Persistence of ex vivo expanded tumour and pathogen specific T-cells after allogeneic stem cell transplant for myeloid malignancies (the INTACT study)

doi:10.21203/rs.3.rs-2962649/v1

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Persistence of ex vivo expanded tumour and pathogen specific T-cells after allogeneic stem cell transplant for myeloid malignancies (the INTACT study)

https://doi.org/10.21203/rs.3.rs-2962649/v1

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published 15 Sep, 2023

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Disease relapse and infections cause significant morbidity and mortality post-allogeneic stem cell transplant (HSCT), which remains the only cure for many myeloid malignancies. We investigated a novel combination of donor-derived, tumour-associated, antigen-specific T-cells targeting Wilm’s tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME), and multipathogen T-cells targeting CMV, EBV, Adenovirus and Aspergillus given prophylactically post-HSCT. Ten patients with acute myeloid leukaemia (n=6) or high risk myelodysplasia (n=4) who overexpressed WT1 and/or PRAME on diagnostic tumour samples received 1 infusion of multipathogen and 1-4 infusions of tumour-specific T-cells (all at 2x10⁷cells/m²). There were no infusion-related severe adverse events. Low level viral reactivations occurred (CMV n=5, EBV n=7, Adenovirus n=1), however none required treatment. There were no cases of viral tissue disease or invasive fungal infections. At a median 2 years post-transplant, overall survival was 80%, all surviving patients were in complete remission and 6/8 patients had ECOG 0/1. Acute GVHD occurred in 2/10 patients, chronic GVHD in another 2/10. Infusion was associated with rapid, sustained reconstitution of pathogen- and tumour-specific immunity as measured by MHC tetramer for CMV and T-cell receptor based clone tracking. This novel combination of T-cell therapies was safe and associated with excellent clinical outcomes.

Health sciences/Medical research/Translational research

Biological sciences/Immunology/Immunotherapy

Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia

Biological sciences/Cancer/Haematological cancer/Myelodysplastic syndrome

Biological sciences/Immunology/Bone marrow transplantation

Adoptive immunotherapy

donor-derived T-cells

virus specific T-cells

fungal specific T-cells

tumour specific T-cells

WT1

PRAME

allogeneic stem cell transplant

Acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) are the two most common indications for allogeneic haemopoietic stem cell transplantation (HSCT) in adults^{1, 2}, and HSCT remains the only curative therapy for MDS and many cases of AML. The primary cause of death post-transplant is disease relapse, accounting for 27% of deaths in the first 100 days and 53% in the first 3 years beyond 100 days¹. Relapse is lower in those with chronic graft versus host disease (GVHD)³ due to the graft versus leukaemia effect, and T-cell depletion has been associated with less GVHD but higher rates of relapse^{4, 5}. The goal of HSCT is to enhance GVL effect while minimising GVHD. Infusions of unmanipulated donor lymphocytes post-infusion have been utilised for this purpose but are limited in efficacy in AML^{5, 6}, and increase the risk of development of GVHD and myelosuppression.

One approach to specifically enhance graft versus leukaemia effect is to infuse lymphocytes enriched for specificity towards leukaemia cell associated antigens. Wilms’ tumour 1 antigen (WT1) is highly expressed in the majority of patients with AML⁷ and its detection following remission and transplant in both AML and MDS correlates with disease relapse^{8, 9}. Preferentially expressed antigen in melanoma (PRAME) is expressed in a variety of tumours including AML¹⁰, Hodgkin lymphoma¹¹, melanoma¹² and renal cell carcinoma¹³. PRAME expression is correlated with better prognosis in AML after chemotherapy and PRAME-reactive lymphocytes are detected in patients in remission after treatment¹⁴. Both WT1 and PRAME have low expression in normal healthy tissue (excepting PRAME expression in gonadal tissue¹⁰), making them attractive targets for immunotherapy. Several previous studies have used donor-derived T-cells targeting WT1, PRAME and other tumour-associated antigens in AML/MDS, showing their safety when administered in the post-transplant^{15, 16} and relapsed¹⁷ settings.

Viral and fungal infections also cause significant morbidity and mortality post-transplant. Infections with multiple organisms increase mortality¹⁸. Conventional antimicrobial pharmacotherapy is not available for all organisms and has limitations, including drug resistance, drug toxicity, cost, and it does not address the underlying immune deficiency. A number of previous studies have shown the safety of donor-derived virus-specific T-cells targeting cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (AdV) and fungal antigens for post-transplant opportunistic infections either as prophylaxis or treatment of established infection ^19–25.

In this Phase 1 trial we examined the safety, engraftment kinetics and clinical effects of a novel combination of donor-derived WT1 and PRAME tumour antigen (TAA) specific T-cells with multipathogen specific T-cells targeting CMV, EBV, AdV and Aspergillus infused prophylactically following allogeneic HSCT for AML or high risk MDS, for the prevention of relapse and infection.

Study design and participants

A prospective, single arm, phase I clinical trial was performed across two participating centres in patients receiving an allogeneic stem cell transplant from an HLA-identical family or unrelated donor for AML or high risk MDS. Detailed inclusion and exclusion criteria can be found in the Supplementary Methods. Written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. This study was approved by the institutional research ethics committee at each site prior to the recruitment of patients and was registered on the US National Institutes of Health Clinical Trials Registry as #NCT02895412.

Testing of diagnostic tissue for WT1/PRAME overexpression

Following informed consent, diagnostic tissue bone marrow samples were tested for WT1 and PRAME expression using an in-house droplet digital PCR (ddPCR) assay (see Supplementary Methods). WT1 overexpression was defined as greater than 250 copies/10⁴ABL copies and PRAME overexpression as greater than 280 copies/10⁴ABL gene copies in the bone marrow. These thresholds were defined based on 1 log above normal highest expression patterns described in previous studies^7,26. Patients who did not overexpress one or both markers or for whom there was no diagnostic sample available were excluded from participation.

Generation of cell products

T-cells for adoptive transfer were manufactured according to previously published methods with some modifications^27,28. T-cell cultures were initiated by stimulating mononuclear cells from transplant donor stem cell harvests with overlapping peptides (CMV pp65, Adenovirus hexon protein, EBV select, WT1 and PRAME (Miltenyi Biotec, Bergisch Gladbach, Germany)) or a water-soluble lysate from Aspergillus fumigatus (Miltenyi Biotech) for 16–24 hours at 37°C. Activated antigen specific T-cells were magnetically selected with clinical grade biotinylated anti-CD137 and anti-biotin microbeads (Miltenyi Biotec). CD137⁺ T-cells were co-cultured with antigen pulsed irradiated autologous mononuclear cells in complete AIM-V supplemented with IL-2, IL-7 and IL-15 for 11 days. Cytokines were replenished every 2–3 days and half media exchanges performed as required. For WT-1 and PRAME T-cell products, cultures were restimulated at day 11 with autologous peptide loaded mononuclear cells and cultured in complete AIM-V supplemented with IL-2, IL-7 and IL-15 for another 7 days. At the end of the culture period the T-cells were cryopreserved in aliquots containing sufficient cells for the target dose of 2x10⁷/m². Pathogen specific T-cells from individual cultures were combined into a multi-pathogen specific T-cell product prior to cryopreservation (equal cell numbers from each pathogen-specific culture to create a multi-component product at the target dose of 2x10⁷/m²). Prior to release, all products underwent quality control testing as described in the Supplementary Methods.

Treatment with TAA and multipathogen T-cells

The study schema is shown in Fig. 1. Patients were administered TAA T-cells (WT1 or PRAME specific, or both) and multipathogen T-cells by intravenous infusion on or after day 28 post-transplant. The cell dose for both T-cell products was 2 x 10⁷/m², and if a patient received both WT1 specific and PRAME specific T-cells, the cell dose was 1 x 10⁷/m² for each TAA product. Up to three further infusions of TAA T-cells were administered at 28-day intervals following this initial infusion. This infusion regimen was chosen because in previous studies by our group and others, a single infusion of multipathogen products was sufficient to generate measurable immune responses^{22, 25, 29, 30}, but up to 4 WT1-specific product infusions were required³¹. Immunosuppression was managed as detailed in the Supplementary Methods. Infusions were delayed for grade II or greater GVHD within 1 week prior to infusion, deranged hepatic or renal function, treatment with anti-lymphocyte globulin or treatment with > 1mg/kg of prednisone or equivalent.

Post-infusion monitoring and outcomes

Patients were monitored clinically for acute infusion reactions and were followed up at regular intervals for a period of 12 months post-transplant. Each review consisted of a full history, physical examination and clinical GVHD assessment and relevant laboratory studies, including assessment for viral reactivation by quantitative PCR and disease relapse with repeat bone marrow examination. Blood samples were also collected for correlative studies (see “Immune monitoring”) below. Patients were followed for relapse and death beyond the 12 month follow up period, until time of data cut off.

Adverse events were graded according to the National Cancer Institute’s Common Terminology Criteria for adverse events (NCI CTCAE), version 4.03. GVHD was graded according to standard criteria for acute³² and chronic³³ GVHD. The primary endpoint was safety of the T-cell infusions. Secondary endpoints are listed in the Supplementary Methods.

Immune monitoring

Post-infusion immune monitoring was performed on batched peripheral blood mononuclear cells (PBMCs) at specific time points for each patient using a mass cytometry panel comprising 40 monoclonal antibodies identifying 75 canonical subsets and a spectral flow cytometry panel of 20 surface markers delineating 49 cellular populations of interest (Supplementary Figure S1) as previously described^34,35. Data were acquired using a Helios Mass Cytometer (Fluidigm, Ontario, Canada) for mass cytometry samples and a BD FACSymphony Flow Cytometer (BD Biosciences, San Jose, CA, USA) for flow cytometry samples. HLA-restricted antigen specificity in post-infusion CD8⁺ T-cell populations was measured using phycoerythrin-conjugated virus-specific iTAg MHC class I human tetramers (MBL International, Woburn, WA, USA) where available.

T-cell receptor sequencing

T-cell receptor (TCR) deep sequencing was performed on donor, recipient (pre-transplant, post-transplant before T-cell infusion and after T-cell infusion) and cell product samples (Supplementary Figure S2) to characterise the cell products and their in vivo fate as well as the immune recovery of recipients. The complementarity determining region 3 (CDR3) of the T-cell receptor beta (TCRβ) chain was amplified and sequenced from the extracted genomic DNA using the ImmunoSEQ hsTCRβ kit (Adaptive Biotechnologies, Seattle, WA, USA) following the manufacturer’s protocol. For a detailed description of the assay and data analysis see Supplementary Methods.

Statistical analysis

D’Agostino & Pearson and Anderson-Darling tests for normal distribution were performed on T-cell product datasets and showed non-Gaussian distribution. Starting absolute cell count and fold expansion between pathogen- and tumour-specific products were compared using a Mann-Whitney test. P < 0.05 was considered significant. Statistical analysis was performed using Prism 9 for macOS (GraphPad Software, Inc., La Jolla, CA).

Participant characteristics

Ten patients were treated on the trial, 9 adult patients and 1 paediatric patient. The median age was 48 years (range 17–67), with 6 patients being transplanted for AML and 4 for high risk MDS. Conditioning was myeloablative in 8 patients and reduced intensity in 2 patients. CIBMTR Disease Risk Index was high for 5/9 adult patients and intermediate for 4/9. Half the patients (5/10) received in vivo T-cell depletion. All except one donor was fully HLA-matched to the recipient (the exception was a sibling donor mismatched at one HLA-DQB1 and one DPB1 locus). The median expression of WT1 on diagnostic bone marrow was 1442 copies/10⁴ copies ABL (0-3870), and PRAME 131 copies/10⁴ copies ABL (4-12300). Full participant characteristics can be found in Table 1.

Table 1

Participant characteristics
Patient	Age, years	Sex	Transplant Indication	Remission status at BMT	Lines of Therapy Pre-BMT	DRI	Conditioning	Donor Type	T-cell depletion	HLA match (mismatch)	Rec/Donor CMV serostatus	Immunosuppression at first TAA/multipathogen infusion	WT1 copies/10^4 ABL	PRAME copies/10^4 ABL
1	26	F	Therapy related AML	CR1	1	Int	MAC	MUD	ATG	11/12 (DPB1pm)	Neg/Neg	CsA	1464	NT
2	45	F	MDS EB1	PD	1	Int	MAC	MUD	ATG	11/12 (DPB1pm)	Pos/Pos	CsA	1750	17
3	53	M	MDS EB1	SD	1	High	MAC	Sib	None	10/10	Neg/Pos	CsA	350	1700
4	31	F	AML	CR1 – flow MRD +	1	Int	MAC	Sib	None	10/10	Pos/Pos	CsA	1140	4.1
5	67	M	AML	CR1	1	Int	RIC	MUD	ATG	8/8	Pos/Pos	CsA	3870	131
6	49	F	MDS/MPN/RS	PR	1	High	MAC	Sib	None	10/10	Neg/Pos	CsA	0	1670
7	52	M	AML	CR1 – flow MRD +	3	High	MAC	Sib	None	10/10	Pos/Pos	CsA	1900	29
8	57	F	AML	CR1	1	High	RIC	Sib	ATG	9/10 (DQB1, DPB1)	Pos/Pos	CsA	1464	131
9	47	F	MDS	SD	1	High	MAC	Sib	ATG	12/12	Pos/Pos	CsA	950	2990
10	17	M	AML	CR2	1	NA – paediatric	MAC	Sib	None	10/10	Neg/Neg	CsA	1420	12300
AML – acute myeloid leukemia, ATG – anti-thymocyte globulin, BMT – bone marrow transplant, CR – complete remission, CsA – cyclosporine, DRI – CIBMTR disease risk index, Int – intermediate, MAC – myeloablative conditioning, MDS – myelodysplastic syndrome, MDS EB1 – myelodysplastic syndrome with excess blasts 1 (5–9% bone marrow blasts), MPN – myeloproliferative disorder, MRD – minimal residual disease, MUD – matched unrelated donor, NA – not applicable, PBSC – peripheral blood stem cell, pm – permissive, NT – not tested, PD – persistent disease, PR – partial remission, RIC- reduced intensity conditioning, SD – stable disease Sib – sibling, SS – Sézary syndrome

Product characteristics

A total of 24 unique products were manufactured, 10 being multipathogen products containing equal proportions of pathogen specific components, 8 WT1-specific products and 6 PRAME-specific products. Two CMV seronegative patients had CMV seronegative donors, so a CMV product was not manufactured for these patients. As per protocol, for the paediatric patient, pathogen-specific T-cells targeting AdV were manufactured and included in the multipathogen product (Fig. 2E).

The median starting absolute cell count of the CD137⁺ fraction was similar for TAA products (0.03x10⁶ CD3⁺CD137⁺ cells per 100x10⁶ mononuclear cells; range 0.0036x10⁶ – 0.31x10⁶) and in the multipathogen products (0.07x10⁶ CD3⁺CD137⁺ cells per 100x10⁶ mononuclear cells; range 0.03x10⁶ – 0.40x10⁶, p = 0.05). Multipathogen and TAA T-cell products expanded briskly from the CD137⁺ fraction, with greater expansion in the TAA products (9188-fold (386-149882) vs 3335-fold (731-49074) respectively, p = 0.008). Median post-thaw viability across all products was 84.4% (60.8% − 92.5%). Median tumour antigen specificity measured by intracellular cytokine detection in the TAA product was 0.8% and 12.4% of CD3⁺ cells for WT1 and PRAME respectively. Median total pathogen specificity in the multipathogen product was approximately 35% (CMV = 11.1%, EBV = 9.4% and AdV = 11.6% of CD3⁺ cells, Asp = 14.2% of CD4⁺ cells). Detailed product information is shown in Fig. 2.

Administration of TAA and multipathogen products

A total of 38 infusions were administered over up to 4 timepoints for each patient. The first timepoint was 28 days post-transplant, provided neutrophil engraftment had occurred and safety criteria for infusion had been met, and subsequent timepoints were every 28 days thereafter. Patients received both multipathogen T-cells and TAA specific T-cells at timepoint 1, and only TAA specific T-cells at subsequent timepoints. Five patients received WT1-specific T-cells and six received PRAME-specific (one received both PRAME and WT1). All infusions were administered at a cell dose of 2 x 10⁷/m² for each of the multipathogen and TAA products. The target cell dose for all multipathogen and PRAME-specific products was reached. Culture failure for WT1 occurred in 4 of 9 (failure to expand n = 1, no antigen specific activity n = 2 or inadequate cell dose n = 1) and these products were not infused.

Safety and toxicity

A single adverse event occurred within the first 24 hours post-infusion, an instance of asymptomatic moderate hypertension that settled rapidly with administration of a calcium channel blocker. All severe adverse events (SAEs) are listed in Table 2. Five out of 10 patients treated reported no SAEs, and none of the reported SAEs in the remaining 5 patients were attributable to the T-cell infusions.

Table 2

Serious Adverse Events
Patient	Day Post-1st infusion	Grade	Caused by Infusion	Event
3	7	3	Unlikely	Chest pain
	18	3	Unlikely	Lower limb cellulitis
	49	5	Unlikely	Enterococcus colitis/sepsis
	49	5	Possible	Acute GVHD (gut)
	49	5	Unlikely	Hepatic veno-occlusive disease
	49	5	Unlikely	Acute renal failure
4	53	3	Possible	Chronic GVHD (lung)
8	40	3	Unikely	Recurrent ESBL sepsis
10	53	3	Unlikely	Aspiration
ESBL – extended-spectrum beta-lactamase, GVHD – graft versus host disease.

Survival, disease response and GVHD

At a median 740 days post-transplant (80-1685), 8 of 10 patients remain alive and in complete disease remission (Fig. 3 and Table 3). There were 2 deaths. Patient 3 had multiple acute post-transplant complications and died on day 82 of Enterococcus sepsis secondary to grade 4 aGVHD of the gut with associated liver and renal failure. Prior to T-cell infusion, this patient had developed grade 3 aGVHD of the gut and skin post-transplant that had been treated with methylprednisolone. Prior to adoptive T-cell therapy symptoms and histological changes had resolved and steroids had been completely weaned. Patient 6 had a high risk MDS/myeloproliferative overlap syndrome with complex cytogenetics and had failed azacitidine treatment pre-transplant. There was persistent disease on bone marrow biopsy performed prior to transplant conditioning, on day 29 following engraftment prior to T-cell infusion, and on all subsequent follow-up bone marrow biopsies post T-cell infusion. Mixed donor/recipient chimerism was seen on these biopsies, the patient was transitioned to palliative care and died on day 272 of persistent disease.

Table 3

– Clinical Outcomes
Patient	Infusions given	Post-infusion course and outcome	Viral reactivations (site, peak cps/mL)	Antiviral treatment post-infusion	aGVHD	cGVHD	Hospital days, first 12 months post-transplant (post 1st infusion), reason	ECOG at 12 months post-transplant (or last follow up if sooner)	Status within follow up period, cause of death (day post transplant)	Days of follow up post-transplant	Long term outcome
1	1 MP 4 WT1	Well, disease remission, no infections or GVHD	BKV (u), EBV (b)^BLQ	None	-	-	30 (0)	0	Completed follow up	1685	Alive
2	1 MP 4 WT1	Well, disease remission, no infections or GVHD	BKV (u), EBV (b)^BLQ, CMV (b)^BLQ	None	-	-	22 (0)	0	Completed follow up	1051	Alive
3	1 MP 1 PRAME	Complicated course post-1st infusion with severe	EBV (b, 515)	None	Pre-infusion grade 3 (GIT, skin), post-infusion grade 5 (GIT)	-	49 (21) aGVHD of the gut, VOD, and Enterococcus sepsis	5	Expired, acute GVHD, sepsis and VOD (82)	82	Deceased
4	1 MP 2 WT1	Well, cGVHD lung	BKV(u, 1.27 x 10^8 cps), CMV (b)^BLQ, HHV6 (b)^BLQ	None	-	Moderate (lung)	21 (3) pneumococcal pneumonia	2	Completed follow up	1289	Alive
5	1 MP 4 WT1 4 PRAME	1st infusion delayed until d63 due to drug-related LFT derangement. Well, disease remission, no infections or GVHD	CMV (b, 1400), EBV (b)^BLQ, HHV6 (b)^BLQ	None (12 days valganciclovir pre-infusion for CMV viraemia)	-	-	19 (0)	0	Completed follow up	795	Alive
6	1 MP 3 PRAME	Persistent MDS pre-infusion, progressive disease	EBV (b)^BLQ, HHV6 (b)^BLQ	None	-	-	59 (32) Palliative care	5	Expired, sepsis secondary to progressive disease (272)	272	Deceased
7	1 MP 1 PRAME	Declined 2nd and subsequent infusions due to COVID pandemic. Well, disease remission, no infections	CMV (b)^BLQ, EBV (b)^BLQ	None	-	Moderate (eyes, oral mucosa, liver)	20 (0)	2	Completed follow up	1002	Alive
8	1 MP 2 WT1	Well, disease remission, no GVHD	HHV6 (b) ^BLQ, CMV (b, 6102), EBV (b, 320)	None	-	-	50 (17) E. Coli urosepsis.	0	Completed follow up	684	Alive
9	1 MP 2 PRAME	Did not receive 3rd/4th infusion due to cytopenias secondary to poor graft function. 100% donor chimerism on BMBx. Well, disease remission, no infections of GVHD.	CMV (b) ^BLQ, EBV (b, 625)	None	-	-	30 (0)	0	Completed follow up	553	Alive
10	1 MP 2 PRAME	Did not receive 3rd/4th infusion due to development of acute GVHD. Well, no infections or disease relapse	EBV (b)^BLQ, HHV6 (b)^BLQ, Adenovirus (b, f)^BLQ	None	Post-infusion grade 2 (liver)	-	73 (4) Liver biopsies x2	1	Completed follow up	390	Alive
aGVHD – acute graft versus host disease, BMBx – bone marrow biopsy, b – blood, cGVHD – chronic graft versus host disease, DVT – deep venous thrombosis, ECOG – Eastern Co-operative Oncology Group score, f – faeces, ESBL – extended-spectrum beta-lactamase GIT – gastrointestinal, GVHD – graft versus host disease, MP – multipathogen, PRAME – Preferentially Expressed Antigen in Melanoma, WT1 – Wilms’ Tumour 1, u - urine, VOD – venoocclusive disease, BLQ - below limit of quantitation

There were no other instances of disease relapse. Two patients developed aGVHD (grades 2–4) and 2 patients cGVHD post T-cell infusion. One patient developed aGVHD prior to infusion (patient 6), and this returned post-infusion as described above. Patient 10 developed acute liver GVHD (grade 2) associated with poor adherence to immunosuppressive medication on day 85 post-transplant, day 51 post-infusion, that improved with steroid treatment but persisted to end of trial follow up. Patient 4 developed moderate cGVHD of the lung, and patient 7 moderate cGVHD involving the eyes, mouth and liver.

Infection

Low level viral reactivations occurred (CMV n = 5, EBV n = 7, AdV n = 1). There were no cases of viral tissue disease or EBV post-transplant lymphoproliferative disorder. There were no invasive fungal infections. The instances of CMV and EBV reactivation and their temporal relationship to the administration of the multipathogen product targeting these infections is shown in Fig. 4. Patient 5 reactivated CMV on day 21 post-transplant, but had persistently abnormal liver function tests (LFTs) that precluded T-cell infusion until day 63. This patient received a total of 21 days of oral valganciclovir to treat the CMV reactivation prior to the multipathogen T-cell infusion. A liver biopsy showed no evidence of CMV infection or GVHD, and the deranged LFTs were attributed to a drug adverse effect. Adenovirus reactivation occurred only in patient 10, detected below the limit of quantitation on day 90, 105, 114, 236 and 254 after transplant, resolving spontaneously and without any clinical manifestations. All other instances of viral reactivation were asymptomatic and resolved spontaneously following administration of the multipathogen product, without administration of antivirals.

Performance status and hospitalisations

Of the 8 patients alive at 12 months, 5 patients had an ECOG status of 0, 1 patient had a status of 1 and 2 patients had a status of 2. The median duration of hospital inpatient stay in the first 12 months post-transplant was 30 days (19–73), and post-infusion 1.5 days (0–30). 5 patients had no hospital inpatient days following their first infusion (Table 3).

Immune reconstitution

Immune reconstitution post T-cell infusion was assessed in 9 of 10 patients using a 20-marker flow cytometry panel. Rapid reconstitution of both CD4⁺ and CD8⁺ T-cells was seen within the first 30 days post-first infusion. The median CD3⁺ pre-T-cell infusion was 0.14x10⁹/L, CD4⁺ 0.039x10⁹/L, CD8⁺ 0.08x10⁹/L, at day 30 post-infusion CD3⁺ 0.56x10⁹/L, CD4⁺ 0.23x10⁹/L, CD8⁺ 0.39x10⁹/L and at day 90 post-infusion CD3⁺ 0.86x10⁹/L, CD4⁺ 0.20x10⁹/L and CD8⁺ 0.53x10⁹/L. The predominant subset within both CD8⁺ and CD4⁺ populations was CD45RA⁻CD62L⁻ effector memory (Tem) cells, with a steady increase over a one year period in CD8⁺CD45RA⁺CD62L⁻ terminally differentiated effector memory (Temra) cells, and CD8⁺CD57⁺ differentiated cytotoxic T-cells³⁶. This is consistent with findings by our group in previous studies^{25, 34, 35, 37, 38}. Progress of all leukocyte populations post-infusion can be seen in Supplemetary Figure S3.

CMV specific immune reconstitution was assessed with MHC tetramer. Four of 6 patients for whom a tetramer was available showed significant CMV-specific tetramer-positive populations throughout the follow up period. Patient 2 had low level CMV reactivation below the level of quantitation first detected on day 11 post-multipathogen T-cell infusion, and an increase in the proportion of CMV tetramer positive CD8⁺ T-cells was observed corresponding with spontaneous clearance of viraemia. No further episodes of reactivation occurred and persistent tetramer-positive T-cells were detectable to the end of follow up (Fig. 5A).

T-cell receptor sequencing

T-cell repertoire characteristics. TCR repertoire characteristics were determined for 8 of 10 patients for whom samples were available and for all cell products infused (Fig. 6 and Supplementary Figure S4). The repertoire space occupancy of various clone sizes was used to characterise the architecture of the repertoire, Simpson’s inverse diversity index to assess sample diversity and the Gini coefficient to describe sample evenness. All T-cell products were dominated by hyperexpanded clones, had low diversity and were more uneven compared to pre-HSCT patient or donor samples, reflecting the ex vivo expansion process. Pathogen-specific products were more polyclonal than TAA products and Aspergillus and AdV products had greater sample diversity and evenness than CMV or EBV products (Figs. 6A and 6B). There was no consistent pattern in the recipients with regard to the global repertoire metrics, likely due to the variety of transplant regimens that included reduced intensity and myeloablative conditioning regimens with or without T-cell depletion.

One of the goals of post-transplant adoptive T-cell therapy is to rapidly restore competent cellular immunity. Morisita’s overlap index was used to assess the change in TCR repertoire over the follow-up period for 6 patients for whom later samples were available (Fig. 6C). The repertoire on the last timepoint was used as the reference sample to assess how quickly this repertoire was established. In patients 1, 2, 4 and 8 in whom T-cells were administered on time at day 28 there was a stepwise change in repertoire between infusions 1 and 2 reflecting the rapid reconstitution of immunity associated with T-cell infusion. In patient 5 who had a late infusion there was a smaller change. There was no observed change in repertoire attributable to T-cell infusion in patient 6, who had persistent MDS throughout the study.

Clone tracking. Clones from the T-cell products were annotated for antigen specificity and tracked post-infusion. Data was available for 8/10 patients (Patient 3 early death, Patient 7 study withdrawal). Some pathogen specific clones for each antigen were detectable before T-cell infusion. These were likely to have come from T-cells infused with the stem cell product. In all cases, clones for each specificity were detectable after infusion apart from patient 2, in whom no WT1-specific T-cell clone could be identified. Product clones were noted to expand immediately after infusion and persisted to the end of follow up. We observed antigen stimulated expansion of product-derived clones in a case of CMV reactivation (Fig. 5B). The rapid rise in CMV-specific T-cells measured by MHC tetramer included product clones that had been identified by TCR sequencing. In vivo expansion after T-cell infusion was also observed in cases of EBV (Fig. 7A) and Aspergillus (Fig. 7B), as well as for tumour-associated antigens (Fig. 7C).

Disease relapse and infection are significant contributors to transplant mortality and this study utilised a novel combination of donor-derived tumour, viral and fungal antigen-specific T-cell products given prophylactically post-allogeneic stem cell transplant to assess safety, clinical course and effects on immune reconstitution. Infusion-related toxicities in the first 24 hours were mild and infrequent, and no SAEs were attributable to T-cell infusion. Rates of acute and chronic GVHD observed during the follow up period were within expectations for this post-allogeneic transplant population. Overall survival outcomes were good, with 8 out of 10 patients alive at a median of more than 2 years post-transplant. Both deaths occurred within the first year post-transplant. Pre-transplant disease risk index was intermediate or high in all patients but only one patient, who had persistent disease throughout, died from their disease. The other patient died from transplant-related complications. The surviving 8 patients have maintained excellent quality of life. These outcomes show that this approach is feasible and that infusion of these products is not associated with adverse outcomes.

In this study, pathogen-specific products targeting CMV, EBV, AdV and Aspergillus spp. were manufactured separately and then pooled into a multipathogen product in equal proportions. In previous studies utilising multipathogen products^{30, 39}, including by our group^{24, 25}, products have been manufactured by stimulating mononuclear cells with multiple antigens in a single culture. Our group’s experience is that products manufactured in this way exhibit dominance of T-cells of certain antigen specificities, particularly of the herpesviruses. The pathogen specific products utilised in this trial had reactivity that reliably crossed all pathogens unlike those of another study in which more than half of products recognised 0 to 3 of the five pathogen targets³⁰. Manufacture of pathogen specific T-cells was robust with no manufacturing failures. PRAME specific T-cell products were successfully manufactured in all cases in which PRAME was overexpressed in the original AML or MDS sample (n = 6), WT1 T-cell manufacture was unsuccessful in 4/9 cases. A broader set of antigen targets or the use of transgenic T-cell receptor modified cells may provide a solution to this manufacturing inconsistency.

The high incidence of infections and increase in mortality that is compounded by multiple infections in the post-transplant setting has been shown in multiple studies^{18, 40–42}. In our patients there were no fungal infections or viral tissue infections, and all viral reactivations post-infusion were at low viral copy number and required no specific treatment. Infusion of T-cells induced rapid early T-cell immune reconstitution that persisted to the end of the study and no reactivations occurred in the follow-up period. Although this Phase 1 study did not have a control group, we have previously observed that patients who have robust immune reconstitution exhibit good viral control³⁴.

Our manufacturing method facilitated the identification of individual pathogen clones and their in vivo tracking. Product clones were detectable in all recipients for most targets and were detectable until the end of follow-up in all patients, confirming previous studies of donor-derived antigen-specific T-cell engraftment²¹. Expansion of pathogen specific clones with antigen stimulation was observed, confirming known biologic activity of CMV and EBV specific adoptive T-cell transfer. A novel finding in this study was the robust in vivo expansion of tumour antigen and Aspergillus specific clones. In contrast to other studies which have tested the persistence of tumour antigen-specific T-cells^{15, 43}, we identified tumour antigen-specific clones after infusion in 7 of 8 cases persisting to the end of follow up. Better engraftment of clones infused was observed when T-cell products were infused early after transplant. There were no cases of relapse in the patients who achieved remission after transplant.

We recognise the limitations of this study which recruited a small number of patients in a non-randomised fashion. Nevertheless, our findings strongly support the engraftment and long term persistence of ex vivo expanded T-cells derived from stem cell donors including those targeting both infectious and malignant antigens.

Acknowledgements

This study was funded by the Australian National Health and Medical Research Council (NHMRC) (PG1088118). E.B. was an NHMRC Early Career Fellow (ECF1089398), a NSW Cancer Institute Early Career Fellow (ECF181576) and a current NSW Health Cell and Gene Therapy Fellow. W.J. was supported by a scholarship from the Haematology Society of Australia and New Zealand and Leukaemia Foundation Australia, and is a current Leukaemia Foundation Australia Fellow. We are grateful to Gillian Huang and Elissa Atkins for clinical trial management. Microchimerism assays, DNA quantification, and flow cytometry were performed at the Westmead Scientific Platforms, which are supported by the Westmead Research Hub, the Westmead Institute for Medical Research the Cancer Institute of New South Wales, the National Health and Medical Research Council, and the Ian Potter Foundation.

Author Contributions

E.B. and D.G. designed the study and wrote the trial protocol. W.J., M.G., C.M.B., K.M., D.J.G. and E.B. recruited and treated patients on the study. S.A., R.S. and L.E.C. performed T-cell product manufacturing. W.J., J.S., K.H.L., G.C-G., R.S., R.B. and H.M.M. performed correlative studies. W.J., S.A., R.S., E.P., A.C., N.M., B.G. and E.B. performed data analysis. All authors contributed to the writing of and approved the final manuscript.

Competing Interests

L.E.C., K.M., E.B., and D.J.G hold patents in adoptive cell therapy for opportunistic infection and malignancy. E.B. declares advisory board membership for IQVIA, Abbvie, MSD, Astellas, Novartis, BMS, Bastion Education and research funding from MSD. The remaining authors declare no competing interests.

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Persistence of ex vivo expanded tumour and pathogen specific T-cells after allogeneic stem cell transplant for myeloid malignancies (the INTACT study)

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Abstract

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Introduction

Methods

Study design and participants

Testing of diagnostic tissue for WT1/PRAME overexpression

Generation of cell products

Treatment with TAA and multipathogen T-cells

Post-infusion monitoring and outcomes

Immune monitoring

T-cell receptor sequencing

Statistical analysis

Results

Participant characteristics

Product characteristics

Administration of TAA and multipathogen products

Safety and toxicity

Survival, disease response and GVHD

Infection

Performance status and hospitalisations

Immune reconstitution

T-cell receptor sequencing

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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