Bacteria and bacterial derivatives as delivery carriers for immunotherapy- Introduction Bacterial strains Non – Pathogenic bacteria Pathogenic bacteria Bacterial derivatives Bacterial outer membrane vesicles (OMVs) Minicells Bacterial ghost Bacterial engineering Therapeutic payloads Payload expression strategies Bioengineering of bacterial derivatives Structural modification of OMV Engineering of bacterial minicells Engineering of BGs Routes of administration Applications Future perspectives

1. BACTERIAAND BACTERIAL DERIVATIVES AS
DELIVERY CARRIERS FOR IMMUNOTHERAPY
PRESENTED BY:
MONIKA PANDARKAR
M.PHARM SEMESTER 1
(PHARMACEUTICS)
GUIDED BY:
Dr N.S.RANPISE
M.Pharm,PhD
SINHGAD COLLEGE OF PHARMACY, VADGAON (BK),PUNE-41
16 Feb 2022
A SEMINAR ON

2. CONTENT
 Introduction
 Bacterial strains
 Non – Pathogenic bacteria
 Pathogenic bacteria
 Bacterial derivatives
 Bacterial outer membrane vesicles
(OMVs)
 Minicells
 Bacterial ghost
 Bacterial engineering
 Therapeutic payloads
 Payload expression strategies
 Bioengineering of bacterial derivatives
 Structural modification of OMV
 Engineering of bacterial minicells
 Engineering of BGs
 Routes of administration
 Applications
 Future perspectives
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16 Feb 2022 Monika J.Pandarkar

3. INTRODUCTION
 Bacteria – ‘Perfect’ anticancer agent
 Specific Tumor targeting
 Cytotoxic
 Self – Propel
 Respond to triggering signals
 Sense local environment
 Externally detectable signals
03
Reference: Forbes, N.S., 2010. Engineering the perfect (bacterial) cancer therapy. Nature Reviews Cancer, 10(11), pp.785-794.
16 Feb 2022 Monika J. Pandarkar

4. BACTERIAL STRAINS
A) Non –Pathogenic bacteria:
 Probiotic bacteria – live
microorganisms localized to GIT
 Prebiotic genera-
lactobacillus,lactococcus,Bifidobacte
ria and Enterococcus
 E.coli Nissle (EcN)- non lactic acid
probiotic
 Non virulence, Modulation of host
immunity and tumor targeting
properties
B) Pathogenic bacteria :
1.Salmonella
2.Clostridium novyi
3.Listeria monocytogenes
Attenuation of bacterial virulence
 Genetic engineering methods
 specific nutrient-dependent
mutations
04
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J.Pandarkar

5. 1.Listeria monocytogenes
 Gram-positive facultative intracellular
bacterium with listeriolysin O (LLO) activity
 Potential vaccine vector that can generate
tumor-specific T-cell responses via fusion of
TAA to LLO
 Strong inhibition of immune tolerance in the
TME
TAA-Tumor associated antigens
TME-Tumor microenvironment
05
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J.Pandarkar

6. 2.Clostridium Novyi
 Gram-positive, endospore-
forming, obligate anaerobic
 Accumulate and proliferate within
the hypoxic and necrotic regions
of tumors
 Triggering immune responses
against tumors without systemic
toxicity
06
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J.Pandarkar

7. 3.Salmonella
 S.typhimurium is motile, easily genetically
manipulated and grows as a facultative anaerobe
in the presence or absence of oxygen
 Migrate towards the tumor by their flagella
 Attracted by the high concentrations of nutrients
available within the TME
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J.Pandarkar
07

8. BACTERIAL DERIVATIVES
 Immune modulation
 Ability to load specific antigens and therapeutic cargoes
 Nano scale sizes of OMV and minicells,which range from 20 to 400 nm
 Highly biocompatible and easy to take up by APCs
 Their non-viability reduces the risk of bacterial transmission and proliferation to non-
target sites
 So,BGs have better safety profiles within their proper dosage ranges than live bacteria
08
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J. Pandarkar

9. 1. Bacterial outer membrane vesicles
(OMVs)
 Spherical lipid-bilayer nanovesicles (20 to 250 nm)
 Derived from the outer membrane (OM) of bacteria
 Gram – negative bacterial species produce OMVs
 Composed of lipopolysaccharide (LPS), phospholipids of the
OM, and OM proteins, periplasmic proteins, cytoplasmic
proteins, nucleic acids, and virulence factors
 Recently involved in horizontal gene transfer ,biofilm
formation,delivery of toxins, resistance to antibiotics and
adherence to host cells
09
Reference: Jan, A.T., 2017. Outer membrane vesicles (OMVs) of gram-negative bacteria: a perspective update. Frontiers in
microbiology, 8, p.1053.
16 Feb 2022 Monika J. Pandarkar

10. 2.Minicells
 Anucleated vesicles (100–400 nm) resulting from the
inactivation of the Min operon
 Controls normal bacterial cell division, thereby depressing polar
sites of cell fission
 Both Gram-positive and Gram-negative bacteria can produce
minicells
 Contain all the molecular structures and organelles present in
their parent cells
 unable to divide or grow
10
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J. Pandarkar

11. 3. Bacterial ghost (BGs)
 Gram- negative bacterial shells with pores
 Cytoplasmic contents - expelled by controlled expression of the
cloned bacteriophage PhiX174 gene E
 With the expression of the lysis gene E, protein E induces
bacterial cell lysis by forming a transmembrane lysis tunnel
structure (40 to 200 nm)
 Retain the structures and components of the native bacterial cell
wall
 Retained surface components- taken up by immune cells and
stimulate innate and adaptive immune responses to the targets
Reference: Chen, H., Ji, H., Kong, X., Lei, P., Yang, Q., Wu, W., Jin, L. and Sun, D., 2021. Bacterial Ghosts-Based Vaccine and Drug
Delivery Systems. Pharmaceutics, 13(11), p.1892.
16 Feb 2022 Monika J.Pandarkar
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12. BACTERIAL DERIVATIVES FOR CANCER
THERAPY
12
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J. Pandarkar

13. Bacterial engineering for target-specific
drug delivery
1.Therapeutic payloads
 In situ production of proteins
 Therapeutic protein
 Immune modulators
 Angiogenesis inhibitors
 Prodrug-converting enzymes
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16 Feb 2022 Monika J. Pandarkar
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.

14. Strategies for payload delivery by live
bacteria
Strategies for payload
delivery
Groups Therapeutic payloads Mechanism
In situ production of
therapeutic proteins
Bacterial
toxins
Pore- forming toxins
clyA(from E.coli)
SAH(from S.aureus)
Listeriolysin O(from
L.monocytogenes
Directly secreted within tumors, form
pores to target cell membrane
Delivery of tumor antigens to dendritic
cells for cancer immunotherapy
Redox protein
Azurin(from
P.aeruginosa
Enhancement of intracellular levels of
p53,which induces apoptosis
In situ production of
immunomodulatory
proteins
Cytokines
Chemokine
s
IL-2,IL-4,IL-12,IL-18
CCL21
Enhancement of immune response,
especially of innate immune system
Enhancement of immune responses
14
Reference: Kang, S.R., Nguyen, D.H., Yoo , S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J.Pandarkar

15. Strategies for payload delivery by live bacteria
Strategies for payload
delivery
Groups Therapeutic payloads Mechanism
In situ production of
proteins targeting tumor
stroma
Endostatin Inhibition of angiogenesis
Enzyme prodrug therapy Exogenous
Enzymes
Herpes simplex virus
thymidine kinase
Phosphorylation of the inactive
prodrug ganciclovir to its activated
form
Endogenous
Enzymes
Purine nucleoside
phosphorylase
Conversion of 6MePdR to 6MeP
15
Reference: Kang, S.R., Reference: Kang, S.R., Nguyen, D.H., Yoo ,S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery
carriers for immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J.Pandarkar

16. Bacterial engineering for target-specific
drug delivery
2.Payload expression strategies :
 Required to avoid or minimize off- target
effects
 Ability to regulate time and location of
payload expression – improve safety
1.External triggering:
 Chemicals like L- arabinose, acetyl salicylic
acid(ASA),and tetracycline; ionizing and
non- ionizing radiation; ultrasound
2. Internal triggering :
 Specific characteristics of the TME , such as
hypoxia and acidic pH
 Hypoxia – inducible promoters are activated
only under anaerobic conditions
 Acidosis- specific promoters are activated
only under acidic conditions
3. Quorum sensing:
 Bacterial quorum-sensing switch
 Bacterial cell-cell communication process
16
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J.Pandarkar

17. Bioengineering of bacterial derivatives
1.Structural modification of OMV
 OMV – based vaccines:
Adjuvants -Aluminum-based,Freunds
complete and incomplete, liposome and CpG
 Immune modulators:
OMVs containing immunostimulatory
components act as Immune-therapeutic
agents against cancer
E.g. OMVs derived from genetically
modified E.coli completely eradicate tumors
through the induction of antitumor cytokines
and IFNγ,without adverse effects
 Drug delivery vehicles :
Bacterial OMVs deliver chemotherapeutic drugs
that stimulate immune response against cancer
 Anti-bacteria adhesion agents:
Bacterial OMVs contains natural adhesion agents,
which competitively adhere to target cells and
block Bacterial adhesion or infection of host cells
E.g. OMs of H.pylori coated with polymeric
nanoparticles
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16 Feb 2022 Monika J. Pandarkar
Reference : Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.

18. Bioengineering of bacterial derivatives
2.Engineering of bacterial minicells
 Drug-packaged minicells conjugated with
targeting ligand on the surface for enhanced
tumor-specific targeting via cancer cell
surface receptors
 Engineered to encapsulate chemotherapeutic
agents as well as to target cancer cells
3.Engineering of BGs
 BGs as adjuvants:
BGs retain the immunostimulating components of
their parent cell,act as adjuvants to enhance
immune response of vaccines
 BGs as a delivery system:
Outstanding loading capacity of the inner space of
BGs allows BGs to be loaded with peptides, drugs
or plasmids
18
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
16 Feb 2022 Monika J.Pandarkar

19. ROUTES OF ADMINISTRATION
1. Intratumoral injection
 Reduces the quantity of medication
administered to each patient
 Direct injection into the tumor result
in high concentration of drug at
desired site
 Reduces off-target toxicities
 Applied to sites in which drug
delivery is limited by BBB
Reference: Shende, P. and Basarkar, V., 2019. Recent trends and advances in microbe-based drug delivery systems. DARU Journal of Pharmaceutical
Sciences, 27(2), pp.799-809.
16 Feb 2022 Monika J. Pandarkar

20. ROUTES OF ADMINISTRATION
2. Oral administration:
 Convenient
 High patient compliance
 Safest
 Limitations:
 Change in pH and enzyme – rich
conditions of digestive tract reduce
viability and activity of bacteria
 To achieve effective immune
response, high dose required
3. Intravenous injection:
 Delivered drug rapidly and
accurately
 Greatest antitumor effect
 Limitation:
 Risk of systemic bacterial infection
22 Feb 2022 Monika J.Pandarkar
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.
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21. APPLICATIONS
21
Cancer
Infections diseases
Gastrointestinal disorders
Obesity and metabolic syndrome
Ischemia
Chronic liver disease
16 Feb 2022 Monika J.Pandarkar
Reference : Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.

22. APPLICATION
S
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16 Feb 2022 Monika J . Pandarkar
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.

23. FUTURE PERSPECTIVES
 Engineered microbes may be able to act as biological thermostats and generate
therapeutic effectors only as needed.
 To realize this vision, Engineered circuits will require validated disease-specific
sensors, rigorous regulation of genetic circuits and optimization of potency.
 Low-cost DNA synthesis, access to various omics databases, and use of artificial
intelligence will enable researchers to build diverse strain libraries .
 Patient acceptance of live bacterial therapeutics will be required.
 Educate the patients, physicians and public on the benefits and safety of these
agents.
23
16 Feb 2022 Monika J.Pandarkar
Reference: Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial derivatives as delivery carriers for
immunotherapy. Advanced drug delivery reviews, p.114085.

24. References
 Kang, S.R., Nguyen, D.H., Yoo, S.W. and Min, J.J., 2021. Bacteria and bacterial
derivatives as delivery carriers for immunotherapy. Advanced drug delivery
reviews, p.114085.
 Jan, A.T., 2017. Outer membrane vesicles (OMVs) of gram-negative bacteria: a
perspective update. Frontiers in microbiology, 8, p.1053.
 Forbes, N.S., 2010. Engineering the perfect (bacterial) cancer therapy. Nature
Reviews Cancer, 10(11), pp.785-794.
 Chen, H., Ji, H., Kong, X., Lei, P., Yang, Q., Wu, W., Jin, L. and Sun, D., 2021.
Bacterial Ghosts-Based Vaccine and Drug Delivery
Systems. Pharmaceutics, 13(11), p.1892.
24
16 Feb 2022 Monika J. Pandarkar

25. References
 Shende, P. and Basarkar, V., 2019. Recent trends and advances in microbe-based
drug delivery systems. DARU Journal of Pharmaceutical Sciences, 27(2), pp.799-
809.
 Marabelle, A., Tselikas, L., De Baere, T. and Houot, R., 2017. Intratumoral
immunotherapy: using the tumor as the remedy. Annals of Oncology, 28, pp.xii33-
xii43.
 Hajam, I.A., Dar, P.A., Won, G. and Lee, J.H., 2017. Bacterial ghosts as adjuvants:
mechanisms and potential. Veterinary Research, 48(1), pp.1-13.
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16 Feb 2022 Monika J. Pandarkar

26. Thank you
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16 Feb 2022 Monika J. Pandarkar