SAM #75N98026Q01031

NIH Solicitation for Nanobody Development Against NPR-C for Growth Disorder Therapeutics

The National Institutes of Health (NIH) is seeking qualified vendors to develop nanobodies (VHH) against human NPR-C for research and potential therapeutic use in growth disorders such as achondroplasia. - Government Buyer: - National Institutes of Health (NIH), Office of Logistics and Acquisition Operations (OLAO) - Scope of Work: - Generation of antigens (including tagged human NPR-C, mouse NPR-C, human NPR2, human IgG1 Fc protein, and human CNP) - Immunization of alpaca/llama - Creation and screening of phage/yeast libraries or single B-cell screening - Functional confirmation of nanobody binding - Production and characterization of VHH-Fc candidates - Research applications: Western blot, immunohistochemistry, ELISA - Animal testing and therapeutic lead generation - Deliverables: - At least 10 unique VHH sequences (purified antibodies, 1mg/antibody) - Serum titer reports, sequence reports, characterization data, and final summary report - Unique Requirements: - Vendor must provide animals, reagents, equipment, and protocols - NIH retains all intellectual property - Compliance with IACUC-equivalent animal welfare standards - Provision of Certificates of Analysis - Avoidance of cross-reactivity with alpaca/llama NPR-C and NPR-B - Explicit exclusion of humanization, bispecific engineering, GMP manufacturing, and cell line development from base scope - No specific OEMs or commercial vendors are named; opportunity is open to qualified providers

Description

See attached Word document.

Project Title: Nanobody against NPR-C for growth disorder

1. Project Overview 1.1 Background We sought to develop nanobodies (VHH) against human NPR-C with high affinity and specificity for future clinical use. These nanobodies could potentially be further developed into therapeutics for treatment of growth disorders such as achondroplasia.   1.2 Main Objectives generation of antigens needed for immunization immunization of Alpaca/Ilama with target antigens generated generation of immunized phage/yeast library and in vitro screening using the library alternatively, single B-cell screening and sequencing instead of phage/yeast library generation/screening. downstream functional confirmation of VHH nanobodies binding to target antigens generation of final VHH-Fc candidates for further validation 1.3 Intended Use of Antibodies Research (Western blot, immunohistochemistry, ELISA)  Animal testing (subcutaneous injection in mice)  Therapeutic lead generation (in combination with other antibody domains, Fc, etc) 

2. Target / Antigen Information Field Detail Target name/ID Human NPR-C (P17342 · ANPRC_HUMAN) Species reactivity required Human / Mouse / Rat / Cross-reactive Target type secreted / extracellular domain Antigens to be provided (amount needed: 0.5-2mg) Human NPR-C, Fc tag (immunization) Human NPR-C, His tag (immunization) Biotinylated human NPR-C, His tag (panning) Mouse NPR-C, His tag (immunization) Human NPR2, mFc tag (counter screening) Human IgG1 Fc Protein, His-Avi Tag (counter screening) Human CNP (blocking screening) Known structural considerations Avoid cross-reactivity with Alpaca/Llama NPR-C and NPR-B

3. Scope of Work Development Platform Track 1: Phage display or Yeast display using immunized library Track 2: Single B-cell cloning 3.2.1 Workstream Breakdown for Track 1 (phage/yeast display)  Phase 1 — Antigen Generation (less than 1 month)  Antigens listed in section 2 to be generated and validated for purity before proceeding to immunization.  Phase 2 — Immunization / Library Generation (1-2 months)  1-2 naïve Alpaca/Llama to be immunized.  Alpaca/Llama shall be immunized four times with antigen over six to eight weeks. Up to two extra boosts shall be performed (preferably at no cost) if the serum titer is below the level required (1:15000) for phage display library construction.  Anti-serum titer to be performed with ELISA against human and mouse NPR-C-His  Peripheral blood mononuclear cell (PBMC) to be collected for library construction after immunization and confirmed serum titer  Phase 3 — Library Construction (up to 1 month)  PBMC collected will be used for library construction  VHH genes to be PCR amplified and cloned into phage or yeast display vector for library construction  A phage/yeast display library with a size of 1x108 or greater is required to proceed to the next step  50 colonies shall be selected by random to check for insertion, an insertion rate of 95% or greater, and a diversity of 90% or greater, is required to proceed to screening  Phase 4 — Screening and Sequencing (1-2 months)  3 rounds of screening for human NPR-C are required  Counter screening should be performed against human NPR-B  Counter-screening (e.g., against homologous proteins, for specificity)  Individual phage clones shall be confirmed using phage ELISA against human and mouse NPR-C  Background ELISA screenings shall be carried out with Human Fc  Positive clones will be sequenced, and sequence diversity analysis shall be performed.  Expected number of positive clones: 20-100 unique clones   Phase 5 — Expression and Purification (less than 1 month)  At least 10 confirmed binders chosen based on sequencing and ELISA shall be produced with human Fc tag (hereafter VHH-Fc) in CHO/Expi293, or comparable mammalian cells.  VHH-Fc shall be purified through Protein A affinity chromatography. SDS-PAGE and SEC-HPLC shall be used as QC for purification. Purified samples are subject to further characterization   Phase 6 — VHH-Fc characterization (less than 1 month)  ELISA shall be performed to confirm dose-dependent binding activity of purified VHH-Fc hits to human and mouse NPR-C protein.  Counter screening to NPR-B by dose-curve ELISA shall be performed.  Ability of VHH-Fc to block CNP binding to NPR-C shall be performed by dose-dependent ELISA blocking assay.  SPR shall be used to evaluate the binding affinity of VHH-Fc to human NPR-C-His  Full KD detection of selected VHH-human Fc antibodies via Biacore (or comparable).   Phase 7 — Project Completion (total expected time: 4-6 months)  At least 10 (ten) unique sequences of VHH antibodies, purified antibodies (1mg/antibody) and plasmids.   A unique sequence is defined as one that has at least 1 unique amino acid in the three CDR regions relative to the sequences of the other clones.  Final project report (sequence report, affinity, ELISA binding results, etc) 

3.2.2 Workstream Breakdown for Track 2 (Single B-cell cloning)  Phase 1 — Antigen Generation (less than 1 month)  Identical to track 1  Phase 2 — Immunization (1-2 months)  Identical to track 1  Phase 3 — Single B-Cell Isolation and Screening (up to 1-2 months)  Antigen-specific B cells shall be isolated from PBMCs/lymphoid tissue via fluorescence-activated cell sorting (FACS) using labeled human NPR-C antigen as bait, or an equivalent single-cell isolation method proposed by the vendor.  Vendor shall report total B cells screened and number of antigen-specific single cells recovered.  Counter-screening against human NPR-B shall be performed at the single-cell stage where the vendor's platform allows, to minimize downstream cross-reactive candidates.  At least 100 antigen-specific single cells are expected to be carried forward for sequencing  Phase 4 — Sequence Recovery and Analysis (up to 1 month)  VHH variable region genes shall be recovered from each confirmed antigen-specific single cell via RT-PCR and sequenced.  Sequence diversity analysis shall be performed to identify unique clones (per the uniqueness definition in Phase 7).  Sequence liability screening (e.g., deamidation, glycosylation, unpaired cysteines) shall be reported for candidate sequences.  Phase 5 — Expression and Purification (less than 1 month)  Identical to Track 1  Phase 6 — VHH-Fc Characterization (less than 1 month)  Identical to Track 1  Phase 7 — Project Completion (total expected time: 4-6 months)  Identical to Track 1 

3.3 Explicitly Out of Scope The following activities are not included in this SOW. Vendors should not include costs for these items in their base proposal but may list them as optional add-ons with separate pricing if desired:  Humanization or affinity maturation of VHH candidates  Bispecific or multi-domain antibody engineering (e.g., fusion to additional Fc domains or antibody formats beyond VHH-Fc)   GMP or GMP-like manufacturing of any antibody material  Cell line development for stable production (beyond transient expression for characterization) 

4. Deliverables # Deliverable Format Acceptance Criteria 1 Serum titer report PDF/Excel titer curves, EC50 values, etc 2 Sequence report PDF + FASTA VHH sequences, annotated CDRs, etc 3 Purified antibody 1 mg, liquid/lyophilized Purity ≥95% by SEC, endotoxin <5 EU/mgExpected affinity for human NPR-C: KD=10nM, or better 4 Characterization data PDF/Excel ELISA, SPR, Biacore data, etc 5 Final summary report PDF Recommendation of 10 lead candidates with data supported rationale

5. Timeline Milestone Target Completion Phase 1: Antigen Generation 2-4 weeks Phase 2: Alpaca Immunization 6-8 weeks Phase 3: Library Generation (track 1), or Single B-Cell Screening (track 2) 2-3 weeks (track 1)   4-6 weeks (track 2)  Phase 4: Phage Library Screening (track 1), or Single B-Cell Sequencing (track 2) 4-6 weeks(track 1)   2-3 weeks (track 2)  Phase 5: VHH-Fc Production 2 weeks Phase 6: Candidate Characterization 2 weeks Project Completion: Total expected time: 4-6 months

6. Materials and Responsibilities 6.1 Provided by Client Reference antibodies, if needed.  6.2 Provided by Vendor Animals, reagents, equipment, protocol  6.3 Intellectual Property The NIH retains all IP ownership of resulting antibodies, sequences, and data. 

7. Quality and Compliance Requirements Animal work must comply with IACUC-equivalent standards; vendor to provide accreditation (e.g., AAALAC)  Data traceability / lab notebook standards  Certificate of Analysis (CoA) required for final deliverables 

8. Pricing Structure Requested Phase Description Payout Total Payout Phase 1 Antigen Generation 10% 10% Phase 2 Alpaca Immunization 20% 30% Phase 3 Phage Library Generation / Single B-Cell Screening 20% 50% Phase 4 Phage Library Screening / Single B-Cell Sequencing 25% 75% Phase 5 VHH-Fc Production 5% 80% Phase 6 Candidate Characterization 10% 90% Phase 7 Project Completion 10% 100%

9. Evaluation Criteria Criterion Weight Technical approach and platform justification 40% Timeline 20% Price 20% Relevant experience / past performance with similar targets 15% Quality certifications 5%

10. Proposal Submission Requirements Vendor response should include:  Project proposal detailing approach narrative (platform choice, screening strategy, risk mitigation for difficult targets, etc)  Proof of Team qualifications / expertise   Detailed timeline with milestones  Itemized pricing  Quality/compliance certifications (if applicable)  Submission format: PDF or microsoft word  Submission method: Email  Q&A Deadline: 8/24/2026  Deadline:  8/28/2026

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