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Production of antibody therapeutic fragments by reduced genome E. coli in continuous culture

Award Number: R44AI155307
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 07/13/2020
PERIOD OF PERFORMANCE END DATE: 02/29/2024

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Production of antibody therapeutic fragments by reduced genome E. coli in continuous culture - Scarab Genomics’ C-Flow™ technology is a highly efficient continuous culture system that can produce kilograms of protein in a few weeks at a mere 10-liter scale. This project will determine whether C-Flow can be further developed as an efficient system for production of single-chain antibody therapeutics. Antibody fragments, especially single-chain variants, are increasingly important for diagnostic and therapeutic use such as toxin and virus neutralization, being relatively easy to manufacture in E. coli, albeit very inefficiently in current techniques. They are of great potential significance as therapies for outbreaks and biothreats, with notable success against Ebola in West Africa. Other antibodies are remarkably successful for e.g. treatment of cancer and autoimmune diseases. These molecules have a variety of structures and indications but severe problems in manufacturing. Lacking glycosylation, antibody fragments are short-lived in vivo, requiring multiple high doses, leading to prohibitively expensive drugs. Therefore, a method of production that is fast, efficient, and low in cost is critically needed. This project will optimize C-Flow production of three structurally distinct single chain fragment antibodies, to evaluate the general applicability of the approach. The goal is high expression levels and sustained production on an unprecedented scale with small-footprint equipment. Correct folding of antibody fragments is critical for function. E. coli mechanisms for expression and delivery into the periplasm, where protein folding occurs, include chaperones, signal sequences, and codon usage and distribution. These mechanisms will be optimized for all three antibody fragments. Computer predictions have the power to reveal folding defects that could be corrected by genetic engineering to replace residues or regions that do not fold well. By testing an example immunotoxin that does not express strongly, the possibility of using software predictions for rational antibody design for increased manufacturability will be explored. Engineered changes suggested by computer predictions will be implemented and evaluated. Such an integrated system of software and production technology could provide a rapid response to a bio-emergency, going from outbreak to therapeutic ready for clinical testing in weeks. The Specific Aims are: 1. Optimize production of a structurally simple antibody fragment, scFv-SG1, at 1-liter then 10-liter C-Flow scales, extending the latter to at least 30 days to evaluate continuous antibody production. 2. Optimize production of a more structurally complex antibody fragment, scFabYMF10 as per Aim 1, purify the antibody and test its function by evaluating scFabYMF10 binding to its target antigen. 3. Enhance expression of the antibody-toxin conjugate B3Fv-PE40 (poorly expressed in E. coli) using physiological and genetic engineering approaches. Determine whether up-front software-assisted design of the protein can be used to predict improved manufacturability.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = $0 )
2024	2022	SCARAB GENOMICS LLC	1202 ANN ST	MADISON	WI	53713	DANE	USA	Allergy and Infectious Diseases Research	000	3	9/18/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2022 ( Subtotal = $961,744 )
2022	2022	SCARAB GENOMICS LLC	1202 ANN ST	MADISON	WI	53713	DANE	USA	Allergy and Infectious Diseases Research	000	3	6/17/2022	NON-COMPETING CONTINUATION	$961,744
														Subtotal = $961,744

Issue Date FY: 2021 ( Subtotal = $944,508 )
2021	2021	SCARAB GENOMICS, LLC	1202 ANN ST	MADISON	WI	53713	DANE	USA	Allergy and Infectious Diseases Research	000	2	6/7/2021	NON-COMPETING CONTINUATION	$944,508
														Subtotal = $944,508

Issue Date FY: 2020 ( Subtotal = $1,000,000 )
2020	2020	SCARAB GENOMICS, LLC	1202 ANN ST	MADISON	WI	53713	DANE	USA	Allergy and Infectious Diseases Research	000	1	7/13/2020	NEW	$1,000,000
														Subtotal = $1,000,000

Grand Total All Awards = $2,906,252

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Production of antibody therapeutic fragments by reduced genome E. coli in continuous culture

Award Number: R44AI155307

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 07/13/2020

PERIOD OF PERFORMANCE END DATE: 02/29/2024

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