Radiopharmaceuticals for immuno-based

theranostics

Dr. Zéna Wimana, PhD

Biomedical Scientist/Radiopharmacy Coordinator

Nuclear Medicine/ Radiopharmacy

Institut Jules Bordet

Université Libre de Bruxelles (U.L.B.)

Development of a radiopharmaceutical

in oncology Target

•↗↗ Expression in cancer cells •Hallmark of cancer

Probe • Specific

Radiolabeling

In vitro • Model: cells (↗↗target)

• Binding/ Internalisation

In vivo • Model: animal

(↗↗target)

• Imaging & ex vivo (distribution, PK,…)

In patients

Preclinical

•Choice of radioisotope

•Choice of the method

•QC

Concept

Regulatory steps:IMPD FAMHP, FANC, ethical committee

Imaging & distribution, PK,…

Radionuclide

• Theranostic

• ImmunoPET β+

• Radioimmunotherapyβ− α

Direct radiolabeling Bifunctional chelator

Targeting moety

• protein

• mAb

• Fab

• Minibodies….

Targeting moety

Nanobodies (15 kDa)

variable domains of camelid heavy-chain-only antibodies

scFv (25kDa)

Ab engineeringAntigen-binding domains into a single polypeptide

Dia/Tribodies (55/80 kDa)

Divalent and multivalent scFvs: spontaneous dimerization/covalent association

Minibodies (75kDa)

fusion proteins of scFv with the hinge region and CH3 domain of IgG

Fab (50kDa)

• digestion Ab by enzyme

• F(ab')2and Fab

Affibodies (15kDa)

non-IgG affinity ligands (58 AA Z-domain scaffold-binding domains of staphylococcal protein A

DARPins (15kDa)

Designed Ankyrin Repeat Proteins

Aptamer

• oligonucleotide or peptide

(m)Ab (150kDa)

• Large MW protein

• secreted from plasma B cell

• identify and remove foreign pathogens such as bacteria and viruses

• Fc: recognition by immune c

• Fab: binding the antigen specificity

Immunoimaging (1990s):

OncoScint® (111In–satumomab pendetide); ProstaScint® (111In–capromab pendetide); CEA-Scan

(99mTc-arcitumomab)

Immunogenicity (murine origin) + SPECT (sensitivity+quantitation)

Protein engineering: human(ized) antibodies(fragments)

ImmunoPET: specificity Ab + high resolution&sensitivity PET

mAb: long residence time (days-weeks)

optimal tumor/non-tumor 2–4 days long half-lives (89Zr, 124I, 86Y,…)

Pretargeting:

(1) unlabeleled mAb tumor + (2) radiolabeled counterpartmAb

(strept)avidin-biotin: extremely high binding affinity, high immunogenicity

DNAcomplementaryDNA binding

bispecific antibodies-haptens

bioorthogonal chemical reaction

Radioimmunotherapy

90Y-Ibritumomab tiuxetan (Zevalin) ;131I-Tositumomab (Bexxar)

mAb main category in immunotheranostics: ↗development/use of mAbs as targeted therapy

Radiolabeled antibodies

Radionuclide

• Theranostic

• ImmunoPET β+

•Radioimmunotherapyβ− α

Direct radiolabeling Bifunctional chelator

Targeting moety

• protein

• mAb

• Fab

• Minibodies….

Radionuclide: immunoPET: Zr-89

Figure : Number of publications on 89Zr in PubMed per year. Search: “Zirconium-89" or “Zr-89" or “89Zr".

23% β+ (mean 395 kEV) , γ (909 keV)

t½ of 78,4h compatible with biological t½ high MW mAb

immunoPET Since then interest +++

Relatively easy produced by cyclotron

89Zr-Oxalate or 89ZrCl

Physiological conditions: +4 oxidation state!!

Bifunctional desferrioxamine B (siderophor) = chelator of choice

Easy labeling procedure

Most used radioisotope for immunoPET

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Radionuclide: immunoPET

T1/2 (h)

Production Targeting moety Advantages Disadvantes

89

Zr 78,4h Cyclotron • mAb

• Others

• Long T1/2 :high MW • High resolution PET • Production

• Long T1/2 + γ 909 keV : radioprotection

• % β+ (23%)

124

I 100,3h

Cyclotron • mAb • Others

• Direct labeling • Perfect pair 124I/131I • Long T1/2 :high MW

• Production (expensive)

• ≠ E photons scatter noise

• % β+ (23%)

64

Cu

12,7 h Cyclotron • mAb • Others

• Range β+ (< 1mm) • central production

• % β+ (19) • Radiation • Also beta, Auger e-

• Moderate T1/2 : limited PK

86

Y 14,74h

Cyclotron • mAb • Others

• Perfect pair 86Y/90Y • % β+ (19)

• Moderate T1/2 : limited PK

68

Ga

1,13h 68Ge/68Ga generator (Cyclotron)

• Fab, Nanobodies • Affibodies,… • mAb if

pretargeting

• Short T1/2

• % β+ (87%)

• Availibility/ Flexibility • cost effect) • Ph. Eur

• Short T1/2: Logistics, low MW

• Chemistry • Spatial resolution:

Range β+ (3mm)

T1/2 (h)

Production Targeting moety Advantages Disadvantes

89

Zr 78,4h Cyclotron • mAb

• Others

• Long T1/2 :high MW • High resolution PET • Production

• Long T1/2 + γ 909 keV : radioprotection

• % β+ (23%)

124

I 100,3h

Cyclotron • mAb • Others

• Direct labeling • Perfect pair 124I/131I • Long T1/2 :high MW

• Production (expensive)

• ≠ E photons scatter noise

• % β+ (23%)

64

Cu

12,7 h Cyclotron • mAb • Others

• Range β+ (< 1mm) • central production

• % β+ (19) • Radiation • Also beta, Auger e-

• Moderate T1/2 : limited PK

86

Y 14,74h

Cyclotron • mAb • Others

• Perfect pair 86Y/90Y • % β+ (19)

• Moderate T1/2 : limited PK

Radionuclide: ImmunoPET T1/2

(h) Production Targeting moety Advantages Disadvantes

89

Zr 78,4h Cyclotron • mAb

• Others

• Long T1/2 :high MW • High resolution PET • Production

• Long T1/2 + γ 909 keV : radioprotection

• % β+ (23%)

124

I 100,3h

Cyclotron • mAb • Others

• Direct labeling • Perfect pair 124I/131I • Long T1/2 :high MW

• Production (expensive)

• ≠ E photons scatter noise

• % β+ (23%)

64

Cu

12,7 h Cyclotron • mAb • Others

• Range β+ (< 1mm) • central production

• % β+ (19) • Radiation • Also beta, Auger e-

• Moderate T1/2 : limited PK

T1/2 (h)

Production Targeting moety Advantages Disadvantes

89

Zr 78,4h Cyclotron • mAb

• Others

• Long T1/2 :high MW • High resolution PET • Production

• Long T1/2 + γ 909 keV : radioprotection

• % β+ (23%)

124

I 100,3h

Cyclotron • mAb • Others

• Direct labeling • Perfect pair 124I/131I • Long T1/2 :high MW

• Production (expensive)

• ≠ E photons scatter noise

• % β+ (23%)

T1/2 (h)

Production Targeting moety Advantages Disadvantes

89

Zr 78,4h Cyclotron • mAb

• Others

• Long T1/2 :high MW • High resolution PET • Production

• Long T1/2 + γ 909 keV : radioprotection

• % β+ (23%)

T1/2 (d) Production Targeting moety

Advantages Disadvantages

131I 8d Reactor • all • Pure theranostic • Direct labeling

• Energy • Dehalogenetion

90Y 2,7d Reactors 90Sr daughter

• all

• Pure β- (radioprotection) • energy (long range) big

lesions • Ph. Eur

• energy (long range) toxicity

177Lu 6,65d Reactor • all

• favorable nuclear characteristics

• amenable chemistry • Ph. Eur • energy toxicity↘ • γ Imaging

• 177mLu waste

Radionuclide: Radioimmunotherapy T1/2 (d) Production Targeting

moety Advantages Disadvantages

131I 8d Reactor • all • Pure theranostic • Direct labeling

• Energy • Dehalogenetion

90Y 2,7d Reactors 90Sr daughter

• all

• Pure β- (radioprotection) • energy (long range) big

lesions • Ph. Eur

• energy (long range) toxicity

177Lu 6,65d Reactor • all

• favorable nuclear characteristics

• amenable chemistry • Ph. Eur • energy toxicity↘ • γ Imaging

• 177mLu waste

213Bi 45,6m 225Ac/213Bi generator

• Generator • 440keV γ

• Recoil • Short half-life

211At 7,2h Cyclotron • 100% α • Cost • T1/2 for low MW

• Availability (30 cyclotrons • Recoil

227Th 19d • Long T1/2

• Long T1/2

• Recoil

T1/2 (d) Production Targeting moety

Advantages Disadvantages

131I 8d Reactor • all • Pure theranostic • Direct labeling

• Energy • Dehalogenetion

90Y 2,7d Reactors 90Sr daughter

• all

• Pure β- (radioprotection) • energy (long range) big

lesions • Ph. Eur

• energy (long range) toxicity

177Lu 6,65d Reactor • all

• favorable nuclear characteristics

• amenable chemistry • Ph. Eur • energy toxicity↘ • γ Imaging

• 177mLu waste

213Bi 45,6m 225Ac/213Bi generator

• Generator • 440keV γ

• Recoil • Short half-life

211At 7,2h Cyclotron • 100% α • Cost • T1/2 for low MW

• Availability (30 cyclotrons • Recoil

T1/2 (d) Production Targeting moety

Advantages Disadvantages

131I 8d Reactor • all • Pure theranostic • Direct labeling

• Energy • Dehalogenetion

90Y 2,7d Reactors 90Sr daughter

• all

• Pure β- (radioprotection) • energy (long range) big

lesions • Ph. Eur

• energy (long range) toxicity

177Lu 6,65d Reactor • all

• favorable nuclear characteristics

• amenable chemistry • Ph. Eur • energy toxicity↘ • γ Imaging

• 177mLu waste

213Bi 45,6m 225Ac/213Bi generator

• Generator • 440keV γ

• Recoil • Short half-life

T1/2 (d) Production Targeting moety

Advantages Disadvantages

131I 8d Reactor • all • Pure theranostic • Direct labeling

• Energy • Dehalogenetion

90Y 2,7d Reactors 90Sr daughter

• all

• Pure β- (radioprotection) • energy (long range) big

lesions • Ph. Eur

• energy (long range) toxicity

T1/2 (d) Production Targeting moety

Advantages Disadvantages

131I 8d Reactor • all • Pure theranostic • Direct labeling

• Energy • Dehalogenetion

Radionuclide:Thorium 227

Propensy of α

Imaging properties? better than Ra-223

t½ of 19d compatible with biological t½ high MW mAb

Physiological conditions: +4 oxidation state!!

HOPO= chelator Several mAb

Radionuclide

• Theranostic

• ImmunoPET β+

•Radioimmunotherapyβ− α

Direct radiolabeling Bifunctional chelator

Targeting moety

• protein

• mAb

• Fab

• Minibodies….

Chelators

Chelator Radioisotopes Advantages Disadvantages

DFO 89Zr, 68Ga • Radiolabeling efficiency

• No heating

• Limited radioisotopes

• Stability: Bone uptake of 89Zr

HOPO

DOTA

NOTA

Chelator Radioisotopes Advantages Disadvantages

DFO 89Zr, 68Ga • Radiolabeling efficiency

• No heating

• Limited radioisotopes

• Stability: Bone uptake of 89Zr

HOPO 89Zr, Thorium • High Stability • Lower bone

uptake of 89Zr-HOPO-mAb

• Lower tumor uptake of 89Zr-HOPO-mAb

Chelator Radioisotopes Advantages Disadvantages

DFO 89Zr, 68Ga • Radiolabeling efficiency

• No heating

• Limited radioisotopes

• Stability: Bone uptake of 89Zr

HOPO 89Zr, Thorium • High Stability • Lower bone

uptake of 89Zr-HOPO-mAb

• Lower tumor uptake of 89Zr-HOPO-mAb

DOTA universal • Outstanding stability

• Heating!!!

Chelator Radioisotopes Advantages Disadvantages

DFO 89Zr, 68Ga • Radiolabeling efficiency

• No heating

• Limited radioisotopes

• Stability: Bone uptake of 89Zr

HOPO 89Zr, Thorium • High Stability • Lower bone

uptake of 89Zr-HOPO-mAb

• Lower tumor uptake of 89Zr-HOPO-mAb

DOTA universal • Outstanding stability

• Heating!!!

NOTA 68Ga, 64Cu • Perfect match for 68Ga

• No/limited heating

• Limited radioisotopes

Radionuclide

• Theranostic

• ImmunoPET β+

•Radioimmunotherapyβ− α

Direct radiolabeling Bifunctional chelator

Targeting moety

• protein

• mAb

• Fab

• Minibodies….

RADIOLABELING

Kits

Sterile, lyophilized, easy-to-use

Avoid the use of expensive equipment & lengthy procedures

Reduction in production cost

Increase in the accessibility

Peptide Radiolabeling = transchelation of cation at high T

mAb Radiolabeling = transchelation of cation, max 40°C

Automated Syntheses

on-line documentation of the manufacturing process (GMP)

disposable sterile cassettes (GMP)

risk of cross-contamination↘ & radiation burden↘robustness↗

Labeling methods

Ready to use

Bexxar

Manual

89Zr-Trastuzumab

4. Conjugation of TFP-N-sucDf-Fe to mAb: 1 mL mAb (5 mg/mL; pH 9.5–9.8( 0.1 M Na2CO3)) + 20 μL of TFP ester solution (1:1 for 54% reaction efficiency) 30 min+50μL gentisic acid (100 mg/mL in 0.32 M Na2CO3) pH is adjusted to 4.3–4.5 (with 0.25 M H2SO4 )

5. removal of Fe(III): +50 μL EDTA (25 mg/mL) 30 min at 35°CPD-10 column elute 2.6 mL 0.9% NaCl/gentisic acid [5 mg/mL] discard elute 2 mL 0.9% NaCl/gentisic acid [5 mg/mL] collected modified mAb

1. synthesis of N-sucDf

2. Complexation of N-sucDf with Fe(III): N-sucDf (9 mg) +(3 mL of saline, containing + 60 μL of 0.1 M Na2CO3) + 300 μL of FeCl3 solution (8 mg/mL in 0.1MHCl)

3. Esterification of N-sucDf-Fe: 10 min + 300 μL TFP solution (200 mg/mL in MeCN) + 120 mg solid EDC 45 min of incubationC18 cartridge (Waters) washing 60 mL H2Owater elution with 1.5 mL of MeCN

Modification of the precursor

Production of 89Zr-T

89Zr

89Zr

Internalisation

Time (h)

Specificity Affinity

Summary & conclusion

• Wide variety of cancer-related targets and probes • RIT: proven to be useful in leukemia and lymphoma, new indications… • ImmunoPET: proven to be useful in gaining information on cancer biology

• Clinical studies on 89Zr-based immuno-PET to predict/monitor treatment (Dr. G. Gebhart)

• Ehrlich in 1913: “corpora non agunt nisi fixata”

(a substance is not (biologically) active unless it is “fixed” (bound by a receptor))

antibodies need to bind for therapeutic (as well as diagnostic) efficacy. not only the mere presence of the target but also its accessibility!!!

Radioisotope: • imaging :SPECT immunoPET 89Zr

• therapy:131I90Y1

77Lu and α

Target: CD20 HER2 VEGF

…

Method : •Direct (124/131I, 99mTc) • IndirectBifunctional chelator (DFO & DOTA)

cancer cell

Probe: Protein: mAb, Fab, nanobodies, affibodies,…

references

Heskamp, Sandra, René Raavé, Otto Boerman, Mark Rijpkema, Victor Goncalves, and Franck Denat. 2017. “89Zr-

Immuno-Positron Emission Tomography in Oncology: State-of-the-Art 89Zr Radiochemistry.” Bioconjugate

Chemistry 28 (9): 2211–23. doi:10.1021/acs.bioconjchem.7b00325.

Moek, Kirsten L., Danique Giesen, Iris C. Kok, Derk Jan A. de Groot, Mathilde Jalving, Rudolf S. N. Fehrmann,

Marjolijn N. Lub-de Hooge, Adrienne H. Brouwers, and Elisabeth G. E. de Vries. 2017. “Theranostics Using

Antibodies and Antibody-Related Therapeutics.” Journal of Nuclear Medicine 58 (Supplement 2): 83S–90S.

doi:10.2967/jnumed.116.186940.

Oehlke, Elisabeth, Cornelia Hoehr, Xinchi Hou, Victoire Hanemaayer, Stefan Zeisler, Michael J. Adam, Thomas J.

Ruth, et al. 2015. “Production of Y-86 and Other Radiometals for Research Purposes Using a Solution Target

System.” Nuclear Medicine and Biology 42 (11): 842–49. doi:10.1016/j.nucmedbio.2015.06.005.

Verel, Iris, Gerard W. M. Visser, Ronald Boellaard, Marijke Stigter-van Walsum, Gordon B. Snow, and Guus A. M. S.

van Dongen. 2003. “89Zr Immuno-PET: Comprehensive Procedures for the Production of 89Zr-Labeled Monoclonal

Antibodies.” Journal of Nuclear Medicine 44 (8): 1271–81.

Verel, Iris, Gerard W.M. Visser, Otto C. Boerman, Julliette E.M. van Eerd, Ron Finn, Ronald Boellaard, Maria J.W.D.

Vosjan, Marijke Stigter-van Walsum, Gordon B. Snow, and Guus E.M. van Dongen. 2003. “Long-Lived Positron

Emitters Zirconium-89 and Iodine-124 for Scouting of Therapeutic Radioimmunoconjugates with PET.” Cancer

Biotherapy and Radiopharmaceuticals 18 (4): 655–61. doi:10.1089/108497803322287745.

Watering, Floor C. J. van de, Mark Rijpkema, Marc Robillard, Wim J. G. Oyen, and Otto C. Boerman. 2014.

“Pretargeted Imaging and Radioimmunotherapy of Cancer Using Antibodies and Bioorthogonal Chemistry.” Frontiers

in Medicine 1 (November). doi:10.3389/fmed.2014.00044.

Radiopharmacy: Prof. G. Ghanem Dr. Z. Wimana Ms. A. De Matos Mr. Pierre Huget radiopharmacie@bordet.be