Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Summary

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCRα or TCRβ of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Abstract

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCRα or TCRβ hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Introduction

T cell receptors (TCRs) are heterodimeric adaptive immune receptors expressed by T lymphocytes, composed of a TCRα and TCRβ chain. They are generated via somatic rearrangement of V(D)J gene segments, which produces a highly diverse repertoire capable of recognizing virtually unlimited configurations of HLA/peptide complexes. Clinically, T cells engineered to express clonotypic TCRs specific for tumor-associated antigens have demonstrated efficacy in a variety of cancers¹. However, many TCRs cloned for this purpose lack sufficient affinity for the antigen of interest, which limit their therapeutic application.

Here, we describe a method to overcome this limitation for existing TCRs by exploiting chain-centricity. It has been reported that one TCR hemichain could play a more dominant role in recognition of the target antigen², here termed centricity. Crystal structural analyses have shown that one centric hemichain of a TCR could account for the majority of the footprint on the MHC/peptide complex^3,4. Using this concept, we have previously demonstrated that the SIG35α TCRα can pair with a diverse repertoire of TCRβ chains and maintain reactivity against the MART1_27-35 peptide presented by HLA-A2⁵. Similar results were obtained with the TAK1 TCR, where the centric TCRβ hemichain paired with various TCRα chains and maintained reactivity for the WT1_235-243 peptide presented by HLA-A24⁶. Both MART1 and WT1 are tumor-associated antigens. Chain-centricity was also applied to study antigen recognition of CD1d-restricted invariant natural killer (iNKT) TCRs, by pairing the invariant Vα24-Jα18 (Vα24i) TCRα chain of human iNKT TCRs with different Vβ11 TCRβ chains⁷.

In all cases, we were able to generate a de novo repertoire of TCRs by transducing the centric TCR hemichain to peripheral blood T cells, where the introduced hemichain paired with the endogenous TCRα or TCRβ counter-chains. In essence, the centric hemichain serves as a bait that can be used to identify the appropriate counter-chains, which when paired together form TCRs that maintain the antigen specificity of interest, yet varying in affinity. From these novel repertoires, we were able to isolate clonotypic TCRs with improved interaction strength against the target antigen compared to pre-existing TCRs. Therefore, we believe this method will accelerate the pipeline of identifying optimal TCRs for clinical application.

Protocol

1. Preparing Retroviral Construct Encoding TCR Hemichain of Interest

1. Linearize pMX vector to allow the insertion of a TCR gene in subsequent steps. Digest the plasmid DNA with EcoRI and NotI restriction enzymes at 37 °C for 3 hr (Table 1)⁸.
2. Carry out electrophoresis of the digested plasmid on 1.2% agarose gel. Excise band of approximately 4,500 base pairs (bps), and elute in 30 μl sterile water using commercially available gel extraction kits⁹.
3. Design 5' and 3' primers for the TCR gene of interest that also encode 15-20 bps overlapping the EcoRI and NotI digestion site of pMX vector, respectively⁸.
4. Amplify TCR gene with primers. Carry out electrophoresis of the PCR product and elute band of approximately 1,000 bps as described in step 1.2.
5. Clone TCR gene into digested vector by combining each linear fragment with commercially available plasmid assembly master mix reagent and incubating at 50 °C for 1 hr.
   NOTE: Refer to manufacturer's protocol for relative volumes of each component. This assembly method is based on the technique originally described by Gibson et al.¹⁰.
6. (Optional) Tag TCR gene to mark transduced cells with the truncated form of human nerve growth factor receptor (ΔNGFR, amino acids 1-277)¹¹ separated by furin cleavage site, serine-glycine linker, and 2A sequences^12,13. Design primers encoding sequences overlapping the fragment ends and assemble plasmid as described in steps 1.3-1.5.
   NOTE: These sequences can be found in references^11-13.
7. Transform chemically competent E. coli with assembled plasmid following manufacturer's protocol¹⁴. Seed transformed bacteria on agar plates (20 mg/ml lysogeny broth (LB), 15 mg/ml agar, and 1 μg/ml ampicillin) and incubate at 37 °C for 18 hr.
8. Culture a single bacterium colony in 50 ml of LB medium (20 mg/ml LB and 1 μg/ml ampicillin) for 16 hr in a shaking incubator at 37 °C and 200 rounds per min.
9. Purify plasmids using commercially available midiprep kits following manufacturer's protocol. Dilute plasmid to concentration of around 1 μg/μl.
   NOTE: The plasmid purification protocol is based on the alkaline extraction method¹⁵.

2. Generating Stable Packaging Cell Line

NOTE: Both 293GPG and PG13 cells are adherent. Culture cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 50 μg/ml gentamicin. Culture 293GPG cells with 1 μg/ml tetracycline before transfection. Incubate cells at 37 °C with 5% CO₂ between all steps.

1. Transfect 293GPG packaging cells¹⁶ cultured in T75 flask with the hemichain-encoding vector obtained in step 1.9, using commercially available transfection reagent following manufacturer's protocol¹⁷. NOTE: Transfect 293GPG cells at 50-60% confluency.
2. Aspirate medium for 293GPG cells and add 10 ml of fresh DMEM medium 1 day post transfection.
3. Harvest transiently produced virus from transfected 293GPG cells 2 days after step 2.2 by transferring culture medium to syringe and passing through 0.45 μm filter.
   NOTE: Virus can be used immediately or stored at -80 °C for future use.
4. Culture 1 x 10⁵ PG13 cells in T25 flask. Count cells using a hemocytometer. After one day, aspirate culture medium and add 1.5 ml of 293GPG-derived virus from step 2.3 and 1.5 ml DMEM medium, along with 8 μg/ml of polybrene.
5. Change the medium as described in step 2.4 once per day for 4 days, to establish stable PG13 packaging cell line producing retrovirus encoding the TCR hemichain of interest.
6. Aspirate medium and replace with fresh DMEM medium for PG13 cell lines 24 hr after last infection for further culture.
   NOTE: Freeze or sub-culture cells by detaching with 0.05% trypsin-EDTA solution.
7. To produce virus from transduced PG13 cell lines, culture 2 x 10⁶ cells in T75 flask with 10 ml DMEM medium, and harvest virus as described in step 2.3 three days after seeding the cells.
   NOTE: Virus is best used immediately but can be stored at -80 °C for up to two months.

3. Activation and Transduction of Human T cells

NOTE: Human samples are obtained and used in accordance with the institutional ethics committee approved protocols. Culture primary human cells in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% human serum instead of FCS and 50 μg/ml gentamicin. Incubate cells at 37 °C with 5% CO₂ between all steps.

1. Isolate human peripheral blood mononuclear cells (PBMC) by density gradient separation following manufacturer's protocol¹⁸.
2. Activate T cells to induce proliferation required for retroviral infection. Culture 2 x 10⁷ fresh or thawed PBMC per well in 6-well plate, in 5 ml of RPMI medium with 100 IU/ml of recombinant human interleukin-2 (rhIL-2) and 50 ng/ml of anti-CD3 monoclonal antibody (clone OKT3).
3. Three days post stimulation, collect T cells by pipetting and centrifuge at 350 x g for 4 min. Discard supernatant and seed 0.5-1 x 10⁶ T cells per well in 24-well plate resuspended in 1 ml of PG13 virus from step 2.7, and 1 ml of RPMI medium supplemented with 200 IU/ml of rhIL-2. Centrifuge plate at 1,000 x g and 32 °C for 1 hr.
4. After 24 hr, collect T cells by pipetting and centrifuge at 350 x g for 4 min. Discard supernatant and resuspend cells in 1 ml of PG13 virus from step 2.7, and 1 ml of RPMI medium supplemented with 200 IU/ml of rhIL-2. Repeat this step for a total of 6 infections.
   NOTE: Number of infections should be optimized depending on titer of virus produced by packaging cell line. If necessary, passage T cells by removing 20-30% of the cells each day to prevent overgrowth.
5. 24 hr after the last infection, collect T cells by pipetting and centrifuge at 350 x g for 4 min. Discard supernatant and resuspend cells in RPMI medium with 100 IU/ml of rhIL-2 for further culture.
6. 2-3 days after step 3.5, stain T cells with HLA multimer at 4 °C for 30 min, then anti-human CD3 and co-receptor monoclonal antibodies (mAbs) at 4 °C for 15 min. Analyze by flow cytometry. Use irrelevant multimer and/or untransduced cells as negative controls (Figure 1-3)¹⁹.
7. If the introduced centric hemichain is a TCRα chain, analyze Vβ usage of the de novo multimer positive cells using commercially available Vβ-specific antibody panel by flow cytometry²⁰.

4. Cloning De Novo TCR Counter-hemichains

1. Sort the de novo multimer positive population in step 3.6 by flow-assisted cell sorting.
2. Isolate RNA from sorted T cells using the acid guanidinium thiocyanate-phenol-chloroform extraction method^21,22.
   NOTE: RNA can be stored at -80 °C, but should be used to generate cDNA as soon as possible.
3. Synthesize cDNA library from extracted RNA using commercially available reverse transcriptase-PCR kits following manufacturer's protocol^23,24.
4. If cloning TCRβ counter-chains, design Vβ gene and TCRβ constant region specific primers, as determined in step 3.7, and clone full-length TCRβ genes as described in steps 1.3-1.9. See step 4.5 if counter-chain is TCRα, otherwise continue to step 5.1.
5. Commercially available Vα-specific antibodies are limited, thus Vα gene usage cannot be determined by flow cytometry. Clone TCRα counter-chains via 5'-rapid amplification of cDNA ends (RACE)²⁵, using commercially available 5' RACE kits⁶.
   1. Synthesize 5' RACE compatible cDNA from RNA extracted in step 4.2 following manufacturer's protocol.
   2. Perform 1^st round PCR as described in Table 2.
   3. Carry out electrophoresis of the PCR product and elute band of approximately 1,100 bps, as described in step 1.2.
   4. Perform 2^nd round PCR as described in Table 3, using 1^st round PCR product as template.
      NOTE: Primer sequences shown under Table 3 were designed for cloning into EcoRI and NotI digested pMX vector.
   5. Carry out electrophoresis of the PCR product and elute band of approximately 1,000 bps, as described in step 1.2.
   6. Clone TCRα genes into EcoRI and NotI digested pMX vector and purify plasmids as described in steps 1.5-1.9.

5. Reconstituting Novel Antigen-specific TCR Clones

NOTE: Culture Jurkat 76 cells and subsequent cell lines in RPMI medium supplemented with 10% FCS and 50 μg/ml gentamicin. Incubate cells at 37 °C with 5% CO₂ between all steps.

1. Transduce Jurkat 76 cells²⁶ (or equivalent TCR^-/- human T cell line) with centric TCR hemichain using 293GPG virus produced in step 2.3. For Jurkat 76, seed 5 x 10⁴ cells per well in 24-well plate with 1 ml of virus and 1 ml of RPMI medium. Centrifuge plate at 1,000 x g and 32 °C for 1 hr.
2. 24 hr after infection, collect hemichain transduced Jurkat 76 cells by pipetting and centrifuge at 350 x g for 4 min. Discard supernatant and resuspend in fresh RPMI medium for further culture.
3. Purify transduced cells if the hemichain is molecularly tagged 2-3 days after step 5.2, by staining with fluorophore conjugated anti-NGFR mAb followed by flow-assisted or magnetic-assisted cell sorting (optional)²⁷.
4. Following steps 2.1 to 2.3, produce 293GPG virus encoding a TCR counter-chain cloned from steps 4.4 or 4.5.
5. To fully reconstitute the TCR, transduce T cell line stably expressing the centric TCR hemichain generated in steps 5.1-5.3 using virus from step 5.4. Perform transduction as described in steps 5.1-5.2.
6. Purify CD3⁺ transfectants using anti-CD3 magnetic-assisted cell sorting following manufacturer's protocol, 2-3 days after step 5.5²⁷.
7. To validate antigen specificity, stain the transfectants expressing clonotypic TCRs composed of the centric TCR hemichain paired with various counter-chains, with anti-CD3 and/or co-receptor mAbs, and HLA multimer, 3-5 days post CD3 purification. Analyze cells by flow cytometry (Figure 4-5)¹⁹.

Results

Without prior knowledge of which hemichain is chain-centric, the TCRα and TCRβ chain should be separately cloned and transduced to peripheral blood T cells, which was done in the case of HLA-A24/WT1 reactive TAK1 TCR (Figure 1). Transduction of TAK1β yielded a noticeably higher frequency of antigen-specific T cells. Conversely, transduction of a non-centric hemichain would not yield de novo multimer positive T cells, as seen with TAK1α chain (...

Discussion

The first requirement for successful application of this method is achieving sufficient transduction efficiency of primary T cells with the hemichain of interest. In our experience, the combination of using PG13 as packaging cell line and pMX as retroviral vector results in stable and efficient expression of the introduced gene in human primary T cells. PG13 packaging cells can be single-cell cloned to select for high-titer packaging cells to improve transduction efficiency. Furthermore, proliferation of T cells is also ...

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	Wisent Bioproducts	325-043-CL
293GPG cells	Generated by Ory et al. (ref 8)
Agar	Wisent Bioproducts	800-010-CG
Agarose	Wisent Bioproducts	800-015-CG
Ampicillin sodium salt	Wisent Bioproducts	400-110-IG
Chloroform	BioShop	CCL402
Deoxynucleotide (dNTP) Solution Mix	New England Biolabs	N0447L
DMEM, high glucose, pyruvate	Life Technologies	11995065
EcoRI	New England Biolabs	R0101S
EZ-10 Spin Column DNA Gel Extraction Kit	BS353
Fetal Bovine Serum	Life Technologies	12483020
Ficoll-Paque Plus	GE Healthcare	17-1440-02
Filter	Corning	431220	0.45 mm pore SFCA membrane
FITC-conjugated anti-human CD271 (NGFR) mAb	Biolegend	345104	clone ME20.4
FITC-conjugated anti-human CD3 mAb	Biolegend	300440	clone UCHT1
Gentamicin	Life Technologies	15750078
Gibson Assembly Master Mix	New England Biolabs	E2611L	used for multi-piece DNA assembly
HLA-A2 pentamer	Proimmune	depends on antigenic peptide	HLA-A2/MART1 multimer used here was purchased from Proimmune
HLA/CD1d monomers	NIH Tetramer Core Facility		multimerize monomers according to protocol provided by NIH tetramer core facility
Human AB serum	Valley Biomedical	HP1022
human CD3 microbeads	Miltenyi Biotec	130-050-101
IOTest Beta Mark TCR V beta Repertoire Kit	Beckman Coulter	IM3497
Jurkat 76 cells	Generated by Heemskerk et al. (ref 10)
LB Broth	Wisent Bioproducts	800-060-LG
LS MACS column	Miltenyi Biotec	130-042-401
NEB 5-alpha Competent E. coli	New England Biolabs	C2987I
NEBuffer 3.1	New England Biolabs	B7203S	used for EcoRI and NotI digestion
NotI	New England Biolabs	R0189S
NucleoBond Xtra Midi	Macherey-Nagel	740410	used for plasmid purification
PC5-conjugated anti-human CD8 mAb	Beckman Coulter	B21205	clone B9.11
PG13 cells	ATCC	CRL-10686
Phusion HF Buffer Pack	New England Biolabs	B0518S
Phusion High-Fidelity DNA Polymerase	New England Biolabs	M0530L
pMX retroviral vector	Cell Biolabs	RTV-010
polybrene	Sigma-Aldrich	H-9268
Proleukin (recombinant human interleukin-2)	Novartis	by Rx only	equivalent product can be purchased from Sigma-Aldrich
Purified anti-human CD3 antibody	Biolegend	317301	clone OKT3, used for T cell stimulation
RPMI 1640	Life Technologies	11875119
SA-PE	Life Technologies	S866	used for multimerizing monomers from NIH tetramer core facility
SMARTer RACE 5'/3'  Kit	Clontech	634858
Sterile water	Wisent Bioproducts	809-115-LL
SuperScript III First-Strand Synthesis System	Invitrogen	18080051	for cDNA synthesis
Syringe	BD	301604	10 ml, slip tip
Tetracycline hydrochloride	Sigma-Aldrich	T7660
TransIT-293	Mirus Bio	MIR 2700	used to transfect 293GPG cells
TRIzol Reagent	Life Technologies	15596026