# Presentation: 143001

##  Strategies and Opportunities for Cancer Therapy with Vaccines Inducing T cells or Antibodies

- Jay A. Berzofsky, M.D., Ph.D.
- Vaccine Branch, CCR, NCI

- NCAB Meeting
- Bethesda, Maryland
- June 17, 2008

- National Cancer Institute

**U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES**
**National Institutes    of Health**

## Rationale for Engineered Vaccines

** **
**However, for cancer or viruses causing chronic infection, such as HIV or hepatitis C virus, the natural disease does not induce sufficient immunity to eradicate the infection. **
** **
**A vaccine must elicit better immunity than the disease itself. **

**Rationale for Engineered Vaccines**

## CD8   Cytotoxic T cells can detect 

**CD8  Cytotoxic T lymphocyte**
**(CTL)**

**Antibody**

**Cell surface protein**

**Endogenous**

**protein**

**Protease**

**Transporter**

**Endoplasmic **

**Reticulum**

**Golgi**

**MHC **

**Class I**

**+**

**CD8   Cytotoxic T cells can detect **

**endogenous antigenic proteins even if not **

**expressed intact on the cell surface**

**+**

**Virus-infected or cancer cell**

## Types of Tumor Antigens

**	****	****	****	****	**** **_**Examples**_
**Overexpressed antigens ****	****	****Her-2/neu, CEA, TARP**
**Altered antigens****	****Shared by many tumors****	****p53, Ras, fusion proteins, MUC1****	****Unique to a single tumor****	****Point mutations in various genes**** ****	****	**
**Tissue-specific antigens****	****	****tyrosinase, MART1, gp100**
**Novel antigens (in adult) ****	****Fetal antigens ****	****	****	****CEA, oncofetal protein**** ****	**** **
** ****	****Viral antigens ****	****	****	****HPV E6 or E7, EBV antigens**
**	****Clonal antigens ****	****	****Idiotype**

## Desirable Characteristics for Tumor Antigens

**1. ****Tumor-selective**
**2. ****Essential to tumor cell survival**
**3. For T-cell antigens**
**	****- ****Processed****   **
**	****- ****Bind MHC**
**	****- ****Immunogenic****  **
**	****        **
**4. For B-Cell antigens**
**	****- ****Cell Surface Expression**
**	****- ****Accessibility of Epitopes**
**	****- ****Immunogenicity****   **
**	****	****	****In vitro**
**	****	****	****In vivo **
**	****	****	****Pre-existing Antibody response**

## Potential Mechanisms of Antibody Action against Tumors

**Antibody-dependent cellular cytotoxicity****(ADCC):  NK or other cells with Fc receptors bind antibodies and use them to target cells for killing.**
**Complement-mediated lysis**
**Inhibition of function of a molecule required for oncogenicity: e.g. HER-2/neu, CD25**
**Success of antibodies to HER-2/neu (Herceptin) and to CD25 (Zenapax) suggests functional targets may be the most effective****.**

## Adeno-neuECTM (Her-2) treatment causes regression of established s.c. TUBO mammary carcinomas

**Adeno-neuECTM (Her-2) treatment causes regression of established s.c. TUBO mammary carcinomas**

**Day 4**

**Day 9**

**Day 11**

**Day 14**

**Day 19**

**Day 26**

**Park et al., *****Cancer Research***** 2008**

**Notes:**
Photos showing regression of a 2 cm tumor over 26 days following a single injection of Ad-neuECTM (108 pfu).

## Adeno-neuECTM (Her-2) vaccine induces regression of established lung tumors from IV injection of TUBO breast cancer cells

**Control, sacrificed day 15**

**Ad-ECTM day 15, **
**Sacr  day 29**

**Control, sacrificed 30**

**Ad-ECTM day 15, **
**Sacr  day 35**

**Ad-ECTM day 15, **
**Sacr  day 48**

**Adeno-neuECTM (Her-2) vaccine induces regression of established lung tumors from IV injection of TUBO breast cancer cells**

**Park et al., **
***Cancer Research***** 2008**

## Ad-neuECTM serum downmodulates ErbB2 (Her-2) and inhibits its phosphorylation

**Phospho ErbB-2**

**Total ErbB-2**

**Proportion of time 0 control**

**conc.of immune sera**

**Hours of culture**

**Hours of culture**

**Ad-neuECTM serum downmodulates ErbB2 (Her-2) ****and inhibits its phosphorylation**

**Park et al., *****Cancer Research***** 2008**

**Notes:**
Incubation of TUBO cells with serum from immunized mice (1% or 5%) inhibits phosphorylation of ErbB2 over 8 hours (left panel), and also downmodulates it from the tumor cell surface right panel).  Normal mouse serum was used to provide an equal total amount of mouse serum in all cultures.  At 8 hrs, inhibition of phosphorylation exceeds downmodulation of ErbB2 at both 1% and 5% serum.  Error bars not visible are smaller than the size of the symbol.

## Advantages of Vaccine over Trastuzumab (Herceptin)

**Antibody induced by vaccine is not dependent on FcRs, but directly inhibits the function of the oncogene product and inhibits tumor growth without other cells. Herceptin requires FcRs.**
**Polyclonal antibodies elicited may target multiple Her-2 epitopes and be less susceptible to escape mutations than a monoclonal antibody to a single epitope.**
**Continuous antibody production avoids the need for repeated expensive monoclonal antibody administration (~$100K/yr).**

## CD8   Cytotoxic T cells can detect 

**CD8  Cytotoxic T lymphocyte**
**(CTL)**

**Antibody**

**Cell surface protein**

**Endogenous**

**protein**

**Protease**

**Transporter**

**Endoplasmic **

**Reticulum**

**Golgi**

**MHC **

**Class I**

**+**

**CD8   Cytotoxic T cells can detect **

**endogenous antigenic proteins even if not **

**expressed intact on the cell surface**

**+**

**Virus-infected or cancer cell**

## Cancer Vaccine Problems & Strategies

| Problems | Strategies to solve |
| --- | --- |
| Self antigens to which host is tolerant
 | Target subdominant epitopes strengthened by epitope enhancement:
Modify the amino acid sequence to improve MHC binding. |
| Downregulation of MHC or of processing machinery | Induce higher avidity T cells that can respond to low densities of peptide-MHC |
| Poor quality or quantity of immune response
For therapeutic vaccines, inadequate CD4+ T help | Use cytokines to improve the  quantity and quality and substitute for CD4+ help:
    |
| Suppression of the immune response | Remove the brakes by blocking negative regulation. |

## PUSH-PULL Approach to Optimizing 

**Antigen**

**Immune**
**Response**

**Optimize**

**Optimize**

**PUSH-PULL Approach to Optimizing **
**Vaccine-induced Immunity**

## Topics:

**Improve CTL quality by increasing avidity****	****with IL-15 **

**Improve CTL quality by using IL-15 to****substitute for CD4****+**** T cell help to induce****long-lived memory CTL**

**Remove the brakes by blocking negative ****regulation:  A new NKT regulatory axis.**

**Improve the antigen:  epitope enhancement**

**Use cytokines to improve the quality and quantity of immune response **

## Sendai Virus Peptide Bound to H-2Kb

**From DH Fremont, M. Matsumura, EA Stura, PA Peterson, **
**& IA Wilson.  Science 257: 919-926, 1992**

- Sendai Virus Peptide Bound to H-2Kb

**Strategy: Epitope Enhancement by Sequence Modification to ****Increase Peptide Affinity for the MHC Molecule**

**Peptide Fragments of Viral Proteins Bind Specifically in the Groove****of Major Histocompatibility Molecules such as HLA-A, B, C**** **

## Major Histocompatibility Molecule (HLA)

**Major Histocompatibility Molecule (HLA**)

**T cell Receptor**

**Peptide**

## Ahlers et al., JCI 108:1677, 2001

**Ahlers et al., *****JCI***** 108:1677, 2001**

## FLRNFSLML = HLA-A2-binding peptideTARP 29-37

**Expressed in prostate and breast cancers, but not in **
**  ****other organs**
**Using different open reading frame from normal TCR******
**Possible role: Oncogenic transformation of the cells**

**Amino Acid Sequence of TARP**

**FLRNFSLML ****= HLA-A2-binding peptide****TARP 29-37**

## TARP		++		--		++

**MCF-7**

**Du145**

**PC3-TARP**

**TARP-29-37**

**TARP-29-37****-3A**

**TARP-29-37****-9V**

**TARP****	****	****++****	****	****--****	****	****++**
**HLA-A2****	****++****	****	****++****	****	**** --**

**Tumor**

**Human CTL raised against an epitope-enhanced TARP peptide ****can kill human tumor cells expressing TARP and HLA-A2**.

## Cytokine Synergies:

**Cytokine Synergies:**
** ****IL-12 and GM-CSF synergized for CTL induction**

## Topics:

**Use of IL-15 in the vaccine to induce high **
**	****functional avidity CTL (recognizing low ****	****densities of peptide-MHC complexes on cells)**** **

## High, Intermediate, and Low Functional Avidity CTL Generated by Stimulation with Different Concentrations of Peptide Antigen

- High, Intermediate, and Low _**Functional Avidity**_ CTL Generated by Stimulation with Different Concentrations of Peptide Antigen

**Alexander-Miller, Leggatt, & Berzofsky, *****PNAS***** 93: 4102,  1996**

- High avidity

- Low avidity

- High avidity

- Low avidity

## Hypothesis: high avidity CTL are more effective at killing tumor cells

**Low **
**avidity**
**CTL**

- Alexander-Miller et al. *PNAS* 1996; Derby et al., *J. Immunol*. 2001; Belyakov et al, *Blood* 2006

- Cancer              Peptide-coated
-     cell                   cell

- Cancer              Peptide-coated
-     cell                   cell

** **

**Specific Killing**

- 0

- %

- 1

- 0

- %

- 2

- 0

- %

- 3

- 0

- %

- 4

- 0

- %

- 5

- 0

- %

- 6

- 0

- %

-  **Peptide-coated cells**

-  **Tumor cells**** **

**Control cells**

**Only HIGH AVIDITY CTL kill tumor cells**

**High Avidity CTL**

**Low Avidity CTL**

**Derby et al, *****J. Immunol.***** 2001**

**HighAv1**

**HighAv2**

**LowAv1**

**LowAv2**

**Type of CTL line**

**Notes:**
12-17-99

## IL-15 (made by DC)

**IL-15 (made by DC)**

**IL-2 (made by T cells)**

**Mast cell **
**proliferation**

**NK cell **
**development**

**Activated**
**T cells**

**Memory**
**CD8 T cells**

**Antigen-**
**Induced**
**Cell Death**

**Maintenance**

## Immunization with antigen + IL-15 induces higher functional avidity memory CD8+ CTL

**0**

**20**

**40**

**60**

**80**

**100**

**120**

**10**

**1**

**0.1**

**0.01**

**0.001**

**0.0001**

**1E-05**

**Control**

**Target cells coated with peptide [P18-I10]**, **M**

**% of maximum lysis**

**vPE16**

**vPE16/****IL-15**

**Immunization with antigen + IL-15 induces higher ****functional avidity memory CD8****+**** CTL**

**2 months after immunization**

**Oh et al., *****PNAS***** 2004**

## IL-15

**Avidity**
**CTL**

**IL-15**

**Complementary Mechanisms for IL-15 in CTL Avidity Maturation**

***Oh et al. PNAS, 2004***

## Topics:

**Use of IL-15 in the vaccine to induce high **
**	****avidity CTL (recognizing low densities **
**	****of peptide-MHC complexes on cells) **

**Improve CTL quality by using IL-15 to****substitute for CD4****+**** T cell help to induce****long-lived memory CTL**

## IL-15 expression by a vaccine vector induced longer-lived

** ****memory CD8****+**** CTL: IFN-gamma-producing cells**** **

**Boosted**

**0**

**1**

**2**

**3**

**4**

**5**

**Unimmunized**

**vPE16**

**vPE16/****VV-IL-15**

**vPE16/****VV-IL-2**

**Oh et al., *****PNAS***** 2003**

**Explained by 1. Higher IL-15R******** expression**
**	****2.  Greater homeostatic proliferation**

## Dendritic Cell

**CD8  Cytotoxic T cell**

**MHC **

**Class I**

**+**

**CD28**

**MHC **

**Class **

**II**

**Dendritic Cell**

**CD4****+**** T-cell Help for CD8****+**** CTL Mediated Through Activation of Dendritic Cell**

**CD40**

**TCR**

**High avidity**
**Longevity**

**Oh et al.,**
***PNAS*****, 2008**

## IL-15 during immunization substitutes for 

**CD4****+**** T cell help to induce long-lived memory CTL**
**(****One year**** after immunization)**

**0**

**0.1**

**0.2**

**0.3**

**0.4**

**Vaccine/Cell Depletion**

**    ****vPE16/**
**Undepleted**

**     ****vPE16/**
**CD4-depleted**

**vPE16****-IL-15****/**
** ****CD4-depleted**

**% of tetramer****+**** CD8****+**** T cells**

**Numbers of antigen-specific CD8****+**** T cells**

**Oh et al., *****PNAS*****, 2008**

## Conclusions for improving CTL quality

**IL-15 in a vaccine:**
**	****-Induces ****longer-lived memory CD8 CTL**
**	****-Induces ****higher avidity CD8 CTL**
**	****-****Overcomes the need for CD4 T cell help**** to elicit prolonged****	****	****CD8 T cell memory **
**	****-Is a critical natural mediator by which CD4 T help ****	****	****elicits  long-lived CD8 memory T cells****	**

**Thus IL-15 is a most promising candidate to enhance the efficacy  **** of vaccines for use in HIV-infected or cancer patients with ****a deficiency of CD4 T cell help (including therapeutic vaccines for ****AIDS or cancer).**

## Topics:

**Use of IL-15 in the vaccine to induce high **
**	****avidity CTL (recognizing low densities **
**	****of peptide-MHC complexes on cells) **

**Improve CTL quality by using IL-15 to****substitute for CD4****+**** T cell help to induce****long-lived memory CTL**

**Remove the brakes by blocking negative ****regulation:  A new NKT regulatory axis.**

## WHY?

**WHY?**

**TUMOR TOLERANCE**

**suppression by tumor**
- (anti-inflammatory
- Cytokines/STAT3 induc)

**absence of danger signals**
- (incorrect presentation);
**Off-signals on T cells**
- (e.g. CTLA-4 or PD-1)

**suppression by **
**immune cells**

- M2 macrophages
- or tumor associated macrophages (TAM)

**CD4****+****CD25****+ ****T regulatory cells (Treg)**

- Myeloid-derived suppressor       cells (MDSC),
- Granulocyte suppressors

**Natural Killer (NK) T cells**

## NKT cells

**NK****T cells**

** ****Unlike NK cells, they express a TCR, but have****unusual restriction to a nonclassical MHC molecule**

**NKT cells**

## CTL

**CD1d-restricted**
**CD4****+****NKT**

**CTL**

**CD11b****+ ****Gr1****+**

**NKT cells and IL-13 suppress CTL tumor immune surveillance though the IL-4R-STAT6 pathway **
**to induce TGF-******** production by CD11b****+****Gr-1****+**** cells**

**Tumor cells**

**tumor lysis**

**APC**

**CD1d**

**glycolipid**

**suppression of CTL activation**

**Terabe et al., *****Nat Immunol*****, 2000., Terabe et al., *****J Exp Med*****, 2003.**

## NKT cells

**NKT CELLS ARE A HETEROGENEOUS CELL POPULATION **

**NKT cells**

## ?

**Treg**

**NK**

**type I**
**NKT**

**type II**
**NKT**

**Infected cells**

**APC**

**CD1d**

**APC**

**CD1d**

**suppression of**
**immunity**

**cross-regulation**

**promotion of **
**immunity**

**lysis**

**lysis**

**Fig. 1**

**?**

_**A new immunoregulatory axis**_

**Terabe & Berzofsky,**
**Trends in Immunol, 2007**

**PD-1**

**CTLA-4**

## PUSH-PULL Approach to Optimizing 

**Antigen**

**Immune**
**Response**

**Optimize**

**Optimize**

**PUSH-PULL Approach to Optimizing **
**Vaccine-induced T-cell Immunity**

- (e.g. **epitope****enhancement**)

**Quantity increase**
**Quality improvement**

## Key Collaborators

- Antibody-inducing Adeno-HER-2/neu vaccine: Jong-Myun Park, Masaki Terabe, Jason Steel, Yoshio Sakai, Guido Forni, John Morris
- Epitope enhancement:  Jeff Ahlers, Takahiro Okazaki, Pablo Sarobe, SangKon Oh, Ira Pastan
- IL-15:  SangKon Oh, Tom Waldmann, Liyanage Perera, Masaki Terabe, Don Burke
- Negative Regulation:  Masaki Terabe, Elena Ambrosino, Jong Myun Park, Susanne Ostrand-Rosenberg, Mark Smyth, Dale Godfrey, Vipin Kumar, Takashi Yamamura

## Slide 41