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Expression and purification of 4-1BB
4-1BB was expressed in mammalian cells with an Fc fusion tag. The protein
was purified on protein A column before cleavage of the Fc-tag.
BACKGROUND
STRUCTURES
COMPLEX PREPARATIONS
BINDING COMPARISON
CONCLUSION
Contact
SARomics Biostructures is a contract research organization (CRO) that offer
structure determination services. Alligator Bioscience is a biotechnology
company that develops innovative tumor-directed antibody-based
immunotherapies. SARomics and Alligator have worked together to determine
the binding epitope of the antibody ATOR-1017 using X-ray crystallography.
ATOR-1017 is an agonistic antibody targeting the co-stimulatory receptor 4-
1BB (CD137) developed to activate tumor specific T cells for immunotherapy
of cancer. This specific antibody was designed and optimized at Alligator to
address the limitations of the monospecific 4-1BB antibodies (Urelumab and
Utomilumab), which have suffered from either being toxic to humans or having
poor efficacy, respectively.
The main goal of the project has been to determine the binding epitope of the
antibody ATOR-1017 using X-ray crystallography and to compare the epitope
with those of Urelumab and Utomilumab that have very different profiles.
Two 4-1BB binders developed by Alligator were studied at SARomics using X-
ray crystallography; ATOR-1017 now in clinical trials phase I as well as Fab-
1618 which has a different binding profile.
VERIFICATION OF THE UNIQUE
FUNCTIONALITY OF ATOR-1017
BY 3D STRUCTURE DETERMINATION
KARIN KETTISEN1, NADIA ROSE1, MARIA HÅKANSSON1 KARIN ENELL SMITH2, REBEKA KOVACIC1, JESSICA PETERSSON2 AND LAURA VON
SCHANTZ2
1SAROMICS BIOSTRUCTURES, LUND, SWEDEN
2ALLIGATOR BIOSCIENCE, LUND, SWEDEN
Preparation of Fab-1618
mAb 1618 was produced in mammalian cells. To prepare the Fab the mAb was
cleaved with papain and purified on protein A column followed by a size
exclusion chromatography step.
Preparation of ATOR-1017 scFv
ATOR-1017 scFv expressed in the periplasm of bacteria was purified on protein
A column followed by a size exclusion chromatography.
Preparation of 4-1BB: fab complexes for structure determination
For complex formation 4-1BB was mixed with either
• Fab-1618 : Fab-1618 : 4-1BB
• ATOR-1017 scFv : ATOR-1017 scFv : 4-1BB
• Fab-1618 and ATOR-1017 scFv : Fab-1618 : 4-1BB : ATOR-1017scFv.
The complexes were purified on size exclusion and crystallization experiments
were set up.
Ø Successful crystallization and structure determination of :
• Fab-1618 : 4-1BB at 2.3 Å
• ATOR-1017 scFv : 4-1BB at 3.1 Å
• Fab-1618 : 4-1BB : ATOR-1017scFv at 3.4 Å
Ø The obtained results:
• confirmed the binding of ATOR-1017 to a unique epitope on 4-1BB
• supported the advancement of ATOR-1017 in clinical studies.
4-1BB
ATOR-1017 scFv
Fab-1618 : 4-1BB1 ATOR-1017 : 4-1BB2
ATOR-1017 : 4-1BB: Fab-1618
4-1BB
ATOR-1017 scFv
Fab-1618
Fab-1618
4-1BB
Nadia Rose, Line Manager, nadia.rose@saromics.com
References
1 Nelson MH, et al. The Bispecific Tumor Antigen-Conditional 4-1BB x 5T4 Agonist, ALG.APV-527,
Mediates Strong T-Cell Activation and Potent Antitumor Activity in Preclinical Studies. Mol Cancer
Ther. 2023;22(1):89-101. doi:10.1158/1535-7163.MCT-22-0395
2 Smith KE, et al. ATOR-1017, an Fc-gamma receptor conditional 4-1BB agonist designed for
optimal safety and efficacy, activates exhausted T cells in combination with anti-PD-. Cancer
Immunol Immunother (Submitted)
Utomilumab ATOR-1017 Fab-1618 Urelumab
Poor efficacy Good safety profile and efficacy High toxicity
Utomilumab
ATOR-1017
Urelumab
Fab-1618
4-1BB
D. 1
D. 2
D. 3
D. 4

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VERIFICATION OF THE UNIQUE FUNCTIONALITY OF ATOR-1017 BY 3D STRUCTURE DETERMINATION

  • 1. Expression and purification of 4-1BB 4-1BB was expressed in mammalian cells with an Fc fusion tag. The protein was purified on protein A column before cleavage of the Fc-tag. BACKGROUND STRUCTURES COMPLEX PREPARATIONS BINDING COMPARISON CONCLUSION Contact SARomics Biostructures is a contract research organization (CRO) that offer structure determination services. Alligator Bioscience is a biotechnology company that develops innovative tumor-directed antibody-based immunotherapies. SARomics and Alligator have worked together to determine the binding epitope of the antibody ATOR-1017 using X-ray crystallography. ATOR-1017 is an agonistic antibody targeting the co-stimulatory receptor 4- 1BB (CD137) developed to activate tumor specific T cells for immunotherapy of cancer. This specific antibody was designed and optimized at Alligator to address the limitations of the monospecific 4-1BB antibodies (Urelumab and Utomilumab), which have suffered from either being toxic to humans or having poor efficacy, respectively. The main goal of the project has been to determine the binding epitope of the antibody ATOR-1017 using X-ray crystallography and to compare the epitope with those of Urelumab and Utomilumab that have very different profiles. Two 4-1BB binders developed by Alligator were studied at SARomics using X- ray crystallography; ATOR-1017 now in clinical trials phase I as well as Fab- 1618 which has a different binding profile. VERIFICATION OF THE UNIQUE FUNCTIONALITY OF ATOR-1017 BY 3D STRUCTURE DETERMINATION KARIN KETTISEN1, NADIA ROSE1, MARIA HÅKANSSON1 KARIN ENELL SMITH2, REBEKA KOVACIC1, JESSICA PETERSSON2 AND LAURA VON SCHANTZ2 1SAROMICS BIOSTRUCTURES, LUND, SWEDEN 2ALLIGATOR BIOSCIENCE, LUND, SWEDEN Preparation of Fab-1618 mAb 1618 was produced in mammalian cells. To prepare the Fab the mAb was cleaved with papain and purified on protein A column followed by a size exclusion chromatography step. Preparation of ATOR-1017 scFv ATOR-1017 scFv expressed in the periplasm of bacteria was purified on protein A column followed by a size exclusion chromatography. Preparation of 4-1BB: fab complexes for structure determination For complex formation 4-1BB was mixed with either • Fab-1618 : Fab-1618 : 4-1BB • ATOR-1017 scFv : ATOR-1017 scFv : 4-1BB • Fab-1618 and ATOR-1017 scFv : Fab-1618 : 4-1BB : ATOR-1017scFv. The complexes were purified on size exclusion and crystallization experiments were set up. Ø Successful crystallization and structure determination of : • Fab-1618 : 4-1BB at 2.3 Å • ATOR-1017 scFv : 4-1BB at 3.1 Å • Fab-1618 : 4-1BB : ATOR-1017scFv at 3.4 Å Ø The obtained results: • confirmed the binding of ATOR-1017 to a unique epitope on 4-1BB • supported the advancement of ATOR-1017 in clinical studies. 4-1BB ATOR-1017 scFv Fab-1618 : 4-1BB1 ATOR-1017 : 4-1BB2 ATOR-1017 : 4-1BB: Fab-1618 4-1BB ATOR-1017 scFv Fab-1618 Fab-1618 4-1BB Nadia Rose, Line Manager, nadia.rose@saromics.com References 1 Nelson MH, et al. The Bispecific Tumor Antigen-Conditional 4-1BB x 5T4 Agonist, ALG.APV-527, Mediates Strong T-Cell Activation and Potent Antitumor Activity in Preclinical Studies. Mol Cancer Ther. 2023;22(1):89-101. doi:10.1158/1535-7163.MCT-22-0395 2 Smith KE, et al. ATOR-1017, an Fc-gamma receptor conditional 4-1BB agonist designed for optimal safety and efficacy, activates exhausted T cells in combination with anti-PD-. Cancer Immunol Immunother (Submitted) Utomilumab ATOR-1017 Fab-1618 Urelumab Poor efficacy Good safety profile and efficacy High toxicity Utomilumab ATOR-1017 Urelumab Fab-1618 4-1BB D. 1 D. 2 D. 3 D. 4