Alternative Strategies for KBMA Tularemia
Vaccine
Cerus TVDC Annual Meeting, October 9th 2007
TVDC Annual Conference, 9 Oct., 2007, Page 1
Two Applications of KBMA Technology
• KBMA bacterial infectious disease agents (e.g: B. anthracis, F.
tularensis)
»
»
»
»
»
Delete uvrAB genes of selected organism
Optimize S-59 and UVA inactivation process
Optimize large scale production
Optimize route and regimen of delivery
Worked well for B. anthracis vaccine not so well for F. tularensis
• KBMA L. monocytogenes expressing antigens
» Clone antigen into L. monocytogenes KBMA platform strain
» Can be applied to viral and cancer, as well as bacterial indications
» Optimization of production, route, and regimen is similar for each
vaccine
» Provides opportunities for synergy with other vaccine development
projects
TVDC Annual Conference, 9 Oct., 2007, Page 2
Rationale for Changing Course on
Development KBMA Tularemia Vaccine
•
Deletion of the uvr genes in Ft novicida or LVS does not significantly increase
the sensitivity to photochemical inactivation or increase the degree of
metabolic activity compared to wild type
•
This suggests that there may be an unknown repair mechanism functioning in
Ft strains that preclude production of potent KBMA Ft vaccines
•
KBMA Ftn provide protection against live Ftn challenge, but this protection was
demonstrated to be humoral and not greater than heat killed Ftn
•
KBMA LVS vaccines provide protection against lethal live LVS challenge, but
not more so than heat-killed LVS
•
KBMA LVS did not provide significant protection against lethal SchuS4
challenge
•
Live LVS may actively inhibit the innate inflammatory response elicited by
Listeria monocytogenes. This response has been demonstrated to be required
for protective T cell immune responses and suggests that LVS may not be a
strong vaccine platform for eliciting a potent memory T cell response
TVDC Annual Conference, 9 Oct., 2007, Page 3
Background on Listeria monocytogenes
Attenuation
Without
loss of potency
Brockstedt et. al
2004
Tilney and Portnoy, 1989
TVDC Annual Conference, 9 Oct., 2007, Page 4
Why Use L. monocytogenes?
• Taking established dogma to develop vaccines
» Potent activator of innate immune effectors
» Taken up by and multiplies within antigen presenting cells
+
» Induces robust CD4+ and CD8 T-cell immunity
+
» CD8 T cells required for protection in mouse listeriosis
model
• Repeat administration
• Practical advantages
TVDC Annual Conference, 9 Oct., 2007, Page 5
UCLA Work on Lm-Based Tularemia Vaccine
• Marcus Horwitz and Qingmei Jia presented poster at the
International Tularemia Meeting in Wood’s Hole
• Used a proteomic approach to identify secreted Ft antigens
• Screened Ft antigens for protection against lethal LVS
challenge either as purified proteins or as expressed from
attenuated (actA) Listeria monocytogenes:
» AcpA, Bfr, DnaK, GroEL, IglC, KatG, Pld
• 2 most protective antigens were IglC and KatG
• These Ft antigens expressed from Listeria monocytogenes
induced a proliferative cellular immune response
TVDC Annual Conference, 9 Oct., 2007, Page 6
UCLA Data Demonstrating Lm-Based
Vaccines Protect Against LVS Challenge
Table 2
Groups
(8 mice/
group)
13
1
2
3
4
5
Vaccine
Prime
Unimmunized Control
LVS
Lm/ΔactA
rLm/iglC
rLm/katG
rLm/iglC + rLm/katG
Vaccine
Boost
Number
Survivors
Survival
Rate (%)
Lm/ΔactA
rLm/iglC
rLm/katG
Lm/iglC + Lm/katG
0
3
5
6
6
8
0
37.5
62.5
75
75
100
For Non-survivors,
Median Time to Death
(Days)
4.9
4.8
4.3
4.5
5
-
• 100% protection against lethal LVS challenge achieved by 2 ID administrations
of a mixture of Lm expressing IglC and KatG
TVDC Annual Conference, 9 Oct., 2007, Page 7
Live-Attenuated Lm-Based Vaccines Can
Protect Against Lethal Schu4 Challenge
Unpublished data provided by Marcus Horwitz
Sup. Fig. 1
Number of survivors
(8 mice/group)
A.
1 x LD100 Schu 4
B.
10 x LD100 Schu 4
8
7
6
5
4
3
2
1
0
8
7
6
5
4
3
2
1
0
1
3
5
7
9
11 13 15 17 19 21
Sham
LVS
rLM / actA
rLM / IgIC
rLM / KatG
1
3
Days post-challenge
• 2 ID vaccinations separated by 4 weeks
• Aerosol challenge 6 weeks post-boost
• Performed by Dr. Bruce Bowen at CSU
TVDC Annual Conference, 9 Oct., 2007, Page 8
5
7
9
11 13 15 17 19 21
Days post-challenge
Advantages of Cerus L. monocytogenes
Platforms
• Listeria monocytogenes is clinically acceptable vaccine platform
» Our live attenuated platform strain (actA inlB) is 1000-fold
attenuated compared to wild type (and causes less liver toxicity than
actA)
» We are currently in one phase I clinical trial with live attenuated Lm
vaccine and have FDA approval to initiate a second phase I trial
» Doses up to 3 x107 Lm administered IV to cancer patients
• Our KBMA platform is actA inlB uvrAB
» Conditions for vaccine production are largely optimized
» We are developing a KBMA Lm vaccine for IND submission in 2008
• We have developed superior antigen expression cassettes
» Our ActAN100 expression cassette is optimized for intracellular
expression and secretion
TVDC Annual Conference, 9 Oct., 2007, Page 9
Cerus Antigen Expression Cassette Produces
Large Quantities of Intracellular IglC
TVDC Annual Conference, 9 Oct., 2007, Page 10
Rationale for Modified Milestones
• KBMA Francisella tularensis vaccines lack potency
• Listeria monocytogenes-based vaccines elicit robust cellular
immune responses
• Listeria monocytogenes is a clinically acceptable platform
• Proof of concept studies using Listeria based vaccines have
been performed by UCLA and demonstrate potency in
SchuS4 challenge model
• Cerus has the ability and capacity to improve the potency of
the existing Lm-based tularemia vaccine and Cerus can
produce KBMA Lm-based tularemia vaccines that can be
acceptable to FDA
TVDC Annual Conference, 9 Oct., 2007, Page 11
Milestone 1: Compare Cellular Immune Responses
Induced by Lm and Ft-Based Tularemia Vaccines
•
Measure cellular immunogenicity of live attenuated vaccine platforms
» Use model ovalbumin epitope to compare Lm-expressing IglC-SIINFEKL and Lm
KatG-SIINFEKL fusion proteins with Ftn-pepO-SIINFEKL and LVS-pepO-SIINFEKL
• Measure the ability of each vaccine to stimulate a CD8 T cell response in vitro using a B3Z
assay
• Measure the cytokine responses elicited by vaccination with each platform in mice
• Compare the CD8 T cell response to SIINFEKL after prime and boost vaccinations in mice
•
Compare immunogenicity of KBMA tularemia vaccine platforms
» Compare KBMA Lm-IglC-SIINFEKL and Lm-KatG-SIINFEKL fusion proteins with
KBMA Ftn-pepO-SIINFEKL and LVS-pepO-SIINFEKL
• Produce 400mL scale lots of each KBMA vaccine
• Measure metabolic activity of each lot of vaccine
• Measure the ability of each vaccine to stimulate a CD8 T cell response in vitro using a B3Z
assay
• Measure the cytokine responses elicited by vaccination with each platform in mice
• Compare the CD8 T cell response to SIINFEKL after prime and boost vaccinations in mice
TVDC Annual Conference, 9 Oct., 2007, Page 12
Milestone 2: Demonstrate that Lm Vaccines Induce
Protective Cellular Immune Responses to Ft Antigens
• Demonstrate that Cerus strains of Live and KBMA Lm-IglC-SIINFEKL
and Lm-KatG-SIINFEKL protect against a SchuS4 challenge
» Produce lots of KBMA vaccine and send to UNM for testing in animal
models (mice and rats)
• Measure the T-cell response to IglC induced by live and KBMA Lm
expressing IglC compared with those elicited by Ftn or LVS
vaccination
» Produce IglC overlapping peptide library 15aa overlapping by 11aa
(211 amino acid long protein)
» Use IglC peptide library for ELISpot assays to measure the IglCspecific T cell responses induced after vaccination with live and KBMA
Lm-IglC and compare to live and KBMA Ftn and LVS vaccination
» Demonstrate mechanism of protection induced by Lm vaccines is
cellular by depletion of T cell populations and passive transfer studies
TVDC Annual Conference, 9 Oct., 2007, Page 13
Milestone 3: Optimization of KBMA Lm Vaccination
Route and Regimen
• Compare various routes of administration including IV, IM, IN, ID and oral
» For oral and ID administration we will mutate the inlA gene of Lm to allow
for binding of murine E-cadherin and mimic the human interaction
» We will compare the potency of the inlA gain of function mutants to our
traditional platform strain
» Routes will be ranked by ability to induce a cellular immune response:
Elispot, in vivo cytotoxity, and ICS
• Optimize dosing regimen of most potent and tolerable route
» Lm expressing IglC and/or KatG will be used
» Initial evaluation will be performed by immunogenicity
• Optimized route and regimen will be confirmed by SchuS4 protection
studies at UNM
TVDC Annual Conference, 9 Oct., 2007, Page 14
Milestone 4: Large Scale GMP-Like
Production of KBMA Lm Tularemia Vaccine
• Optimize scalable KBMA vaccine production at 4L scale
• Produce a 30L lot of most potent KBMA Lm vaccine under
GMP-like conditions at a contract manufacturer for protection
studies in non-human primates
• Develop quality assays to support release and stability testing
of KBMA Lm vaccine lots
• Perform toxicology studies using KBMA Lm platform
» Histopathology of administration sites
» Biodistribution studies (using PCR based methods)
TVDC Annual Conference, 9 Oct., 2007, Page 15
Optional Milestone 5: Use Lm Platform For
Delivery of Ft Antigens Discovered by TVDC
• Cerus could potentially make available the Lm platform
• Clone up to 10 Ft antigens identified by TVDC group into Lm
expression cassettes
• Characterize the intracellular expression levels of various Ft
antigens (and SIINFEKL immunogenicity)
• Rank potency of each vaccine candidate by sharing with UNM
for protection studies
TVDC Annual Conference, 9 Oct., 2007, Page 16
Benefits of Modifying Aims
• We cannot evaluate various KBMA Ftn strains by Ftn protection studies
since HK is nearly as potent at inducing a humoral response. Thus, we
cannot use Ftn as a model to inform the construction of an attenuated
KBMA SchuS4 vaccine.
• KBMA LVS vaccine failed to protect mice against SchuS4 challenge.
• Through our collaboration with UCLA, we have identified an alternative
vaccine platform (Listeria monocytogenes) and 2 antigens that have
succeeded in pivotal POC protection studies.
• Cerus has a superior platform strain and antigen expression cassette
than those initially selected by UCLA, and the ability to produce KBMA
vaccines based on this platform.
• Cerus has a large degree of expertise in developing Lm-based vaccines
and seeing them through the regulatory process of IND filing.
• Switching our focus to a Lm based vaccine now allows us to take this
opportunity before we spend a lot more time trying to optimize a suboptimal platform without incurring extra costs.
TVDC Annual Conference, 9 Oct., 2007, Page 17
Questions after Presentation
•
Rick: push hard on subunit vaccine. Justin and Tom have a
collaboration for proof of concept of subunit vaccine, which is the best
so far. A subunit vaccine providing protection is very important. Justin
made a cogent argument for not pursuing the KBMA on the FT platform.
Is a LVS a good platform for delivering a vaccine, even its own
antigens? Could compare LVS to LM delivery of Ft antigen for their
relative antigencity. Elements could add to knowledge in the community
•
Kathy: This subunit approach, Lm platform for Ft antigens and UNM T
cell responses triangulate the studies of protection. Are the same
antigens picked up in Terry’s T cell assays, Stephen’s expression
assays and Lm vaccine delivery assay? Would be interesting.
•
Tom; Cerus has done 15x11 plate assays and would love some of
ASU’s 200aa peptides to test.
•
Kathy: stimulatory regions of peptides may be overlapping
TVDC Annual Conference, 9 Oct., 2007, Page 18
Questions after Presentation
• SAJ: Don’t ignore the fact that antibody/humoral protection
appears to be occurring in KBMA results to date. Don’t see any
use for an i.v. route because won’t be practical in humans for an
Ft vaccine, though understands why the iv route was used for
cancer delivery.
• Rick: A drug just came out with a regimen of once per year i.v.
injection actually being used.
• Stephen: i.v. is probably not likely for the Ft vaccination purpose.
• Justin: i.m doesn’t work as well, and i.v. route is a control route
known to work well. Justin said the iv route is a research gold
standard and not intended for Ft vaccination clinically.
• Tom; Can protect with multiple i.m. vaccinations actually.
TVDC Annual Conference, 9 Oct., 2007, Page 19