Plant and algal systems show promise for antibody production as an alternative to mammalian systems. Case study 1 demonstrates successful production of an anti-cancer idiotype vaccine in tobacco plants. Case study 2 describes the production of an immunotoxin targeting B-cell tumors in algae. The algal-produced immunotoxin was shown to selectively bind and kill target cancer cells while inhibiting tumor growth in mice, demonstrating the potential of algal systems for antibody production. Overall, plant and algal systems provide cost-effective and safe means for large-scale antibody manufacturing and several candidates are advancing through clinical trials.

1. Antibody production in plants and green
algae
Malavika M. R.
2015-09-013
B. Sc. – M. Sc. (Integrated) Biotechnology
1

2. CONTENTS
 Introduction
 Conventional method of antibody
production
 Inconvenience of mammalian system
 Plant systems
 Case study 1
 Algal systems
 Case study 2
 Conclusion
2

3. Introduction
• Antibodies are immunoglobulin
proteins produced by immune
cells of different organisms to
fight against pathogens.
• 50 monoclonal antibodies are
currently approved by the FDA
for treatment of different human
diseases.
• 1975 – Hybridoma Technology in
mouse
• First monoclonal antibody
approved by FDA - 1986
3

4. 4

5. Inconvenience of mammalian system
 Low production capacity
 Expensive
 Decreased viral safety
 Risk of contamination
5

6. PLANT
SYSTEMS
6

7. Plant systems
 Cost effective and highly scalable
 Safe
 Plants can also perform posttranslational
modifications of target proteins, including:
Disulfide bond formation
N-glycosylation, which is similar to mammalian
cells.
7

8. 8
Comparison of different expression systems
Short

9. Plant
viral expression vectors
Agrobacterium
tumefaciens
Agroinfiltration
Harvesting and extraction
Purification
StepsformAbproduction
9

10. EBOLA FEVER
10

11. • One of the world’s most
deadly pathogens
• Guinea, West Africa
– Liberia
– Sierra Leone and
– Nigeria
• infecting 28,512 people
with 11,313 fatalities
11

12. Survival of two American health aid
workers
• Contracted Ebola at the Liberian
hospital
12
Dr. Kent Brantly Nancy Writebol

13. The drug is a cocktail of three chimeric mAbs made
in Nicotiana benthamiana plants
Development of ZMapp started at Arizona State
University (ASU)
13

14. 14
Video on mAB production

15. 15
CASESTUDY1

16. Non-Hodgkin’s lymphoma (NHL)
 90% of NHL is B-cell lymphomas
 Most common indolent B-cell lymphoma
is follicular lymphoma (FL)
 Successfully treated with chemotherapy
 Recurrence is common
16

17. Worldwide incidence of NHL is
estimated
17
MALE FEMALE
Affected Rate(per 1
lakh)
6.1 4.0
Mortality Rate(per 1
lakh)
3.5 2.3

18. Aim of Study
 Primary objective: safety and tolerability of
the produced Id vaccines
 Secondary objective:
 Assessment of humoral idiotype-specific
immune response
 Assessment of cellular idiotype-specific
immune response
 Long-term safety/tolerability to the
vaccines
18

19.  Identity of Investigational Product:
Genetic material required was produced
from lymphocytes taken from the
patients
• The resultant recombinant vaccine was
supplied to the clinics
19

20. Vaccine Design
 Fusing the variable region of the patient’s
tumor-specific Id with a generic constant
domain of a human IgG1
 Heavy chain expressed in TMV(Tobacco
Mosaic Virus )
 Light chain in PVX(Potato Virus X)
 magnICON vectors
 Purified mAb – linked with KLH(keyhole
limpet hemocyanin)
20

21. 21
Study design

22.  Vaccines are safe
 Induce tumor idiotype-specific immune responses
 >40% of subject - humoral immune responses
 >50% of subjects - cellular immune responses
 82% response rate in plant systems(Tus´e et al., 2015)
22

23. Major mAB product candidates produced in
plants
23ClinicalTrials.gov.2015

24. 24
ClinicalTrials.gov.2015

25. Why Tobacco
 High leaf biomass yield
 Rapid scaleup
 Expression level - stems was
similar to that of leaves
 Whole tobacco plant
biomass can be used for
production
25

26. • Excluding human pathogen
contamination, which reduces
biosafety concerns
• Tobacco contains nicotine or other
toxic alkaloids, removed using an
additional extraction step
26

27. Organisms used for mAb production
• Plant systems:
– tobacco
– alfalfa
– maize
– soybean
– Chinese cabbage
27

28. 28

29. Advantages
 Generate stable lines fairly rapidly.
 Advances in engineering
– photosynthesis
– cell metabolism
 Ability to grow algae in contained
photobioreactors
 Reduced downstream processing costs
 Safer algal products
29

30. Organisms used
• Algal systems:
– Chlamydomonas reinhardtii,
– Dunaliella salina
30

31. Bacterial systems
 Lack the ability to fold complex multi-domain
proteins
 But lack the chaperones
 Attempts have been made to co-express these
chaperones with recombinant proteins of
interest - limited success (Outchkourov et
al.,2011)
31

32. CASE STUDY 2
32

33. Aim of study
• To demonstrated that algal-produced immunotoxins
accumulate as soluble and enzymatically active proteins that
that bind target B cells tumors and efficiently kill them in vitro.
33

34. Construct Design
34
Created a
recombinant gene
Single
chain
antibody
(scFv)
CD22
Genetically
fused to
domains II
and III of
Exotoxin A
(PE40)
Pseudomonas
aeruginosa
αCD22PE40

35. 35
αCD22PE40
short serum
half-life
hinge
CH2
CH3
Human IgG1
Placed between the
αCD22 scFv antibody
and PE40
αCD22CH23PE40

36. 36
(A) Single-chain
antibody (scFv)
(B) CD22-scFv is genetically linked to P.
aeruginosa
exotoxin A domains 2 and 3
(C) CD22-scFv genetically fused to the hinge and constant
domains of an IgG1 and to exotoxin A domains 2 and 3

37. Algal Transformations and Selection
37
Wild type
Kanamycin resistant gene
Particle Bombardment
PCR analysis
Lane 1 : wild type
Lane 2: α CD22
Lane 3: αCD22PE40
Lane 4: αCD22CH23PE40

38. ADP Ribosyltransferase Assays
 Biotinylated nicotinamide adenine
dinucleotide (NAD+) - substrate for
ribosyltransferases
 Incubated with eEF2
 Purified algal produced immunotoxins
 Active PE40 molecules are capable of
transferring biotinylated-ADP molecules from
biotinylated-NAD+ onto eEF2
 Biotin on EF2 with an anti-biotin
alkaline phosphatase-conjugated antibody
38
Lane 1: control α CD22
Lane 2: αCD22PE40
Lane 3: αCD22CH23PE40
Western Blot Analysis

39. Flow cytometry
 CA-46 B cells, Ramos B cells, and Jurkat T
cells
 Incubated in the presence of algal-produced
αCD22PE40 or αCD22HCH23PE40
 Incubated with an anti-exotoxin A antibody
produced in rabbit
 Incubated with an anti-rabbit DyLight 488-
conjugated antibody
39

40. Flow cytometry
40
Log of Fluorescence
CellCounts

41. Cytotoxic Cell-Viability Assay
• CA-46
• Ramos
• Jurkat
• CA-46
• Ramos
• Jurkat
• CA-46
• Ramos
• Jurkat
41
αCD22
αCD22PE40
αCD22HCH23PE40
Human Serum Albumin (HSA) 0.2%
Negative control
Determine the baseline for 100% survival
10 μg/mL of cycloheximide
Resulting in 100% cell death
Positive control

42. Cytotoxic Cell-Viability Assay
42

43. Antitumor Efficacy of Algal-Produced
Immunotoxins
• Female mice - lack adaptive immunity and
natural killer cells
• Ramos cells (3 × 107) were transplanted
• Tumors reached a mean diameter of 5 mm –
4th day after transplantation
• Injected with 240 μg/kg of αCD22,
αCD22PE40, αCD22CH23PE40
• Tumors were measured every day for up to
25th day
43

44. 44
αCD22
αCD22PE40
αCD22CH23PE40

45. mAB produced by algal systems
45

46. Conclusion
46
High market
demand for
mAbs
Cost effective
and safe drugs Several mAB
reached
prerclinical
&clinical
devepments
mAB
production
need to be
addressed
Expecting more
commercialized
drugs in the
coming years

47. 47