Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

Figures & data

Figure 1. Schematic representation of the workflow used for determination of the light and heavy chain variable domain sequences.

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. 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.¹⁸ CDRs are shown in bold and were defined according to the sequence definition of Kabat et al.,¹⁸ except for CDR-H1, which is the combined sequence and structural definition.¹⁹

Table 1. Ambiguous regions of sequence and strategy for resolving these.

Sequence region	Mass (Da)	^†Possible Sequence(s)	Fix or randomize sequence?	Rationale
3H3-VL: 2, 10, 33, 40, 46, 48, 58, 75, 78 3H3-VH: 4, 45, 51, 69, 80 LOB12.3-VL: 2, 15, 18, 21, 46, 48, 54, 73, 75, 83, 85, 106 LOB12.3-VH: 4, 20, 45, 78, 80, 82c	113	Leu or Ile	Fix	Fix to Leu or Ile according to germline homology
3H3-VL: 11, 21, 47, 54, 73, 104, 106 3H3-VH: 11, 20, 78, 82c LOB12.3-VL: 11, 47, 78, 104 LOB12.3-VH: 11, 18	113	Leu or Ile	Fix	Fix as Leu based on germline homology & chymotryptic cleavage at these positions
LOB12.3-VH: 98-99	114	GlyGly or Asn	Fix	Fix as GlyGly as “YGGYSE” motif is exact match to +1 translation of IGHD1-6 D-segment germline
LOB12.3-VL: 29, 33, 89 LOB12.3-VH: 51	113	Leu or Ile	Randomize	Although germline homology could have been used to fix as Leu or Ile, these site were randomized given the CDR location and low overall complexity of LOB12.3 library
3H3-VH: 29, 94 LOB12.3-VH: 28, 56, 100c	113	Leu or Ile	Randomize	Localized in or near CDR. Germline homology non-guiding, or not applicable. Randomize to Leu/Ile in library (in case of 3H3-VH29, Leu and Ile were encoded in two separate libraries)
3H3-VH: 55-56	158	AlaSer, SerAla, GlyThr, ThrGly	Randomize	Localized in CDRs. Germline homology non-guiding, therefore randomize in library
3H3-VH: 61-62	216	GluSer, AspThr	Randomize
3H3-VH: 98-99	114	GlyGly or Asn	Randomize
3H3-VH: 100a-100b	144	SerGly or GlySer	Randomize

^† Based on mass constraints and germline homology considerations.

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. 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.¹⁸ CDRs are shown in bold and were defined according to the sequence definition of Kabat et al.,¹⁸ except for CDR-H1, which is the combined sequence and structural definition.¹⁹

Table 2. Distribution of sequences following 2 (R2) or 3 (R3) rounds of phage display selection.

Sequence region	†Possible sequence(s)	Sequences at R2	Sequences at R3
*3H3-VH: 94	Leu or Ile	15% Leu, 85% Ile	4% Leu, 96% Ile
*3H3-VH:55-56	GlyThr, AlaSer, SerAla, ThrGly	74% GlyThr, 16% AlaSer, 5% SerAla, 5% ThrGly ---------------------------- 28% GlyThr, 14% GlyAla, 14% GlySer, 11% ThrSer, 7% ThrThr, 6% AlaSer, 5% AlaAla, 5% SerThr, 2% AlaThr, 2% ThrAla, 2% SerSer, 2% SerAla, 2% ThrGly	49% GlyThr, 31% AlaSer, 20% SerAla ---------------------------- 19% GlyThr, 19% ThrSer, 15% GlyAla, 15% ThrThr, 12% AlaSer, 8% SerAla, 8% GlySer, 4% ThrAla
*3H3-VH: 61-62	GluSer, AspThr	94% GluSer, 6% AspThr ---------------------------- 46% GluSer, 45% AspSer, 6% GluThr, 3% AspThr	95% GluSer, 5% AspThr ---------------------------- 58% AspSer, 40% GluSer, 2% AspThr
*3H3-VH: 98-99	GlyGly or Asn	100% GlyGly	100% GlyGly
*3H3-VH: 100a-100b	SerGly or GlySer	95% SerGly, 5% GlySer ---------------------------- 39% SerGly, 31% SerSer, 29% GlyGly, 2% GlySer	100% SerGly ---------------------------- 54% SerSer, 27% SerGly 19% GlyGly
LOB12.3-VL: 29	Leu or Ile	14% Leu, 86% Ile	0% Leu, 100% Ile
LOB12.3-VL: 33	Leu or Ile	39% Leu, 61% Ile	36% Leu, 64% Ile
LOB12.3-VL: 89	Leu or Ile	90% Leu, 10% Ile	100% Leu, 0% Ile
LOB12.3-VH: 28	Leu or Ile	28% Leu, 72% Ile	18% Leu, 82% Ile
LOB12.3-VH: 51	Leu or Ile	31% Leu, 69% Ile	0% Leu, 100% Ile
LOB12.3-VH: 56	Leu or Ile	22% Leu, 78% Ile	18% Leu, 82% Ile
LOB12.3-VH: 100c	Leu or Ile	57% Leu, 43% Ile	64% Leu, 36% Ile

Frequency of sequences satisfying MS-data and germline homology constraints are shown. Consensus sequences are shown in bold, and represent positions where >70% of clones had an identical sequence within these constraints. For completeness, where the library encoded additional sequences outside of these constraints, the full set of sequence distributions is shown in italic text.

^† Based on MS-data constraints and germline homology considerations.

* As both 3H3 libraries (which differed only at VH29) gave similar sequencing outputs after CD137 selection, the combined data are represented in the table.

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 = Fab_p). Binding kinetics calculated from the full set of surface plasmon resonance data are shown in Table 3.

Table 3. Binding affinities of reverse-engineered 3H3 and LOB12.3 Fab variants to CD137.

Fab sample	ka (x 10^5M^−1s^−1)	kd (x 10^−3s^−1)	KD (nM)
LOB12.3-Fab1 (VH-I100c)	4.29	3.68	8.60
LOB12.3-Fab2 (VH-L100c)	5.29	3.75	7.06
LOB12.3-Fabp	4.50	3.74	8.30
3H3-Fab1 (VH-I29)	25.4	4.63	1.82
3H3-Fab2 (VH-L29)	28.8	4.83	1.68
3H3-Fabp	22.1	4.51	2.05

LOB12.3-Fab1, -Fab2 and 3H3-Fab1, -Fab2 were reverse-engineered Fab variants, while LOB12.3-Fab_p and 3H3-Fab_p were Fabs derived by partial proteolysis of the parental (i.e., commercially obtained) IgGs. Standard errors for fitting of data to a 1:1 binding model were less than one unit in the third significant digit for all computed k_a, k_d and K_D values.

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