Att 1 - Nanobody Affinity Maturation SOW 071825.pdf

Attachment 1 STATEMENT OF WORK (SOW)

Nanobody Affinity Maturation Using Phage Display Selection

Independently and not as an agent of the Government, the Contractor shall furnish all necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the

Government, as needed to perform the Statement of Work.

1.0 BACKGROUND

The National Cancer Institute/Center for Cancer Research (NCI/CCR), Molecular Imaging Branch

(MIB) is engaged in the development of radiolabeled nanobodies targeting clinically relevant tumor antigens for diagnostic and therapeutic applications. One of the current programs focuses on targeting B-cell maturation antigen (BCMA) in multiple myeloma. A parental anti-human BCMA nanobody was purchased and demonstrated promising preliminary binding in vitro and in vivo, but further affinity enhancement is required to support translational applications. The goal is to generate high affinity nanobody variants (based on the parental one), suitable for imaging and radiotherapy through affinity maturation using phage display.

2.0 OBJECTIVE

The Contractor shall perform targeted affinity maturation of an anti-human BCMA nanobody via phage display library construction and selection. The objective is to deliver five affinity-optimized nanobody sequences along with sufficient purified protein for in vitro and in vivo testing.

3.0 SCOPE

The Contractor shall perform nanobody affinity maturation using established phage display techniques. The project will be based on nanobody sequence provided by the Government and will encompass a series of integrated activities aimed at enhancing binding affinity to the BCMA target antigen. The Contractor will initiate the work by producing recombinant nanobody protein in a mammalian expression system to confirm expression and proper folding. The binding activity of the parental nanobody to BCMA will be evaluated using immunoassays such as ELISA, followed by quantitative affinity measurements using validated platforms (e.g., BLI or SPR).

Subsequently, the parental nanobody will be cloned into a suitable phagemid vector for display and functional validation. Mutant libraries will be constructed through in silico-guided design, utilizing error-prone PCR and semi-rational mutagenesis to introduce diversity across relevant regions of the nanobody.

Affinity selection will be conducted through multiple rounds of biopanning against soluble BCMA antigen to enrich for variants with improved binding characteristics. Candidate clones from enriched libraries will be screened via ELISA or equivalent binding assays, and selected high-affinity binders will be sequenced and analyzed.

Top-performing nanobody variants will be recombinantly expressed in mammalian cells, purified, and subjected to standard quality control procedures. Binding affinities of the matured clones will be re-evaluated to confirm improvements over the parental nanobody.

Up to five optimized nanobody clones will be delivered to the Government, including the corresponding amino acid and nucleotide sequences. These five clones will be provided to the MIB for downstream evaluation. At least one of the five delivered clones must demonstrate a minimum

10-fold improvement in binding affinity relative to the parental nanobody.

The Government shall not exercise any supervision or control over the contract service providers performing the services herein. Such contract service providers shall be accountable solely to the

Contractor who, in turn is responsible to the Government. The Contractor shall provide all personnel, labor, facilities, materials and equipment necessary to generate the synthesized compounds. All development and production will be performed off-site.

4.0 CONTRACT REQUIREMENTS / PERSONNEL QUALIFICATIONS

The Contractor must have demonstrated expertise in nanobody engineering, phage display library construction, and affinity maturation, with a proven track record in delivering affinity-optimized nanobody reagents for therapeutic or diagnostic applications. The Contractor shall perform nanobody affinity maturation using phage display methods, based on sequences provided by the Government.

The Contractor shall perform the following tasks:

1. Parent VHH Recombinant Production

Based on the Government provided parental nanobody sequence, the Contractor shall produce 1 mg of purified VHH protein using mammalian cells (293 or CHO).

2. Parent VHH ELISA Validation

Evaluate the binding activity of the recombinant VHH to the target BCMA. The

Government-supplied VHH protein will serve as a control.

3. Parent VHH Binding Affinity Measurement

Measure the binding affinity of the parent nanobody to the BCMA antigen using Octet BLI or Biacore SPR. The results shall be shared with the Government for decision-making prior to continuing.

4. Parent Phage VHH Construction and Binding Test

Clone the parent VHH into a phagemid vector for phage display and validate for target binding.

5. Design and Construction of Mutant VHH Phage Display Libraries

Based on in silico analysis of the parent VHH, construct multiple mutant libraries (VHmut-

VLwt, VHwt-VLmut, VHmut-VLmut, and hotspot library) using error-prone PCR and semi-rational design approaches.

6. Biopanning Against Target Antigen Using Mutant Libraries

Perform three to four rounds of in-solution panning using soluble BCMA antigen to enrich nanobody variants with increased affinity.

7. Phage ELISA and Validation

Evaluate up to 300 phage clones from the enriched pool. Clones with superior binding will be selected, sequenced, and analyzed.

8. Recombinant Production of Selected Nanobodies

Use mammalian systems to express the five top-ranked affinity-matured nanobody clones.

Each will be produced at a scale of 2 mg, QC-tested by SDS-PAGE, and delivered to the

Government.

9. Affinity Measurement of Matured Nanobodies

Meausre the binding affinities of the five purified clones using Octet BLI or Biacore SPR to determine improved KD values.

10. Milestone 2 - Deliver up to 5 new clones (2mg from each), with at least 1 clone showing a

10-fold improvement in affinity.

11. Internalization Evaluation - Evaluate internalization performance on target

Cells, and confirm the new nanobody sequences have better internalization than the parent nanobody used for the affinity maturation.

5.0 TYPE OF ORDER

This is a Firm Fixed Price (FFP) purchase order.

6.0 PERIOD OF PERFORMANCE

The period of performance shall be for six (6) months from the date of award.

7.0 PLACE OF PERFORMANCE

Work shall be performed at the Contractor’s facility.

8.0 REPORT(S)/DELIVERABLES AND DELIVERY SCHEDULE

The NCI shall review the deliverables submitted by the Contractor for acceptance within 14 days of receipt and approve the submitted deliverables. At the completion of the review, the NCI’s technical point of contact shall notify the Contractor as to the acceptability of the deliverable. If any changes or revisions are required of the Contractor, the revised deliverables shall be submitted to NCI within 5 days of the date of notification. If no comments or request for revisions are provided within 14 business days, the deliverables will be considered acceptable. In general, NCI’s technical point of contact will provide technical scientific review and approval of all deliverables submitted by the

Contractor.

Deliverable Description Due Date Delivery Location

#1 Five (5) new clones

The newly generated nanobody sequences must be distinct from the parental sequence, with at least one variant demonstrating a minimum

10-fold improvement in binding affinity. All selected clones should exhibit low nanomolar affinities, comparable to or exceeding that of the parental nanobody, and

Due within thirty calendar (30) days prior to expiration date

Central Point of Distribution:

ncibranchbinvoices@mail.nih.gov

Contracting Officer (CO):

michelle.nguyen2@nih.gov

Contracting Officer’s

Representative (COR) & SME:

pchoyke@mail.nih.gov orit.jacobsonweiss@nih.gov mailto:ncibranchbinvoices@mail.nih.gov mailto:michelle.nguyen2@nih.gov mailto:pchoyke@mail.nih.gov mailto:orit.jacobsonweiss@nih.gov achieve internalization rates greater than 60%.

#2 Purified

Protein

Each of the five newly generated nanobodies must be provided at a quantity of 2 mg in PBS, with a minimum purity of

>95%.

Due within thirty calendar (30) days prior to expiration date pchoyke@mail.nih.gov orit.jacobsonweiss@nih.gov

#3 Experimental

Report

The report shall include quality control data, purity assessment, mass spectrometry results, binding assay data and analysis, as well as the full amino acid and nucleotide sequences of the five newly generated nanobodies.

Due within thirty calendar (30) days prior to expiration date pchoyke@mail.nih.gov orit.jacobsonweiss@nih.gov

9.0 PAYMENT

Payment shall be made with a purchase order. Payment authorization requires submission and approval of invoices to the COR and NIH OFM, in accordance with the attached payment provisions listed below:

The following clause is applicable to all Purchase Orders, Task or Delivery Orders, and Blanket

Purchase Agreement (BPA) Calls: PROMPT PAYMENT (JAN 2017) FAR 52.232-25.

mailto:ncibranchbinvoices@mail.nih.gov mailto:michelle.nguyen2@nih.gov mailto:pchoyke@mail.nih.gov mailto:orit.jacobsonweiss@nih.gov mailto:ncibranchbinvoices@mail.nih.gov mailto:michelle.nguyen2@nih.gov mailto:pchoyke@mail.nih.gov mailto:orit.jacobsonweiss@nih.gov