Methods of making active antibodies from biological fluids
Inventors
SAJADI, Mohammad • DEVICO, Anthony • Lewis, George
Assignees
US Department of Veterans Affairs
Publication Number
US-11067583-B2
Publication Date
2021-07-20
Expiration Date
2037-05-25
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Abstract
The present invention provides a method of making an antibody by identifying a circulating antibody with activity from a subject comprising i) subjecting biological fluid selected from the group consisting of blood, plasma and serum and combinations thereof from the subject to one or more rounds of affinity chromatography to purify the circulating antibody; ii) optionally further subjecting the circulating antibody to isoelectric focusing to purify the circulating antibody based on charge; iii) testing the purified circulating antibody for activity; iv) digesting the purified circulating antibody from parts i) or ii) to create an antibody fragment; v) subjecting the antibody fragment to mass spectrometry to generate a mass assignment and a deduced amino acid sequence of the antibody fragment; vi) comparing the deduced amino acid sequence with an amino acid sequence of an antibody generated from the subject's B-cells to identify an antibody sequence that matches the deduced amino acid sequence; vii) generating an antibody comprising light chain and heavy chain CDR sequences of the B-cell antibody that matches the deducted amino acid sequence of party vi); and viii) testing the antibody of part vii) for activity.
Core Innovation
The invention provides a method of making an antibody by identifying a circulating antibody with activity from a subject. The process includes subjecting biological fluid from the subject to affinity chromatography to purify circulating antibodies, optionally further purifying the antibody by isoelectric focusing based on charge, testing for activity, digesting to create antibody fragments, and using mass spectrometry to deduce amino acid sequences of these fragments. These sequences are then compared with antibody sequences generated from the subject’s B-cells to identify matching antibodies, followed by generating antibodies with the matching light and heavy chain complementarity determining regions (CDRs) and testing these antibodies for activity.
The problem addressed is the limited understanding and efficient identification of circulating, active, polyclonal antibody responses in infectious diseases, particularly HIV. Current methods rely heavily on memory B cell-derived antibodies, which may not correspond to circulating antibodies or may not exist at functional levels, limiting the capacity to fully define circulating neutralizing antibodies contributing to viral inhibition. Furthermore, existing studies of proteomics-based deconvolution of circulating antibodies face challenges in accurately reflecting authentic heavy and light chain pairings.
Claims Coverage
The patent includes two independent claims covering methods related to identifying and making antibodies from circulating biological fluids. They focus on steps involving purification, sequencing, and generating antibodies matching sequences from B-cells of the subject.
Method of making an antibody by identifying circulating antibody with activity
A method comprising: (1) purifying circulating antibody from subject's biological fluid via affinity chromatography; (2) optionally using isoelectric focusing for further purification based on charge; (3) testing purified antibody for activity; (4) digesting antibody to create antibody fragments; (5) performing mass spectrometry on fragments to deduce amino acid sequences; (6) comparing these sequences to those derived from subject's B-cells to identify matches; (7) generating an antibody incorporating light and heavy chain CDRs matching the deduced sequences; and (8) testing the generated antibody for activity. The method specifies that the circulating antibody binds HIV antigens such as gp120, p24, and others, and that B-cells have specific phenotypes including CD38hi and variations of CD19 and CD138.
Method of identifying a circulating antibody with activity from a subject
A method comprising: (1) purifying circulating antibody from the subject's biological fluid by affinity chromatography; (2) optionally further purifying by isoelectric focusing based on charge; (3) testing purified antibody for activity; (4) digesting the purified antibody into fragments; (5) subjecting the fragments to mass spectrometry to obtain amino acid sequences; and (6) comparing these sequences with antibody sequences generated from the subject's B-cells to identify a matching antibody sequence. This claim also involves circulating antibodies binding HIV antigens and B-cells having phenotypes including CD38hi and variable CD19 and CD138 expression.
The patent claims encompass detailed methods for purifying circulating antibodies from biological fluids, characterizing their sequences via mass spectrometry, matching these sequences to antibody sequences from the subject’s B-cells, and using the matched sequences to generate and test new antibodies. The claims specify the types of antibodies, antigens targeted, and phenotypes of source B-cells, reflecting a comprehensive approach to derive active antibodies, particularly related to HIV antigens.
Stated Advantages
Broadly neutralizing antibodies are functional and persistent in plasma despite representing a minor fraction of total antibodies.
The method permits accurate identification and reconstruction of authentic heavy and light chain pairings of circulating antibodies.
The technique enables the discovery and production of antibodies that correspond to circulating functional antibodies, overcoming limitations of memory B cell-derived antibody methods.
Documented Applications
Treatment or prevention of HIV infection by administering antibodies generated by the described methods.
Development of therapeutics to treat and prevent infectious diseases using antibodies identified from circulating plasma antibodies.
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