Marker Therapeutics : 2019 ASBMT Presentations

Administering AML-directed DLIs to

patients with AML or MDS Post-

Allogeneic HSCT Relapse

Premal Lulla, Swati Naik, Ifigeneia Tzannou, Shivani Mukhi, Manik Kuvalekar, Catherine Robertson, Carlos A Ramos, George Carrum, Rammurti Kamble, Jasleen Randhawa, Adrian P Gee, Bambi Grilley Malcolm K Brenner, Helen E Heslop, Juan F Vera and Ann M Leen

Acute Myeloid Leukemia

16%Cured

20%

AML	Adverse risk/relapsed
~26,000/yr

80%

Allo-HSCT

potentially curative

Koreth J et al, DeAngelo DJ JAMA2009

Adverse risks

>CR2

Complex cytogenetics

Monosomy 7

MDS→AML

t-AML

MLL-r

FLT3/DNMT3A etc

Outcomes of AML patients post-alloHSCT

18 month Relapse rate post-HSCT

MAC-SCT

14%

RIC-SCT

48%

1-year survival after relapse: 23%

BMT-CTN 0901. Scott et al J Clin Oncol.2017

Bejanyan et al BBMT2015

SCT recipient SCT donor

Donor lymphocytes

Blood draw	Antigen specificity

Adoptive T cell

transfer

Infusion

Cell expansion

Tumor-specific T cells

MultiTAA-TcellsforAML/MDS

TAAFreq.

WT172-90%

PRAME 40-60%

Survivin 90-100%

NY-ESO10-36%

Our approach

• Simultaneously target multiple TAAs

MultiTAA Manufacture

Overlapping pepmixes

DC

PBMCs

MultiTAA T cells

	Profile of MultiTAA-T cells
			Phenotype			30%	Alloreactive
	100%						potential
cells	80%					lysis	25%
60%					20%
Positive					Specific
40%					15%
%						%	10%
20%
							5%
	0%
	CD3+	CD4+	CD8+ RO+/	RO+/	RO+/		0%
			62L+/ 62L+/ 62L-/
			CCR7-	CCR7+ CCR7-			n=24

MultiTAA T cell specificity

SFC/2x105

PRAMENYESO1Survivin WT1

1000

100

10

1

0.1

MultiTAA T cell specificity

SFC/2x105

1000

100

10

1

0.1

PRAMENYESO1Survivin WT1

100000

10000

										clones	1000
										100
										# of
										10

1

Line clones

mean = 5382 clones

(1697 - 16227)

n=12

Phase I trial - ADSPAM

Any patient with AML/MDS post allo-HSCT

Donor-derived multiTAA T cells

GROUP A - Adjuvant

	AML/MDS patients		Dose Escalation
	≥30 days post allo-HSCT		DL1	5x106	cells/m2
			DL2	1x107	cells/m2
GROUP B - Active disease
DL3	2x107	cells/m2
	AML/MDS patients
	≥30 days post allo-HSCT

Clinical trial: Current status

Patients Enrolled:

27 patients (24 AML and 3 MDS)

Patients Treated:

20 patients (0.5-2x107cells/m2)

Grade II or lesser	Grade III
3 (all grade I elevation in1 grade III LFT elevation (resolved with
LFTs	0.5 mg/kg prednisone)

Patients infused - ARM A

ID	Age/	Disease		Prior Treatments
G
1*	57/F	FLT3-ITD	CIA →Sorafenib →CIAx2 →RIC-SCT
2	18/F	FLT3-ITD	Bortezomib/Dauno EC →sorafenib →MAC-SCT
5	55/F	MLL-r	7+3	→HiDAC →MAC-SCT
6	70/F	AML CR3	7+3	→HiDAC →CIA →RIC-SCT-Relapse→7+3
7	53/F	DNMT3a	7+3	→HiDAC →MAC-SCT
10	65/M	MLL-r	7+3x2 →5-Azax11→RIC-SCT
11	55/M	t-AML	Mitoxantrone/Ara-C→RIC-SCT→Relapse→7+3
12	45/M	Ph+AML	7+3+imatinib→MAC-SCT
13	51/F	AML CR2	7+3	→HiDAC →Relapse→FLA →HiDAC →MAC-SCT
14	54/F	Complex-rIPSS:	5-azax11→Transf-dep→RIC-SCT
Int-2
15	58/M	RAEB-1	Decitabine→RIC-SCT→Relapse with RAEB →CIA→relapse as
rIPSS: Int-2	MDS→DLIx4
16	53/F	CR2 (MRD+)	7+3	→HiDAC →Relapse→FLA →MRD+→MAC-SCT
18	18/F	FLT3-ITD/MRD+	AAML1031 →Relapse--.CPX-351→FLAG →Ara-C/Peg/Midostaurin→
refractory→Venetoclax/Decitabine→Residual disease→MAC-SCT

Outcomes - ARM A

ID	Disease	Wk 4 Marrow	Relapse?	Status at last f/up
(% blasts)
1*	FLT3-ITD	0	No, but bone	Treated on ARM B (9 mo's post-
relapse	infusion)
2	FLT3-ITD	0	No	Alive in CR(2.5 years)
5	MLL-r	0	No,CNS relapse,	Alive in CR(2 years)
Local Rx alone
6	AML CR3	0	No,CNS relapse	Alive in CR(1.5 years)
Local Rx alone
7	DNMT3a	0	No	Alive in CR(1.5 years)
10	MLL-r	0	No	Alive in CR(6 mo)
11	t-AML	0	No	Alive in CR(6 mo)
12	Ph+AML	0	No	Alive in CR(9 mo)
13	AML CR2	0	No	Alive in CR(9 mo)
14	MDS	0	No	Died in CR(1 year)
15	MDS	0	Yes (8 months)	2ndtransplant, alive in relapse
(1.5 years)
16	CR2 MRD+	0	No	Alive in CR(6 mo)
18	FLT3-ITD/MRD+	0	No	Alive in CR(week 6)

Patients infused - ARM B

GROUP B:Active AML:7 patients treated for active AML

ID	Ag	Disease	Prior Treatments
e/G
3	70/M	IDH1mut	7+3 →decitabine →IDH inhib →cutis relapse →CIA →RIC-SCT→Relapse
4	16/M	MDS→AML	Double cord SCT →AML Relapse→C →haplo-SCTx2→Relapse
1*	57/F	FLT3-ITD	CIA →Sorafenib →CIAx2 →RIC-SCT→mTAA-T cells →steroids →Relapse
8	55/M	Induc. failure	7+3 →HiDAC x4 →RIC-SCT→Relapse→DLIx4 →MEC →5-aza→Relapse
9	23/M	Del 17p	CIAx3 →haplo-SCT→Relapse→CIA-decitabine→haplo-SCT→5-aza→
Nivolumab →CD123 BiTE →MEC-decitabine→midostaurin →Relapse
			CIAx3 →haplo-SCT#1→Relapse→CIA-decitabine→haplo-SCT#2→5-aza
9*	23/M	Del 17p	→Nivolumab →CD123 BiTE →MEC-decitabine→midostaurin →Relapse→
			mTAA T cells →haplo-SCT#3→Relapse
17	20/F	FLT3-ITD	7+3 →HiDAC→MAC-SCT→Relapse→CIA →Relapse

Outcomes - ARM B

ID	Disease	Day 0	Week 4	Response
3	IDH1mut	Skin relapse	Stable skin	NR
lesion
4	MDS→AML	30% blasts	30%	NR
1*	FLT3-ITD	4 bone lesions	All resolved	CR
8	Induc. failure	50% blasts	15%	PR
9	Del 17p	30% blasts	30%	NR
9*	Del 17p	30% blasts	N/E	N/E
17	FLT3-ITD	70% blasts	45%	NR

Outcomes - ARM B

ID	Disease	Day 0	Week 4	Response	Status at last f/up
3	IDH1mut	Skin relapse	Stable skin	NR	PD (3 mo)→HiDAC chemo
lesion
4	MDS→AML	30% blasts	30%	NR	PD (4 wks) →Hospice
1*	FLT3-ITD	4 bone lesions	All resolved	CR	CR (13 mo)→Relapse →7+3 chemo
8	Induc. failure	50% blasts	15%	PR	PR (4 mo) →2ndalloHSCT
9	Del 17p	30% blasts	30%	NR	SD (2 mo)→Chemo/venetoclax →
4thalloHSCT
9*	Del 17p	30% blasts	N/E	N/E	PD (3 wks) →Ara-Cchemo
17	FLT3-ITD	70% blasts	45%	NR	SD (2 mo)→Chemo/venetoclax

Tracking infused clones in vivo

Rationale:

• Infused lymphocytes are not gene modified
• Leukemia specific T cell clones enriched in infused line

In vivoexpansion of line clones

baseline)

Overall

2

382 line-exclusive

Marrow

60%

0.5% line-exclusive

Fold change (from

1.5		clones
1							n=12
0.5

Productive Frequency

40%

20%

0%

clones

n=4

By week 4

Week 4

Responders vs Non-responders

%Repertoire

		By disease response
60%						Relapse/progress n=3
					No relapse/progression n=4

40%

20%

0%

Preinfusion

By week 4

Clinical course - Pt#1

FLT3mut AML received T cells as adjuvant (120 days post HSCT)

SFC/5x105

16

14

12

10

8

6

4

2

0

6.1%	7%	1.8%
Relapse

Prednisone

Prame

NYESO1

Survivin

WT1

Lesions-4

pre inf 1

Week 8

Clinical course - Pt#1

Tumor - antigen expression

		WT1
H+E

Clinical course - Pt#1

Post-decitabine (Mo.10)

Post-T cell (Mo.11)

1.45%

2.47%

3.07%

20	240	• CASSSGQAYEQYF
			240
15		• CASSQVFPNTGELFF
160	WT1	160
10		Survivin
	NYESO-1
	80
5	PRAME	80
0	0		0
	Post decitabine	CR	Marrow

Clinical course - Pt#8

TAA expression	% cells	Intensity
PRAME	50-75%	2+
Survivin	<10%	2+
NYESO1	<10%	1+
WT1	<10%	1+

Blasts%

Summary

course - SM

• Leukemia-directeddonor T cell infusions are safe
• Mediateanti-tumor effects
• In vivo expansion superior in responders
• Antigen spreading studies ongoing
• Investigation of immune escape mechanisms

Administering AML-directed DLIs to patients with AML or MDS Post-

Allogeneic HSCT Relapse

Premal Lulla, Swati Naik, Ifigeneia Tzannou, Shivani Mukhi, Manik Kuvalekar, Catherine Robertson, Carlos A Ramos, George Carrum, Rammurti Kamble, Jasleen Randhawa, Adrian P Gee, Bambi Grilley Malcolm K Brenner, Helen E Heslop, Juan F Vera and Ann M Leen

Funding:

Evans MDS discovery research grant, Leukemia Texas, Leukemia and

Lymphoma SCOR, Lymphoma SPORE, ASBMT New Investigator Award, ASH Scholar Award, BCM Junior Faculty Seed Funding Award, EPCRS- DLDCC, LLS/Rising Tide, ARC-Coalition

Targeting Lymphomas Using Non-

Engineered, Multi-Antigen Specific T Cells

George Carrum, Premal Lulla, Ifigeneia Tzannou, Ayumi Watanabe, Manik Kuvalekar, Munu Bilgi, Tao Wang, Rammurti Kamble, Carlos A. Ramos, Rayne Rouce, Bambi J. Grilley, Adrian P. Gee,

Malcolm K. Brenner, Helen E. Heslop, Cliona M. Rooney, Juan F. Vera and Ann M. Leen

Patient

PBMCs

Blood draw	Antigen
	Specificity

Adoptive T cell

transfer

Infusion

Tumor-specific T cells

Our approach

• Simultaneously target multiple TAAs

MultiTAA Tcelltherapyforlymphoma

MAGEA4 PRAME Survivin

NYESO1 SSX2

MultiTAA T cells

MultiTAA-T Cell manufacture

Overlapping pepmixes

DC

Expansion

MultiTAA T cells

MultiTAA-T Cell Phenotype

% Positive cells

100

80

60

40

20

n=39

0

CD3 CD4 CD8 NK DC TCM TEM

MultiTAA-T Cell Specificity

SFC/2x105cells

1000

100

10

1

0.1

0	PRAME	SSX2	MAGEA4 NYESO1	Survivin	6
1	2	3	4	5

Multi TAA-T Cell Autoreactivity

20%

% Specific Lysis

10%

0%

0

0.5

E:T of120:1

1.5

2

Clinical Trial: Eligibility

Any patient >18 yrs with HL or NHL

Active disease

• in 2ndor subsequent relapse
• in 1strelapse for indolent lymphoma after 1stline therapy for relapse
• in 1strelapse if immunosuppressive chemotherapy contraindicated
• primary refractory disease or persistent disease after 1stline therapy
• multiply relapsed patients in remission at a high risk of relapse
• lymphoma as a second malignancy e.g. Richters

After autologous or syngeneic SCT (adjuvant therapy)

Infusion of multiTAA-T cells specific for

PRAME, SSX2, MAGEA4, NYESO1, Survivin

Safety of MultiTAA T cells - Antigen escalation

Antigen Escalation Phase = fixed dose 5x106/m2 -2 pts/stage:

Day 0: PRAME-specific T cells

Day 28: PRAME and SSX-specific T cells

Stage Two:

Day 0: PRAME and SSX-specific T cells

Day 28: PRAME/SSX/MAGE-specific T cells

Stage Three:

Day 0: PRAME/SSX/MAGE-specific T cells

Day 28: PRAME/SSX/MAGE/NYESO1-specific T cells

Stage Four:

Day 0: PRAME/SSX/MAGE/NYESO1-specific T cells

Day 28: PRAME/SSX/MAGE/NYESO1/Survivin-specific T cells

Safety of MultiTAA T cells - Dose escalation

PRAME/SSX/MAGE/NYESO1/Survivin-specific T cells:

2-4 pts at each level, 2 infusions 14 days apart

Dose Level 1:

Day 0 and 14: 5x106cells/m2

Dose Level 2:

Day 0 and 14: 1x107cells/m2

Dose Level 3:

Day 0 and 14: 2x107cells/m2

Clinical Trial: Treatment

• 33 patients infused

Clinical Trial: Treatment

• 33 patients infused

Antigen escalation (n=4)

Group A:

In remission

Group B:

Active lymphoma (failed prior lines)

Dose escalation

(n=14)

Dose escalation

(n=11)

Antigen escalation (n=4)

Clinical Trial: Treatment

-33 patients infused (0.5-2x107cells/m2)

• • 12 HL
  • 19 aggressive NHL (DLBCL/mantle/peripheral T)
  • 2 with composite lymphoma
• No lymphodepletion
• No adverse events

Pt1 (HL) - Clinical and Immune effects

A	Pre T cells

Post CTLPost+ radT cellsiation

B

SFC/2x10F5x10e5

					Tar et	d Antigens
50					Targeted antigens
90
40
80																																		SSX2
30
																																	PRAME
70
20
60
10
50
				Pre												Post
40					Non-targeted				antigens
				Non-targeted	antigens
140
12030																																		AFP
100
																																	NYESO1
20
80
60
10
40
20
0
0					Prame						SSX2	MAGE A4			NYESO-1
					Pre															Post

Pt2 (NHL) - Clinical and Immune effects

PreMth-Infusion39

SFC/2x105

45

40				MAGEC1

35

30

25

20

15

10

5

0

Pre Mth3 Mth9

Clinical Outcomes - Adjuvant

- 18 patients infused as adjuvant

-15/18 in remission (median 19 months)

Clinical Outcomes - Adjuvant

ID	Age/Sex	Disease	Prior Therapies	Response to T cell therapy (duration)
1*	39/M	HL & DLBCL	ABVD →RICE →ASCT	CCR (>3 years)
2*	78/F	DLBCL	R→RCHOP	In remission (8 mo) →relapse
3*	78/F	DLBCL	R→RCHOP →multiTAA T cells →R-Bendamustine	CCR (>3 years)
4*	21/M	HL	ABVD →Brentuximab →Nav/Gem →ASCT	CCR (>4 years)
5	34/M	HL	ABVD →ICE →ASCT + XRT →Brentuximab	In remission (12 mo) →relapse
6	54/M	DLBCL	RCHOP →R-EPOCH→R-DHAP→ASCT	In remission (19 mo) →relapse
7	61/M	DLBCL	R-EPOCH→ASCT →XRT	CCR (>2 years)
8	41/F	HL	ABVD + XRT →ICE →ASCT →XRT →Brentuximab →DHAP	CCR (>4 years)
9	62/M	T cell	CHOP + XRT →ASCT	CCR (>3 years)
10	53/M	Mantle	R-HyperCVAD→R-Bendamustine→R-Ibrutinib→ASCT + XRT	CCR (>2 years)
11	39 not 67/M	Mantle	R-Bendamustine-Ara-C→ASCT	CCR (>3 years)
12	65/F	DLBCL	R-EPOCH→ASCT	CCR (>2 years)
13	35/M	HL	ABVD →Brentuximab+Bendamustine →ASCT →XRT	CCR (> 2 years)
14	73/F	DLBCL	R-CHOP→XRT →ESHAP →RIE	CCR (>1 year)
15	50/F	DLBCL	HyperCVAD →ASCT	CCR (9 mo)
16	41/M	DLBCL	ABVD →R-ICE→ASCT	CCR (> 1 year)
17	32/F	T cell ALCL	CHOP →Brentuximab →Crizotinib →CD30 CAR T cells→Crizoinib	CCR (9 mo)
18	25/M	HL	ABVD →Brentuximab →ICE →ASCT	CCR ( >1 year)

Clinical Outcomes - Active disease

• 15 patients treated for active disease
• • 6 CRs; 4 SD; 5 PD

Clinical Outcomes - Active disease

ID	Age/Sex	Disease						Prior Therapies			Response to multiTAA T cells (duration)
1*	31/F	HL	ABVD →ICE →Cis-Gem→XRT →ASCT→		Stable disease (5 mo) →Off study [Revilimid (5 mo) →PD1]
EBV T cells	→	Brentuximab	→	Yttrium90	→	CART-CD30
2*	55/F	HL/NHL	RCHOP + XRT →ICE →ASCT				CR (4 mo) Died of pneumonia
3*	38/M	HL	ABVD →XRT →IGEV →ESHAP →ASCT →GVD →XRT	CR (>2 years ongoing)
4*	44/F	HL	ABVD →ICE →ASCT →Brentuximab				CR (>5 years ongoing)
5	46/M	HL	ABVD →ICE →ASCT + XRT →Brentuximab	CR (>2 years ongoing)
6	46/F	DLBCL	RCHOP →GDC →ASCT						CR (>3 years ongoing)
7	31/F	HL	ABVD →XRT →ICE →Nav/Gem →ASCT →	Stable disease (5 mo) →PD
HDACi	→	Brentuximab	→	Bendamustine	→	PD1i
8	69/M	NHL	EPOCH →Romidepsin →ASCT				Stable disease (>2 years)
9	54/M	DLBCL	RCHOP →R-ICE→ASCT						Stable disease (6 mo) →PD →Started PD1i - >2 years; Alive
10	18/F	HL	ABVE-PC→XRT →IVBor →Brentuximab →PD1i	Stable disease (9 mo) →PD
11	48/M	DLBCL	EPOCH-R→R-ICE→ASCT →XRT				CR (>1 year)
12	49/M	HL	ABVD →ICE →ASCT →XRT→Brentuximab →	PD (3 mo)
Nivolumab	→	Bendamustine
13	54/M	DLBCL	EPOCH-R→ICE-R→XRT →ASCT				SD (9 mo)
14	64/M	DLBCL	R-CHOP→Bendamustine/Rituxan→RICE→RIE→ASCT	PD (9 mo)
15	68/M	DLBCL	RCHOP→GDP→ASCT							Stable disease (4 mo) →CD19-CAR-T

Summary to date

• Safe to date
• Feasible adjuvant and treatment
• In vivo expansion of T cells directed to targeted antigens
• Antigen/Epitope spreading
• Clinical benefit

Targeting Lymphomas Using Non-

Engineered, Multi-Antigen Specific T Cells

George Carrum, Premal Lulla, Ifigeneia Tzannou, Ayumi Watanabe, Manik Kuvalekar, Munu Bilgi, Tao Wang, Rammurti Kamble, Carlos A. Ramos, Rayne Rouce, Bambi J. Grilley, Adrian P. Gee,

Malcolm K. Brenner, Helen E. Heslop, Cliona M. Rooney, Juan F. Vera and Ann M. Leen

Adoptive T cell therapy for ALL targeting

multiple tumor associated antigens

Swati Naik, Premal Lulla, Ifigeneia Tzannou, Shivani Mukhi, Manik Kuvalekar, Catherine Robertson, George Carrum, Rammurti Kamble, Adrian P Gee, Bambi Grilley, Robert Krance, Malcolm K Brenner, Helen E Heslop, Juan F Vera, Stephen Gottschalk and Ann M Leen

ALL Relapse after HSCT

• Leukemic relapse is major cause of treatment failure after HSCT
• • Incidence of relapse:24-35%
• Poor prognosis for pts who relapse
• • Particularly those who relapse earlypost-HSCT
  • Overall survival: 7- 32%

Fagioli Hematologica 2013

Porter et al , BBMT 2011

Arellano, BBMT 2006

Prevention of ALL relapse

• Strategies to prevent relapse
• • Prophylactic use of targeted agents (e.g. TKIs)
  • Modulation of immune suppression
  • • Promote immune reconstitution resulting in GvL effect
  • Immunotherapeutic intervention with DLIs
  • • Enhance GvL effect

Wayne, Hematology 2017

De Lima,BBMT 2013

Alyea et al, BBMT, 2010

SCT recipient SCT donor

Blood draw

Donor lymphocytes

Adoptive T cell

transfer

	•	Low tumor-specific T cell
Infusion		frequency
	•	High frequency of alloreactive
		cells (GvHD)

SCT recipient SCT donor

Donor lymphocytes

Blood draw	Antigen specificity

Adoptive T cell

transfer

Infusion

Cell expansion

Tumor-specific T cells

MultiTAATcelltherapyforAML

TAAFreq.

WT1 70%

PRAME 65%

Survivin 40%

MultiTAA-T Cell manufacture

Pepmix spanning full length

WT1, PRAME, Survivin

DC

Expansion

PBMCs

MultiTAA T cells

MultiTAA T cell profile

	120%		Phenotype			40%	Safety
cells					lysis
					30%
Positive	80%					Specific	20%
40%					10%
%						%	0%
	0%						-10%	n=11
	CD3	CD4	CD8	CD3+/	CD3+/			20:1
				RO+/	RO+/
				62L+	62L-

MultiTAA T cell profile

SFC/2x105

1000Specificity

100

10

1

0	1	2	3	4
	Prame	Survivin	WT1

Study design (STELLA)

Any patient with ALL who received an allogeneic SCT from a family donor

DL1	5x106	cells/m2
DL2	1x107cells/m2
DL3	2x107	cells/m2

Given after day +30 post-transplant

Patients infused - STELLA

ID	Age/G	Disease	Prior Treatments	Dose level
1	5/F	Ph+ ALL	Induction chemo →Primary induction failure →MRD SCT	1
2	18/F	HR- ALL	Completed therapy for HR- ALL →Relapse →MRD SCT	1
3	18/F	Ph+ ALL	Completed therapy for HR- ALL →Relapse →MRDSCT→	1
Relapse →Chemo→CD34+ top -off
4	41/M	HR- ALL	HyperCVAD + Ofatumumab x 5 cycles →MRD SCT	1
5	8/M	Ph+ ALL	Completed therapy for HR- ALL →Relapse →MRD SCT	1
6	48/F	HR- ALL	Induction chemo →Primary induction failure →MRD SCT	2
9	12/F	T-cell ALL	Completed therapy for T- ALL →Relapse →MRD SCT	2
10	18/M	HR-ALL	Induction chemo →Primary induction failure →MRD SCT	2
11	12/F	MPAL	Induction chemotherapy →MRD SCT	3
12	16/M	Ph+ ALL	Relapsed on therapy for HR- ALL →MRD SCT	3

n=10

Safety

• No Dose Limiting Toxicities (DLTs)
• No GVHD
• No CRS/neurotoxicity or other adverse events

Clinical outcomes

ID	Age/G	Disease	Dose level	Clinical course
2	18/F	HR- ALL	1	CR with mixed chimerism for 6 months→Relapse
3	18/F	Ph+ ALL	1	Alive in CR (22 months post-infusion)
4	41/M	HR- ALL	1	Alive in CR (28 months post-infusion)
5	8/M	Ph+ ALL	1	Died in CR (9 months post-infusion)
9	12/F	T-cell ALL	2	Alive in CR (17 months post-infusion)
10	18/M	HR-ALL	2	Alive in CR (15 months post -infusion)
11	12/F	MPAL	3	Alive in CR (4 months post-infusion)

Median follow-up 16 months (range 4-28 months)

Immune Reconstitution

	350	Pt 3 - CR	35	Pt 4 - CR		20	Pt 2 - Relapse
	300			30				18
						16
SFC/5x105			SFC/5x105				SFC/5x105
250		25			14
200		20		WT1	12		WT1
		WT1
				10
	150		Survivin	15		Survivin			Survivin
		Prame		Prame		8		Prame
	100			10				6
	50			5				4
						2
	0	Preinf	Wk 4	0				0
			Preinf	Wk 4			Preinf	Wk 4

Tumor antigen expression and

T cell expansion

Pre

WT1PRAME

neg

4+

SFC/5x105

350

300

250

200

150

100

50

0

Immune Reconstitution

Pt 3

WT1

Survivin

Prame

Preinf

Wk 4

Antigen Spreading

Pt 3

Target Antigens

Antigen spreading

	350		WT1	500	AFP
SFC/5x105	300			MART1
	Survivin	400
250
		NYESO1
		Prame		MC1
	200		300	MA3
				MA2B
	150			200
			MA1
	100
			100
	50			mageA4
	0			0	SSX2
	Preinf	Wk 4		Preinf	Wk 4

Summary

• Feasible for bothB-cell and T-cell ALL
• Safe to date,well-tolerated
• In vivo expansion oftumor-antigen associated T-cells directed to target antigens
• Evidence of antigen spreading which may contribute to relapse prevention
• May present a safe and effective strategy to prevent leukemic relapsepost-HSCT

Safety and efficacy of multi-TAA-T cells for Myeloma

Premal Lulla, Ifigeneia Tzannou, George Carrum, Carlos A. Ramos, Rammurti Kamble, Mrinalini Bilgi, Adrian P. Gee Shivani Mukhi, Betty Chung, Ayumi Watanabe, Manik Kuvalekar, Bambi Grilley, Malcolm K. Brenner, Helen E. Heslop, Juan F. Vera and Ann M. Leen

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital, and Texas Children's Hospital, Houston, Texas, USA

Introduction

Despite an array of approved agents for the treatment of multiple myeloma (MM), most patients eventually relapse after conventional treatments. The adoptive transfer of tumor- targeted T cells has demonstrated efficacy in the treatment of patients with chemo- refractory hematological malignancies including MM. While the majority of T cell-based therapies in the clinic explore genetically modified T cells that target a single tumor- expressed antigen, we have developed a strategy to generate non-engineered T cell lines that simultaneously target a number of MM-expressed antigens, thereby reducing the risk of tumor immune evasion. We manufacture multiTAA-specific T cells targeting the tumor antigens PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin (Table 1) by culturing patient- derived PBMCs with DCs loaded with pepmixes spanning all 5 target antigens in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail (Figure 2).

T lymphocytes

	Blood draw	Antigen	Table 1: Expression of TAAs on
		Specificity		lymphoma cells
			Antigen	Expression in lympomas
Lymphoma patient	Adoptive T cell		Survivin	90-100%
transfer		SSX2	35-61%
			PRAME	36-48%
			NY-ESO-1	25-31%
			MAGE-A4	17-30%
	Infusion	Cell
	expansion

TAA-specific T cells

Figure 1

Characteristics of mTAA-T cells

We have successfully generated multi-antigen-targeted lines for 19 patients, comprising a polyclonal mixture of CD4+ (28.9±7.2%) and CD8+ (56.6±7.2%) T cells (Figure 3) reactive against 2 to 5 of the target antigens (Figure 4), with no activity against non- malignant autologous targets (2±3% specific lysis; E:T 20:1). We assessed the clonal diversity using TCR vβ deep sequencing analysis and found that the majority (mean 79%; range: 59 to 95%; Figure 5) represented rare T cell clones that were unique to the ex vivoexpanded cell line, thereby enabling in vivotracking studies.

Activating Cytokines

Expansion

7 days
Figure 2- Manufacturing process		n=19
	Figure 3-Phenotype

n=19

Figure 4-Specificity in an ELISPOT Assay	Figure 5 TCR clonality

We have initated a phase I/II clinical trial to explore the safety and efficacy of mTAA- directed T cells administerd to patients with myeloma who have failed at least one line of prior therapy. The schema for enrollment is shown in Figure 6. We have treated 20 patients (Group A: 11, Group B: 9) so far: 12 with active myeloma and 8 with myeloma in at doses of 0.5-2x107multiTAA-T cells/m2in 2 infusions 2 weeks apart without prior lymphodepletingchemotherapy.

Group A:		Group B:
>90 days post autologous		<90 days post autologous
transplant or no transplant		transplant

Clinical Outcomes

To date we have infused 20 patients who had received a median of 4 lines of prior therapy at cell doses ranging from 0.5-2x107/m2. 12 patients were refractory to their latest therapy and had active MM, while 8 were in remission at the time of infusion. Of the 8 patients in CR at the time of T cell infusion, all remained in CCR at the week 6 disease assessment and of the 6 evaluable patients who are >1 year post T cells, only 1 has relapsed.

Table 2: Clinical outcomes of patients treated on group A

ID	Age/G	Prior Treatments	Marrow Week 6 Response	Mo 12
1	53/M	Bor/Dex →ASCT	10%	Unknown	SD	PR
6	61/M	RVD →ASCT	0%	0%	CCR	CCR
7	44/M	CyBorD →ASCT	0%	0%	CCR	CCR
14	47/M	RVD →ASCT	0%	0%	SD	SD
(MRD+)	(MRD+)
		RVD →ASCT →CyBorD →Carf/D		PD
3*	65/F	90%	85%	SD
→ASCT	(2m)
13	31/F	VD	4%	0%	SD	SD
10	69/F	VD →ASCT→R→Pom/Carf/D	10%	10%	SD	PD (7m)
		RVD →ASCT →R-vidaza→Pom/D
15	70/M	→ibrutinib/Carf →dinaciclib/VD →	80%	80%	SD	PD (3m)
		CyBorD →Daratumumab →RD-Elot
		→Ixa/RD
2*	40/M	RVD →ASCT →	15%	15%	SD	SD
Pom/Carf/D→ASCT→mTAA T cells	(3m)
18	50/F	VD →ASCT →Dara/VD →XRT →	0%	0%	CCR	CCR
ASCT	(8m)
		5%	3%
		RVD →ASCT →R →VD →Pom/D		SD
20	57/M	(0.97	(0.53	SD
→KPD →ASCT →Ixa →Dara/D	(3m)
		g/dl)	g/dl)

Ten patients were refractory to their latest therapy and had active MM, while 8 were in remission at the time of infusion. At the 6 week assessment, of the 10 patients infused to

	treat active disease, 1 had a CR, 1 had a PR and 8 had SD. Seven of these 10 patients
In this fashion we have
	were infused >1 year ago. Although 2 of the 7 subsequently had disease progression, the
now ide tified the HLA-
restriction	for	four
	remaining 5 continue to respond, with sustained CR (1), PR (2) or SD (2). (Tables 2, 3).
	None of the treated patients developed cytokine release syndrome,
additional		immuno-
dominant epitopes that
	neurotoxicity or any other infusion related adverse events.
			Table 3: Clinical outcomes of patients treated on group B
	ID	Age/G	Prior Treatments	Marrow Week 6 Response Mo 12
	2	40/M	RVD →ASCT →Pom/Carf/D →	20%		0%	CR	CR
	ASCT
	3	65/F	RVD →ASCT →CyBorD →Carf/D	15%		10%	SD	PD
	→ASCT		(6m)
	5	76/M	CyBorD →ASCT	20%		15%	SD	PR
	8	57/M	VTD →ASCT →Rd →Cy/Carf/D	0%		0%	CCR	CCR
	→ASCT
	9	50/F	RVD →ASCT	0%		0%	CCR	CCR
	11	53/M	VD →RVD →ASCT	0%		0%	CCR	Relapse
		(7m)
	12	54/M	RVD/rituximab →Rd →ASCT	0%		0%	CCR	CCR
	17	44/F	VRD →KD →ASCT	0% (0.4		0% (0.2	PR	PR
	g/dl		g/dl)	(6m)
	19	70/M	XRT →VD →ASCT →R →VD →	0%		0%	CCR	CCR
	KPD →ASCT		(6m)

Clinical responses correlated with the emergence and persistence (>6mths) of "line-exclusive"tumor-reactive T cells in patient peripheral blood (Figure 6A) and marrow (6B), as assessed by TCR deep sequencing. The expansion of product-derived clones was higher among patients with active MM than those in remission (6A). This matched the pattern of expansion of TAA-directed T cells as measured by an IFN-Υ ELISPOT assay (6C & D)

Figure 6 T cell expansion/persistence

Responses in patients

Figure 7: Complete responses in a patient (ID:#2) with lambda light chain myeloma

correlates with expansion of infused mTAA-T cells

Shown in Figure 7is an example of a patient with lambda light chain myelomawith residual marrow disease despite undergoing several lines of prior therapies. Six weeks post-infusion, this patient entered a CR as measured by paraprotein levels as well as by marrow findings concomitant with an increase in the circulating frequency of TAA-(MAGE-A4)-specific T cells in both the blood as well as the bone marrow. The same pattern of expansion was observed when monitoring for the T cell clones present in the infused T cell line but absent in the patient prior to infusion (Figure 7).

Figure 8: Immune escape in a patient (ID#3) with treatment refractory multiple myeloma

Patient #3 had active multiple myeloma despite recently undergoing an autologous HSCT. At baseline the patients tumor cells expressed Survivin, MAGE-A4 and PRAME as assessed by immunohistochemistry analysis (Figure 8). Within 3 months of T cell infusion, there was an increase in the circulating frequency of T cells specific for the targeted TAAs as well as non-targeted TAAs (antigen spreading) in the blood and the bone marrow. However by month 6 the patient developed progressive disease along with loss of TAA-specific T cells within the marrow. Coincident with relapse the patients tumor lost expression of Survivin, MAGEA4 and PRAME in the presence of circulating Survivin, PRAME and MAGE-A4 specific T cells (Fig 8). Furthermore, mRNA sequencing demonstrated an increase in immune inhibitory markers (CTLA4 and LAG3) and an upregulation of >400 cell cycle promoters.

Conclusions

Thus, infusion of autologous multiTAA-targeted T cells directed to PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin has been safe and provided durable clinical benefit to patients with lymphomas. Responses in all six patients who entered a CR were durable and associated with an expansion of infused T cells as well as the induction of antigen spreading.

AL, JFV, MKB and HH are co-founders of Marker Therapeutics that aspires to commercialize the described approach to cell therapy