Genetically Engineered T Cells

Selecting for high affinity receptors

Extreme toxicitiesNormal T cell receptor/antigen/MCH complexes are relatively low affinity

On-target, off tumor

Addressing the Issue of Acute Toxicity of Genetically Engineered T cells

N

CN

C

C

C

N

N

C

O

H2N

H

CH2

O

CH2

CH2HO

ATP ADP

HSV thymidine kinase

N

CN

C

C

C

N

N

C

O

H2N

H

CH2

O

CH2

CH2OP

O

O-

O-

ACV-TPcellular kinases

• acyclovir, first specific antiviral drug• used to treat herpes simplex I and II and varicella zoster virus infections

Antiviral Drugs

promiscuous, broad substrate specificity

active drugcellular TK does not phosphorylate ACV

PPP-

O-C

H2

O

GC4

PPi

HSV DNA polymerase

* * * *

3′

C4*

P

-O-C

H2

O

Gchain

termination

Acyclovir: Mechanism of ActionACV binds with a 30-fold higher affinity to HSV DNA polymerase relative to cellular polymerase

OHOH

• ACV selectively phosphorylated by HSV TK• ACV-TP binds selectively to HSV DNA pol• ACV functions as a chain terminator

3′ - 5′ phosphodiester bonds

Addressing the Issue of Acute Toxicity of Genetically Engineered T cells

Autologous Adoptive Cell Therapy

Immunosuppressive environment

Even after reinfusion of expanded and activated T

cells the immunosuppressive environment of tumors and

natural T cell regulatory mechanisms limit their

effectiveness, particularly in solid tumors

What if Rather than Go through all the Bioengineering Involved in this Thought Experiment We Instead Reactivated the Natural

Diverse T Cells Already Present in the Tumor in the Body?

An Alternative Approach to T Cell Therapy: Immuno-Regulation

• Soon after T cells are activated a regulatory mechanism kicks in that begins to turn them off

CTLA-4 is a member of the CD28 family of co-receptors except it inhibits rather than activates T cells

CTLA = cytotoxic lymphocyte antigen

Upregulated 48 to 72

hours after activation

Prevents pathogenic

autoimmune disorders

T Regulatory Cells Also Use CTLA-4 in Regulating T Cell Activation

• Increased CTLA-4• Increased CD25• No production of IL-2

Tregs

CTLA-4

B7, CD80

Tregs

antigen presenting

cell

• Interaction results in internalization and degradation of CD80 on antigen presenting cells

• APCs can no longer provide signal 2 for T cell activation

Ag

MHC

T Regulatory Cells Also Serve as a Sponge for IL-2

Tregs• Tregs do not express

IL-2 but produce large amounts of CD25, a high affinity IL-2 receptor

IL-2

IL-2IL-2

IL-2

• Binding of IL-2 to Tregs stimulates Treg proliferation and deprives other T cell types of IL-2

Lympho-Depletion Prior to Adoptive T Cell Therapy

Lympho-depletion may work in part by reducing the number of immunosuppressive lymphocytes

Trials Using Ipilimumab, an antibody to CTLA-4, for Metastatic Melanoma

Gp100 vaccine

Ipi alone

Ipi + gp100 vaccine

It’s not much, but it is a start

676 patients comparing Ipilimumab with and without vaccination with GP100

GP100, a major TAA in melanoma

Hodi et al NEJM 2010

Combining Standard Chemotherapy With and Without Ipilimumab

Bondarenko et al NEJM 2011

• Immunotherapies often work better with combined with cytotoxic chemotherapies

• Standard of care chemotherapy for melanoma is dacarbazine

Combining Ipilimumab with Standard Chemotherapy: Overall Survival

Effect of Ipilimumab on overall survival disappointing

Dacarbazine is an Imadazotetrazine Prodrug

Imadazotetrazines are alkylating agents

temozolomide

dacarbazine

Methyldiazonium ions active alkylating agents

Alkylating Agent Efficacy can be Influenced by DNA Repair Capacity

alkylating agents

N

CN

C

C

C

N

N

C1

23

4

56

7

8

9

O

H2N

CH3O6-methyl G can base pair with C or T alkylation at N7 can

lead to depurination

promotes cleavage of N-glycosidic bond

10,000 depurination events per day even in the absence of alkylating agents

MGMT – methylguanine methyl transferase

Temozolomide – alkylating agent used to treat glioblastoma

overexpression of MGMT leads to temozolomide resistance

accepts methyl group from base inactivating enzyme

gaps can be repaired but if damage too great, the guardian of the genome, p53, can induce cell death

DNA repair -

good

DNA repair -

bad

Dacarbazine (or temozolomide)

There’s More to the Tumor Immunosuppressive Environment than the Down-Regulation of T Cells by

CTLA-4

• PD-1 is also a negative regulator of T cell activation

The SHP Proteins are Tyrosine Phosphatases

Activation of SHP Phosphatases Disrupt Numerous Signaling Pathways

Nivolumab Trials for Advanced Melanoma

Combining Anti-CTLA-4 and Anti-PD-1 Therapies for Melanoma

Outcomes of the Trial

BRAF a tyrosine kinase involved in melanoma, which can be targeted by a TKI

Adverse Events With New Immuno-modulatory Agents

Immune related adverse events

Systems affected

• Skin – 3-4 weeks

• GI tract/liver – 6-7 weeks

• endocrine – 9 weeks

Patients who develop anti-CTLA-4 associated endocrinopathies may present with nonspecific symptoms such as:

• fatigue• headache • nausea • behavioral changes, visual impairments,

memory loss, decreased libido, • anorexia, • insomnia, • cold or heat intolerance.

hypothalamic-pituitary-thyroidal axis