pool used in step a of item 29, the method according to item 29 or 30. 34.a. Combining component A with at least one component C' and / or E', where one or two or more components C' and / or E' encode one or two or more aAMs, and combining with an integration factor, and the following: b. Selecting for loss of a genomic acceptor site selection marker c. Selecting for acquisition of expression of one or two or more aAMs d. Selecting for acquisition of one or two or more integration selection markers The method for producing eAPC-a as defined in item 25, comprising at least one of the above. 35. The method according to item 34, including b and d.

[0174] 36.1 or two or more components C' and / or E' encode a single aAM in step a of item 34, the method according to item 34 or 35. 37. The method according to item 36, which is performed multiple times to obtain a discrete and defined library of eAPC-a, and each step a of item 34 is performed using a unique aAM so that a unique eAPC-a is obtained. 38.1 or two or more components C' and / or E' encode a mixture pool of two or more unique aAMs in step a of item 34 to obtain a library, where the library comprises a mixed population of eAPC-a that each express a single aAM from the pool used in step a of item 34, according to the method of item 34 or 35. 39. a. Combining an eAPC-a with at least one component C' or E', where one or two or more components C' or E' encode one or two or more aAPX ORFs, and combining with an integration factor, and the following: b. Selecting for loss of a genomic acceptor site selection marker c. Selecting for acquisition of surface expression of one or two or more aAPXs d. Selecting for acquisition of one or two or more integration selection markers A method for producing an eAPC-pa as defined in item 28, comprising at least one of the following. 40. The method according to item 39, including b, c, and d. 41. The method according to item 39 or 40, where one or two or more components C' or E' encode a single aAPX in step a of item 39. 42. The method according to item 41, performed multiple times to obtain a library of discrete and defined eAPC-pa, where each step a of item 39 is performed with a unique aAPX such that a unique eAPC-pa is obtained. 43. The method according to item 39 or 40, where one or two or more components C' or E' encode a mixture pool of two or more unique aAPXs in step a of item 39 to obtain a library, where the library comprises a mixed population of eAPC-pa that each express a single aAPX from the pool used in step a of item 39.

[0175] 44. a. Combining an eAPC-p with at least one component C' or E', where one or two or more components C' or E' encode one or two or more aAM ORFs, and combining with an integration factor, and the following: b. Selecting for loss of a genomic acceptor site selection marker c. Selecting for acquisition of expression of one or more aAMs d. Selecting for acquisition of one or more integration selection markers A method for producing an eAPC-pa as defined in item 27, comprising at least one of the following: 45. The method according to item 44, comprising b and d 46. The method according to item 44 or 45, wherein one or more components C' or E' encode a single aAM in step a of item 44 47. The method according to item 46, which is performed multiple times to obtain a library of discrete and defined eAPC-pas, and wherein step a of item 44 is performed with a unique aAM each time so that a unique eAPC-pa is obtained 48. The method according to item 44 or 45, wherein one or more components C' or E' encode a mixed pool of two or more unique aAMs in step a of item 44 for obtaining a library, and wherein the library comprises a mixed population of eAPC-pas in which each eAPC-pa expresses a single aAM from the pool used in step a of item 44 49. a. Combining an EAPC with at least one component C' or E', wherein one or more components C' and / or E' encode one or more aAM ORFs and one or more aAPX ORFs, and combining with an integration factor, and the following: b. Selecting for loss of a genomic acceptor site selection marker c. Selecting for acquisition of expression of one or more aAMs and / or surface expression of one or more aAPXs d. Selecting for acquisition of one or more integration selection markers A method for producing an eAPC-pa as defined in item 26, comprising at least one of the following:

[0176] 50. The method according to item 49, comprising b, c and d 51. The method according to item 49 or 50, wherein one or more components C' and / or E' encode a single aAM and a single aAPX in step a of item 49. 52. The method according to item 51, which is performed multiple times to obtain a library of discrete and defined eAPC-pa, and each step a of item 49 is performed using at least one unique aAM and / or unique aAPX such that a unique eAPC-pa is obtained. 53. The method according to item 49 or 50, wherein one or more components C' and / or E' encode a mixed pool of two or more unique aAMs and / or two or more unique aAPXs in step a of item 49 to obtain a library, and the library comprises a mixed population of eAPC-pa, wherein each eAPC-pa expresses a single aAM and a single aAPX from the pool used in step a of item 49. 54. The following characterization: a. The specificity of the expressed analyte antigen for an analyte affinity reagent, and / or b. The affinity of the expressed analyte antigen for an analyte affinity reagent c. A signal response of an analyte cell (analyte TC) expressing one or more analyte TCRs to the expressed analyte antigen, wherein the analyte antigen is selected from aAPX:aAM and / or aAM and / or aAPX and / or aAPX:CM, and the analyte eAPC is selected from eAPC-p and / or eAPC-a and / or eAPC-pa, and the analyte eAPC is obtained from the multi-component system according to any one of items 1 to 28 for use.

[0177] 55. A method of selecting one or more analyte eAPC from an input library of analyte eAPC or analyte eAPC, to obtain one or more analyte eAPC that bind to one or more analyte TCR, the following a. Combining one or more analyte eAPC with one or more analyte TCR to cause contact between the analyte antigen presented by the analyte eAPC and the analyte TCR b. When formed, measuring the formation of a complex between one or more analytes and one or more analyte TCRs, and / or c. When induced, measuring the signal response of one or more analyte eAPCs induced by complex formation between the analyte antigen and one or more analyte TCRs, and / or d. When induced, measuring the signal response of one or more analyte TCs induced by complex formation between the analyte antigen and one or more analyte TCRs expressed by one or more analyte TCs, and e. Measuring one or more analyte eAPCs that are step b, where the selection is made by positive and / or negative measurement comprising, wherein the analyte antigen is selected from aAPX:aAM and / or aAM and / or aAPX and / or aAPX:CM, the analyte eAPC is selected from eAPC-p and / or eAPC-a and / or eAPC-pa, the analyte TCR is at least one form of a TCR chain pair or a TCR-mimicking affinity reagent presented by the following: a soluble reagent, a non-cell-based particle (NCBP), an immobilized reagent presented by a cell surface (TC), and the cell can be selected from primary T cells and / or recombinant T cells and / or genetically engineered cells, method.

[0178] 56. The method according to item 55, wherein the selection step is performed by single cell sorting and / or cell sorting into a pool. 57. The method according to item 56, wherein the sorting precedes the expansion of the selected single cells. 58. The method according to item 56, wherein the sorting precedes the expansion of the selected cell pool. 59. The method according to any one of items 56 to 58, further comprising the step of sequencing component B' and / or component D' of the selected and / or expanded cells. 60. The sequencing step is as follows: a. Extracting genomic DNA, and / or b. Extracting RNA transcripts of component B' and / or component D', and / or c. Amplifying the DNA and / or RNA transcripts of component B' and / or component D' by PCR and / or RT-PCR The method according to item 59, subsequent to this. 61. The method according to item 59 or 60, wherein the sequencing step is destructive to the cell and the obtained sequencing information is used to produce the analyte eAPC selected in step e of item 55. 62. The selected analyte eAPC is subjected to affinity analysis to determine the affinity of the analyte antigen for the analyte TCR, as follows: a. Labeling the selected analyte eAPC with the analyte TCR at a range of concentrations b. Performing FACS analysis on the labeled analyte eAPC of step a c. Determining the intensity of the fluorescent label of the analyte eAPC over a concentration range of the analyte affinity reagent d. Calculating the affinity of the analyte antigen for the analyte TCR The method according to any one of items 55, 56, 57, 58, 61, further comprising the above.

[0179] 63. The method according to item 62, wherein steps b - c are performed using a labeled reference, and step d calculates the affinity using the ratio of the fluorescent intensity of the analyte affinity reagent to the fluorescent intensity of the reference. 64. The labeled reference is a. Analyte eAPC labeled with an affinity reagent for the analyte antigen b. A cell or particle presenting the labeled reference analyte antigen The method according to item 63, selected from the above. 65. The selected analyte eAPC is subjected to characterization of the signal response, as follows: a. Determining the native signal transduction response, and / or b. Determining the synthetic signal transduction response The method according to any one of items 55, 56, 57, 58, 61, further comprising the above. 66. The induced signal response is as follows compared to the non-induced signal response state: a. Secreted biomolecules b. Secreted chemical substances c. Intracellular biomolecules d. Intracellular chemical substances e. Surface-expressed biomolecules f. Cytotoxic effect of analyte TC on analyte eAPC g. Paracrine effect of analyte TC on analyte eAPC (where the signal response is induced in analyte eAPC and determined by detection of an increase or decrease in any of a - e) h. Amplification of analyte TC i. Immunological synapse formation between analyte TC and analyte eAPC The method according to item 65, determined by detecting one or more increases or decreases of

[0180] 67. A method of selecting one or more analyte TCRs from an input analyte TCR or library of analyte TCRs (where the analyte TCR binds to one or more analyte eAPCs), obtaining the sequences of one or a pair or more pairs of TCR chains encoded by the analyte TCR, and / or obtaining the analyte TCR, comprising: a. Combining one or more analyte eAPCs with one or more analyte TCRs to effect contact between the analyte antigen presented by the analyte eAPC and the one or more analyte TCRs; b. Measuring the formation of a complex between the analyte antigen and the one or more analyte TCRs, if formed; and / or c. Measuring the signal response of one or more analyte TCs induced by complex formation between the analyte antigen and one or more TCRs expressed by the one or more analyte TCs, if induced; and / or d. Measuring the signal response of one or more analyte eAPCs induced by complex formation between the analyte antigen and the one or more analyte TCRs, if induced; and e. Selecting one or more analyte TCRs from procedures b, c, and / or d, where the selection is made by positive and / or negative measurements comprising, wherein the analyte antigen is selected from aAPX:aAM and / or aAM and / or aAPX and / or aAPX:CM, the analyte eAPC is selected from eAPC-p and / or eAPC-a and / or eAPC-pa, the analyte TCR is a TCR chain pair or a TCR-mimicking affinity reagent in at least one form of an immobilized reagent presented by a soluble reagent or a non-cell-based particle (NCBP) or on the cell surface (TC), and the cells can be selected from primary T cells and / or recombinant T cells and / or genetically engineered cells, a method.

[0181] 68. The method according to item 67, wherein the selection step is performed by single cell sorting and / or cell sorting into a pool. 69. The method according to item 68, wherein the sorting precedes the expansion of the selected single cells. 70. The method according to item 68, wherein the sorting precedes the expansion of the selected cell pool. 71. The method according to any one of items 67 to 70, further comprising sequencing the analyte TCR chains of the selected and / or expanded cells. 72. The sequencing step is as follows a. Extracting genomic DNA, and / or b. Extracting analyte TCR chain RNA transcripts, and / or c. Amplifying the DNA and / or RNA transcripts of the analyte TCR chain by PCR and / or RT-PCR, the method according to item 71. 73. The selected analyte TC is characterized for signal response, as follows a. Determining the native signal transduction response, and / or b. Determining the synthetic signal transduction response The method according to any one of items 67, 68, 69, 70, further comprising.

[0182] 74. The induced signal response is as follows compared to the non-induced signal response state: a. Secreted biomolecules b. Secreted chemical substances c. Intracellular biomolecules d. Intracellular chemical substances e. Surface-expressed biomolecules f. Cytotoxic effect of analyte TC on analyte eAPC g. Paracrine effect of analyte TC on analyte eAPC (where the signal response is induced in analyte eAPC and determined by detection of an increase or decrease in any of a - e) h. Amplification of analyte TC i. Immunological synapse between analyte TC and analyte eAPC The method according to item 73, determined by detecting an increase or decrease in one or more of the following: 75. A method for selecting and identifying an aAM load or a CM load, wherein the load is a metabolite and / or peptide loaded on aAPX of analyte eAPC, as follows: a. Isolating aAPX:aAM or aAPX:CM or loaded aM or loaded CM, and b. Identifying the loaded load The method comprising the following.

[0183] 76. The method according to item 75, wherein step b comprises subjecting the isolated aAPX:aAM or aAPX:CM to one or more of the following: a. Mass spectrometry b. Peptide sequencing analysis The method according to item 75, comprising subjecting the isolated aAPX:aAM or aAPX:CM to one or more of the following: 77. A TCR chain pair sequence or a library of TCR chain pair sequences selected by the method defined in items 67 - 74 for use in at least one of the following: a. Diagnosis b. Medicine c. Cosmetics d. Research and development The method according to item 67 - 74, selected for use in at least one of the following: 78. The following: a. Diagnosis b. Medicine c. Cosmetics d. Research and development An antigenic molecule selected by the method defined in items 55 to 66 or 75, 76 and / or an ORF encoding said antigenic molecule or a library thereof for use in at least one of 79. The following: a. Diagnosis b. Medicine c. Cosmetology d. Research and development An antigen-presenting complex loaded with an antigenic molecule selected by the method defined in items 55 to 66 or 75, 76 and / or an ORF encoding said complex or a library thereof for use in at least one of

[0184] 80. The following: a. Diagnosis b. Medicine c. Cosmetology d. Research and development An eAPC or a library of eAPCs selected by the method defined in items 55 to 66 for use in at least one of 81. The following: a. Diagnosis b. Medicine c. Cosmetology d. Research and development A cell or a library thereof that expresses a TCR selected by the method defined in items 67 to 74 with CD3 complexed on the cell surface for use in at least one of 82. The following: a. Diagnosis b. Medicine c. Cosmetology d. Research and development The multi-component system according to any one of items 1 to 28 for use in at least one of

[0185] 83. The following: a. Diagnosis b. Medicine c. Cosmetology d. Research and development A TCR-mimicking affinity reagent sequence or a library of TCR-mimicking affinity reagent sequences selected by the method defined in items 67-74 for use in at least one of 84. The following a. Diagnosis b. Medicine c. Cosmetics An NCBP having a TCR pair or a TCR-mimicking affinity reagent selected by the method defined in items 67-74 or a library of NCBPs having a TCR pair or a TCR-mimicking affinity reagent for use in at least one of

[0186] SEQUENCE LISTING <110> Genovie AB <120> An Engineered Multi-component System for Identification and Characterisation of T-cell receptors and T-cell antigens <130> P018243PCT1 <160> 72 <170> BiSSAP 1.3 <210> 1 <211> 13 <212> PRT <213> Homo sapiens <220> <223> Analyte Antigenic Molecule <400> 1 Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr 1 5 10 <210> 2 <211> 4 <212> PRT <213> Homo sapiens <220> <223> Analyte Antigenic Molecule <400> 2 Pro Lys Tyr Val 1 <210> 3 <211> 17 <212> PRT <213> Homo sapiens <220> <223> Analyte Antigenic Molecule, APD-2 <400> 3 Asn Leu Val Pro Met Val Ala Thr Asn Leu Val Pro Met Val Ala Thr 1 5 10 15 Val <210> 4 <211> 9 <212> PRT <213> Homo sapiens <220> <223> Analyte Antigenic Molecule, APD-21 <400> 4 Asn Leu Gly Pro Met Ala Ala Gly Val 1 5 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <220> <223> Analyte Antigenic Molecule, APD-11 <400> 5 Val Tyr Ala Leu Pro Leu Lys Met Leu 1 5 <210> 6 <211> 6071 <212> DNA <213> Artificial Sequence <220> <223> V1.A.4 pcDNA3.1_GFP <400> 6 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaactt aagcttggta ccgccaccat ggaatccgat gagtctggcc tgcccgccat 960 ggaaatcgag tgcagaatca ccggcaccct gaacggcgtg gaatttgagc tcgtgggcgg 1020 aggcgagggc acacctgaac agggcagaat gaccaacaag atgaagtcca ccaagggggc 1080 cctgaccttc agcccctacc tgctgtctca cgtgatgggc tacggcttct accacttcgg 1140 cacctacccc agcggctacg agaacccttt cctgcacgcc atcaacaacg gcggctacac 1200 caacacccgg atcgagaagt acgaggacgg cggcgtgctg cacgtgtcct tcagctacag 1260 atacgaggcc ggcagagtga tcggcgactt caaagtgatg ggcaccggat tccccgagga 1320 cagcgtgatc ttcaccgaca agatcatccg gtccaacgcc accgtggaac atctgcaccc 1380 catgggcgac aacgacctgg acggcagctt caccagaacc ttctccctgc gggatggcgg 1440 ctactacagc agcgtggtgg acagccacat gcacttcaag agcgccatcc accccagcat 1500 cctccagaac ggcggaccca tgttcgcctt cagacgggtg gaagaggacc acagcaacac 1560 cgagctgggc atcgtggaat accagcacgc cttcaagacc cccgatgccg atgccggcga 1620 ggaatgagtc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc 1680 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 1740 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 1800 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 1860 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg 1920 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 1980 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 2040 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 2100 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 2160 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 2220 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 2280 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 2340 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt 2400 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 2460 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 2520 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 2580 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 2640 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 2700 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca 2760 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct 2820 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc 2880 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc 2940 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc 3000 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg 3060 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc 3120 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac 3180 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat 3240 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc 3300 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg 3360 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg 3420 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc 3480 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc 3540 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg 3600 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa 3660 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 3720 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 3780 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 3840 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 3900 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 3960 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 4020 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 4080 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 4140 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4200 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4260 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4320 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4380 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 4440 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 4500 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 4560 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 4620 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 4620 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 4680 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 4680 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 4740 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 4740 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 4800 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 4800 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggttttt 4860 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggttttt 4860 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 4920 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 4920 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 4980 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 4980 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 5040 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 5040 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 5100 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 5100 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 5160 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 5160 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 5220 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 5280 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 5340 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 5400 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 5460 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 5520 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 5580 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 5640 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 5700 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 5760 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 5820 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 5880 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 5940 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 6000 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 6060 cacctgacgt c 6071 <210> 7 <211> 10428 <212> DNA <213> Artificial Sequence <220> <223> SpCas9-2A-GFP Vector V1.A.8 <400> 7 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaactt aagcttggta ccgccaccat ggactataag gaccacgacg gagactacaa 960 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa 1020 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg 1080 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt 1140 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct 1200 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata 1260 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa 1320 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa 1380 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta 1440 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg 1500 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg 1560 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta 1620 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct 1680 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga 1740 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt 1800 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga 1860 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc 1920 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat 1980 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct 2040 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt 2100 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt 2160 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa 2220 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca 2280 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt 2340 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt 2400 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac 2460 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat 2520 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag 2580 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag 2580 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga 2640 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga 2640 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct 2700 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct 2700 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat 2760 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat 2760 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg 2820 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg 2820 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa 2880 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa 2880 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat 2940 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat 2940 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa 3000 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa 3000 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga 3060 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga 3060 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa 3120 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa 3120 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca 3180 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc 3240 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg 3300 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa 3360 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg 3420 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta 3480 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg 3540 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat 3600 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc 3660 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat 3720 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga 3780 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc 3840 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga 3900 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt 3960 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt 4020 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga 4080 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc 4140 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct 4200 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat 4260 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt 4320 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc 4380 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg 4440 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg 4500 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag 4560 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa 4620 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag 4680 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata 4740 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa 4800 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca 4860 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc 4920 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct 4980 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga 5040 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat 5100 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc 5160 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggaa ttcggcagtg gagagggcag 5220 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccagtga gcaagggcga 5280 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 5340 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa 5400 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac 5460 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa 5520 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa 5580 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 5640 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 5700 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt 5760 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 5820 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 5880 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 5940 cgccgccggg atcactctcg gcatggacga gctgtacaag gaattctaac gctagagggc 6000 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 6060 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 6120 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 6180 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg 6240 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg 6300 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 6360 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 6420 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 6480 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat 6540 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 6600 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag 6660 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 6720 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc 6780 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc 6840 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt 6900 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc 6960 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct 7020 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg 7080 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat 7140 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta 7200 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca 7260 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga 7320 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga 7380 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct 7440 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg 7500 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga 7560 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca 7620 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga 7680 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg 7740 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc 7800 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt 7860 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga 7920 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca 7980 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc 8040 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac 8100 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc 8160 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta 8220 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag 8280 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 8340 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 8400 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 8460 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 8520 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 8580 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 8640 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 8700 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 8760 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 8820 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 8880 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 8940 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 9000 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 9060 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca 9120 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 9180 tgatccggca aacaaaccac cgctggtagc ggtttttttg tttgcaagca gcagattacg 9240 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 9300 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 9360 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 9420 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 9480 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 9540 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 9600 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 9660 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 9720 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 9780 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 9840 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 9900 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 9960 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 10020 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 10080 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 10140 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 10200 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 10260 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 10320 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 10380 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc 10428 <210> 8 <211> 2508 <212> DNA <213> Artificial Sequence <220> <223> pMA-SV40pA vector V1.C.2 <400> 8 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca 360 aggccgcatg aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa 420 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 480 tgtccaaact catcaatgta tcttatcatg tctggatctg cggatccaat ctcgagctgg 540 gcctcatggg ccttccgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 600 cattaacatg gtcatagctg tttccttgcg tattgggcgc tctccgcttc ctcgctcact 660 gactcgctgc gctcggtcgt tcgggtaaag cctggggtgc ctaatgagca aaaggccagc 720 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 780 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 840 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 900 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 960 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 1020 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 1080 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 1140 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 1200 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 1260 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 1320 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 1380 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 1440 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 1500 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 1560 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 1620 agggcttacc atctggcccc agtgctgcaa tgataccgcg agaaccacgc tcaccggctc 1680 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 1740 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 1800 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 1860 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 1920 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 1980 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 2040 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 2100 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 2160 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 2220 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 2280 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 2340 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 2400 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 2460 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac 2508 <210> 9 <211> 4341 <212> DNA <213> Artificial Sequence <220> <223> HLA-A 02:01 6xHis + Exon2 / 3-HA-L+R vector V1.C.6 <400> 9 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca 360 aggccgcatg aattcgctac cggtatagta atcaattacg gggtcattag ttcatagccc 420 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 480 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 540 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 600 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 660 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 720 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 780 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 840 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 900 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca 960 gatcaggtac catggccgtc atggcgcccc gaaccctcgt cctgctactc tcgggggctc 1020 tggccctgac ccagacctgg gcgggctctc actccatgag gtatttcttc acatccgtgt 1080 ctcggccagg acgcggagag ccacgcttca tcgcagtggg ctacgtggac gacacgcagt 1140 tcgtgcggtt cgacagcgac gccgcgagcc agaggatgga gccgcgggcg ccgtggatag 1200 agcaggaggg tccggagtat tgggacgggg agacacggaa agtgaaggcc cactcacaga 1260 ctcaccgagt ggacctgggg accctgcgcg gctactacaa ccagagcgag gccggttctc 1320 acaccgtcca gaggatgtat ggctgcgacg tggggtcgga ctggcgcttc ctccgcggat 1380 accaccagta cgcctacgac ggcaaggatt acatcgccct gaaagaggac ctgcgctctt 1440 ggaccgcggc ggacatggca gctcagacca ccaagcacaa gtgggaggcg gcccatgtgg 1500 cggagcagtt gagagcctac ctggagggca cgtgcgtgga gtggctccgc agatacctgg 1560 agaacgggaa ggagacgctg cagcgcacgg acgcccccaa aacgcatatg actcaccacg 1620 ctgtctctga ccatgaagcc accctgaggt gctgggccct gagcttctac cctgcggaga 1680 tcacactgac ctggcagcgg gatggggagg accagaccca ggacacggag ctcgtggaga 1740 ccaggcctgc aggggatgga accttccaga agtgggcggc tgtggtggtg ccttctggac 1800 aggagcagag atacacctgc catgtgcagc atgagggttt gcccaagccc ctcaccctga 1860 gatgggagcc gtcttcccag cccaccatcc ccatcgtggg catcattgct ggcctggttc 1920 tctttggagc tgtgatcact ggagctgtgg tcgctgctgt gatgtggagg aggaagagct 1980 cagatagaaa aggagggagc tactctcagg ctgcaagcag tgacagtgcc cagggctctg 2040 atgtgtctct cacagcttgt aaagtgcccg ggcatcatca ccatcaccac tgactatagt 2100 cgtctagacc tgatcataat caagccatat cacatctgta gaggtttact tgctttaaaa 2160 aacctccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact 2220 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 2280 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 2340 atgtctggat ctgcggatcc aatctcgagc tgggcctcat gggccttccg ctcactgccc 2400 gctttccagt cgggaaacct gtcgtgccag ctgcattaac atggtcatag ctgtttcctt 2460 gcgtattggg cgctctccgc ttcctcgctc actgactcgc tgcgctcggt cgttcgggta 2520 aagcctgggg tgcctaatga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 2580 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 2640 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 2700 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 2760 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 2820 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 2880 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 2940 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 3000 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 3060 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 3120 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 3180 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 3240 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 3300 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 3360 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 3420 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 3480 caatgatacc gcgagaacca cgctcaccgg ctccagattt atcagcaata aaccagccag 3540 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 3600 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 3660 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 3720 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 3780 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 3840 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 3900 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 3960 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 4020 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 4080 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 4140 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 4200 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 4260 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 4320 catttccccg aaaagtgcca c 4341 <210> 10 <211> 4332 <212> DNA <213> Artificial Sequence <220> <223> HLA-B 35:01 6xHis + Exon2 / 3-HA-L+R vector V1.C.9 <400> 10 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca 360 aggccgcatg aattcgctac cggtatagta atcaattacg gggtcattag ttcatagccc 420 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 480 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 540 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 600 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 660 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 720 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 780 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 840 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 900 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca 960 gatcaggtac catgcgggtc acggcgcccc gaaccgtcct cctgctgctc tggggggcag 1020 tggccctgac cgagacctgg gccggctccc actccatgag gtatttctac accgccatgt 1080 cccggccagg acgcggagag ccacgcttca tcgcagtggg ctacgtggac gacacccagt 1140 tcgtgaggtt cgacagcgac gccgcgagtc cgaggacgga gcctcgggcg ccatggatag 1200 agcaggaggg gccggagtat tgggaccgga acacacagat cttcaagacc aacacacaga 1260 cttaccgaga gagcctgcgg aacctgcgcg gctactacaa ccagagcgag gccgggtctc 1320 acatcatcca gaggatgtat ggctgcgacc tggggcccga cgggcgcctc ctccgcgggc 1380 atgaccagtc cgcctacgac ggcaaggatt acatcgccct gaacgaggac ctgagctcct 1440 ggaccgcggc ggacaccgcg gctcagatca cccagcgcaa gtgggaggcg gcccgtgtgg 1500 cggagcagct gagagcctac ctggagggcc tgtgcgtgga gtggctccgc agatacctgg 1560 agaacgggaa ggagactctt cagcgcgcag atcctccaaa gacacacgtg acccaccacc 1620 ccgtctctga ccatgaggcc accctgaggt gctgggccct gggcttctac cctgcggaga 1680 tcacactgac ctggcagcgg gatggcgagg accaaactca ggacactgag cttgtggaga 1740 tcacactgac ctggcagcgg gatggcgagg accaaactca ggacactgag cttgtggaga 1740 ccagaccagc aggagataga accttccaga agtgggcagc tgtggtggtg ccttctggag 1800 ccagaccagc aggagataga accttccaga agtgggcagc tgtggtggtg ccttctggag 1800 aagagcagag atacacatgc catgtacagc atgaggggct gccgaagccc ctcaccctga 1860 aagagcagag atacacatgc catgtacagc atgaggggct gccgaagccc ctcaccctga 1860 gatgggagcc atcttcccag tccaccatcc ccatcgtggg cattgttgct ggcctggctg 1920 gatgggagcc atcttcccag tccaccatcc ccatcgtggg cattgttgct ggcctggctg 1920 tcctagcagt tgtggtcatc ggagctgtgg tcgctactgt gatgtgtagg aggaagagct 1980 tcctagcagt tgtggtcatc ggagctgtgg tcgctactgt gatgtgtagg aggaagagct 1980 caggtggaaa aggagggagc tactctcagg ctgcgtccag cgacagtgcc cagggctctg 2040 caggtggaaa aggagggagc tactctcagg ctgcgtccag cgacagtgcc cagggctctg 2040 atgtgtctct cacagctccc gggcatcatc accatcacca ctgactatag tcgtctagac 2100 atgtgtctct cacagctccc gggcatcatc accatcacca ctgactatag tcgtctagac 2100 ctgatcataa tcaagccata tcacatctgt agaggtttac ttgctttaaa aaacctccac 2160 ctgatcataa tcaagccata tcacatctgt agaggtttac ttgctttaaa aaacctccac 2160 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg 2220 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg 2220 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 2280 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 2280 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga 2340 tctgcggatc caatctcgag ctgggcctca tgggccttcc gctcactgcc cgctttccag 2400 tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct tgcgtattgg 2460 gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt aaagcctggg 2520 gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 2580 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 2640 ggtggcgaaa cccgacagga ctataaagat accaggc...

Claims

1. A multi-component system in which a first component is a genetically engineered antigen presenting cell (eAPC) (referred to as component A) and a second component is a gene donor vector for delivery of one or more ORFs encoding an analyte antigen presenting complex (aAPX) and / or an analyte antigenic molecule (aAM) (referred to as component C), wherein component A is a. lacking endogenous surface expression of at least one family of aAPXs and / or aAMs; b. containing at least two genomic acceptor sites (designated components B and D) for the integration of at least one ORF, each encoding at least one aAPX and / or aAM; Component C matches component B, c. a single ORF encoding at least one aAPX and / or aAM; or d. Two or more ORFs encoding at least one aAPX and / or aAM It is designed to deliver A multi-component system in which components B and D are synthetic constructs designed for recombinase-mediated exchange (RMCE), and an analyte aAPX and / or analyte aAM can be expressed by component A and identified based on the reactivity of a TCR or other affinity reactant (or analyte TC) to the APX and / or aAM expressed by component A.

2. 2. The multicomponent system of claim 1, wherein c and / or d code for a selection marker for integration such that the ORF can be stably integrated into component B and aAPX and / or aAM are expressed.

3. A further component (called E) is a gene vector that matches D, and component E a. a single ORF encoding at least one aAPX and / or aAM; or b. Two or more ORFs encoding at least one aAPX and / or aAM 3. The multicomponent system according to claim 1 or 2, which is designed to deliver

4. 4. The multicomponent system of claim 3, wherein the ORF can be stably integrated into component D, and a and / or b optionally encode an integrated selectable marker such that aAPX and / or aAM are expressed.

5. A multicomponent system according to any one of claims 1 to 4, wherein one or more additional genomic acceptor sites and matching gene donor vectors are added as additional components.

6. aAPX is: a. One or more members of HLA class I b. One or more members of HLA class II c. one or more non-HLA antigen-presenting complexes, or d. Any combination of a, b, and c The multi-component system according to any one of claims 1 to 5,

7. aAM is below: a. A polypeptide or a complex of polypeptides that serves as the analyte antigen b. A peptide derived from a polypeptide provided as an analyte antigen c. Peptides serving as analyte antigens d. Metabolites serving as analyte antigens e. A polypeptide or a complex of polypeptides translated from the analyte antigenic molecule ORF. f. Peptides derived from a polypeptide translated from the analyte antigenic molecule ORF g. Peptides derived from alterations of the component A proteome h. Polypeptides resulting from alterations of the component A proteome i. Metabolites resulting from alterations of the Component A metabolome and / or combinations thereof The multicomponent system according to any one of claims 1 to 6, selected from:

8. Components B and / or D are the following genetic elements: a. Heterospecific recombinase site b. homology arm c. Eukaryotic promoters d. Eukaryotic conditional regulatory elements e. Eukaryotic terminators f. Selection Marker g. splice acceptor site h. splice donor site i. Non-protein coding genes j. Insulator k. Mobile Genetic Elements l. Meganuclease recognition site m. Internal ribosome entry site (IRES) n. Viral self-cleaving peptide elements o. Kozak consensus sequence A multi-component system according to any one of claims 1 to 7, comprising at least one of:

9. Components B and / or D are for RMCE integration of a single ORF, comprising: a. Eukaryotic promoters b. A pair of heterospecific recombinase sites c. Kozak consensus sequence d. Selection Marker e. Eukaryotic terminators The multi-component system according to any one of claims 1 to 8, comprising:

10. Component E is present and components C and / or E are for RMCE integration of a single ORF, comprising the following genetic elements: a. A pair of heterospecific recombinase sites b. Kozak consensus sequence c. Antibiotic Resistance Cassette d. bacterial origin of replication e. A cloning site for the introduction of a single ORF encoding one or more aAPX and / or aAM and / or a selection marker for integration. The multi-component system according to any one of claims 3 to 9, comprising:

11. A multicomponent system according to any one of claims 3 to 10, wherein components C and / or E comprise at least one ORF encoding at least one aAPX and / or aAM.

12. A multi-component system as described in claim 11, wherein two or more components C each contain an ORF encoding a different aAPX and / or a different aAM, and / or two or more components E each contain an ORF encoding a different aAPX and / or aAM, such that two or more components C and / or two or more components E are capable of delivering multiple ORFs encoding a library of aAPXs and / or aAMs to components B and / or D.

13. A multicomponent system as described in claim 11 or 12, wherein component C contains one or more aAPX ORFs for obtaining cells (referred to as eAPC-p) expressing aAPX on the cell surface.

14. A multicomponent system as described in claim 11 or 12, wherein component C contains one or more aAM ORFs for obtaining cells (referred to as eAPC-a) expressing aAM on the cell surface or intracellularly.

15. A multicomponent system as described in claim 11 or 12, wherein components C and E contain one or more aAPX ORFs and one or more aAM ORFs to obtain cells expressing aAPX and aAM and / or aAPX:aAM (referred to as eAPC-pa).

16. a. combining component A with one or more components C, in combination with integration factors, wherein the one or more components C contain one or more ORFs encoding one or more aAPXs; and b. Selecting for loss of the genomic acceptor site selection marker c. Selecting for gain of surface expression of one or more aAPXs. d. Selecting for the acquisition of one or more integrated selectable markers. At least one of A method for producing eAPC-p as defined in claim 13, comprising:

17. 17. The method of claim 16 comprising b, c and d.

18. 18. The method of claim 16 or 17, wherein one or more components C comprise an ORF encoding a single aAPX.

19. The method of claim 18, wherein step a is performed multiple times to obtain a library of discrete and defined eAPC-p, and each time step a is performed using a different aAPX so as to obtain a different eAPC-p.

20. The method of claim 16 or 17, wherein the one or more components C comprise two or more ORFs encoding two or more distinct aAPXs in step a to obtain a library, wherein the library comprises a mixed population of eAPC-p, each eAPC-p expressing a single aAPX.

21. a. combining component A with one or more components C, in combination with integration factors, wherein the one or more components C contain one or more ORFs encoding one or more aAMs; and b. Selecting for loss of the genomic acceptor site selection marker c. Selecting for gain of surface expression of one or more aAPXs. d. Selecting for the acquisition of one or more integrated selectable markers. A method for producing eAPC-a as defined in claim 14, comprising at least one of the following:

22. 22. The method of claim 21 comprising b and d.

23. 23. The method of claim 21 or 22, wherein the one or more components C comprise an ORF encoding a single aAM in step a.

24. The method of claim 23, wherein step a is performed multiple times to obtain a library of discrete and defined eAPC-a, and each time step a is performed using a different aAM so as to obtain a different eAPC-a.

25. The method of claim 21 or 22, wherein one or more components C comprise two or more ORFs encoding two or more distinct aAMs in step a to obtain a library, wherein the library comprises a mixed population of eAPC-a, each eAPC-a expressing a single aAM.

26. a. combining component A with one or more of components C and E, and combining with integration factors, wherein one or more of components C and E contain two or more ORFs encoding one or more aAM ORFs and one or more aAPX ORFs; b. Selecting for loss of the genomic acceptor site selection marker c. Selecting for expression of one or more aAMs and / or gain of surface expression of one or more aAPXs. d. Selecting for the acquisition of one or more integrated selectable markers. A method for producing eAPC-pa as defined in claim 15, comprising:

27. 27. The method of claim 26 comprising b, c and d.

28. 28. The method of claim 26 or 27, wherein one or more of components C and E comprise one or more ORFs encoding a single aAM and a single aAPX in step a.

29. The method according to claim 28, wherein step a is performed multiple times to obtain a library of discrete and defined eAPC-pas, and each time step a is performed using at least one different aAM and / or different aAPX so as to obtain different eAPC-pas.

30. The method of claim 26 or 27, wherein one or more components C and E comprise one or more ORFs encoding two or more distinct aAMs and / or two or more distinct aAPXs in step a to obtain a library, wherein the library comprises a mixed population of eAPC-pas, each eAPC-pa expressing a single aAM and a single aAPX from the pool used in step a.

31. Use of a multicomponent system according to any one of claims 1 to 15 for the production of one or more analyte eAPCs selected from eAPC-p and / or eAPCa and / or eAPC-pa.

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