Nanobody against NPR-C for growth disorder

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 na�ve 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/mg Expected 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