High Affinity Humanized Antibodies without Making Hybridomas; Immunization Paired with Mammalian Cell Display and <em>In Vitro</em> Somatic Hypermutation

<div><p>A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID). Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.</p> </div

Discovery and affinity maturation of C5-C345C-specific antibodies.

<p>(<i>A</i>) FACS scattergrams depicting isolation of a C5-C345C-specific population from the sub-library pool containing IGHV3-30-3 and IGHV4-34 (panels I–III), and subsequent affinity maturation of APE777 in strategy B (panels IV–VI). Approximately 5×10⁷ cells were sorted per round. IgG expression is shown on the y-axis and C5-C345C binding on the x-axis. (I) The first sort round (5 µM C5-C345C-Myc) showed little detectable binding; (II) round 4 (100 nM C5-C345C-Myc); and (III) round 9 (3 nM C5-C345C-Myc) which included a room-temperature incubation to increase stringency. Affinity maturation FACS scattergrams of isolated C5-C345C-specific antibody, APE777, are shown for strategy B starting with (IV) round 1 (20 nM C5-C345C-Myc), (V) round 5 (1.5 nM C5-C345C-Dyl-650), and (VI) round 8 (100 pM C5-C345C-Dyl-650). Each sort round consisted of AID-induced SHM and selection of highest antigen binding and antibody expressing cells by FACS. Note emergence of new cell populations that exhibited increased antigen binding relative to the original population over time, and which were able to recognize sequentially decreasing antigen concentrations. (<i>B</i>) Plot comparing stability late (value corresponds to amount of antibody-bound antigen) versus binding late (value corresponds to off-rate, k_d) capture-adjusted report points for Biacore 4000 screen of single cell clone supernatants. Higher values correspond to more antigen bound per unit antibody (binding late) and slower off-rates (stability late). Highlighted points indicate highest affinity C5-C345C binding clones further characterized by Biacore T200 and sequencing. (<i>C</i>) Full kinetic analysis of APE777 binding to C5-C345C (first panel) with a K_D of 200 nM (k_a = 1.4×10⁵ M⁻¹ s⁻¹, k_d = 2.4×10⁻² s⁻¹). Binding to C5 and C5b6 complement proteins was detectible (second and third panels), while APE777 shows no significant binding to the C7 negative control.</p

Summary of observed enriching mutations.

<p>Table of HC and LC point mutation, insertion, and deletion events observed during affinity maturation of APE777. The first column indicates the amino acid mutation, listed with Kabat numbering. The second column indicates whether the mutation is in the HC or LC. Columns three and four show the starting and ending nucleotide sequence surrounding the site of mutation, with the mutated nucleotides underlined. Columns five and six indicate the starting and ending nucleotide for point mutations. The last two columns indicate which strategy the mutation was observed in, and at which round it was enriched. Ten of the 11 point mutations were initiated at G or C nucleotides; one of the 11 was initiated at a T. Seven of the point mutations, the deletion, and one of the insertions occurred at known AID hotspots (WRCH, highlighted in <b>bold</b>) located within CDRs 1, 2, or 3. Seven of the 11 point mutations were nucleotide transitions, and four were nucleotide transversions.</p>1<p>Enrichment observed by Sanger sequencing of 40 HC and LC variable regions post-sort round.</p

Isolation of high-affinity monoclonal antibodies from immunized mice.

<p>Spleen and draining lymph nodes were harvested from mice immunized with the C345C fragment of complement protein C5. After RNA isolation and cDNA synthesis, (D)J diversity was amplified from HCs and cloned into nine human germline variable regions (IGHV 1-2, 1-69, 3-7, 3-23, 3-30, 4-34, 4-59, 5-51, and 6-1). These were paired with a fully human LC library, composed of five germline IGκV regions (IGKV 1-33, 1D-39, 2D-30, 3-20, and 4-1), combined with CDR3 diversity obtained from a pool of normal human donors. Libraries were transfected into HEK 293 c18 cells as four sub-library pools for cell surface display and FACS selection of antigen specific clones. Subsequent affinity maturation by AID-induced mutagenesis and FACS resulted in a high-affinity antibody with demonstrated functional activity.</p