NIHLM2015582_STATEMENT_OF_WORK.pdf

STATEMENT OF WORK

(07-14-2015)

Purchase Order Title Generation and sequencing of llama monoclonal antibodies against the E. coli BamA protein.

1.0 Background Information

Unlike most mammalian antibodies, camelid monoclonal antibodies, called vHHs or nanobodies, generated in llama, camel or alpaca consist of a single heavy chain. In recent years, nanobodies have been increasingly used in structural studies and in biotechnological and pharmaceutical applications. Nanobodies are small in size and can access novel and difficult-to-reach protein epitopes that cannot be accessed by traditional antibodies. Access to smaller epitopes often leads to receptor/active-site neutralization and enzyme inhibition. A lot of research published to date demonstrates remarkable results using nanobodies against flexible multi-domain proteins, large protein complexes and membrane proteins. While the results and applications have been exceptional, the generation of nanobodies is still rather expensive.

2.0 Purpose and Objectives for the Procurement

Unlike conventional antibodies, the very nature of nanobodies makes them ideal for expression in bacteria. In our case, we intend to generate a nanobody against an essential component of a multi-protein outer membrane complex in E. coli called the Bam (barrel assembly machinery) complex. This complex is essential for the biogenesis of outer membrane proteins and hence is critical for cell viability. As an essential factor the Bam complex is a potential target for the development of novel antibiotics. To date, no simple and reliable method of inactivating the Bam complex and studying the subsequent effects on cellular processes has been developed. We would like to generate a nanobody that binds effectively to a key component of the Bam complex called BamA in vivo and inactivates it. For this purpose, we need the services of a commercial vendor who specializes in llama nanobody production. We will supply the purified antigen (BamA) to the vendor who will use it to immunize a llama, generate the nanobodies, and perform screening and sequencing of the best nanobodies. At the end of the contract period, they will deliver the gene sequences of the best nanobodies against BamA to us. The gene encoding the nanobody will be cloned into an appropriate plasmid and expressed with an N-terminal signal sequence in E. coli in our laboratory.

3.0 Contractor Requirements

The vendor is expected to have a well-established track record in llama nanobody production and commercial market presence with state-of-the-art inspected llama facilities and research laboratories. Initially, our laboratory at NIH will generate the antigen in the form of purified BamA protein. We will supply the antigen to the contracting vendor, who will carry out the subsequent steps. They will use the antigen supplied by us to immunize a naïve llama at their animal facilities. Once the llama generates a stable repertoire of antibodies against BamA, lymphocytes will be collected, the mRNAs for the nanobodies will be extracted and a phage-display vector based cDNA library will be generated by synthesizing cDNA from mRNA followed by PCR amplification and cloning into a standard phage display vector. The generated library of phages will display a different nanobody for each phage. The best binding nanobody clones will be screened from the phage-display library by multiple rounds of panning using an ELISA based screening technique. Only the phages displaying specific nanobodies that bind to BamA with high efficiency will be isolated and the nanobody DNA sequenced from the corresponding vector. The vendor will finally deliver the gene sequences of multiple unique nanobody clones to us in a suitable cloning plasmid and we will perform the subsequent steps of sub-cloning and expression in our laboratory.

4.0 Period of Performance

The entire process from llama immunization to sequencing of the best nanobody clones will be performed by the vendor over a time frame of approximately 4-6 months in their own facilities.

Once we receive the DNA sequences from the vendor, the sub-cloning and expression of the nanobodies in E.coli will be done in our laboratory indefinitely depending on the experimental application.

5.0 Government Responsibilities

We will clone, express and purify BamA from E.coli in our laboratory and provide the purified protein to the vendor according to their requirements for immunization and ELISA based screening. The vendor will carry out all the subsequent steps from llama immunization to nanobody screening and sequencing in their own laboratories and facilities. They will provide weekly or bi-weekly progress reports on the project as the immune response in llama progresses.

We will monitor the immune response and decide at what phase of post-immunization, the blood cells (lymphocytes) from the llama will be isolated for the subsequent nanobody phage display library generation. Additionally, we will decide how many nanobody clones we will need sequenced and cloned from the vendor based on the screening results from the ELISA.

6.0 Reporting Requirements and Deliverables

Below is an approximate timeframe of events/phases of the entire project to be carried out by the vendor and will change depending on the vendor. However, the vendor is expected to report back to us at weekly or bi-weekly intervals depending on the phase of events. The commencement of the next phase will rely solely on whether the previous phase was a success and that decision will rely solely with our laboratory.

The deliverables will consist of nanobody gene sequences in a vendor-supplied cloning vector.

7.0 Attachments

An illustrated overview of the llama nanobody generation, screening and sequencing process is attached below.