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, 8 (3), 501-12

Combining Phage Display With De Novo Protein Sequencing for Reverse Engineering of Monoclonal Antibodies

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Combining Phage Display With De Novo Protein Sequencing for Reverse Engineering of Monoclonal Antibodies

Keith W Rickert et al. MAbs.

Abstract

The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

Keywords: Antibody sequencing; mass spectrometry; phage display; proteomics; reverse engineering.

Figures

Figure 1.
Figure 1.
Schematic representation of the workflow used for determination of the light and heavy chain variable domain sequences.
Figure 2.
Figure 2.
Mass spectra and location of protease cleavage sites during LOB12.3 and 3H3 Fab generation. (A) Intact mass spectra of reduced Fab generated by papain digestion of LOB12.3 IgG. The rat IgG1 hinge region sequence is shown above the spectra and sites of cleavage by papain (open triangles) or SpeB (filled triangle) are shown. (B) Intact mass spectra of reduced Fab generated by papain digestion of 3H3 IgG. The rat IgG2a hinge region sequence is shown above the spectra and sites of cleavage by papain are shown.
Figure 3.
Figure 3.
LOB12.3 variable domain sequences described in this work. LOB12.3-Lv1/Hv1 represent the initial assembly of proteomic data into light and heavy V-domain sequences, where X represents ambiguous regions of sequence (gray highlighting = 113 Da; green = 114 Da). Alignment of this draft to closest V, D and J translated germline gene segments is shown below. LOB12.3-Lv2/Hv2 were refined draft sequences, as defined in Table 1, and ambiguous regions in this light/heavy pair were programmed into a Fab phage display library. Further refinement after phage display led to LOB12.3-Lv3/Hv3, and finally 2 unique light/heavy pairings (LOB12.3-Lv4/Hv4 and LOB12.3-Lv4/Hv5) that were part of recombinant Fab fragments. Sequence numbering is according to Kabat et al. CDRs are shown in bold and were defined according to the sequence definition of Kabat et al., except for CDR-H1, which is the combined sequence and structural definition.
Figure 4.
Figure 4.
MS/MS spectrum of a 3H3 chymotryptic peptide containing part of CDR-H3. Interpretation of the fragmentation observed from the 728.3 Da parent ion led to assignment of the sequence as CT(L/I)DGY where C is modified with a carbamidomethyl group. Additional peaks arise primarily by water loss from the labeled b ion peaks.
Figure 5.
Figure 5.
3H3 variable domain sequences described in this work. 3H3-Lv1/Hv1 represent the initial assembly of proteomic data into light and heavy V-domain sequences, where X represents ambiguous regions of sequence (gray highlighting = 113 Da; yellow = 158 Da; blue = 216 Da; green = 114 Da; magenta = 144 Da). An alignment of this draft to closest V and J translated germline gene segments is show below (a germline D-segment could not be assigned based on the initial draft sequence). 3H3-Lv2/Hv2 were refined draft sequences, as described in Table 1, and ambiguous regions in this light/heavy pair were programmed into 2 Fab phage display libraries. Further refinement after phage display led to 3H3-Lv2/Hv3, and finally 2 unique light/heavy pairings (3H3-Lv2/Hv4 and 3H3-Lv2/Hv5) that were part of recombinant Fabs. Sequence numbering is according to Kabat et al. CDRs are shown in bold and were defined according to the sequence definition of Kabat et al., except for CDR-H1, which is the combined sequence and structural definition.
Figure 6.
Figure 6.
Binding of reverse-engineered LOB12.3 and 3H3 Fab variants to murine CD137. A) Competition phage ELISA measuring relative CD137 binding affinities to phage displayed LOB12.3 or 3H3 Fab variants. LOB12.3 Fab variants contained LOB12.3-Lv3/Hv3 light/heavy V-region pairs, where the sequence at VL33/VH100c was Leu/Leu (green), Leu/Ile (blue), Ile/Leu (magenta) or Ile/Ile (red). IC50 values for displacement of 50% of bound Fab-phage was between 2.0 – 3.9 nM for all 4 variants. 3H3 Fab variants contained 3H3-Lv2/Hv3 V-region pairs, where the sequence at VH29 was Ile (blue), or contained a VH-E61D substitution and Ile (red) or Leu (magenta) at VH29. IC50 values for displacement of 50% of bound Fab-phage was between 3.1 – 6.2 nM for all 3 variants. B) Sensorgram traces showing near identical binding kinetics for 11.1 nM injections of Fab samples over an immobilized CD137 surface. Reverse-engineered Fab variants (blue = Fab1; green = Fab2) are compared to the corresponding reference Fabs generated by partial proteolysis of the commercially-sourced parental IgG (black = Fabp). Binding kinetics calculated from the full set of surface plasmon resonance data are shown in Table 3.

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