key: cord-0871938-13zwa3gx
authors: Leung, Wing; Soh, Teck Guan; Linn, Yeh Ching; Low, Jenny Guek‐Hong; Loh, Jiashen; Chan, Marieta; Chng, Wee Joo; Koh, Liang Piu; Poon, Michelle Li‐Mei; Ng, King Pan; Kuick, Chik Hong; Tan, Thuan Tong; Tan, Lip Kun; Seng, Michaela Su‐fern
title: Rapid production of clinical‐grade SARS‐CoV‐2 specific T cells
date: 2020-07-31
journal: Adv Cell Gene Ther
DOI: 10.1002/acg2.101
sha: b7f9f28c947dda23a940325fcb2e69f7d4bf7d35
doc_id: 871938
cord_uid: 13zwa3gx

OBJECTIVES: To determine whether the frequencies of SARS‐CoV‐2‐specific T cells are sufficiently high in the blood of convalescent donors and whether it is technically feasible to manufacture clinical‐grade products overnight for T‐cell therapy and assessment of COVID‐19 immunity. METHODS: One unit of whole blood or leukapheresis was collected from each donor following standard blood bank practices. The leukocytes were stimulated using overlapping peptides of SARS‐CoV‐2, covering the immunodominant sequence domains of the S protein and the complete sequence of the N and M proteins. Thereafter, functionally reactive cells were enriched overnight using an automated device capturing IFNγ‐secreting cells. RESULTS: From 1 × 10(9) leukocytes, a median of 0.98 × 10(6) (range 0.56‐2.95) IFNγ + T cells were produced from each of the six donors, suggesting a high frequency of SARS‐CoV‐2 reactive T cells in their blood, even though only one donor had severe COVID‐19 requiring mechanical ventilation whereas the other five donors had minor symptoms. A median of 57% of the enriched T cells were IFNγ+ (range 20%‐74%), with preferential enrichment of CD56+ T cells and effector memory T cells. TCRVβ‐spectratyping confirmed distinctively tall oligoclonal peaks in final products. With just six donors, the probability that a recipient would share at least one HLA allele with one of the donors is >88% among Caucasian, >95% among Chinese, >97% among Malay, and >99% among Indian populations. CONCLUSIONS: High frequencies of rapid antigen‐reactive T cells were found in convalescent donors, regardless of severity of COVID‐19. The feasibility of clinical‐grade production of SARS‐CoV‐2‐specific T cells overnight for therapeutics and diagnostics is revealed.

A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 . 1, 2 No specific treatment has been proven to be effective, although promising preliminary data suggested that certain non-specific immunomodulating agents and antivirals can be repurposed to shorten the duration of illness. [3] [4] [5] While specific vaccines are being developed, passive immunity can be acquired immediately by infusion of plasma from convalescent donors into newly infected patients. 6,7 SARS-CoV-2-specific T cells, which are another key component of adaptive immunity, have not been used therapeutically, primarily because of the concerns of low frequencies of virus-specific T cells, technical feasibility, and long manufacturing time (>1 month).

The aim of this study is to determine whether the frequencies of SARS-CoV-2 specific T cells are sufficiently high in the blood of convalescent donors and whether it is technically feasible to manufacture clinical-grade products overnight for T-cell therapy and assessment of COVID-19 immunity during a fast-progressing pandemic. The specific hypothesis is that SARS-CoV-2 specific T cells can be isolated from the blood of convalescent donors rapidly and efficiently using SARS-CoV-2 specific peptides and a functional selection strategy.

Convalescent donors were referred by their infectious disease physicians. Eligibility criteria included age 21-65, a history of COVID-19 with documented positive test for SARS-CoV-2 in the past but the test had become negative since, fulfillment of all blood donation criteria per standard blood bank practices including a negative blood PCR testing for SARS-CoV-2, and an informed consent with approval by the hospital Institutional Review Board (clinicaltrials.gov NCT04351659). One unit of whole blood or leukapheresis was collected from each donor once. The medical history and type of cells collected from the six donors are summarized in Table 1 .

After plasma extraction and buffy coat preparation (if the collection was whole blood), the leukocytes were processed using the automated CliniMACS Prodigy IFN-γ Cytokine Capture System® (CCS) (Miltenyi Biotec, Germany). Briefly, 1 × 10 9 cells were stimulated using overlapping peptides of SARS-CoV-2, covering in combination the immunodominant sequence domains of the S protein, and the complete sequence of the N and M proteins (GenBank MN908947.3, Protein QHD43416.1, Protein QHD43423.2, and Protein QHD43419.1). The peptide pools were short 15-mer peptides with 11-amino-acid overlaps, which can bind to MHC class I and class II complexes and thus were able to stimulate both CD4+ and CD8+ T cells. Thereafter, the cells were labeled with the Catchmatrix Reagent containing bispecific antibodies for CD45 and IFN-γ, which was secreted by the stimulated target cells during the secretion period. After the secretion phase, the cell surface-bound IFN-γ was targeted by the Enrichment Reagent, which contained IFN-γ-specific antibody conjugated to superparamagnetic iron dextran particles (MACS® MicroBeads), thus allowing subsequent immunomagnetic cell separation. The unlabeled cells (CCS negative fraction) passed through the built-in magnetic column, whereas the Microbead-labeled cells (CCS positive fraction) were retained in the magnetic field. Afterwards, the magnetic field was turned off and the target cells were eluted into the target cell bag. The entire process took only 12 hours for cell manufacturing.

For the analysis of cell compositions, two antibody panels were used. 

To assess TCR spectra, the CDR3-encoding region of the TCRVβ gene was amplified using 24 TCRVβ subfamily-specific primers and a carboxyfluorescein (FAM)-conjugated TCRVβ constant region-specific primer for the first four clinical products. 8 The PCR products were denatured with Hi-Di formamide (Applied Biosystems, Carlsbad, CA) and electrophoresed along with Gene Scan-600 LIZ size standard (Applied Biosystems) on a SeqStudio Genetic Analyzer (Applied Biosystems).

From 1 × 10 9 leukocytes, containing a median of 0.78 × 10 9 (range 0.72-0.90) lymphocytes ( (Table 1) .

A median of 57% of the enriched T cells were IFNγ+ (range 20%-74%),

indicating their functional competency in rapid IFNγ secretion even with just a brief 4-hour exposure to SARS-CoV-2 peptides ( Figure 1A ), compatible with a memory recall response. 

Before cell enrichment, the median CD4:CD8 ratio was 60:40 and only 7% of the T cells were CD56+ ( Table 2) IFNγ+ CD4+ cells (median 82%, range 68%-89%) were TEM and most IFNγ+ CD8+ cells (median 76%, range 58%-98%) were TEM or TEMRA ( Figure 1B) . Persistent function and specificity were observed after interval restimulation ( Figure 1C ).

Before cell enrichment, polyclonal spectra of TCRVβ in general

Gaussian distribution were observed (Figure 2 ). After enrichment, distinctively tall oligoclonal peaks were observed in the final products, with restricted TCRVβ representations both across subfamilies and within subfamily, suggesting high purity of SARS-CoV-2 specific T cells in general and high-degree of TCR specificity towards SARS-CoV-2 peptides in particular. As expected, the TCRVβ spectra in the negative fraction were similar to those before cell isolation with general Gaussian distribution.

Three of the donors are Chinese Singapore residents, and one each of Caucasian, Indian, and mixed Chinese and European descent. With just six donors, the probability that a recipient would share at least one HLA allele with one of the donors is >88% among Caucasian, >95% among Chinese, >97% among Malay, and >99% among Indian populations (Table S1 ). The probability of sharing one haplotype could be as high as >15% among Caucasian, Chinese, or Malay residents.

COVID-19 is a rapidly progressing pandemic with no specific vac- Because the convalescent SARS-CoV-2 specific T cells are allogeneic, bidirectional alloreactivity is a concern. For graft-ver- proportions of memory subsets collected from all six donors are displayed for CD3 + T cells before cell selection and in the IFNγ + populations after cell enrichment. (C) Cells from the product of Donor 5, which had the lowest purity, were cultured for 2 weeks to return to steady state. Thereafter, the cells were restimulated with SARS-CoV-2 peptides or CMV peptides or without peptide, demonstrating persistent functionality and specificity Pharmacokinetically, the level of antibodies universally drops after infusion because of consumption or natural metabolism. In contrast, T cells are a "living drug" and can expand in vivo. Accordingly, a unit of donor's blood may potentially treat more patients and the therapeutic effects of T cells may be more durable than that of plasma therapy. For a dose of 5x10 3 SARS-CoV-2 T cells/kg used in prior studies, 12 one unit of whole blood from each of our donor could generate sufficient cells overnight for 3 to 6 adult recipients and one blood-volume leukapheresis for 20 recipients, without the requirement for ex vivo culture for cell expansion. Pharmacodynamically, F I G U R E 2 TCRVβ spectratyping. TCRVβ spectratyping for cells before enrichment, after enrichment in the positive fraction, and in the negative fraction SARS-CoV-2 specific T cells may be used alone or synergistically with plasma therapy to establish immediately an adaptive immune status mimicking that after successful vaccination. Under the protection of adaptive immunity, the body will not depend solely on innate immune response, which may contribute to the cytokine storm and SARS pathogenesis. [19] [20] [21] Coronavirus-specific T cells have been shown to be crucial for virus clearance and may dampen further damage to the lung by the dysregulated, overactive innate immunity mediated by neutrophils, macrophages, and dendritic cells. [22] [23] [24] [25] For the SARS-CoV-2 specific T cells to be effective, the donor cells must share some HLA with the recipient for proper viral antigen presentation to the donor TCRs. Previous studies have demonstrated that viral-specific T cells from donors who shared only one HLA were efficacious. 26 With just six random donors, we estimated that > 88% of Caucasian and > 95% of Asian Singapore residents will share at least one HLA allele with the manufactured cells, suggesting that our approach is feasible for both small-scale and large-scale, offthe-shelf delivery model and manufacture-on-demand model with simple ethnic-group matching. [26] [27] [28] Extending from therapeutics to diagnostics, our data showing that virus-specific T cells can be detected easily regardless of the severity of COVID-19 suggest that a diagnostic assay can be devel- 

This protocol has been reviewed by the SingHealth Centralised Institutional Review Board for ethics approval. This approval is mutually recognized by National Healthcare Group Domain Specific

Review Board (DSRB).

We thank the donors for their blood donation; the staff at Singapore 

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