HOW DO I….manufacture T cells for cellular therapies

Low-dose memory T cell DLI for infection prophylaxis

Michael Maschan, MD

Dmitriy Rogachev National Medical Center

of pediatric hematology, oncology and immunology

EBMT 2019, Frankfurt-am-Maine

Conflict of interest disclosure

• Lecturer fee from Miltenyi Biotec

Conflict of interest II

I do not manufacture memory T cells

Graft processing and apheresis

Flow cytometry

Introduction to αβ T cell depletion

T cellsMonocytes

Myeloid precursors

CD34+ stem cells

γδ T

αβ T

γδ T

αβ T

• Developed by R.Handgretinger (Chaleff, Cytotherapy, 2006)

• Used successfully across the globe in different indications (Bertaina, Blood, 2014; Lang, BMT, 2015; Balashov, BBMT, 2015; Maschan, BMT, 2016; Im, BMT, 2016; Jacoby, PBC, 2017; Locatelli, Blood, 2017)

αβ T cell depletion: overall results in malignant indications, 2012-2018

0

0.2

0.4

0.6

0.8

1

0 1 2 3 4 5 6 7

time, years

HAPLO MUD

OS HAPLO @3y 76%, n 216OS MUD @3y 65%, n 91

other JMML NHL

T-ALL AML B-ALL

A.Bogoyavlenskaya, EBMT2019, Pediatrics 3, Tuesday, March 26th

αβ T cell depletion: overall results in non-malignant indications (excl. SCID)

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0.2

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Time, years

OS HAPLO @3y 85%, n 57OS MUD @3y 82%, n 186

A.Laberko, EBMT2019, Pediatrics 4-5, Tuesday, March 26th A.Maschan, EBMT2019, OS17, Wednesday, March 27th

2%13%

23%

62%

Thalassemia

IBMF

SAA

PID

TCR alpha/beta depletion transplant-related mortality

0

0.2

0.4

0.6

0.8

1

0 1 2 3 4

years after HSCT

Transplant-related mortality

TRM 15±2%, n=317

Viral41%

Bacterial20%

Other inf14%

MOF11%

GVHD9%

IM5%

Causes of treatment-related death

TCR alpha/beta depletion: impact of viral infections

• N = 182

• CMV: still problematic

• 90% of cases recipient cmv+

• D-/R+ HR 3,7

• CMV disease in 6%

• In 5 pts cause of death

0

0.2

0.4

0.6

0.8

1

0 10 20 30 40 50 60

weeks after HSCT

Cum Inc CMV 0,51 (95% CI 0,44-0,6)Cum Inc EBV 0,33 (95% CI 0,26-0,42)

CMV EBV

A.Laberko, BBMT2016

0

0.2

0.4

0.6

0.8

1

0 10 20 30 40 50 60weeks after HSCT

Cum Inc CMV_GVHD 67% (95% CI 56-80)Cum Inc CMV_no GVHD 40% (95% CI 32-50)

GVHD no GVHD

p=0,003

TCR alpha/beta depletion: impact of viral infections

Adeno: fulminant killer

• 50% of fatal viral infections • Fulminant• Often without GVHD• Monitoring problematic• Preemptive therapy not very robust

A patient after bone marrow transplantation,liver biopsy with features of fulminant necrotisinghepatitis with positive IHC staining against AdV.

TCR alpha/beta depletion immune recovery: qualitative

• Naïve T-cell count/ml TCR Vβ diversity vs time

I.Zvyagin, Leukemia 2016D.Balashov, BBMT 2015M.Maschan, BMT 2016

Next goal

Improve pathogen-specific immune reconstitution to prevent TRM caused by common post-transplant pathogens

Cell therapy for viral infections post-transplant

• Based on ex-vivo stimulation/expansion (Rooney, Lancet, 1995)• Single virus• Multi-virus

• Based on direct selection from donor peripheral blood (Feuchtinger, BJH, 2006)

• Both methods de facto are based on selecting/expanding memory T cells

• Demand certain level of technical expertise and GMP facility and investment

CD45RA depletion: rationale

• Fundamental principle: alloreactivity is concentrated in the the naive- T cell compartment (Anderson et al, JCI,2003; Chen et al, Blood, 2004)

• T-cell fraction depleted of CD45RA has decreased (>1 log) alloreactive potential (Distler, Haematologica, 2011; Teschner, BMT, 2013)

• Retains broad pathogen reactivity (Bleakley,BBMT, 2014, Teschner, BMT, 2013)

• CD45RA depletion procedure was developed as a graft processing method for prevention of GVHD

Use of low-dose donor memory T cell to foster immune recovery and provide protection from common (viral) pathogens ?

• Lund case report – EBMT 2014

• SCID patient, severe multi-virus infection

• No immune recovery• 24k CD3/kg infusion at d+84

(Brodszki et al. Orphanet Journal of Rare Diseases, 2016)

• Safe dose?• Persistence?• Technical practicality?• Cost?

Rationale behind memory cell approach

• Prophylactic use• Frequency of the event of interest (viremia) >50%• High direct and (especially) indirect costs of control• Risk of pathology due to tissue damage in the setting of late

therapy (Immune reconstitution inflammatory syndrome, IRIS)

• Memory cell (naïve (CD45RA+) T cell depletion)• Polyclonal• Multi-pathogen reactive• Represents repertoire of T cells reactive towards common

pathogens within family (haplo setting)• Automatic single-step production from whole

blood/apheresis, no clean room, no culture

• Small dose• Physiologic immune response starts from few (single?) T cells• Less GVHD risk (non zero risk!)• ? Effective enough in the prophylactic setting• Cost containment

CD45RA depleted DLI: End-points

• Safety• CI of aGVHD/cGVHD

• Laboratory correlates• CMV-reactive T-cells in PB (pp65 peptivator, Miltenyi Biotec, γIFN

Immunospot assay)

• CMV DNA in PB

• Clinical• Transplant-related mortality

• GVHD characteristics

CD45RA depleted DLI: the Moscow experiencePilot trial schema

CMV PCR monitoring

ELISPOT 1 ELISPOT 2 ELISPOT 3 ELISPOT 4 ELISPOT 5

HSCT DLI 1 DLI 2 DLI 3

day 0 30 60 90 120 360

Overall schema

DLI dosing

Dose 1 Dose 2 Dose 3

MUD 100x103/kg 200x103/kg 300x103/kg

HAPLO 25x103/kg 50x103/kg 100x103/kg

CD45RA depleted DLI post-engraftment: pilot trial population

• 56 patients

• April 2014 – October 2015

• MUD n-33

• Haplo n – 23

• 60% CMV PCR+ before DLI

• Follow-up – 15mo (8-24)

• 143 infusions

IBMF4% Lymphoma

8%

PID8%

MDS/MPD6%

SAA16%

ALL21%

AML37%

Maschan et. al., BMT, March 2018

CD45RA depletion: technical results

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1

2

3

4

5

6

Log depletion CD45RA

0

20

40

60

80

100

Recovery CD45RO, %

≈ 2% of donorsmedian = 3,8 (3-5,3) median = 37% (4-80)

DLI composition after CD45RA depletion

CD45RA depletion

CD45RA depleted DLI pilot trialSafety: graft-versus-host disease

43 pts without prior aGVHD10 pts without prior aGVHD

Chronic GVHD

CD45RA depleted DLI pilot trialSafety: GVHD characteristics and outcomes

Case Grade Site GVHDbefore

Time, day postDLI

Response Outcome Commentary

1,MUD

3 GI no 113 post DLI2 200/kg

Yes Death, sepsis

2, Haplo

2 Skin Yes ongoing no Death, adeno

Prolonged adeno beforeDLI

3, Haplo

2 Skin+GI Yes 14 post DLI 3100/kg

Yes alive

4, MUD

2 Skin No 59 post DLI1 100/kg

Yes Death, adeno

Fulminant, on steroids

5,Haplo

2 Skin+GI Yes 18 post DLI2 50/kg

Yes Alive, mild skin cGVHD

6, Haplo

2 GI Yes Ongoing Yes Alive Concomitant adeno colitis

CD45RA depleted DLILaboratory correlates: CMV pp65 peptide pool reactivity by gamma-IFN ELISPOT

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200

400

600

800

1000

1200

1400

before DLI after DLI

median spots/300k MNC

before after2 304

p=0,009

CD45RA depleted DLIantigen exposure correlates with CMV-reactive T cell expansion

CMV ELISPOT+ CMV ELISPOT- Fisher’s

CMV PCR+ 26 0 p <0,0005

CMV PCR- 5 7

0

200

400

600

800

1000

1200

1400

cmv_0 cmv_1

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200

400

600

800

1000

1200

1400

cmv_0 cmv_3

< 10 spots/300K MNC at start > 10 spots/300K MNC at start

Naïve T lymphocyte recovery

Final success still highly dependent on thymus function

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200

400

600

800

1000

30 60 90 120 180 360

Me

dia

n T

na

ïve

Time, days

CMV-

CMV (0) EBV (335) ADV (261)

CMV+

CMV (387) EBV (0) ADV (1)

EBV-

CMV (446) EBV (1) ADV (19)

EBV+

CMV (1) EBV (36) ADV (1)

AdV-

CMV (351) EBV (265) ADV (0)

AdV+

CMV (0) EBV (0) ADV (38)

Reactivity towards common viral pathogens among healthy donors (n= 147)

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10

100

1000

CM

V

CMV ELISPOT, spots/300 000

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100

1000

EB

V

EBV ELISPOT, spots/300 000

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10

100

1000

Ad

V

AdV ELISPOT, spots/300 000

156

2954

CMV, EBV

6% CMV, AdV

9%

EBV, AdV

4%

CMV

10%

AdV

2%

CMV, EBV,

AdV

61%

Non-reactive

8%

Positive = >5 spots/300 000 MNC

CMV

158EBV

75ADV

63

Allo

59PHA

Y. Bayzanova, Poster EBMT2019, Monday, March 25th

R.Nikolaev, Poster, EBMT2019, Tuesday, March 26th

γ-ELISPOT with common viruses and haploidentical recipient stimulator cells

CMV EBV ADV Allo

1 158 75 63 59

2 317 21 44 8

3 138 88 82 354

4 135 308 76 5

5 213 86 - 8

Ongoing randomized trial of memory (CD45RA) DLI

• To evaluate the safety and efficacy of co-infusion and planned DLI of low-dose donor memory T cells

• NCT02942173, Start Oct 2016, >100 recruited, ongoing

High-risk leukemiaαβ T-depleted HSCT R

CD45RA- DLI

Control

Day 0 Day 60Day 30 Day 90

25k/kg 50k/kg 50k/kg50k/kg

? Safety (aGVHD)? Efficiency (prevention of CMV viremia)

Zhanna Shekhovtsova, EBMT2019 Oral Abstract, OS19, Tuesday, March 27th

ATG serotherapy and αβ T-depleted HSCT

• Most groups use ATG• To secure engraftment (depends on the

intensity of preparative regimen)• To guarantee GVHD prevention

• ATGAM • 50 mg/kg (Balashov, BBMT, 2015)

• Fresenius• 12mg/kg (Locatelli, Blood, 2017)• 15mg/kg (Lang, BMT, 2015)• 30mg/kg

• Thymoglobulin (Maschan, BMT, 2018)• 5mg/kg

Anti-lymphocyte serum

• Non-selective lymphodepletion

• Variable PK

• Optimal dose ?• Weight-based?• ALC-based?

• Therapeutic drug monitoring?

Choice of targeted immunomodulation to replace ATG

Abatacept

• IgG1–CTLA4 fusion

• Blocks CD80/86 on APC

• GVHD prophylaxis (Jaiswal et al, TranspalntImmunology, 2017; Watkins et al. ASH2017 abs#212)

Tocilizumab

• Antibody to IL-6 receptor

• GVHD prophylaxis (Kennedy et.al, Lancet Oncol. 2014 Dec)

Key features- non-lymphodepleting- less interference with

- memory T responses- NK cytotoxicity- γδ T responses

Retrospective analysis

• To compare outcomes among children with acute leukemia, transplanted with versus without anti-thymocyte globulin

• To evaluate effect of the omission of ATG on the immune recovery at day+30

• Includes patients from two prospective trials and extended clinical cohort

• Inclusion criteria:• Acute leukemia

• Complete remission at Tx

• αβ-depleted HSCT

ASH2018 Oral abstract #481, Sunday, December 2, 2018

Preparative regimen and GVHD prevention

Basic regimen

Drug Σ Dose Days

Treosulfan(TBI 12 Gy in ALL > 3 y.o.)

42 g/m2 -5,-4,-3

Thiotepa 10 mg/m2 -6, -5

Fludarabine 150 mg/m2 -6,-5,-4,-3,-2

Rituximab 100 mg/m2 -1

Bortezomib 1.3 mg/m2 -5,-2,+2, +5

Drug Σ Dose Days

ATG rabbit (Genzyme) 5 mg/kg -5,-4

Drug Σ Dose Days

Tocilizumab 8 mg/kg -1

Abatacept 10 mg/kg -1 ,7, 14, 28

Basic characteristics of the compared groups

Group 1 (Thymoglobulin), n=98 Group 2 (Tocilizumab + abatacept), n = 67

Age 8,6 9,1

Gender m:f 57:41 40:27

αβ T dose, k/kg 17 14

CD34+ dose, mio/kg 8 10

AML : ALL 49:49 18:49

Treosulfan:TBI 69:29 23:44

Haplo, % 73 93

CD45RA DLI +, % 80 52

(post-engraftment) (day 0 AND post-engraftment)

Follow-up, years 2,6 (0,9-4,5) 1,2 (0,3-3)

p

ns

ns

ns

ns

0,003

0,0001

0,002

0,002

0,001

Outcomes 1 Engraftment

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0 10 20 30

Time, days

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0 10 20 30

Time, days

ATG+ (n=98) ATG- (n=67)

Death before d+30 3 0

No engraftment 1 1

Engraftment 93 (99%) 66 (99%)

Median day ANC > 500 +13 +12

Outcomes 2 graft-versus-host disease

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0 20 40 60 80 100

Time, days

aGVHD II-IV ATG+ 12%

aGVHD II-IV no ATG 12%

p=0,9

ATG+ (n=98)

ATG-(n=67)

aGVHD II-IV 11 8

aGVHD III-IV 4 4

cGVHD 10 7

cGVHD severe 2 2

on IST >1 y 2 20

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1

0 1 2 3 4 5

Time, years

cGVHD@2y ATG + 10%

cGVHD@2y no ATG 9%

p=0,9

Outcomes 3 Transplant-related mortality and relapse incidence

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Time, years

TRM@2y ATG+ 12%

TRM@2y ATG- 1,5%

p=0,015

ATG+ (n=98)

ATG-(n=67)

TRM 12 1

TRM 100d 8 1

Infection 10 1

GvHD 1 0

Other 1 0

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1

0 1 2 3 4 5

Time, TRM

CIR@2y ATG+ 22%

CIR@2y ATG- 19%

p=0,82

ATG+ (n=98)

ATG-(n=67)

Relapse 22 11

Median, m 5,5 5

K- PHA CMV (283) EBV (76) AdV (157)

Donor peripheral blood

K- K+ CMV (386) EBV (0) AdV (8)

Recipient day 32 after HSCT

K- K+ CMV (70) EBV (0) AdV (0)

Recipient day +46

Progressive disseminated adenoviral disease

CMV (34)K- PHA EBV (40) AdV (9)

СD45RA-depleted DLI after thawing

K- PHA CMV (283) EBV (76) AdV (157)

Donor peripheral blood

αβ ATG- αβ ATG+

3rd Quartile 0,126 0,062

Median 0,038 0,003

1st Quartile 0,013 0,000

Mann-Whitney p=0,0003

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5 No ATG ATG+

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0.6

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1

0 1 2 3 4 5

Time, years

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0.4

0.6

0.8

1

0 1 2 3 4 5

Time, years

CIR αβ T > med 19%CIR αβ T < med 32% p 0,08

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0.6

0.8

1

0 1 2 3 4 5

Time, years

TRM αβ T > med 12%TRM αβ T < med 0% p 0,005EFS αβ T > med 81%

EFS αβ T < med 56% p 0,002

cells/mcl

Immune reconstitution and outcome αβ T cells day+30

• polyclonal serotherapy is not an essential component of

αβ T-depleted HSCT in leukemia

• replacement of ATG (thymoglobulin) with toci/abata

• associated with improved early recovery of T cells and lower TRM

• does not compromise engraftment (at least with intensive conditioning) and GVHD control

• first results of the randomized trial will be presented by ZhannaShekhovtsova, EBMT2019 Oral Abstract, OS19, March 27th

Interim conclusions

Key practical points for CD45RA-negative DLI

• Can be prepared from PB, stimulated and unstimulated apheresis

• Final results of the manual and automatic procedures are equal

• Donor should be tested for• T-cell reactivity to common pathogens (CMV, Adeno, EBV)

• RA/RO distribution plot

• Preferably cell product should be also tested

• Can be used both fresh (preferred for small doses) and thawed

Key practical points for CD45RA-negative DLI

• Safe at doses 25-100k/kg in haplo after engraftment

• Safe at doses 100-300k/kg in MUD after engraftment

• Safe at 25k per kg at day 0

• To be used at day 0 the problem of ATG should be addressed (PK monitoring, timing, substitution)

• In vivo expansion critically depends on antigen exposure

• May cause IRIS – inflammatory damage to infected tissue – hard to differentiate from GVHD

H&E IHC Adeno

Acknowledgements