Artificial antigen presenting cell line

Abstract

The present invention provides an artificial antigen presenting cell line.

Description

[0001] Artificial Antigen presenting cell line

[0002] The present invention provides an artificial antigen presenting cell line (aAPC) and its use in MHC -associated peptide proteomics sequencing assays.

[0003] A critical step in the immunogenicity cascade is the ability of dendritic cells (DCs) to present therapeutic monoclonal antibody (mAb)-derived peptides via major histocompatibility complex (MHC) class II receptors to naive CD4+ T cells. Recognition and binding of the MHC -II receptor-peptide complexes to the T cell receptor (TCR) leads to the activation and proliferation of CD4+ T cells. In turn, T cell-dependent events are initiated, such as the downstream activation and proliferation of B cells. The B cells specific to the therapeutic mAb differentiate into either memory cells or antidrug antibody (ADA)-secreting plasma cells.

[0004] MHC -associated peptide proteomics (MAPPs) is a technique used to identify peptides presented by Major Histocompatibility Complex (MHC) molecules on the surface of cells. The current MAPPs methodology faces challenges due to the inherent biological variability for MHC haplotypes of the donor material and the labor-intensive process of dendritic cell (DC) generation. Additionally, the input material is expensive and requires continuous replenishment.

[0005] Applying MAPPs early in the design of large molecule therapeutics provides a link between in silico predictive assessments and in vitro data, but variability and lengthy turn-around times can hinder timely decision-making.

[0006] There is a need for artificial antigen presenting cells (aAPC) which can be used in MAPPs.

[0007] Summary of the invention

[0008] In a first aspect, the present invention provides a HLA deficient B cell line which has been modified to express at least exogenous proteins selected from the group consisting of CD64, CD74, HLA-DM and at least one protein selected from the group consisting of HLA-DR, HLA-DP and HLA-DQ.

[0009] In an embodiment of the present invention, HLA deficient B cell line is a K562 cell line.

[0010] In an embodiment of the present invention, the expressed proteins are human proteins.

[0011] In an embodiment of the present invention, the HLA deficient B cell is modified by lentiviral transduction to introduce nucleic acids encoding the exogenous proteins.

[0012] In an embodiment of the present invention, the lentiviral transduction comprises three different lentiviral vectors.

[0013] In an embodiment of the present invention, the lentiviral transduction comprises more than one transduction which are performed sequentially.

[0014] In an embodiment of the present invention, the lentiviral transduction comprises three transductions.

[0015] In an embodiment of the present invention, the first lentiviral transduction introduces the nucleic acid encoding the CD 64 protein.

[0016] In an embodiment of the present invention, the second lentiviral transduction introduces the nucleic acids encoding the HLA-DM and CD74 proteins.

[0017] In an embodiment of the present invention, the third lentiviral transduction introduces the nucleic acid encoding the HLA-DR, HLA-DP or HLA-DQ protein.In a further embodiment, the present invention provides the use of the HLA deficient B cell line of the present as an antigen presenting cell (APC).

[0018] In a further embodiment, the present invention provides the use of the HLA deficient B cell line of the present invention in an MHC -associated peptide proteomics sequencing assay.

[0019] In a second aspect, the present invention provides a method for the generation of an artificial antigen presenting cell line (APC) which expresses at least exogenous proteins selected from the group consisting of CD64, CD74, HLA-DM and at least one protein selected from the group consisting of HLA-DR, HLA-DP and HLA-DQ, the method comprising the following steps:

[0020] a) Transducing an HLA deficient B cell line with a lentiviral vector comprising a nucleic acid encoding a CD64 protein,

[0021] b) Transducing the cell of step a) with a lentiviral vector comprising nucleic acids encoding the HLA-DM and CD74 proteins and

[0022] c) Transducing the cell of step b) with a lentiviral vector comprising nucleic acid encoding the HLA-DR, HLA-DP or HLA-DQ protein.

[0023] In an embodiment of the method of the present invention, the transduced cells are checked for expression of the exogenous protein at the end of each step.

[0024] In an embodiment of the method of the present invention, the HLA deficient B cell line is a K562 cell line.

[0025] In an embodiment of the present invention, the transductions in steps a) to c) are performed sequentially.

[0026] The present invention provides artificial antigen-presenting cells (aAPCs) with defined single HLA-II alleles. This approach offers unlimited cell availability through regular cell culture expansion without the need for differentiation and full control over the interrogated HLA-II space.

[0027] Short description of the figures:

[0028] Fig. 1 shows the expression of the exogenous proteins CD64, CD74, HLA-CM and DR-B1 in an HLA deficient B cell line modified according to the present invention;

[0029] Fig. 2A shows the length distribution of MHC bound peptides identified in MHC -associated peptide proteomics (MAPP) assay using the HLA deficient B cell line of the present invention as an artificial antigen presenting cell line with KLH (Keyhole Limpet Hemocyanin) and a monoclonal antibody as antigens;

[0030] Fig. 2B shows the amino acid sequence of the MHC bound peptides identified in an MHC-asso-ciated peptide proteomics sequencing (MAPPs) assay using the HLA deficient B cell line of the present invention as an artificial antigen presenting cell line and KLH (Keyhole Limpet Hemocyanin) and a monoclonal antibody as antigens.

[0031] Definitions:

[0032] CD64 is a high-affinity Fc gamma receptor (FcyRI) found on the surface of certain immune cells, such as macrophages and monocytes. It binds to the Fc region of IgG antibodies, facilitating phagocytosis and the activation of immune responses.

[0033] CD74, also known as the invariant chain, is a protein associated with the Major Histocompatibility Complex (MHC) class II molecules. It plays a crucial role in the assembly and transport of MHC class II molecules and helps prevent premature binding of peptides in the endoplasmic reticulum.

[0034] HLA (Human Leukocyte Antigen) refers to a group of genes that play a critical role in the immune system's ability to recognize and respond to foreign substances.The HLA genes are part of the major histocompatibility complex (MHC) in humans. The MHC is divided into two main classes:

[0035] • Class I MHC: Includes HLA-A, HLA-B, and HLA-C. These molecules are present on all nucleated cells and present peptide fragments from proteins inside the cell to cytotoxic T cells (CD8+ T cells).

[0036] • Class II MHC: Includes HLA-DR, HLA-DP, and HLA-DQ. These molecules are primarily found on antigen-presenting cells like macrophages, dendritic cells, and B cells, and present extracellular protein fragments to helper T cells (CD4+ T cells).

[0037] HLA-DM is a non-classical MHC class II molecule that aids in the loading of antigenic peptides onto MHC class II molecules. HLA-DM is a heterodimer consisting of two chains: the alpha (a) chain and the beta (P) chain. It facilitates the exchange of CLIP (Class Il-associated invariant chain peptide) with antigenic peptides, ensuring that MHC class II molecules present relevant antigens to CD4+ T cells.

[0038] HLA-DR, HLA-DP, and HLA-DQ are all classical MHC class II molecules. They present exogenously derived peptides to CD4+ T helper cells, which is a critical step in the adaptive immune response. Each of these molecules (HLA-DR, HLA-DP, HLA-DQ) is composed of two chains (alpha and beta) that form a binding groove for peptide antigens. The DR-B chain is highly polymorphic, encoded by the HLA-DRB1 gene, among others (such as DRB3, DRB4, and DRB5).The HLA-DP chains are encoded by the HLA-DPA1 and HLA-DPB1 genes, respectively. The HLA DQ chains are encoded by separate genes: HLA-DQA1 and HLA-DQB1. Both the DQA1 and DQB1 genes exhibit significant polymorphism, resulting in a wide variety of HLA-DQ alleles.

[0039] The term “protein” refers to molecules composed of one or more chains of amino acids.

[0040] The terms "cell," "cell line," and "cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but may contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0041] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0042] The term "MAPPs" stands for "MHC -associated peptide proteomics" and it is a technique used to identify peptides presented by Major Histocompatibility Complex (MHC) molecules on the surface of cells. This information is crucial for evaluating the potential immunogenicity of biopharmaceuticals.

[0043] The MAPPs assay is used to predict and assess the immunogenic potential of therapeutic proteins by identifying which peptides are naturally presented by MHC molecules on antigen-presenting cells (APCs).

[0044] MAPPs Process:

[0045] Cell Culture: The therapeutic protein of interest is incubated with APCs, such as dendritic cells or B cells.Peptide Isolation: MHC molecules are isolated from the surface of these cells, and the peptides bound to MHC are extracted. This involves immunoprecipitation techniques to selectively capture the MHC -peptide complexes.

[0046] Mass Spectrometry: The isolated peptides are then analyzed using mass spectrometry, which allows for the identification and sequencing of the peptides.

[0047] Data Analysis: The resulting peptide sequences are analyzed to determine which peptides are derived from the therapeutic protein and assess their potential to trigger an immune response.

[0048] Example

[0049] Generation of aAPC

[0050] Cells:

[0051] K562 cell line was obtained from Roche cell repository (Speciment ID CLBA00261). Cells were cultured in FBS free condition, based on RPMI-1640 medium supplemented with 20pg / mL Insulin, Hpg / mL Transferrin, 77nMNa Selenite, 50nM Hydrocortisone, lOng / mL EGF, lOpM Ethanolamine, lOpM O-PEA, 75pM triiodothyronine, 0,1% bovine albumin, ImM Na Pyruvate, lOmM HEPES and 2mM L-Glutamine. For the purpose of selective pressure lOpg / mL Puromycin or 200pg / mL Hygromycin were applied on cells post transduction depending on Lentiviral construct.

[0052] Lentiviral Vector Description:

[0053] For the expression of all the components of the HLA-II presentation machinery three different lentiviral vectors have been generated. CD64 transcript variant 2 (NM 000566.4) was used for the first lentiviral vector. For the second vector the transcript from HLA-DMB (NM 002118.5) was placed upstream of HLA-DMA (NM 001025159.3) separated by P2A self-cleaving peptide for equimolar expression. HLA-DMA followed downstream an internal ribosomal entry side (IREMS) upstream of the transcript for HLA class II alpha chain-like-product CD74 (X6744.1). The third vector expresses the hetero dimeric HLA complex and consists of the two HLA-DR, DP or DQ transcripts of choice separated by a P2A self-cleaving peptide for equimolar expression.

[0054] Lentiviral transduction:

[0055] Lentiviral constructs acquired as customized purchase (Cellomics Technologies, Halethorpe, MD, USA). Each transduction was carried out by spinoculation. Cells were incubated with a mix of Polybrene at 8pg / mL (Tocris, UK) and desired MOI of each particular LV construct for 20’ at RT, then centrifuged for 30’ at 32°C and 800g. Supernatants were aspirated, cell pellets were resuspended in fresh growth medium and allowed to proliferate for 48h before the start of antibiotic selection.

[0056] Cell line generation:

[0057] K652 cells were first transduced with the lentivirus expressing CD64 for enhanced uptake of exogenous proteins. Single cell sorting was applied on polyclonal populations post antibiotic selection. Stable single clones with different CD64 expression levels (low, medium, high) were transduced withthe second lentiviral vector coding for HLA-DMa / b and CD74. Single cell sorting was applied on polyclonal populations post antibiotic selection. Stable single clones with different CD64, HLA-DMa / b and CD74 were transduced using the third lentiviral vector coding for the desired HLA alpha and beta chains.

[0058] FACS:

[0059] Single cell sorting was applied on polyclonal populations post antibiotic selection. Cells were stained with mAbs recognizing human CD64 (recombinant, Miltenyi, Kbln, Germany), conjugated to APC fluorophore or HLA-DRB1 (clone L243, RayBiotech, Peachtree Comers, Georgia, USA), conjugated to PE fluorophore and sorted for high, medium and low protein expression on Cytoflex SRT (Beckman Coulter, Indianapolis, USA) with 1 cell / well into 384 well plates, prefdled with 50pL / well of conditioned medium. Positive selection for HLA-DM / CD74 expression was carried over on living cells via CD64 expression. Single cell colonies were expanded to a bigger amount of cells before cryopreservation and quality check.

[0060] Flow cytometry analysis or high content imaging:

[0061] Living single cell clones and subclones were stained with mAbs recognizing surface antigens human CD64 (recombinant, Miltenyi, Kbln, Germany) or HLA-DRB1 (clone L243, RayBiotech, Peachtree Comers, Georgia, USA) and appropriate isotype control (clone RPC5.4, RayBiotech, Peachtree Comers, Georgia, USA). For intracellular detection of HLA-DM / CD74 expression, cells were fixed, permeabilized with Cytofix / Cytoperm Fixation / Permeabilisation Kit (BD Biosciences, San Diego, CA, USA) and stained with mABs recognizing human HLA-DM or CD74, conjugated to APC fluorophore (recombinant, Miltenyi, Kbln, Germany). Flow cytometry analysis was applied on For-tessa LX-10 (BD Biosciences, San Diego, CA, USA), Median fluorescence intensities were extracted with FLowJo software.

[0062] For visualization of the staining intensity, cells were counterstained with Hoechst3332 (Thermo Fisher, Waltham, MA, USA) or DAPI dyes (Thermo Fisher, Waltham, MA, USA). Images were acquired on Opera Phenix (Perkin Elmer, Shelton, CT, USA).

[0063] dPCR:

[0064] To assess stability of transduced genes on RNA level in generated single cell clones or subclones, dPCR analysis was done on cells, lysed in RLT buffer (Qiagen, Hilden, Germany). Total RNA was isolated with RNeasy Mini kit (Qiagen, Hilden, Germany), Ipg of total RNA was converted into cDNA with cDNA high capacity reverse transcriptase kit (Thermo Fisher, Waltham, MA, USA). Duplex qPCR reactions were assembled by reaction mix of Ing cDNA, qPCR Master Mix (Qiagen, Hilden, Germany) and primers for HKG and genes of interest in one reaction. dPCR mns were performed in 96-well nanoplates with 8500 partitions on Qiaquity One device (Qiagen, Hilden, Germany).MAPPs:

[0065] Cells are first resuspended in the culture flasks. If necessary, cells are detached from the bottom of the flask. Twelve milliliters of each cell suspension are collected into a 15 mL Falcon tube and centrifuged at 300g for 5 minutes. Following centrifugation, the cell pellets are resuspended in 5 mL of fresh SFMPH or SFM for the K562 parental cell line.

[0066] Next, the cells are counted and the suspension is diluted to 0.33 x 10A6 cells / mL in 10 mL of fresh medium in a new 15 mL Falcon tube. The diluted cells are then distributed into three wells of a 6-well plate, with 3 mL per well (equivalent to 1 million cells per well). The plates are placed into the incubator until treatment.

[0067] For the treatment application, samples are added to the cells at 67 pM for mAB or 22 pM for KLH. After the test compounds are added, the contents of the plates are mixed crosswise. The plates are then incubated for 24 hours.

[0068] Post-treatment, the side of the 6-well plate is gently tapped to dislodge the cells. The treated cells are collected into a 15 mL Falcon tube and each well is washed with an additional 3 mL of DPBS, which is added to the corresponding Falcon tube. The cells are centrifuged at 300g for 5 minutes, the supernatant is removed, and the cell pellet is resuspended in 10 mL of DPBS. This centrifugation step is repeated at 300g for 5 minutes. After the supernatant is removed, the cells are resuspended in 1 mL of DPBS.

[0069] The cells are counted and the cell suspension is transferred into a 1.5 mL protein low bind tube. The cells are then centrifuged at 1200 rpm for 5 minutes. The supernatant is carefully removed and 95 pL of ice-cold complete Cell Lysis Buffer is added per pellet. The sample is vortexed for a few seconds and incubated in a Thermomix at 4°C and 1100 rpm for at least 1 hour. Following incubation, the sample is vortexed again for a few seconds and centrifuged at 13200 rpm for 10 minutes at 4°C. The supernatant (approximately 110-120 pL) is transferred into a new 1.5 mL protein low bind tube and the sample is frozen at -80°C.

[0070] Samples are thawed at room temperature (RT). To each sample, 50 pL of a 2.5-fold pre-diluted anti HLA-DX biotin antibody in lysis buffer (without detergent / inhibitors) is added. The samples (lysate + anti-HLA-DX biotin antibody) are incubated in a Thermomixer for 2 hours at 37°C and 1100 rpm. After incubation, the samples are transferred into a 96 deep well plate.

[0071] The Bravo robotic system is initialized, and the affinity purification protocol is executed. Once the run is completed, the plate with samples (18 pL per sample) is spun down at 1000 rpm for 15 seconds and stored at +4°C until loading onto EVO tips.

[0072] For the EVO tips preparation, 20 pL of Solution B (0.1% FA in Acetonitrile) is transferred into the tips. The tips are spun down at 800g for 1 minute, then soaked in isopropanol for 15 seconds. Acetonitrile is washed out from the tips by loading them twice with 20 pL of Solution A (0.1% FA in water). A 96-well plate is prepared, filled with 100 pL / well of Solution A, and the EVO tips adaptor is placed onto this plate with the equilibrated tips on top of the adaptor.Samples (18 pL per sample) are loaded into the EVO tips. The tips are spun down at 800g for 1 minute. The loaded EVO tips are washed four times with 20 pL of Solution A (0.1% FA in water). Then, 200 pL of Solution A is added into the EVO tips, which are spun down for 10 seconds at 800g.

[0073] Finally, the tips are placed in a box fdled with Solution A, ensuring that the tip filters are covered with Solution A from both the top and bottom. The tips are then stored in the refrigerator until mass spectrometry analysis.

[0074] Samples are acquired on a timsTOF HT, and data is analyzed with PEAKS X.

Claims

Claims1. An HL A deficient B cell line which has been modified to express at least exogenous proteins selected from the group consisting of CD64, CD74, HLA-DM and at least one protein selected from the group consisting of HLA-DR, HLA-DP and HLA-DQ.

2. The HLA deficient B cell line of claim 1, wherein said cell is a K562 cell line.

3. The HLA deficient B cell line of claim 1 or 2, wherein the expressed proteins are human proteins.

4. The HLA deficient B cell line of claims 1 - 3, wherein the HLA deficient B cell is modified by lentiviral transduction to introduce nucleic acids encoding the exogenous proteins.

5. The HLA deficient B cell line of claims 1 - 4, wherein the lentiviral transduction comprises three different lentiviral vectors.

6. The HLA deficient B cell line of claims 1 - 5, wherein the lentiviral transduction comprises more than one transduction which are performed sequentially.

7. The HLA deficient B cell line of claims 1 - 6, wherein the lentiviral transduction comprises three transductions.

8. The HLA deficient B cell line of claims 1 - 7, wherein the first lentiviral transduction introduces the nucleic acid encoding the CD64 protein.

9. The HLA deficient B cell line of claims 1 - 8, wherein the second lentiviral transduction introduces the nucleic acids encoding the HLA-DM and CD74 proteins.

10. The HLA deficient B cell line of claims 1 -9, wherein the third lentiviral transduction introduces the nucleic acid encoding the HLA-DR, HLA-DP or HLA-DQ protein.

11. The HLA deficient B cell line of claims 1 - 10 for use as an antigen presenting cell (APC).

12. The HLA deficient B cell line of claims 1 - 10 for use in an MHC -associated peptide proteomics sequencing assay.

13. A method for the generation of an artificial antigen presenting cell (APC) which expresses at least exogenous proteins selected from the group consisting of CD64, CD74, HLA-DM and at least one protein selected from the group consisting of HLA-DR, HLA-DP and HLA-DQ, the method comprising the following steps:a) Transducing an HLA deficient B cell line with a lentiviral vector comprising a nucleic acid encoding a CD64 protein,b) Transducing the cell of step a) with a lentiviral vector comprising nucleic acids encoding the HLA-DM and CD74 proteins andc) Transducing the cell of step b) with a lentiviral vector comprising nucleic acid encoding the HLA-DR, HLA-DP or HLA-DQ protein.

14. The method of claim 13, wherein the transduced cells are checked for expression of the exogenous protein at the end of each step.

15. The method of claim 13 or 14, wherein the HLA deficient B cell line is a K562 cell line.

16. The method of claims 12 - 15, wherein the transductions in steps a) to c) are performed sequentially.9

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