Biologics, Biosimilars, and Bioconjugates
Christoph Rader, The Scripps Research Institute (Jupiter, FL)
Chemical Biology I, October 16, 2019
Outline
What are biologics?
The rise of biosimilars
Bioconjugates
Cysteine-based bioconjugates
Lysine-based bioconjugates
Bioconjugates based on unnatural amino acids
Antibody-based cancer therapy
Small molecule drugs
C9H8O4
C20H21FN2O
C33H35FN2O5
aspirin
(180 g/mol)
escitalopram
(324 g/mol)
atorvastatin
(559 g/mol)
Biologics
C845H1343N223O343S9
filgrastim
(18,803 g/mol)
C6416H9874N1688O1987S44
rituximab
(143,860 g/mol)
Biologics as defined by the FDA
Biologics can be composed of sugars, proteins, or nucleic acids or
complex combinations of these substances, or may be living entities such
as cells and tissues.
Small molecule drugs vs. biologics
Small molecule drug
Biologic
Size
small
large
Manufacturing
chemical synthesis
living system
Process
not important
important
Characterization
complete
incomplete
Generics
yes
no
Costs
low
high
Administration
oral
parenteral
Best selling drugs in 2018
60% biologics
CVCVCVCV
CVCVCVCV
Outline
What are biologics?
The rise of biosimilars
Bioconjugates
Cysteine-based bioconjugates
Lysine-based bioconjugates
Bioconjugates based on unnatural amino acids
Antibody-based cancer therapy
Generic vs. similar
=
≠
Posttranslational modifications!
Why are biosimilars not biogenerics?
•
•
•
•
•
•
Differential N-glycosylation
N-terminal PyroGlu formation from Glu or Gln
Asn deamidation and Asp isomerization
Met oxidation
Trp oxidation
C-terminal Lys clipping
≠
Why are biosimilars not biogenerics?
•
•
•
•
•
•
Differential N-glycosylation
N-terminal PyroGlu formation from Glu or Gln
Asn deamidation/Asp isomerization
Met oxidation
Trp oxidation
C-terminal Lys clipping
≠
Frequent amino acid modifications
Asn deamidation/Asp isomerization
N-terminal PyroGlu formation from Glu or Gln
H 2O
NH 3
O
H 2N
Protein
Asp
β
O
H
N
O
O
Protein
H 2N
𝛂
Protein
β
β
𝛂
H 2N
O
N-terminal
N-terminal
Gln Gln
HO
N-terminal
pyroGlu
N-terminal
pyroGlu
𝛂
O
β
𝛂
N-terminal
Glu
N-terminal Glu
Asn
succinimide
isoAsp
Met oxidation
Trp oxidation
Met-S-sulfoxide
Met
Met-sulfone
Met-R-sulfoxide
Trp
Oxindolyl-Ala
N-Formyl-kynurenine
Kynurenine
23 FDA-approved biosimilars
Name
filgrastim
(Neupogen®)
filgrastim-sndz (Zarxio®)
filgrastim-aafi
(Nivestym®)
adalimumab (Humira®)
adalimumab-atto
(Amjevita®)
adalimumab-adbm (Cyltezo®)
(Hyrimoz®)
adalimumab-adaz
adalimumab-bwwd (Hadlima®)
infliximab
(Remicade®)
infliximab-dyyb (Inflectra®)
infliximab-abda
(Renflexis®)
infliximab-qbtx (Ixifi®)
etanercept
(Enbrel®)
etanercept-szzs (Erelzi®)
etanercept-ykro
(Eticovo®)
bevacizumab (Avastin®)
bevacizumab-awwb
(Mvasi®)
bevacizumab-bvzr (Zirabev®)
trastuzumab
(Herceptin®)
trastuzumab-dkst (Ogivri®)
(Herzuma®)
trastuzumab-pkrb
trastuzumab-dttb (Ontruzant®)
trastuzumab-qyyp
(Trazimera®)
trastuzumab-anns (Kanjinti®)
epoietin alfa
(Epogen®)
epoetin alfa-epbx
(Retacrit®)
pegfilgrastim (Neulasta®)
pegfilgrastim-jmdb
(Fulphila®)
pegfilgrastim-cbqv (Udenyca®)
rituximab
(Rituxan®)
rituximab-abbs (Truxima®)
rituximab-pvvr
(Ruxience®)
Manufacturer
Biologics class
Target
Indication
Approval
Amgen
cytokine
GCSF-R
neutropenia
1991
Sandoz
cytokine
GCSF-R
neutropenia
2015
Pfizer
cytokine
GCSF-R
neutropenia
2018
AbbVie
mAb
TNFα
inflammatory diseases
2002
Amgen
mAb
TNFα
inflammatory diseases
2016
Boehringer Ingelheim
mAb
TNFα
inflammatory diseases
2017
Sandoz
mAb
TNFα
inflammatory diseases
2018
Samsung
mAb
TNFα
inflammatory diseases
2019
Janssen
mAb
TNFα
inflammatory diseases
1998
Celltrion
mAb
TNFα
inflammatory diseases
2016
Samsung
mAb
TNFα
inflammatory diseases
2017
Pfizer
mAb
TNFα
inflammatory diseases
2017
Amgen
decoy receptor
TNFα
inflammatory diseases
1998
Sandoz
decoy receptor
TNFα
inflammatory diseases
2016
Samsung
decoy receptor
TNFα
inflammatory diseases
2019
Roche
mAb
VEGF
cancer
2004
Amgen
mAb
VEGF
cancer
2017
Pfizer
mAb
VEGF
cancer
2019
Genentech
mAb
HER2
cancer
1998
Biocon
mAb
HER2
cancer
2017
Celltrion
mAb
HER2
cancer
2018
Samsung
mAb
HER2
cancer
2019
Pfizer
mAb
HER2
cancer
2019
Amgen
mAb
HER2
cancer
2019
Amgen
cytokine
EPO-R
anemia
1989
Pfizer
cytokine
EPO-R
anemia
2018
Amgen
cytokine
GCSF-R
neutropenia
2002
Mylan
cytokine
GCSF-R
neutropenia
2018
Coherus
cytokine
GCSF-R
neutropenia
2018
Roche
mAb
CD20
cancer, inflammatory diseases
1997
Celltrion
mAb
CD20
cancer
2018
Pfizer
mAb
CD20
cancer, inflammatory diseases
2019
Outline
What are biologics?
The rise of biosimilars
Bioconjugates
Cysteine-based bioconjugates
Lysine-based bioconjugates
Bioconjugates based on unnatural amino acids
Antibody-based cancer therapy
Bioconjugates
Bioconjugation is a chemical strategy to form a stable covalent link
between two molecules, at least one of which is a biomolecule, such
as a sugar, protein, or nucleic acid.
From filgrastim to pegfilgrastim
Heterogeneous antibody-drug conjugates (ADCs)
C
C
Adcetris®
Polivy ®
C
C C
C C
C
Kadcyla®
Besponsa®
Mylotarg®
K
K
K
K
K
K
K
K
Homogeneous ADCs
natural
amino acids
Arg
Lys
Sec
Cys
Cys-X-Pro-X-Arg
unnatural
amino acids
Leu-Leu-Gln-Gly
Leu-Pro-X-Thr-Gly
p-acetyl-Phe
(formyl-Gly)-X-Pro-X-Arg
chemical or enzymatic conjugation to drugs
p-azidomethyl-Phe
azidoethoxy-Lys
Homogeneous ADCs
natural
amino acids
Arg
Lys
Sec
Cys
Cys-X-Pro-X-Arg
unnatural
amino acids
Leu-Leu-Gln-Gly
(formyl-Gly)-X-Pro-X-Arg
Leu-Pro-X-Thr-Gly
p-acetyl-Phe
p-azidomethyl-Phe
azidoethoxy-Lys
Outline
What are biologics?
The rise of biosimilars
Bioconjugates
Cysteine-based bioconjugates
Lysine-based bioconjugates
Bioconjugates based on unnatural amino acids
Antibody-based cancer therapy
Random cysteine conjugation
C
C
C
C C
C C
IgG1
C
C
DTT or TCEP
C
C
C C
C C
C
IgG1-(drug)4 (0-8)
Adcetris® and Polivy®
anti-CD30/
CD79B IgG1
C
C
maleimidocaproyl
cleavable
Val-Cit PABC
monomethyl
auristatin E (MMAE)
C
C C
C C
C
cathepsin B cleavage
1,6 elimination
free MMAE
Site-specific cysteine conjugation
C
C
C
C
C C
C C
thiomab
IgG1
C
C
DTT or TCEP
CuSO4 or
dehydroascorbic acid
C
C
C
C
C C
C C
C
C
thiomab
IgG1-(drug)2
Junutula et al., Nat. Biotechnol. 2008
Stability of thiomab-drug conjugates
C
C
C
C
>>
>
C
V205C
antibody
A114C
S396C
albumin
antibody
C
albumin
antibody
Shen et al., Nat. Biotechnol. 2012
Improving the stability of thiomab-drug conjugates
fast
antibody
antibody
slow
antibody
Lyon et al., Nat. Biotechnol. 2014
Selenocysteine
H
H
pKa 5.2
pKa 8.3
Se
S
O
+
H3N
O
U
-
O
+
H3N
O
C
-
Genetic code
center
G
A
G
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
Ile
Ile
Ile
Met
Val
Val
Val
Val
Ser
Ser
Ser
Ser
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
Tyr
Tyr
stop
stop
His
His
Gln
Gln
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu
Cys
Cys
stop
Trp
Arg
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
Ile
Ile
Ile
Met
Val
Val
Val
Val
Ser
Ser
Ser
Ser
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
Tyr
Tyr
stop
stop
His
His
Gln
Gln
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu
Cys
Cys
Sec
Trp
Arg
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
3’
A
C
3’
5’
C
U
5’
U
center
Selenocysteine incorporation machinery
Sec
AAA
AUG
UGA
UAA
ACU
AAA
Met
[…]
Leu
Ser
Pro
Gly
Ala
Sec
ACU
AUG
UGA
UAA
Site-specific selenocysteine conjugation
DTT, NaOAc (pH 5.2)
U
U
selenomabIgG1
(S396U)
U
U
selenomabIgG1-(drug)2
Li et al., Cell Chem. Biol. 2017
Outline
What are biologics?
The rise of biosimilars
Bioconjugates
Cysteine-based bioconjugates
Lysine-based bioconjugates
Bioconjugates based on unnatural amino acids
Antibody-based cancer therapy
Random lysine conjugation
K
K
K
K
IgG1
IgG1-(drug)3.5 (0-8)
non-cleavable
anti-HER2 IgG1
thioether
maytansine
K
K
K
K
lysosomal degradation
lysine
Kadcyla®
anti-CD22/CD33
IgG1
hydrazone
disulfide linker
Besponsa® and Mylotarg®
K
K
K
endosomal hydrolysis
calicheamicin
Site-specific lysine conjugation
β-lactam hapten
K
monomethyl
auristatin F (MMAF)
K
+
DVD-IgG1
K
PBS (pH 7.4)
4 h, RT
non-cleavable
linker
K
DVD-IgG1-(drug)2
Nanna et al., Nat. Commun. 2017
Outline
What are biologics?
The rise of biosimilars
Bioconjugates
Cysteine-based bioconjugates
Lysine-based bioconjugates
Bioconjugates based on unnatural amino acids
Antibody-based cancer therapy
Site-specific p-acetyl-phenylalanine conjugation
F*
F*
F* =
antibody
IgG1
(A114F*)
F*
F*
NH4OAc (pH 4.5)
16-48 h, 37°C
+
auristatin F
antibody
non-cleavable
linker
IgG1 (A114F*)(drug)2
alkoxyamine
Axup et al., Proc. Natl. Acad. Sci. U. S. A. 2012
DUPA
Fab
Site-specific p-acetyl-phenylalanine conjugation
T
cell
CCW702
Clinical trial: NCT04077021
F*
anti-CD3 Fab
(K138F*)-DUPA
Cancer
cell
Kim et al., Proc. Natl. Acad. Sci. U. S. A. 2013
Outline
What are biologics?
The rise of biosimilars
Bioconjugates
Cysteine-based bioconjugates
Lysine-based bioconjugates
Bioconjugates based on unnatural amino acids
Antibody-based cancer therapy
36 FDA-approved and marketed antibody-based cancer therapies
Name
rituximab
Format
(Rituxan®)
trastuzumab (Herceptin®)
ibritumomab tiuxetan
(Zevalin®)
cetuximab (Erbitux®)
bevacizumab
(Avastin®)
Payload
Target
Cancer
FDA approval
chimeric mouse/human IgG1κ
none
CD20
B-NHL, CLL
1997, 2010
humanized IgG1κ
none
HER2
breast, stomach
1998, 2010
mouse IgG1κ
90Y
CD20
B-NHL
2002
chimeric mouse/human IgG1κ
none
EGFR
colorectal, h & n
2004, 2006
humanized IgG1κ
none
VEGF
colorectal, lung, brain, kidney, cervical, ovarian, fallopian tube, peritoneal
2004-2014, 2018
panitumumab (Vectibix®)
human IgG2κ
none
EGFR
colorectal
2006
ofatumumab (Arzerra®)
human IgG1κ
none
CD20
CLL
2009
human IgG1κ
none
CTLA4
melanoma, kidney, MSI-H/dMMR colorectal
2011, 2018
chimeric mouse/human IgG1κ
MMAE
CD30
HL, T-NHL
2011
humanized IgG1κ
none
HER2
breast
2012
humanized IgG1κ
DM1
HER2
breast
2013
ipilimumab
(Yervoy®)
brentuximab vedotin (Adcetris®)
pertuzumab
(Perjeta®)
ado-trastuzumab emtansine (Kadcyla®)
obinutuzumab
(Gazyva®)
humanized IgG1κ (glycoengineered Fc)
none
CD20
CLL, B-NHL
2013, 2017
ramucirumab (Cyramza®)
human IgG1κ
none
VEGFR2
stomach, colorectal, liver
2014, 2015, 2019
pembrolizumab (Keytruda®)
humanized IgG4κ
none
PD1
melanoma, lung, h & n, HL, bladder, MSI-H/dMMR, stomach, cervical, B-NHL, liver, kidney
2014-2019
mouse (scFv)2 (BiTE)
none
CD19 x CD3
ALL
2014
human IgG4κ
none
PD1
melanoma, lung, kidney, HL, h & n, bladder, MSI-H/dMMR colorectal, liver
2014-2018
blinatumomab
(Blincyto®)
nivolumab (Opdivo®)
dinutuximab
(Unituxin®)
chimeric mouse/human IgG1κ
none
GD2
neuroblastoma
2015
daratumumab (Darzalex®)
human IgG1κ
none
CD38
multiple myeloma
2015
(Portrazza®)
necitumumab
human IgG1κ
none
EGFR
lung
2015
elotuzumab (Empliciti®)
humanized IgG1κ
none
SLAMF7
multiple myeloma
2015
atezolizumab (Tecentriq®)
humanized IgG1κ (aglycosylated Fc)
none
PDL1
bladder, lung, breast
2016, 2019
human IgG1κ
none
PDGFRA
sarcoma
2016
human IgG1λ
none
PDL1
Merkel cell carcinoma, bladder, kidney
2017, 2019
olaratumab
(Lartruvo®)
avelumab (Bavencio®)
durvalumab
(Imfinzi®)
human IgG1κ (engineered Fc)
none
PDL1
bladder, lung
2017, 2018
inotuzumab ozogamicin (Besponsa®)
humanized IgG4κ
calicheamicin
CD22
ALL
2017
tisagenlecleucel (Kymriah®)
mouse scFv-based CAR-T
T cell
CD19
ALL, B-NHL
2017, 2018
gemtuzumab ozogamicin
(Mylotarg®)
humanized IgG4κ
calicheamicin
CD33
AML
2017
bevacizumab-awwb (Mvasi®)
humanized IgG1κ
none
VEGF
colorectal, lung, brain, kidney, cervical
2017
axicabtagene ciloleucel (Yescarta®)
mouse scFv-based CAR-T
T cell
CD19
B-NHL
2017
trastuzumab-dkst (Ogivri®)
humanized IgG1κ
none
HER2
breast, stomach
2017
mogamulizumab-kpkc
(Poteligeo®)
humanized IgG1κ (afucosylated Fc)
none
CCR4
T-NHL
2018
moxetumomab pasudotox-tdfk (Lumoxiti®)
mouse dsFv
bacterial toxin
CD22
B-NHL
2018
cemiplimab-rwlc (Libtayo®)
human IgG4κ (S228P hinge)
none
PD1
cutaneous squamous cell carcinoma
2018
chimeric mouse/human IgG1κ
none
CD20
B-NHL
2018
humanized IgG1κ
none
HER2
breast
2018
humanized IgG1κ
MMAE
CD79B
B-NHL
2019
rituximab-abbs
(Truxima®)
trastuzumab-pkrb (Herzuma®)
polatuzumab vedotin-piiq
(Polivy®)
Natural vs. synthetic
Natural antibody molecule
IgG1
VL
VH
CL
F(ab’)2
CH1
CH2
Fc
CH3
High specificity
Nanomolar affinity
Long circulatory half-life
Effector functions
Multiple and programmable specificity
Picomolar affinity
Tunable circulatory half-life
Tunable effector functions
Natural effector functions
ADCP
MØ
ADCC
NK
cell
Cancer
cell
CDC
Natural vs. synthetic antibody molecule
IgG1
VL
VH
CL
F(ab’)2
CH1
CH2
Fc
CH3
High specificity
Nanomolar affinity
Long circulatory half-life
Effector functions
Multiple and programmable specificity
Picomolar affinity
Tunable circulatory half-life
Tunable effector functions
Delivery of cytotoxic payloads
ADCs
C
C
Adcetris®
Polivy ®
C
C C
C C
C
Kadcyla®
Besponsa®
Mylotarg®
K
K
K
K
K
K
K
K
Natural vs. synthetic antibody molecule
IgG1
VL
VH
CL
F(ab’)2
CH1
CH2
Fc
CH3
High specificity
Nanomolar affinity
Long circulatory half-life
Effector functions
Multiple and programmable specificity
Picomolar affinity
Tunable circulatory half-life
Tunable effector functions
Delivery of cytotoxic payloads
Recruitment and activation of T cells
T cells attacking cancer cell
Antibody-based cancer immunotherapy
T
cell
Blincyto®
Tecentriq®
Bavencio®
Imfinzi®
Yervoy®
Keytruda®
Opdivo®
Libtayo®
Cancer
cell
T
cell
Kymriah®
Yescarta®
T
cell
Kymriah®, a living drug!
36 FDA-approved and marketed antibody-based cancer therapies
Name
rituximab
Format
(Rituxan®)
trastuzumab (Herceptin®)
ibritumomab tiuxetan
(Zevalin®)
cetuximab (Erbitux®)
bevacizumab
(Avastin®)
Payload
Target
Cancer
FDA approval
chimeric mouse/human IgG1κ
none
CD20
B-NHL, CLL
1997, 2010
humanized IgG1κ
none
HER2
breast, stomach
1998, 2010
mouse IgG1κ
90Y
CD20
B-NHL
2002
chimeric mouse/human IgG1κ
none
EGFR
colorectal, h & n
2004, 2006
humanized IgG1κ
none
VEGF
colorectal, lung, brain, kidney, cervical, ovarian, fallopian tube, peritoneal
2004-2014, 2018
panitumumab (Vectibix®)
human IgG2κ
none
EGFR
colorectal
2006
ofatumumab (Arzerra®)
human IgG1κ
none
CD20
CLL
2009
human IgG1κ
none
CTLA4
melanoma, kidney, MSI-H/dMMR colorectal
2011, 2018
chimeric mouse/human IgG1κ
MMAE
CD30
HL, T-NHL
2011
humanized IgG1κ
none
HER2
breast
2012
humanized IgG1κ
DM1
HER2
breast
2013
ipilimumab
(Yervoy®)
brentuximab vedotin (Adcetris®)
pertuzumab
(Perjeta®)
ado-trastuzumab emtansine (Kadcyla®)
obinutuzumab
(Gazyva®)
humanized IgG1κ (glycoengineered Fc)
none
CD20
CLL, B-NHL
2013, 2017
ramucirumab (Cyramza®)
human IgG1κ
none
VEGFR2
stomach, colorectal, liver
2014, 2015, 2019
pembrolizumab (Keytruda®)
humanized IgG4κ
none
PD1
melanoma, lung, h & n, HL, bladder, MSI-H/dMMR, stomach, cervical, B-NHL, liver, kidney
2014-2019
mouse (scFv)2 (BiTE)
none
CD19 x CD3
ALL
2014
human IgG4κ
none
PD1
melanoma, lung, kidney, HL, h & n, bladder, MSI-H/dMMR colorectal, liver
2014-2018
blinatumomab
(Blincyto®)
nivolumab (Opdivo®)
dinutuximab
(Unituxin®)
chimeric mouse/human IgG1κ
none
GD2
neuroblastoma
2015
daratumumab (Darzalex®)
human IgG1κ
none
CD38
multiple myeloma
2015
necitumumab (Portrazza®)
human IgG1κ
none
EGFR
lung
2015
humanized IgG1κ
none
SLAMF7
multiple myeloma
2015
humanized IgG1κ (aglycosylated Fc)
none
PDL1
bladder, lung, breast
2016, 2019
human IgG1κ
none
PDGFRA
sarcoma
2016
human IgG1λ
none
PDL1
Merkel cell carcinoma, bladder, kidney
2017, 2019
(Empliciti®)
elotuzumab
atezolizumab (Tecentriq®)
olaratumab
(Lartruvo®)
avelumab (Bavencio®)
durvalumab
(Imfinzi®)
human IgG1κ (engineered Fc)
none
PDL1
bladder, lung
2017, 2018
inotuzumab ozogamicin (Besponsa®)
humanized IgG4κ
calicheamicin
CD22
ALL
2017
tisagenlecleucel (Kymriah®)
mouse scFv-based CAR-T
T cell
CD19
ALL, B-NHL
2017, 2018
gemtuzumab ozogamicin
(Mylotarg®)
humanized IgG4κ
calicheamicin
CD33
AML
2017
bevacizumab-awwb (Mvasi®)
humanized IgG1κ
none
VEGF
colorectal, lung, brain, kidney, cervical
2017
axicabtagene ciloleucel (Yescarta®)
mouse scFv-based CAR-T
T cell
CD19
B-NHL
2017
trastuzumab-dkst (Ogivri®)
humanized IgG1κ
none
HER2
breast, stomach
2017
mogamulizumab-kpkc (Poteligeo®)
humanized IgG1κ (afucosylated Fc)
none
CCR4
T-NHL
2018
mouse dsFv
bacterial toxin
CD22
B-NHL
2018
human IgG4κ (S228P hinge)
none
PD1
cutaneous squamous cell carcinoma
2018
chimeric mouse/human IgG1κ
none
CD20
B-NHL
2018
humanized IgG1κ
none
HER2
breast
2018
humanized IgG1κ
MMAE
CD79B
B-NHL
2019
moxetumomab pasudotox-tdfk
(Lumoxiti®)
cemiplimab-rwlc (Libtayo®)
rituximab-abbs
(Truxima®)
trastuzumab-pkrb (Herzuma®)
polatuzumab vedotin-piiq
(Polivy®)
Biologics as defined by the FDA
Biologics can be composed of sugars, proteins, or nucleic acids or
complex combinations of these substances, or may be living entities such
as cells and tissues.