EcoCRM TM

A Recombinant CRM197 Carrier Protein

A. Lees ,
1 R. Simon ,
2 S. Baliban ,
2 I. Krauss ,
3 D. Nguyen ,
3 M. Pravetoni ,
4,5 N. Oganesyan 1

1, 2Fina Biosolutions, Rockville, MD; 2U Maryland School of Med, Baltimore; 3Brandeis U, Waltham, MA;
4Minneapolis Medical Research Foundation (MMRF); 5Center for Immunology, U Minnesota

S. Typhimurium Core-OPS-EcoCRMTM Conjugate

Development of a glycoconjugate vaccine to prevent invasive Salmonella Typhimurium
EcoCRMTM Affordable CRM197 Carrier Protein infections in sub-Saharan Africa. Baliban et al. PLoS Negl Trop Dis 11(4):e0005493, 2017

CRM197 , a genetically detoxified diphtheria toxin, is widely used as a carrier protein

in conjugate vaccines. It was originally expressed as a secreted protein in
Corynebacterium diphtheriae. Until recently, both “native” and recombinant CRM197
STm-COPS was covalently linked
has been difficult to obtain and/or expensive. to EcoCRMTM via its KDO group

Fina BioSolutions has developed a new CRM197, EcoCRMTM, produced in E.coli.

EcoCRMTM is expressed as soluble protein in the cytoplasm of a widely-used E.coli
expression strain. High expression of EcoCRMTM in a BL21 strain and a simple
purification method allows low cost production and the promise of significantly
reducing the cost of this component of conjugate vaccines. S. Typhimurium core O-
STm-COPSKDO-EcoCRMTM
neoglycoconjugate
polysaccharide (STm-COPS)

The data presented show that EcoCRMTM is an excellent carrier protein for a
variety of antigens. P < 0 .0 0 0 1 P < 0 .0 0 0 1
100
P < 0 .0 0 0 1

S u rv iv a l (% )
7
10 80 P B S (lo w d o s e )

a -P S s e ru m Ig G
6
10
10 5 60 P B S ( h ig h d o s e )

( E U /m L )
4
10 40 P S - C R M (lo w d o s e )
3
10
P S -C R M (h ig h d o s e )
10 2 20
1
10
0
0
10
0 2 4 6 8 10 12 14
PBS PBS P S -C R M P S -C R M
T im e p o s t- c h a lle n g e ( d a y s )
(lo w d o s e ) (h ig h d o s e ) (lo w d o s e ) (h ig h d o s e )

Kaplan-Meier survival curves of mice immunized with

Serum IgG titers against STm-COPS from mice (n =
SEC HPLC of EcoCRMTM
SDS PAGE EcoCRMTM
>99% purity. No nicking evident. Dimer is <0.6%. Purity >99%.
20/group) immunized with 2.5 µg STm-COPSKDO:CRM197
or PBS. Solid bars indicate the GMT.
HIV Glycopeptide Conjugate
STm-COPSKDO-CRM197 (circle) or PBS (diamond) after
challenge (n = 20/group) with 1 or 5x106 CFU of STm D65.

Synthesis of multivalent glycopeptide

S. Typhimurium COPS-EcoCRMconjugatesTM
that mimic an HIV epitope
conjugate
provided Bailey
100%etprotection
al. Tetrahedronin72:6092, 12016 model
this animal

Therapeutic Vaccines for Treatment of

HIV Glycopeptide-EcoCRMTM Conjugate
Opioid Abuse and Overdose
Synthesis of multivalent glycopeptide conjugates that mimic an HIV epitope.
The Pravetoni lab has developed a series of conjugate vaccines that elicit opioid-specific IgG Bailey et al. Tetrahedron. 72:6091, 2016.
antibodies that reduce opioid distribution to the brain. Immunization selectively blocks opioid-
induced behaviors, such as opioid self-administration, and prevents opioid-induced respiratory Thio-ether conjugation is frequently used for linking peptides, oligosaccharides
depression and bradycardia in mice and rats. Current efforts focus on readying for FDA approval and other antigens to a carrier protein such as CRM197.
and Phase I clinical trial a vaccine consisting of oxycodone (OXY) conjugated to a GMP
monomer KLH carrier protein. Due to its mullosk origin and poor characterization, native KLH is
not an ideal carrier. This study compared oxycodone conjugates using EcoCRMTM and KLH.

The haptenization ratio of

oxycodone-EcoCRMTM can
be characterized by MALDI-
TOF.
EcoCRMTM was analyzed before and
after conjugation to the OXY hapten. A Thiol-glycopeptide was conjugated to the
EcoCRMTM was labeled with an excess of the NHS maleimide
ratio of 24 haptens per CRM was EMCS-labeled CRM197. Mass spec of the
reagent sulfo-EMCS. The MW of EcoCRMTM increased from
calculated from the MW difference. We
were unable to characterize the
58,413 to 62,525, (delta=4100). Each linker adds a mass of 208, conjugates indicated ~7 glycopeptides/CRM.
indicating about 20 of CRM’s 39 lysines have been labeled.
conjugates containing native KLH.

IN VIVO VACCINE EFFICACY Induction of oxycodone-specific IgG antibody Glycopeptide conjugates of EcoCRMTM and
A
prevents oxycodone distribution to the brain CRM from P. fluorescens (“Pfenex”) were
B
400 1000 in mice. prepared. Rabbits were immunized twice.
Oxycodone-specific IgG titer (x103)

** After the final bleed,IgG anti-glycopeptide

BALB/c mice (n ≥ 4/group) were immunized s.c. with 100 µg titers were measured using glycopeptide-BSA
Brain oxycodone (ng/mL)

800
300 ***
unconjugated KLH (control), OXY-KLH, or OXY-EcoCRMTM, as the ELISA antigen.
600 (58%)

200
**** formulated with 1 mg of alum adjuvant on days 0, 14 and 28. On
400 (73%)
day 35, mice were challenged with 2.25 mg/kg oxycodone, and
**** then brain and serum collected for analysis of vaccine efficacy.
100
200
Glycopeptide conjugates with
A) Oxycodone-specific serum IgG titers analyzed by ELISA.
0 0
B) Oxycodone concentration in the brain 30-min after oxycodone EcoCRMTM & Pfenex CRM induced
M
LH

LH

M
MT

LH

LH

MT
K

-K

-K

comparable anti-glycopeptide titers

R
XY

challenge, analyzed by GC/MS. Above bars, percentages (%)

XY
oC

C
O

co
O
c
-E

-E
XY

XY

indicate decrease in brain oxycodone compared to KLH. in rabbits.

O

Pfenex
O

C D
0.0020 ** 0.0060 **
Induction of oxycodone-specific B cells
Oxycodone-specific IgM+ B cells

OXY-specific GC B cells

0.0015 0.0045
BALB/c mice (n ≥ 4/group) were immunized i.m. with 100 µg
(% of lymphocytes)

(% of lymphocytes)

KLH, OXY-KLH, or OXY-EcoCRMTM, formulated with 200 µg of

0.0010 ** 0.0030
alum. At 7 days post-immunization, OXY-specific B cell
populations from spleen and lymph nodes were isolated by
0.0005 0.0015
antigen-based magnetic enrichment paired with multi-parameter
flow cytometry.
0.0000 0.0000
M

M
LH

LH

LH

LH
MT

MT
K

-K

-K

C) Oxycodone-specific IgM+ B cells.

EcoCRMTM is an effective carrier protein for
R

R
XY

XY
C

C
co
O

co
O
-E

-E

D) Oxycodone-specific germinal center (GC) B cells.

XY

XY
O

conjugate vaccines and is an alternative to

The oxycodone-EcoCRMTM conjugate showed superior efficacy than the previously
CRM197 from Corynebacterium and
established oxycodone-KLH. In contrast to KLH conjugates, EcoCRMTM conjugates are
An oxycodone-EcoCRMTMeasier conjugate demonstrated similar or superior
to characterize. Pseudomonas. A simple expression system,
efficacy against oxycodone compared to the standard KLH carrier protein. Fina Biosolutions high yields and the efficient purification of
The Pravetoni group is currently developing vaccines and monoclonal antibodies EcoCRMTM can help reduce the cost of
against heroin, oxycodone, and fentanyl abuse and overdose (Pubmed, “Pravetoni M”). www.FinaBio.com conjugate vaccines.
For collaborations or partnership, please contact Info@FinaBio.com
Dr. Marco Pravetoni at prave001@umn.edu.

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