N02CO82598-8_SOW.pdf

STATEMENT OF WORK (SOW)

N02CO82598-8

1.0 TITLE

High resolution X-ray crystal structures of the extracellular domains of Chimeric Antigen Receptor (CAR) proteins and other immunologically relevant surface receptors and co-receptors, and their interactions with their tumor antigens.

2.0 BACKGROUND

Clinical researchers at the U.S. Department of Health and Human Services, National Institutes of Health, National Cancer Institute (NCI), Center for Cancer Research (CCR) have been working for many years to harness the patient’s own immune system to fight their cancer. A central direction for NCI/CCR in this cancer immunotherapy approach has been “Adoptive Cell Transfer” (ACT). New T-cell-based immunotherapies use the patient’s T-cells, enriched with genetically engineered proteins called Chimeric Antigen Receptors (CARs), to target antigens on the surface of tumor cells. These cells are then expanded in large numbers in vitro and infused back into the patient to attack cancer cells. Moreover, as the cells expand and persist in the patient they serve as a long-term drug (a “living drug”) to keep the tumor in check. These therapies have shown great promise and recently, as a result of the success of CAR T-cell clinical trials, the NCI received FDA approval for CAR T-cell therapy to treat large-B-cell lymphoma.

CAR T-cell therapy involves isolating T-cells from the patient’s blood and genetically engineering them to express a surface protein called a chimeric antigen receptor (CAR). This receptor protein is made from different domains of native proteins of the immune system and has the ability to recognize antigens expressed on the surface of tumor cells. As T-cells are the immune system’s natural killing cells, binding of a CAR to the tumor antigen also initiates a cascade of cellular events that results in clearing of tumor cells.

While both CAR T-cell hold great promise in the clinic, the biochemical and biophysical basis for what makes an effective CAR protein remains unexplored. The three-dimensional structure of these CAR molecules that give the genetically engineered T-cells the ability to identify target antigens on the surface of tumor cells, has never been studied. Structural analysis of the CAR proteins either alone or interacting with their target antigens on tumor cells, can enable clinicians to rationally design and improve CAR therapies.

CCR clinicians are also using other genetically engineered receptor proteins as immunotherapeutic molecules for recognition of tumor-specific neoantigen complexes as targets on solid tumors in epithelial cancers.

Comprehensive structural analysis of extracellular domains of individual CAR proteins or other immunologically relevant receptor proteins alone, and bound to the extracellular domains of their specific antigen or neoantigen complex respectively, as well as their co-receptors can help elucidate how receptors on the surface of T-cells engage with antigens or neoantigen complexes to lead to effective T-cell activation. This can enable NCI researchers to rationally design more effective ACT immunotherapies that target multiple types of cancer. Structure determination of immunologically relevant surface receptors (proteins) require considerable expertise, and structural analysis can only be performed once atomic coordinates of the protein structures become available.

The NCI/CCR requires protein structures of the extracellular domains of anti-BCMA CAR and anti- CD19 CAR proteins, using X-ray crystallography, both alone and in complex with the extracellular domains of their respective antigen. The results from this work will assist CCR computational biologists elucidate how the different extracellular structural domains/regions of individual CAR proteins contribute to CAR-surface antigen interactions, and reveal how this contributes to effective T-cell activation.

An additional requirement is to generate high resolution crystal structures after specific domains of the extracellular domains of these CAR proteins are altered, to obtain structural information on these new “variant” proteins. This can help determine if these new variants could be more effective receptors within engineered T-cells.

3.0 SCOPE

The CCR requires services to generate high resolution X-ray crystal structures of the extracellular regions of individual CAR proteins, either alone or bound to the extracellular domain of their respective surface antigen. The CCR shall provide the Contractor with dematerialized information in the form of a sequence of a protein. The Contractor shall then provide all materials and supplies, and all technical services and scientific expertise that will be required to successfully obtain high-resolution crystal structures of these proteins. This includes, preparing a plan for structural determination and performing all of the following: cloning of the specific extracellular domain of the selected proteins, protein expression, protein purification, crystallization screening, crystal optimization, X-ray diffraction studies, structure determination, to deliver dematerialized information as 3D positions of atoms of the protein, with a detailed expert description in of the crystal structure.

The Contractor shall provide all required technical and scientific expertise for all aspects of this work, from the planning stages through to interpretation of the results. The Contractor shall provide expertise by Ph.D. scientists with +5years of experience in structural biology, and can exhibit a record of publishing structural analysis of immunologically-relevant receptor proteins in a peer-reviewed journal.

For each task, the Contractor shall provide a project plan that includes the extracellular regions of the target protein domain and details on their strategies for DNA cloning, protein expression and protein purification. Upon approval of each plan, by the NCI Project Officer, the Contractor shall proceed with the implementation of the project plan, which may initially involve some optimization of protein expression and purification, before proceeding to large scale protein purification to enable protein crystallization. Initial protein crystallization shall be conducted using sparse matrix screening to help identify optimal crystallization conditions. Once optimal crystallization conditions are identified, the Contractor shall proceed to large scale protein crystallization that can be used for X-ray diffraction studies using synchrotron beamlines and structural determination using conventional crystallographic structure determination and structure refinement software.

As a final product, CCR/NCI requires atomic coordinates for crystal structures to be delivered as electronic files in PDB format, and structure factors corresponding to atomic coordinates delivered as electronic files in MTZ format, to the NCI Project Officer.

4.0 CONTRACT REQUIREMENTS

The Contractor shall perform the following tasks in the BASE PERIOD:

4.1 Generate high-resolution crystal structures of the extracellular domain of anti-B-cell maturation antigen (BCMA) CAR both alone and in complex with the extracellular domain of BCMA. The details for the anti-BCMA CAR molecule (11D5-3-CD828Z) are referenced in Ali SA, et al. 2016: Blood 128:1688-1700.

4.1.1 All work shall be carried out on the Contractor’s site or an affiliated site, using their own resources. These shall include:

a) Facilities to perform cloning, bacterial or eukaryotic protein expression, and protein purification including cold room capabilities to enable optimal protein purification and crystallization.

b) Access to automated chromatography systems to produce high purity protein suitable for crystallization studies.

c) Facilities equipped with specialized robotic technologies incorporated in an integrated workflow that enables protein crystallization and optimization.

d) In-house X-ray equipment for diffraction studies as well as dedicated access to a beamline at an internationally recognized laboratory with a Synchrotron light source.

4.1.2 The Contractor shall prepare a plan to perform structural determination of extracellular domain of anti-B-cell maturation antigen (BCMA) CAR both alone and in complex with the extracellular domain of BCMA. The plan shall include the exact amino acid sequence of the extracellular domain of the anti-B-cell maturation antigen (BCMA) CAR and the extracellular domain of BCMA. The project plan prepared by the Contractor shall include a cloning, expression and purification strategy for the extracellular domain of the anti-BCMA CAR and a cloning, expression and purification strategy for the extracellular domain of BCMA. The cloning, expression and purification strategies shall take into consideration whether the domains are best expressed in eukaryotic or bacterial expression systems and the appropriate leader sequence(s) that can enable effective expression, secretion and purification. All protein purification shall proceed under optimal conditions, (4-degree C), to ensure native folding of the protein.

4.1.3 Upon approval of the plan by the NCI Project Officer, the Contractor shall proceed to cloning, expression and purification of the extracellular domains of the anti- BCMA CAR and the extracellular domain of BCMA. The results of the small-scale protein purification(s) shall be provided to the NCI Project Officer to ensure that optimal protein isolation can be obtained before production of sufficient quantities of protein for crystallization studies. Results shall be communicated electronically to the NCI Project Officer as a brief written report.

4.1.4 Upon approval by the NCI Project Officer, the Contractor shall commence on expression and purification of the anti-BCMA CAR and the extracellular domain of BCMA, that is sufficient for protein crystallization.

4.1.5 Initial protein crystallization shall be conducted using sparse matrix screening to help identify optimal crystallization conditions. This will ensure that multiple formulations can be tried, using different salts, polymers, organics, buffers, pH, to identify the best conditions for protein crystallization. X-ray diffraction of these crystals from the matrix screen shall help identify the appropriate conditions for crystal growth.

4.1.6 Once initial crystallization conditions are identified, the Contractor shall proceed to optimization of crystallization for structure determination. The protein crystals shall be used for X-ray diffraction studies using a micro-diffraction undulator synchrotron beamline.

4.1.7 A progress report on the project that includes any intermediary result shall be delivered as electronic files to the NCI Project Officer.

4.1.8 Once x-ray diffraction data has been collected, the Contractor shall determine structures using molecular replacement methods with appropriate search models available in the Protein Data Bank (PDB) which is the repository of 3D protein structures, or through de novo phasing methods. All structural analyses shall identify and describe in detail important intramolecular and intermolecular interactions, hydrogen bond patterns, salt bridges, intra and intermolecular cysteine bridges, as well as ordered crystallographic waters. Regions of no electron density in critical regions of interest shall receive special attention if they could benefit from a new structure determination strategy.

4.1.9 A final project report that includes the atomic coordinates for crystal structures shall be delivered as electronic files in PDB format, and structural factors corresponding to atomic coordinates shall be delivered as electronic files in the MTZ format, the file format used for storage of crystallographic data, to the NCI Project Officer.

4.2 Generate high-resolution crystal structures of the extracellular domain of the anti-CD19 CAR

(47G4-CD828Z, referenced in Patent CA2951045A1)

4.2.1 All work shall be carried out on the Contractor’s site or an affiliated site, using their

a) Facilities to perform cloning, bacterial or eukaryotic protein expression, and protein purification including cold room capabilities to enable optimal protein purification and crystallization.

b) Access to automated chromatography systems to produce high purity protein suitable for crystallization studies.

c) Facilities equipped with specialized robotic technologies incorporated in an integrated workflow that enables protein crystallization and optimization.

d) In-house X-ray equipment for diffraction studies as well as dedicated access to a beamline at an internationally recognized laboratory with a Synchrotron light source.

4.2.2 The Contractor shall prepare a plan to perform structural determination of extracellular domain of the anti-CD19 CAR. The plan shall include the exact amino acid sequence of the extracellular domain of the anti-CD19 CAR and shall include a cloning, expression and purification strategy. The cloning, expression and purification strategies shall take into consideration whether the domain is best expressed in eukaryotic or bacterial expression systems and the appropriate leader sequence(s) that can enable effective expression, secretion and purification. All protein purification shall proceed under optimal conditions, (4-degree C), to ensure optimal native folding of the protein.

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0ahUKEwjPlqWtoLDaAhXKT98KHcNYADwQFggnMAA&url=https%3A%2F%2Fwww.google.com%2Fpatents%2FCA2951045A1%3Fcl%3Den&usg=AOvVaw1GKrxTn8iv6Jtst7A1fOlE

4.2.3 Upon approval of the plan by the NCI Project Officer, the Contractor shall proceed to cloning, expression and purification of the extracellular domain of the anti-CD19 CAR. The results of the small-scale protein purification(s) shall be provided to the NCI Project Officer to ensure that optimal protein isolation can be obtained before production of sufficient quantities of protein for crystallization studies. Results shall be communicated electronically to the NCI Project Officer as a brief written report.

4.2.4 Upon approval by the NCI Project Officer, the Contractor shall commence on expression and purification of the extracellular domain of anti-CD19 CAR, that is sufficient for protein crystallization.

4.2.5 Initial protein crystallization shall be conducted using sparse matrix screening to help identify optimal crystallization conditions. This will ensure that multiple formulations can be tried, using different salts, polymers, organics, buffers, pH, to identify the best conditions for protein crystallization. X-ray diffraction of these crystals from the matrix screen shall help identify the appropriate conditions for crystal growth.

4.2.6 Once initial crystallization conditions are identified, the Contractor shall proceed to optimization of crystallization for structure determination. The protein crystals shall be used for X-ray diffraction studies using a micro-diffraction undulator synchrotron beamline.

4.2.7 A progress report on the project that includes any intermediary result shall be delivered as electronic files to the NCI Project Officer.

4.2.8 Once x-ray diffraction data has been collected, the Contractor shall determine structures using molecular replacement methods with appropriate search models available in the Protein Data Bank (PDB) which is the repository of 3D protein structures, or through de novo phasing methods. All structural analyses shall identify and describe in detail important intramolecular and intermolecular interactions, hydrogen bond patterns, salt bridges, intra and intermolecular cysteine bridges, as well as ordered crystallographic waters. Regions of no electron density in critical regions of interest shall receive special attention if they could benefit from a new structure determination strategy.

4.2.9 A final project report that includes the atomic coordinates for crystal structures shall be delivered as electronic files in PDB format, and structural factors corresponding to

4.3 Generate high-resolution crystal structures of the extracellular domain of anti-BCMA CAR alone and in complex with extracellular domain of BCMA, in which a specific part of a domain or linker, or sequence of a domain of the anti-BCMA CAR has been modified (insertion, deletion or change of amino acids), to become a “variant” (modified) protein, as specified by the NCI Project Officer. This variation will be determined after obtaining the results from Task 4.1 to include the scFv portion with or without a specific variant of the hinge region).

4.3.1 All work shall be carried out on the Contractor’s site or an affiliated site, using their own resources. These shall include:

a) Facilities to perform cloning, bacterial or eukaryotic protein expression, and protein purification including cold room capabilities to enable optimal protein purification and crystallization.

b) Access to automated chromatography systems to produce high purity protein suitable for crystallization studies.

c) Facilities equipped with specialized robotic technologies incorporated in an integrated workflow that enables protein crystallization and optimization.

d) In-house X-ray equipment for diffraction studies as well as dedicated access to a beamline at an internationally recognized laboratory with a Synchrotron light source.

4.3.2 The Contractor shall prepare a plan to perform structural determination of the extracellular domain of the specific variant of anti-BCMA CAR variant and the amino acid sequence of the extracellular domain of BCMA. The plan shall include how the amino acid sequence of the extracellular domain of the anti-BCMA CAR is being modified in the variant. The project plan prepared by the Contractor shall include a cloning, expression and purification strategy for the extracellular domain of the variant anti-BCMA CAR variant and the cloning, expression and purification strategy for the extracellular domain of BCMA. The cloning, expression and purification strategies shall take into consideration whether the domains are best expressed in eukaryotic or bacterial expression systems and the appropriate leader sequence(s) that can enable effective expression, secretion and purification. All protein purification shall proceed under conditions to ensure native folding of the protein domains and peptide complexes.

4.3.3 Upon approval of the plan by the NCI Project Officer, the Contractor shall proceed to cloning, expression and purification of the domains of the variant anti-BCMA CAR and the extracellular domain of BCMA. The results of the small-scale protein purification(s) shall be provided to the NCI Project Officer to ensure that optimal protein isolation can be obtained before production of sufficient quantities of protein for crystallization studies. Results shall be communicated electronically to the NCI Project Officer as a brief written report.

4.3.4 Upon approval by the NCI Project Officer, the Contractor shall commence on expression and purification of the variant anti-BCMA CAR and the extracellular domain of BCMA, that is sufficient for protein crystallization.

4.3.5 Initial protein crystallization shall be conducted using sparse matrix screening to help identify optimal crystallization conditions. This will ensure that multiple formulations can be tried, using different salts, polymers, organics, buffers, pH, to identify the best conditions for protein crystallization. X-ray diffraction of these crystals from the matrix screen shall help identify the appropriate conditions for crystal growth.

4.3.6 Once initial crystallization conditions are identified, the Contractor shall proceed to optimization of crystallization for structure determination. The protein crystals shall be used for X-ray diffraction studies using a micro-diffraction undulator synchrotron beamline.

4.3.7 A progress report on the project that includes any intermediary result shall be delivered as electronic files to the NCI Project Officer.

4.3.8 Once x-ray diffraction data has been collected, the Contractor shall determine structures using molecular replacement methods with appropriate search models available in the Protein Data Bank (PDB) which is the repository of 3D protein structures, or through de novo phasing methods. All structural analyses shall identify and describe in detail important intramolecular and intermolecular interactions, hydrogen bond patterns, salt bridges, intra and intermolecular cysteine bridges, as well as ordered crystallographic waters. Regions of no electron density in critical regions of interest shall receive special attention if they could benefit from a new structure determination strategy.

4.3.9 A final project report that includes the atomic coordinates for crystal structures shall be delivered as electronic files in PDB format, and structural factors corresponding to

4.4 Generate high-resolution crystal structures of a specific variant of the extracellular domain of anti-BCMA CAR in which there is a three residue AAA insertion between the last strand of the scFv and the beginning of the hinge.

4.4.1 All work shall be carried out on the Contractor’s site or an affiliated site, using their own resources. These shall include:

a) Facilities to perform cloning, bacterial or eukaryotic protein expression, and protein purification including cold room capabilities to enable optimal protein purification and crystallization.

b) Access to automated chromatography systems to produce high purity protein suitable for crystallization studies.

c) Facilities equipped with specialized robotic technologies incorporated in an integrated workflow that enables protein crystallization and optimization.

d) In-house X-ray equipment for diffraction studies as well as dedicated access to a beamline at an internationally recognized laboratory with a Synchrotron light source.

4.4.2 The Contractor shall prepare a plan to perform structural determination of this specific variant of the extracellular domain of anti-B-cell maturation antigen (BCMA) CAR in which there is a three residue AAA insertion between the last strand of the scFv and the beginning of the hinge. The project plan shall include the exact amino acid sequence of this AAA-inserted variant of the extracellular domain of the anti- BCMA CAR and shall include a cloning, expression and purification strategy. The cloning, expression and purification strategies shall take into consideration whether the domains are best expressed in eukaryotic or bacterial expression systems and the appropriate leader sequence(s) that can enable effective expression, secretion and purification. All protein purification shall proceed under optimal conditions, (4-degree C), to ensure optimal native folding of the protein.

4.4.3 Upon approval of the plan by the NCI Project Officer, the Contractor shall proceed to cloning, expression and purification of this AAA-inserted variant of the extracellular domain of the anti-BCMA CAR. The results of the small-scale protein purification(s) shall be provided to the NCI Project Officer to ensure that optimal protein isolation can be obtained before production of sufficient quantities of protein for crystallization studies. Results shall be communicated electronically to the NCI Project Officer as a brief written report.

4.4.4 Upon approval by the NCI Project Officer, the Contractor shall commence on expression and purification of this AAA-inserted variant of the extracellular domain of the anti-BCMA CAR, that is sufficient for protein crystallization.

4.4.5 Initial protein crystallization shall be conducted using sparse matrix screening to help identify optimal crystallization conditions. This will ensure that multiple formulations can be tried, using different salts, polymers, organics, buffers, pH, to identify the best conditions for protein crystallization. X-ray diffraction of these crystals from the matrix screen shall help identify the appropriate conditions for optimal crystal growth.

4.4.6 Once initial crystallization conditions are identified, the Contractor shall proceed to optimization of crystallization for structure determination. The protein crystals shall be used for X-ray diffraction studies using a micro-diffraction undulator synchrotron beamline.

4.4.7 A progress report on the project that includes any intermediary result shall be delivered as electronic files to the NCI Project Officer.

4.4.8 Once x-ray diffraction data has been collected, the Contractor shall determine structures using molecular replacement methods with appropriate search models available in the Protein Data Bank (PDB) which is the repository of 3D protein structures, or through de novo phasing methods. All structural analyses shall identify and describe in detail important intramolecular and intermolecular interactions, hydrogen bond patterns, salt bridges, intra and intermolecular cysteine bridges, as well as ordered crystallographic waters. Regions of no electron density in critical regions of interest shall receive special attention if they could benefit from a new structure determination strategy.

4.4.9 A final project report that includes the atomic coordinates for crystal structures shall be delivered as electronic files in PDB format, and structural factors corresponding to

The Contractor shall perform the following tasks in OPTION PERIOD 1:

4.5 Generate high-resolution crystal structures of the extracellular domain of anti-B-cell maturation antigen (BCMA) CAR both alone and in complex with the extracellular domain of BCMA, where the antigen binding domain in the anti-BCMA CAR (which is the scFv in Task 4.1), is replaced by a single Immunoglobulin domain, as specified by the Project Officer.

4.5.1 All work shall be carried out on the Contractor’s site or an affiliated site, using their own resources. These shall include:

a) Facilities to perform cloning, bacterial or eukaryotic protein expression, and protein purification including cold room capabilities to enable optimal protein purification and crystallization.

b) Access to automated chromatography systems to produce high purity protein suitable for crystallization studies.

c) Facilities equipped with specialized robotic technologies incorporated in an integrated workflow that enables protein crystallization and optimization.

d) In-house X-ray equipment for diffraction studies as well as dedicated access to a beamline at an internationally recognized laboratory with a Synchrotron light source.

4.5.2 The Contractor shall prepare a plan to perform structural determination of the extracellular domain of the specific variant of anti-BCMA CAR where the antigen binding domain in the anti-BCMA CAR is replaced by a single Immunoglobulin domain, and the amino acid sequence of the extracellular domain of BCMA. The plan shall include details on how the extracellular domain of the anti-BCMA CAR is being modified in the variant. The project plan prepared by the Contractor shall include a cloning, expression and purification strategy for the extracellular domain of the variant anti-BCMA CAR and the cloning, expression and purification strategy for the extracellular domain of BCMA. The cloning, expression and purification strategies shall take into consideration whether the domains are best expressed in eukaryotic or bacterial expression systems and the appropriate leader sequence(s) that can enable effective expression, secretion and purification. All protein purification shall proceed under conditions to ensure native folding of the protein domains and peptide complexes.

4.5.3 Upon approval of the plan by the NCI Project Officer, the Contractor shall proceed to cloning, expression and purification of the domains of the variant anti-BCMA CAR and the extracellular domain of BCMA. The results of the small-scale protein purification(s) shall be provided to the NCI Project Officer to ensure that optimal protein isolation can be obtained before production of sufficient quantities of protein for crystallization studies. Results shall be communicated electronically to the NCI Project Officer as a brief written report.

4.5.4 Upon approval by the NCI Project Officer, the Contractor shall commence on expression and purification of the variant anti-BCMA CAR and the extracellular domain of BCMA, that is sufficient for protein crystallization.

4.5.5 Initial protein crystallization shall be conducted using sparse matrix screening to help identify optimal crystallization conditions. This will ensure that multiple formulations can be tried, using different salts, polymers, organics, buffers, pH, to identify the best conditions for protein crystallization. X-ray diffraction of these crystals from the matrix screen shall help identify the appropriate conditions for crystal growth.

4.5.6 Once initial crystallization conditions are identified, the Contractor shall proceed to optimization of crystallization for structure determination. The protein crystals shall be used for X-ray diffraction studies using a micro-diffraction undulator synchrotron beamline.

4.5.7 A progress report on the project that includes any intermediary result shall be delivered as electronic files to the NCI Project Officer.

4.5.8 Once x-ray diffraction data has been collected, the Contractor shall determine structures using molecular replacement methods with appropriate search models available in the Protein Data Bank (PDB) which is the repository of 3D protein structures, or through de novo phasing methods. All structural analyses shall identify and describe in detail important intramolecular and intermolecular interactions, hydrogen bond patterns, salt bridges, intra and intermolecular cysteine bridges, as well as ordered crystallographic waters. Regions of no electron density in critical regions of interest shall receive special attention if they could benefit from a new structure determination strategy.

4.5.9 A final project report that includes the atomic coordinates for crystal structures shall be delivered as electronic files in PDB format, and structural factors corresponding to

4.6 Generate a high-resolution crystal structure of the extracellular domain of the anti-CD19

CAR (47G4-CD828Z, referenced in Patent CA2951045A1) in complex with the extracellular domain CD19.

4.6.1 All work shall be carried out on the Contractor’s site or an affiliated site, using their own resources. These shall include:

a) Facilities to perform cloning, bacterial or eukaryotic protein expression, and protein purification including cold room capabilities to enable optimal protein purification and crystallization.

b) Access to automated chromatography systems to produce high purity protein suitable for crystallization studies.

c) Facilities equipped with specialized robotic technologies incorporated in an integrated workflow that enables protein crystallization and optimization.

d) In-house X-ray equipment for diffraction studies as well as dedicated access to a beamline at an internationally recognized laboratory with a Synchrotron light source.

4.6.2 The Contractor shall prepare a plan to perform structural determination of extracellular domain of the anti-CD19 CAR in complex with the extracellular domain CD19. The plan shall include the exact amino acid sequence of the extracellular domain of the anti-CD19 CAR and the extracellular domain of CD19. The project plan prepared by the Contractor shall include a cloning, expression and purification strategy for the extracellular domains of the anti-CD19 CAR and a cloning, expression and purification strategy for the extracellular domain of CD19. The cloning, expression and purification strategies shall take into consideration whether the domains are best expressed in eukaryotic or bacterial expression systems and the appropriate leader sequence(s) that can enable effective expression, secretion and purification. All protein purification shall proceed under optimal conditions, (4-degree C), to ensure native folding of the protein.

4.6.3 Upon approval of the plan by the NCI Project Officer, the Contractor shall proceed to cloning, expression and purification of the extracellular domains of the anti-CD19 CAR and the extracellular domain of CD19. The results of the small-scale protein purification(s) shall be provided to the NCI Project Officer to ensure that optimal protein isolation can be obtained before production of sufficient quantities of protein for crystallization studies. Results shall be communicated electronically to the NCI Project Officer as a brief written report.

4.6.4 Upon approval by the NCI Project Officer, the Contractor shall commence on expression and purification of the anti-CD19 CAR and the extracellular domain of CD19, that is sufficient for protein crystallization.

4.6.5 Initial protein crystallization shall be conducted using sparse matrix screening to help identify optimal crystallization conditions. This will ensure that multiple formulations can be tried, using different salts, polymers, organics, buffers, pH, to identify the best conditions for protein crystallization. X-ray diffraction of these crystals from the matrix screen shall help identify the appropriate conditions for crystal growth.

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0ahUKEwjPlqWtoLDaAhXKT98KHcNYADwQFggnMAA&url=https%3A%2F%2Fwww.google.com%2Fpatents%2FCA2951045A1%3Fcl%3Den&usg=AOvVaw1GKrxTn8iv6Jtst7A1fOlE

4.6.6 Once initial crystallization conditions are identified, the Contractor shall proceed to optimization of crystallization for structure determination. The protein crystals shall be used for X-ray diffraction studies using a micro-diffraction undulator synchrotron beamline.

4.6.7 A progress report on the project that includes any intermediary result shall be delivered as electronic files to the NCI Project Officer.

4.6.8 Once x-ray diffraction data has been collected, the Contractor shall determine structures using molecular replacement methods with appropriate search models available in the Protein Data Bank (PDB) which is the repository of 3D protein structures, or through de novo phasing methods. All structural analyses shall identify and describe in detail important intramolecular and intermolecular interactions, hydrogen bond patterns, salt bridges, intra and intermolecular cysteine bridges, as well as ordered crystallographic waters. Regions of no electron density in critical regions of interest shall receive special attention if they could benefit from a new structure determination strategy.

4.6.9 A final project report that includes the atomic coordinates for crystal structures shall be delivered as electronic files in PDB format, and structural factors corresponding to

4.7 Generate high-resolution crystal structures of the extracellular domain of anti-BCMA CAR alone and in complex with extracellular domain of BCMA, in which a specific part of a domain or linker, or sequence of a domain of the anti-BCMA CAR has been modified (insertion, deletion or change of amino acids), to become a “variant” (modified) protein, as specified by the NCI Project Officer. The variation will be determined after obtaining the results from Task 4.5 (to include the portion with or without a specific variant of the hinge region).

4.7.1 All work shall be carried out on the Contractor’s site or an affiliated site, using their own resources. These shall include:

a) Facilities to perform cloning, bacterial or eukaryotic protein expression, and protein purification including cold room capabilities to enable optimal protein purification and crystallization.

b) Access to automated chromatography systems to produce high purity protein suitable for crystallization studies.

c) Facilities equipped with specialized robotic technologies incorporated in an integrated workflow that enables protein crystallization and optimization.

d) In-house X-ray equipment for diffraction studies as well as dedicated access to a beamline at an internationally recognized laboratory with a Synchrotron light source.

4.7.2 The Contractor shall prepare a plan to perform structural determination of the extracellular domain of the specific variant of anti-BCMA CAR, and the amino acid sequence of the extracellular domain of BCMA. The plan shall include details on how the extracellular domain of the anti-BCMA CAR is being modified in the variant.

The project plan prepared by the Contractor shall include a cloning, expression and purification strategy for the extracellular domain of the variant anti-BCMA CAR and the cloning, expression and purification strategy for the extracellular domain of

BCMA. The cloning, expression and purification strategies shall take into consideration whether the domains are best expressed in eukaryotic or bacterial expression systems and the appropriate leader sequence(s) that can enable effective expression, secretion and purification. All protein purification shall proceed under conditions to ensure native folding of the protein domains and peptide complexes.

4.7.3 Upon approval of the plan by the NCI Project Officer, the Contractor shall proceed to cloning, expression and purification of the domains of the variant anti-BCMA CAR and the extracellular domain of BCMA. The results of the small-scale protein purification(s) shall be provided to the NCI Project Officer to ensure that optimal protein isolation can be obtained before production of sufficient quantities of protein for crystallization studies. Results shall be communicated electronically to the NCI Project Officer as a brief written report.

4.7.4 Upon approval by the NCI Project Officer, the Contractor shall commence on expression and purification of the variant anti-BCMA CAR and the extracellular domain of BCMA, that is sufficient for protein crystallization.

4.7.5 Initial protein crystallization shall be conducted using sparse matrix screening to help identify optimal crystallization conditions. This will ensure that multiple formulations can be tried, using different salts, polymers, organics, buffers, pH, to identify the best conditions for protein crystallization. X-ray diffraction of these crystals from the matrix screen shall help identify the appropriate conditions for crystal growth.

4.7.6 Once initial crystallization conditions are identified, the Contractor shall proceed to optimization of crystallization for structure determination. The protein crystals shall be used for X-ray diffraction studies using a micro-diffraction undulator synchrotron beamline.

4.7.7 A progress report on the project that includes any intermediary result shall be delivered as electronic files to the NCI Project Officer.

4.7.8 Once x-ray diffraction data has been collected, the Contractor shall determine structures using molecular replacement methods with appropriate search models available in the Protein Data Bank (PDB) which is the repository of 3D protein structures, or through de novo phasing methods. All structural analyses shall identify and describe in detail important intramolecular and intermolecular interactions, hydrogen bond patterns, salt bridges, intra and intermolecular cysteine bridges, as well as ordered crystallographic waters. Regions of no electron density in critical regions of interest shall receive special attention if they could benefit from a new structure determination strategy.

4.7.9 A final project report that includes the atomic coordinates for crystal structures shall be delivered as electronic files in PDB format, and structural factors corresponding to

4.8 Generate high-resolution crystal structures of the extracellular domain of anti-B-cell maturation antigen (BCMA) CAR both alone and in complex with the extracellular domain of BCMA, where the antigen binding domain in the anti-BCMA CAR (which is the scFv in Task 4.1), is replaced by a single Immunoglobulin domain, and there is an insertion or deletion in the hinge region as specified by the Project Officer.

4.8.1 All work shall be carried out on the Contractor’s site or an affiliated site, using their

a) Facilities to perform cloning, bacterial or eukaryotic protein expression, and protein purification including cold room capabilities to enable optimal protein purification and crystallization.

b) Access to automated chromatography systems to produce high purity protein suitable for crystallization studies.

c) Facilities equipped with specialized robotic technologies incorporated in an integrated workflow that enables protein crystallization and optimization.

d) In-house X-ray equipment for diffraction studies as well as dedicated access to a beamline at an internationally recognized laboratory with a Synchrotron light source.

4.8.2 The Contractor shall prepare a plan to perform structural determination of the extracellular domain of the specific variant of anti-BCMA CAR where the antigen binding domain in the anti-BCMA CAR is replaced by a single Immunoglobulin domain, and the amino acid sequence of the extracellular domain of BCMA. The plan shall include details on how the extracellular domain of the anti-BCMA CAR is being modified in the variant. The project plan prepared by the Contractor shall include a cloning, expression and purification strategy for the extracellular domain of the variant anti-BCMA CAR and the cloning, expression and purification strategy for the extracellular domain of BCMA. The cloning, expression and purification strategies shall take into consideration whether the domains are best expressed in eukaryotic or bacterial expression systems and the appropriate leader sequence(s) that can enable effective expression, secretion and purification. All protein purification shall proceed under conditions to ensure native folding of the protein domains and peptide complexes.

4.8.3 Upon approval of the plan by the NCI Project Officer, the Contractor shall proceed to cloning, expression and purification of the domains of the variant anti-BCMA CAR and the extracellular domain of BCMA. The results of the small-scale protein purification(s) shall be provided to the NCI Project Officer to ensure that optimal protein isolation can be obtained before production of sufficient quantities of protein for crystallization studies. Results shall be communicated electronically to the NCI Project Officer as a brief written report.

4.8.4 Upon approval by the NCI Project Officer, the Contractor shall commence on expression and purification of the variant anti-BCMA CAR and the extracellular domain of BCMA, that is sufficient for protein crystallization.

4.8.5 Initial protein crystallization shall be conducted using sparse matrix screening to help identify optimal crystallization conditions. This will ensure that multiple formulations can be tried, using different salts, polymers, organics, buffers, pH, to identify the best conditions for protein crystallization. X-ray diffraction of these crystals from the matrix screen shall help identify the appropriate conditions for crystal growth.

4.8.6 Once initial crystallization conditions are identified, the Contractor shall proceed to optimization of crystallization for structure determination. The protein crystals shall be used for X-ray diffraction studies using a micro-diffraction undulator synchrotron beamline.

4.8.7 A progress report on the project that includes any intermediary result shall be delivered as electronic files to the NCI Project Officer.

4.8.8 Once x-ray diffraction data has been collected, the Contractor shall determine structures using molecular replacement methods with appropriate search models available in the Protein Data Bank (PDB) which is the repository of 3D protein structures, or through de novo phasing methods. All structural analyses shall identify and describe in detail important intramolecular and intermolecular interactions, hydrogen bond patterns, salt bridges, intra and intermolecular cysteine bridges, as well as ordered crystallographic waters. Regions of no electron density in critical regions of interest shall receive special attention if they could benefit from a new structure determination strategy.

4.8.9 A final project report that includes the atomic coordinates for crystal structures shall be delivered as electronic files in PDB format, and structural factors corresponding to

5.0 PERIOD OF PERFORMANCE

The anticipated period of performance shall consist of a 12-month base period, plus one 12-month option period, from the date of award.

6.0 PLACE OF PERFORMANCE

All work shall be performed at the Contractor’s facility.

7.0 REPORT(S)/DELIVERABLES AND DELIVERY SCHEDULE

BASE PERIOD DELIVERABLES:

DELIVERABLE DELIVERABLE DESCRIPTION / FORMAT

REQUIREMENTS DUE DATE

#1

Task 4.1.2 and Task 4.2.2

Scientific project plan in Word or PDF format outlining step-by-step plan to perform structural determination of the extracellular domain of anti- BCMA CAR alone and in complex with the extracellular domain of BCMA. The plan shall be submitted to the NCI Project Officer. It shall include the exact amino acid sequence of the extracellular domains of the CAR protein and the amino acid sequence of the extracellular domain of BCMA. The plan shall also include a cloning, expression and purification strategies.

Scientific project plan in Word or PDF format outlining step-by-step plan to perform structural determination of the extracellular domain of anti- CD19 CAR. The plan shall be submitted to the NCI Project Officer. It shall include the exact amino acid

30 days after award sequence of the extracellular domain and shall include a cloning, expression and purification strategies.

#2

Task 4.1.3 and Task 4.2.3

Electronically transmitted brief written report summarizing results of the small-scale protein purification(s) of the extracellular domains for anti- BCMA CAR and BCMA and recommendation to proceed or plan for trouble-shooting if unsuccessful.

Electronically transmitted brief written report summarizing results of the small-scale protein purification(s) of the extracellular domains for anti- CD19 CAR and recommendation to proceed or plan for trouble-shooting if unsuccessful.

90 days after award

#3

Task 4.1.7 and Task 4.2.7

Progress reports on tasks that include any intermediary results shall be delivered as electronic files to the NCI Project Officer.

180 days after award

#4

Task 4.1.9 and Task 4.2.9

Final reports shall be delivered to the Project Officer.

Final report or structural determination of the extracellular domain of anti-BCMA CAR alone and in complex with the extracellular domain of BCMA shall include the atomic coordinates for the crystal structures delivered as electronic files in PDB format, and structural factors corresponding to atomic coordinates shall be delivered as electronic files in MTZ format. Reports shall also include details of the structural analysis performed in 4.1.8.

Final report for structural determination of the extracellular domain of anti-CD19 CAR shall include the atomic coordinates for the crystal structures delivered as electronic files in PDB format, and structural factors corresponding to atomic coordinates shall be delivered as electronic files in MTZ format. Reports shall also include details of the structural analysis performed in 4.2.8.

completion of Task 4.1.8 and 4.2.8, not to exceed 12 months from date of award.

#5

Task 4.3.2 and Task 4.4.2 determination of a specific variant of the extracellular domain of anti-BCMA CAR alone and in complex with the extracellular domain of BCMA.

The plan shall be submitted to the NCI Project Officer. It shall include the exact amino acid sequence of the variant extracellular domain of the CAR protein and the amino acid sequence of the

120 days after award extracellular domain of BCMA. The plan shall also strategies.

Scientific project plan in Word or PDF format outlining step-by-step plan to perform structural determination of a specific variant of the extracellular domain of anti-BCMA CAR in which there is a three residue AAA insertion between the last strand of the scFv and the beginning of the hinge. The plan shall be submitted to the NCI Project Officer. It shall include the exact amino acid sequence of the extracellular domain and shall include a cloning, expression and purification strategies.

#6

Task 4.3.3 and Task 4.4.3

Electronically transmitted brief written report summarizing results of the small-scale protein purifications of the specific variant of the extracellular domain of anti-BCMA CAR and the extracellular domain of BCMA, and recommendation to proceed or plan for trouble-shooting if unsuccessful.

Electronically transmitted brief written report summarizing results of the small-scale protein purification of the AAA-inserted variant of the extracellular domain of the anti-BCMA CAR and recommendation to proceed or plan for trouble-shooting if unsuccessful.

180 days after award

#7

Task 4.3.7 and Task 4.4.7

Progress reports on both tasks that includes any files to the NCI Project Officer.

270 days after award

#8

Task 4.3.9 and Task 4.4.9

Final reports shall be delivered to the Project Officer.

Final report on structural determination of the specific variant of the extracellular domain of anti- BCMA CAR alone and in complex with the extracellular domain of BCMA, shall include the atomic coordinates for the crystal structures delivered as electronic files in PDB format, and structural factors corresponding to atomic coordinates shall be delivered as electronic files in MTZ format. Reports shall also include details of the structural analysis performed in 4.3.8.

Final report for structural determination of the AAA-inserted variant of the extracellular domain of the

30 days after completion of Task 4.3.8 and 4.4.8, not to exceed 12 months from date of award.

anti-BCMA CAR shall include the atomic coordinates for the crystal structures delivered as electronic files in PDB format, and structural factors corresponding to atomic coordinates shall be delivered as electronic files in MTZ format. Reports shall also include details of the structural analysis performed in 4.4.8.

OPTION PERIOD 1 DELIVERABLES:

DELIVERABLE DELIVERABLE DESCRIPTION / FORMAT

REQUIREMENTS DUE DATE

#1

Task 4.5.2 and Task 4.6.2…