T CELL GENE THERAPY WITH

DESIGNER T CELLS:

A LOOK AT THE FUTURE FOR IMMUNO-ONCOLOGY?

Richard P Junghans, PhD, MD

​

Director, Biotherapeutics Development Lab

Associate Professor of Surgery and Medicine

Boston University School of Medicine

Chief, Division of Surgical Research �Roger Williams Medical Center�Providence, RI, USA

No commercial relationships to disclose.

June 17, 2010

What To Learn?

• What are the parts of the immune system?
• What are T cells? What do they do?
• What is a tumor antigen?
• What is gene therapy? How can gene therapy teach T cells to attack cancer?
• How does an idea lead to a new therapy? What is a clinical trial?
• How does a clinical trial stimulate lab studies that modify the therapy?

​

​

History of Immune Therapies

HUMORAL

CELLULAR

SPECIFICITY

AFFINITY

ADAPTABILITY

CYTOTOXICITY

SELF RENEW

ACCESS

Leukemia

Lymphoma

Melanoma

Colorectal

Breast

Renal Cell

Melanoma

BIFUNCTIONAL Abs

TUMOR VACCINES

Ab2 IMMUNIZATION

DESIGNER T CELLS

(IL2)

LAK

TIL

T cells

(Antibodies)

T cells attack virus-infected cells

T cells

• Evolved to kill our own cells infected by virus
• Specificity mediated through variable T cell receptor (TCR)
• Tolerant to self, including cancer
• How to overcome this tolerance?
• I.e., how to “fool” the T cells into “thinking” the tumor has a virus infection????

TCR

Gene-Modified TCR

Anti-Cancer T Cell Gene Therapy

Carcinoembryonic antigen (CEA)

• Surface membrane glycoprotein 180,000 Daltons
• Expression high on tumor, low on normal
• High clinical relevance:
• Colorectal, pancreas, breast, lung, others
• More than 100,000 deaths/yr with CEA+ tumors

​

Antigen:

“Anything that can be recognized by an immune system”

​

Tumor Antigen:

Clinical Retroviral Vector

• Vector = “carries something in”
• Retroviral vector = virus used to infect T cells
• Inserts new gene “transgene” into host chromosome
• Virus dies after it infects once
• Stable gene expression; T cells permanently modified

⁺

LTR

anti-CEA IgTCR

LTR

Gene Therapy:

“Transgene”

“The transfer of new or modified genes into cells.”

CFSE stained total T cells

Rhodamine staining of IgTCR+ T cells

Total and

IgTCR-modified

T cells

M21 + Normal T cells

M21 + Designer T cells

Ganglioside GD3 on Melanoma

Binding

DESIGNER T CELLS BIND TO

ANTIGEN+ TUMOR CELLS

Activation: Cytotoxicity

DESIGNER CD8 T CELLS KILL

Ag+ TUMOR CELLS

250

200

150

100

50

0

0

1

2

3

4

5

Days

Ag+

Ag-

Viable Tumor Cells x 10^-3

Phase I Study of Anti-CEA Designer T Cells in Adenocarcinoma�(“1st generation”)

FDA BB IND 7301

Clinical Data: 1^st Generation

What is a clinical trial?

“A test of hypotheses of drug activity in human subjects.”

TCR

Gene-Modified TCR

Anti-Cancer T Cell Gene Therapy

Clinical Summary

• Number of doses administered (24)
• Patients treated (7): 5 colorectal, 2 breast
• Doses sizes administered
• 1 x 10⁹, 3 x 10⁹, 1 x 10¹⁰, 3 x 10¹⁰, 1 x 10¹¹ cells

Pharmacokinetics�“Drug disappearance in the body”

Rapid Systemic Loss…

Response: CEA Profile on Patient GT

Increasing pain

Pain resolved

1600

1400

1200

1000

800

600

400

200

0

-28

-21

-14

-7

0

7

14

21

28

Day of Treatment

CEA (NG/ML)

T Cells

CEA

BUT! Time-Limited in Duration…

Conclusions: �1st Gen Phase I Study

• Adequate safety
• Proof-of-principle biologic response
• But… Time-limited efficacy profile
• ​
• WHY DIDN’T IT CURE???

Immunology 101

“T cells evolved to kill virus-infected cells.”

Remember!

T Cell Activation

• Gene expression

- Cytokines (IL-2, 4, IFN-, etc)

- Surface molecules (CD25, CD40L, etc)

• Cytotoxicity
• Proliferation

​

B7

MHC

TCR

CD28

Antigen

Presenting Cell

T Cell

“1”

“2”

Designer T Cells – First Generation

• IgTCR – chimeric immunoglobulin – T cell receptor

CD28

Advantage: IgTCR provides Signal 1: adequate T cell cytotoxicity.

Disadvantage: Lacking Signal 2, undergoes Activation-Induced Cell Death (AICD) after killing target cells. [HYPOTHESIS]

Ig

Antigen

TCR

Modified

T Cell

Tumor Cell

“1”

MIPCEA

Signals

• Signal 1+2:
• T cells kill tumor >> and proliferate

​

• Signal 1:
• T cells kill tumor >> and die by AICD
• ​
• ​

Hypothesis test: �Tumor Modified to Give Signal 2

B7

CD28

Ig

Antigen

TCR

Modified

T Cell

Tumor Cell

“1”

“2”

MIPCEA-B7

Comparing Signal 1 with Signal 1+2�(IL2 present)

10⁹

10⁸

10⁷

10⁶

10⁵

0 5 10 15 20 25 30

Time (days)

Viable Cells

10⁹

10⁸

10⁷

10⁶

10⁵

0 5 10 15 20 25 30

Time (days)

Total Viable Cells

10⁹

10⁸

10⁷

10⁶

10⁵

0 5 10 15 20 25 30

Time (days)

Total Viable Cells

T cells on MIP-CEA tumor

T cells on MIP-CEA-B7 tumor

T cells on MIP-CEA tumor

Fresh CEA tumor

T cells on MIP-CEA-B7 tumor

Fresh MIP-CEA-B7 tumor

A

B

C

Signal 1-only = AICD Signal 1+2 = Proliferation Proliferation = Increased tumor cell killing

--> EFFICACY HAMPERED BY PROLIFERATION DEFECT

Causes of Activity Loss

T

T

T

T

#1

#2

Cancer

Blood

Liver, etc

Tissues

*

*

Infuse

Hypothesis

• Tumor eradication would follow if

EITHER

• #1. T cells persisted systemically
• (“bypass co-stimulation”)
• OR
• #2. T cells persisted/expanded intratumorally
• (“provide co-stimulation”)

​

Strategies to Overcome AICD/Proliferative Defect

1. Provide co-stimulation

​

2. Bypass co-stimulation

Strategy 1

Provide co-stimulation

​

Incorporate Signal 2 into designer T cells (2nd generation)

​

1^st and 2^nd Gen Constructs

Signal 1

Signal 1+2

Expansion on MIPCEA

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1.0E+07

Day 0

Day 3

Day 7

Day

T cell number

UnTd

IgTCR

Tandem

Expansion on MIP101

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1.0E+07

Day 0

Day 3

Day 7

Day

T cell number

UnTd

IgTCR

Tandem

2nd Gen T Cell Tumor-Induced Proliferation

CEA(-)

CEA(+)

2^nd Gen Designer T Cells are Selectively Expanded

1st Gen

2nd Gen

Control

0

50

100

150

200

250

300

1

3

5

7

9

11

13

Day post tumor cell injection

Untransduced

IgTCR

Tandem

0

50

100

150

200

250

300

1

3

5

7

9

11

13

Day post tumor cell injection

Tumor size (mm )

2

Untransduced

IgTCR

Tandem

Tumor size (mm )

2

MIP101

MIPCEA

Superior in vivo Tumor Suppression by 2nd Gen T Cells

Adjuvant model:

CEA(-)

CEA(+)

Mvc-007f

CEA-

CEA+

2 Tumors in a mouse-2

CEA-

CEA+

MVC 004f

CEA-

CEA+

45d post Tx

70d postTx-2 Tandem

CEA+ (only)

A Established Tumor - Pretherapy

B Established Tumor - UnTd T cells, 10 days

C Established Tumor - PR in CEA+, 10 days

D Established Tumor - Tandem, CR 70 days

Phase Ia/Ib Trial of 2^nd Generation �Anti-CEA Designer T Cells in Adenocarcinoma

FDA BB IND 10791

What is a clinical trial?

“A test of hypotheses of drug activity in human subjects.”

Hypotheses

• 2nd gen “Signal 1+2” designer T cells will escape AICD and proliferate intratumorally
• Expanding designer T cells in tumor will maintain anti-tumor activity until tumor elimination

Treatment Plan

• T cell harvest, Designer T cell preparation
• Designer T cell infusion (x1)
• Monitoring
• Safety
• Designer T cell persistence/expansion
• in blood
• in tumor (paired biopsies)
• Tumor response

Eligibility

• Gastric, breast or colon cancer
• CEA+
• Failed first line chemotherapy
• Measurable disease
• PS 0-1
• >4 months life expectancy

​

​

Patients are listed by numbers. Enrollment is nominal, for no DLT. Tumor biopsies (Bx) to assess in situ designer T cell expansion.

-IL2

Pt #

Cohort

1x10 ⁹

1x10¹⁰

1x10¹¹

#1

#2

#3

#4

#5

#6

#7

#8

#9

X

I

X

X

X

II

(Bx)

III

X

X

X

X

X

Study Design

T Cell Dose, Number of Cells

BIOPSIES

Day 2 = designer T cell trafficking

Day 10 = designer T cell survival/ proliferation

Cell proliferation index (CPI) = d10/d2

​

Phase Ia “Safety”

Progress

• 2nd gen designer T cells created and validated
• Approvals
• FDA, IND filed (11/02)
• BB IND 10791, “release from clinical hold” (5/03)
• RAC/OBA/NIH approval (4/03)
• IRB, IBC approvals DFCI, RWMC (4/03, 11/04)
• Clinical vector preparation – NGVL/NCRR/NIH
• supernatant available (10/07)
• Trial funding
• Phase Ia, Orphan Products Office/FDA $600,000 [gastric]
• Phase Ib/Pilot, seeking from NCI and DOD
• Phase Ia clinical trial start (12/07)

Escalation Plan

T Cell Dose, Number of Cells

Pt # Cohort -IL2 +IL2

1x10⁹ 1x10¹⁰ 1x10¹¹ 1x10¹¹

#1 X

#2 I X

#3 X

#4 X

#5 II X

#6 X

#7 X

#8 III X

#9 X

Completes Phase Ia goals (-IL2)

(Bx)

#10 | X

#11 | X

#12 R IV X

#13 A X

#14 N X

#15 D X

O (Bx)

#16 M X

#17 I X

#18 Z V X

#19 E X

#20 | X

#21 | X

Completes Phase Ib goals (OBD –IL2/+IL2)

Summary 2^nd Generation CEA

• 5 patients treated
• 1^st cohort completed (10^9 cells)
• 2^nd cohort in process (10^10 cells)
• Safety, no SAEs (no new risks with 2^nd gen)
• Responses
• one “minor” (brain and lung)
• one SD 12+ months
• Continue in escalation
• Need to assess value of IL2 supplementation

Strategy 2

Bypass co-stimulation

​

“Designer Auto-graft”

Auto-Transplant: Engraft designer T cells via lympho-expansive capacities of the body after lympho-depletion treatments

Melanoma: TILs

Melanoma

CD8+�TIL

Rosenberg et al NEJM 1988;319:1676.

Tumor Harvest

~5% cure

20% major response

Responses not durable

Only melanoma, limited numbers

Technically challenging, antigen(s) unknown

Not reproducible in other studies

But:

Melanoma: NMA + TILs

Melanoma

CD8+�TIL

Hematologic Recovery

Tumor Response

Dudley et al, Science 2002;298:850

Tumor Harvest

Non-myeloablative (NMA) Conditioning

X

50% major responses

Designer T Cell Engraftment

Ex vivo gene therapy

T Cell Harvest

Hematologic Recovery

Tumor Response

Anti-PSMA designer T cells

Non-myeloablative (NMA) Conditioning

CD3+

CIR+

CIR+

+IL2 low dose (outpatient)

Phase Ia/Ib Trial of Anti-PSMA Designer T Cells in Advanced Prostate Cancer after Non-Myeloablative Conditioning

FDA BB-IND 12084

What is a clinical trial?

….

Prostate Specific Membrane Antigen (PSMA)

• Surface membrane glycoprotein 100,000 Daltons
• On normal prostate epithelium and prostatic vasculature
• Elevated expression in metastatic lesions and hormone refractory disease
• High clinical relevance:
• 25,000 deaths per year from PSMA+ prostate tumors
• ​

Tumor Antigen:

Hypotheses

• After chemotherapy, designer T cells will engraft as stable fraction of blood cells
• Systemic engraftment will support unremitting infiltration of designer T cells into tumor
• Sustained infiltration will lead to sustained anti-tumor activity until tumor elimination

-- irrespective of activation-induced T cell death,

bypassing co-stimulation.

Eligibility

• Prostate cancer
• Failed androgen ablation therapy
• Elevated serum PSA
• PS 0-1
• >4 months life expectancy

​

​

Treatment Schema

​

-------- modify T cells ------ microbiologic testing ---------

​

• NMA T cell infusion
• T cell collection ------ G-CSF ----- PSC collection ------- chemotherapy ----------- start IL2 ------ end IL2
• -21* -20* -16* -7 -1 0 +28

​

• Study Day

​

Biopsy

CTX 60 mg/kg d-7, d-6 Fludarabine 25 mg/m2 d-5 to d-1

Designer T cells

---- Rescue Pack ----

Phase Ia/Ib Study Enrollment Plan

​

• T Cell Dose, Number of Cells
• Pt # Cohort
• 10.9 10.10 10.11 .
• #1 X
• #2 I X
• #3 X

​

• #4 X
• #5 II X
• #6 X
• #7 X
• #8 X
• #9 X

(Bx)

• #10 X
• #11 III X
• #12 X
• #13 X
• #14 X
• #15 X

​

• Monitoring
• Safety
• Designer T cell persistence/expansion
• in blood
• In tumor
• Tumor response

1.1%

CD3

2.5%

Engraftment

Blood sample

Day +14

Dose

61%

7.3%

CIR+

CIR+

CD3+

#1

#2

PSA Response

Conditioning d-8 to d-2

T cells infused d0

Low dose IL2 d0 to d28+

T cells

chemo

chemo

T cells

Conclusions

• Recursive/iterative process:
• Bench to bedside, bedside to bench, bench to bedside…
• Application of lab correlates to improve clinical outcomes
• Define defect of 1^st gen vector, Signal 1-only = AICD
• Define defect of 1^st gen administration method, simple infusion = poor engraftment
• Study modifications address defects
• NEXT STEP: Sustained anti-tumor immune response.

​

• Future: Combination of 2^nd gen vector and method?

​

​

What To Learn?

• What are the parts of the immune system?
• What are T cells? What do they do?
• What is a tumor antigen?
• What is gene therapy? How can gene therapy teach T cells to attack cancer?
• How does an idea lead to a new therapy? What is a clinical trial?
• How does a clinical trial stimulate lab studies that modify the therapy?

​

​

The Goal:�T cells killing cancer cells

Clinical Trials Currently Recruiting

• Breast cancer
• Colon cancer
• Gastric cancer
• Prostate cancer

​

​

Contact Dr Junghans’s office

at Roger Williams Hospital

401 456 2507

T cells homing in on target