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9 • Developability in Antibody Discovery 231
tremelimumab, and vesencumab) exhibited evidence for high self‑interaction as deter‑
mined by AC‑SINS. We had chosen vesencumab to be in our test set upon inferring that
it may correspond to a poorly behaved anti‑NRP1 antibody highlighted in the afore‑
mentioned BVP assay article15 after learning that an anti‑NRP1 antibody published by
a group, also from Genentech, showed unexpected differences in pharmacokinetics in
monkey vs. other species.
33
A proposal to adapt AC‑SINS to assess hydrophobicity, by performing the assay
under a salt gradient, was reported34 and evaluated using 32 named antibody samples,
including vesencumab. The assay results were shown to correlate with HIC (hydropho‑
bic interaction chromatography) retention times, and like in work with previous assays,
a few of the “clinical samples” showed increased hydrophobicity relative to a human
polyclonal antibody sample with potential undesirable consequences for purication
and aggregation.
35,36
In collaboration with the Wittrup group at MIT, we explored a potential relationship
between a set of high‑throughput assays and pharmacokinetics in mice.
21,37
In the rst
study, 16 samples, including four named (constructed using variable region sequences
from INN‑assigned, clinical stage, antibodies, and expressed transiently in human
embryonic (HEK) cells) were tested for clearance after administration to wild‑type
mice. In parallel, the corresponding samples were assayed for PSR, AC‑SINS, CSI‑BLI,
CIC, size‑exclusion chromatography (SEC), and accelerated stability (AS). Signicant
correlations to antibody clearance were noted for PSR, CIC, and marginally, AS. In a
similar timeframe, a team at Merck put forward a hypothesis tying differential bind‑
ing to FcRn at neutral pH to pharmacokinetics38 and was furthered by a team at Roche
to help explain the difference in reported clearance in human patients between two
antibodies against the same target, ustekinumab and briakinumab, both against the
common subunit of IL‑12 and IL‑23.39 Ustekinumab, an approved drug, shows normal
pharmacokinetics with terminal elimination half‑life in the order of 20 days; in contrast,
briakinumab, whose development was apparently terminated in 2011 after the sponsor
withdrew its regulatory submission, has a reported half‑life of under 10 days. In this
work,39 a correlation is observed between FcRn interaction at neutral pH and clearance
in humans or animal models. In our work, we aimed to distinguish this mechanism with
a simpler explanation involving nonspecic interactions.37 Simplistically, if an antibody
binds to FcRn at neutral pH, it may bind multiple off‑target moieties which contribute to
the observed poor half‑life. First, it was noted that ustekinumab exhibited more favorable
scores or results for four metrics associated with polyspecicity and/or self‑interaction,
namely, AC‑SINS, BVP, CIC, and PSR. Second, pharmacokinetics experiments were
carried out in FcRn‑knockout mice, thus removing FcRn as a factor. As expected, both
antibodies presented much faster clearance in comparison to their behavior in wild‑type
mice (it should be noted that antibodies with human Fc sequences bind mouse FcRn).
However, the clearance for the highly polyspecic antibody briakinumab was substan‑
tially higher than that for ustekinumab: 207 vs. 125mL/day/kg, which was statistically
signicant. Similarly, the terminal half‑lives were 9.8 and 13.8 hours, respectively, for
briakinumab and ustekinumab, also statistically signicant.
At this stage, we had accumulated a series of intriguing results involving a relatively
small number of samples where attempts to correlate tended to be limited. Inspired in
232 Biopharmaceutical Informatics
(B) Hydrophobicity (A) Polyspecificity
part by studies involving much larger numbers of antibodies,
11,15
we initiated a project
aimed at collecting data on “all” antibodies known to have reached at least phase II
of clinical development and with reliably known sequence information (INN designa‑
tions). This was eventually published in 2017,12 and some of the results are reviewed and
discussed in the next section of this chapter.
9.3 CLINICAL ANTIBODY DATA SET
In this comprehensive analysis,12 antibody samples were generated using sequences cor‑
responding to 137molecules with INN designations and at various clinical stages. The
antibodies were cloned into a common human IgG1 expression vector, with light chains
built using human kappa or lambda constant regions as appropriate and expressed tran‑
siently in HEK cells. The analysis of these samples included 12 assays probing the
biophysical properties of the resulting molecules. Perhaps, unsurprisingly, there was a
trend for more clinically advanced molecules, all the way to approval, to have gener‑
ally better biophysical proles than those in earlier stages of clinical development. By
looking at the properties of approved antibodies, it was possible to dene thresholds for
10 of the 12 assays (see Table1 of Jain etal.12) by which antibodies were agged for
potential aberrant behavior. At the same time, rank‑order results between assays were
compared to identify clusters of assays measuring redundant biophysical properties.
The 12 assays could be grouped into 5 clusters (Figure9.1), Two clusters, which include
six of the assays, could in turn be merged into a larger cluster, group A, by using a differ‑
ent cut point in the cluster analysis. The assays in group A are generally understood as
FIGURE9.1 Clustering of biophysical properties recalculated from published data.
12
9 • Developability in Antibody Discovery 233
PSR score
HIC retention time (min)
ed
reecting a measure of polyspecicity while the assays in the other main cluster, termed
group B, assess hydrophobicity. By denition, clustering suggests group A (polyspeci‑
city) assays and group B (hydrophobicity) assays are not highly correlated, but beyond
that, an interesting observation from this analysis is that very few antibodies in the set
of 137 were agged as having unfavorable measures in both group A assays and group
B assays.
As we utilize extensively the PSR binding and HIC assays in our discovery work‑
ow, we focus on them as prototypical group A and group B assays, respectively, to
further exemplify this observation.
A bivariate plot of HIC retention time (RT) vs. PSR score with the thresholds
dened originally (0.27 for PSR score and 11.7minutes for HIC RT) reveals a single
antibody, with both metrics above the thresholds, among the 137 antibodies used in that
study (Figure9.2). This outlier corresponds to the anti‑IGFR1 antibody cixutumumab,
which reached phase II of clinical development targeting cancer indications, but further
work has been reported as discontinued as of September, 2018. This may well be a target
class effect: of nine antibodies to this target with INN designations, seven have been
terminated and the surviving ones, including one approved, are aimed at thyroid eye
disease. Very similar bivariate plots are obtained when pairing other assays from group
A with those from group B. (Data not shown here, but obtainable from the supplemental
data in Jain etal.12).
The observation that the high PSR, high HIC quadrant is virtually unpopulated by
our surrogate clinical antibody set may mean this would be a good metric by which to
screen early candidates, or simply that antibodies in general just do not tend to possess
these characteristics. Analysis of additional data sets below will distinguish these two
possibilities.
It is worth mentioning in this section a handful of published studies that used the
same or similar set of samples for analysis of other properties. These include chemical
degradation,
with heme.
8,9
extensiona l ow,40 heparin and FcRn chromatography,41 and interaction
42
16
14
12
10
8
0.0 0.2 0.4 0.60.8 1.0
FIGURE9.2 Bivariate plot of HIC retention time vs. PSR score for 137 samples made with
sequences from clinical-stage antibodies, thresholds at 0.27 for PSR and 11.7minutes for
HIC shown as vertical and horizontal lines, respectively. Data from Jain etal.12 Here and
elsewhere, HIC retention time values were set at 16minutes for cases when the sample did
not elute from the column.
Phase II
Phase III
Approv
234 Biopharmaceutical Informatics
TABLE9.1 Spearman (rank-order) correlations to FcRn and Heparin
retention measures41 with prior work
METRIC
CIC 0.86 0.35
AC-SINS 0.83 0.50
CSI_BLI 0.67 0.45
PSR score 0.63 0.44
SGAC-SINS 0.55 −0.07
BVP Score 0.43 0.37
ELISA 0.41 0.40
Heparin relative retention 0.48 1.00
HIC retention time 0.30 −0.34
SMAC 0.30 −0.30
Accelerated stability 0.17 0.12
Estimated Fab pI 0.39 0.78
12
FCRN RELATIVE
RETENTION
HEPARIN RELATIVE
RETENTION
Since the work of Kraft etal.41 included data for 130 samples common to the 137 in
Jain etal., it is instructive to see how the results of their assays correlate with the previ‑
ously reported metrics. Rank‑order correlation coefcients of their FcRn and heparin
relative retention metrics with ten of the assays from Jain etal.12 are shown in Table9.1.
Notably, very high correlations are observed between FcRn relative retention measures
and some assays in group A (polyspecicity category), most prominently CIC retention
times, AC‑SINS, and the PSR scores. We added to this analysis a simple sequence‑based
estimation of the isoelectric point for the Fab portion (Fab pI) and showed a high cor‑
relation (0.78) with heparin relative retention.
9.4 HUMAN B‑CELL‑DERIVED ANTIBODIES
In another Adimab study, Shehata etal.20 isolated 400 antibodies from several B‑cell
compartments. As described in detail in the original publication, antibody heavy‑ and
light‑chain variable regions were PCR‑amplied from single B cells, and then cloned
and expressed as IgG1 antibodies in an engineered yeast strain. It is important to note
that for identical sequences expressed in this system and transiently in CHO cells, we
observe a high correlation between PSR score and HIC retention time measurements. For
example, in a recent comparative head‑to‑head set comprising 51 cases, the PSR scores
show linear correlation, Pearson R2, of 0.96, and rank‑order correlation, Spearman ρ, of
0.97. For HIC retention time, the correlation coefcients are 0.97 and 0.98, respectively,
2
and ρ (our unpublished results). This establishes that for these two metrics, it is
for R
possible to compare data obtained with IgG samples made in our engineered yeast with
those produced in HEK or CHO cells.
9 • Developability in Antibody Discovery 235
16
PSR score
HIC retention time (min)
s
Naive
14
12
10
8
0.00.2 0.40.6 0.81.0
N=5 (1.4%)
IgM memory
IgG memory
Long-lived plasma cell
FIGURE9. 3 Bivariate plot of HIC retention time vs. PSR score for 349 samples from human
B cells. Thresholds at 0.27 for PSR and 11.7minutes for HIC are shown as vertical and horizontal lines, respectively. Data from Shehata etal.
20
Of the 400 antibodies included in the original study, 349 have data reported for both
the PSR and HIC assays. The results in Figure9.3 show that very few antibodies map to
the high PSR scores and high HIC RT region dened by the thresholds reported in the
clinical antibody study.12 Specically, only 5 of the 349 antibodies, or 1.4%, exhibited
this behavior, which is virtually absent in the clinical antibody study. In accordance
with the general theme of the Shehata etal. investigation, four of these ve came from
naïve or IgM memory compartments, with only one from plasma cells. As discussed
therein, it appears that by these two metrics, natively paired human antibodies have
developability characteristics comparable to those of bona de therapeutic antibodies
in clinical development. Of course, this is not a comprehensive developability assess‑
ment; for example, in the human‑derived antibodies, more mutated sequences (relative
to germline) tended to show more favorable polyspecicity scores, but this often came
at the expense of reduced thermal stability assessed using differential scanning uo‑
rescence.43 Nonetheless, antibodies derived directly from human sources via analogous
means have been developed for prophylactic and/or therapeutic use for Ebolavirus and
SARS‑CoV‑2 infections.
44,45
More generally, it is notable that of the eight antibodies
(corresponding to ve distinct products, as three of these are mixtures of two antibod‑
ies) receiving emergency use authorization (EUA) in the United States for therapeutic
or prophylactic use against SARS2‑CoV‑2, seven came from human donors
46 –51
; the one
exception being one component of the two‑antibody mix from Regeneron, which came
from transgenic mouse immunizations.
9.5 CONTROL ANTIBODIES
More recently, while creating control antibodies for a variety of targets, we have accu‑
mulated a subset of 156 cases with measured PSR scores and HIC retention times. To
make the samples, genes encoding literature amino acid sequences for the variable
regions were sourced and the resulting material was cloned into human IgG1, kappa, or
lambda, vectors for expression in our engineered yeast strain, as described in Shehata
236 Biopharmaceutical Informatics
PSR score
Approved
HIC retention time (min)
Approved
HIC retention time (min)
16
(a)
(b)
etal.20 In addition, these antibodies represent a cross‑section of sequences disclosed in
the patent literature, most of which, to our knowledge, have not reached clinical develop‑
ment status. They come from a variety of institutions ranging from academia and small
biotechnology companies, all the way to large biopharma. As shown in Figure9.4a, the
relative number of antibodies scoring poorly (above the dened thresholds) for both
the PSR and HIC metrics is now much higher than in the sets discussed previously: 11
out of 156 or about 7%, compared to about 0.7% and 1.4% for the clinical and human
B‑cell sets, respectively. Breaking down the results by inferred clinical stage is also
informative. This analysis shows that molecules constructed with sequences known or
believed to have reached clinical development tend to lie outside the undesirable upper
None
14
12
10
HIC retention time (min)
8
0.0 0.5 1.0
N=11 (7.0%)
PSR score
Phase I
Phase II
Phase III
Approved
Chimeric
16
14
12
10
8
0.00.2 0.40.6 0.81.0
N= 4 (12.5%)
PSR score
None
Phase II
Phase III
16
14
12
10
HIC retention time (min)
8
0.00.2 0.40.6 0.81.0
In vitro
16
14
12
10
8
0.00.5 1.0
N= 1 (4%) N= 2 (5%)
PSR score
None
Phase I
Phase II
Phase III
16
14
12
10
HIC retention time (min)
8
0.00.2 0.40.6 0.81.0
Humanized
N= 4 (6%)
PSR score
Transgenic
None
Phase I
Phase II
Phase III
None
Phase I
Phase II
FIGURE9.4 Bivariate plot of HIC retention time vs. PSR score for 156 samples corresponding to control antibodies, color-coded according to the estimated stage of clinical development (a); same data broken down by antibody type (b). Thresholds at 0.27 for PSR and
11.7minutes for HIC shown as vertical and horizontal lines, respectively.
9 • Developability in Antibody Discovery 237
right quadrant dened by the PSR and HIC thresholds. The sole exception is a sample
made from variable region sequences derived from an antibody believed to be in phase
II clinical stage, but not one with an INN designation. It should be noted that the lack of
association with an INN creates some uncertainty on the connection between sequence
and clinical candidate.
Figure9.4b breaks out the data by antibody type, dened as chimeric, humanized,
in vitro (from libraries), or from humanized transgenic animals. Although numbers are
now necessarily smaller, it is notable that antibodies from in vivo sources are not always
better, by these metrics, than those emerging from in vitro discovery. Chimeric antibod‑
ies have a slightly higher tendency to map to the high PSR, high HIC quadrant than
others in this data set: 12.5% vs. 4%–6% for the other antibody types.
One clear result from this set is the observation that the high PSR and high HIC
quadrant can be populated by antibodies in general. This suggests that their relative
absence in clinical and natively paired human antibodies is the result of direct or indi‑
rect ltering away from this dual property.
9.6 ASSAYS RESULTS FOR HUMAN
ANTIBODIES FROM DE NOVO
DISCOVERY CAMPAIGNS
Before examining the antibody properties, we describe here some basic elements of our
discovery process for additional context. The primary aim in early selection rounds is
to enrich binders to the target of interest. However, during this process, concomitant
enrichment of antibodies with high PSR binding can occur. Therefore, tracking and
deselecting such antibodies is essential for identifying target‑specic output with low
overall polyspecicity. The process is illustrated in Figure9.5a. As seen in Figure9.5b,
naïve libraries have a small percentage of PSR binders; these can get enriched in the
selection process depending on the biophysical properties of the target antigen. After
positively selecting for a binding population in the third round of successive enrichment
(Figure9.5a), a subsequent fourth round of selection on the PSR nonbinding population
depletes the population of high PSR binders, which can then be observed in round 5
(Figure9.5b).
Figure9.5b shows that the percentage of PSR binders in the naïve library, prior to
any selection, depends on the germlines present, and it correlates with the output as seen
in the right‑hand side panels. For example, we see a higher proportion of PSR binders in
the unselected VH4germline family libraries, which would result in more polyreactive
(in this work, following historical precedent, we use the terms polyspecic and poly‑
reactive interchangeably, although, a recent review52 has proposed to ascribe distinct
meanings to the terms) antibodies post‑selection if no PSR negative round is performed.
To offer a glimpse of sequence correlates of polyspecicity and hydrophobicity,
as assessed by the PSR binding and HIC assays, we collected data on several 1,000
samples spread over close to 100 projects and break them down by Variable domain
238 Biopharmaceutical Informatics
(a)
(b)
R2 MACSR1 MACS
Antigenselection
PSR
Negative selection
Antigenselection
VH1
VH3
VH4
Antibody expression
Antigenbinding
Library
Secondaryonly
P1: 0.07% P1: 2.67%
P1: 0.09% P1: 1.31%
P1: 0.08%
PSR
P1: 3.07%
PSR
Antigenbinding
R3 FACS R4 FACS
PSR
P1: 2.89%
P1: 1.40%
P1: 4.0%
PSR
P1: 3.13%
P1: 0.43%
P1: 26.2%
PSR
R5 FACS
PSR
P1: 0.30%
P1: 0.53%
P1: 0.49%
FIGURE9.5 (a) Representative selection process. Two rounds of magnetic bead enrichment (MACS) with the target antigen, followed by three rounds of selections on FACS.
Round 3 (R3): incubation with the target and selection of the binding population. R4: incubation with the polyspecicity reagent (PSR) and negative selection. R5: incubation with
the antigen and selection for strong binders for plating and sequencing. (b)Tracking of
PSR binding during selection. PSR binding of naïve libraries, and during R3, R4, and R5 of
the selection process. The percentage of clones in the P1gate (PSR binding population) is
shown for each plot.
of Heavy chain (VH) or Variable domain of Light chain (VL) germline. They cor‑
respond to antibodies from the earliest stages of discovery using input libraries with
human‑like synthetic diversity. While a detailed description of the design of these
9 • Developability in Antibody Discovery 239
TABLE9.2 Summary of PSR binding and HIC data for top ve occurring VH and VL
germlines
PERCENT
MEAN PSR
GERMLINE NUMBER
VH1–2 572 0.07 5.6% 9.3 0.3%
VH1–69 1,686 0.04 2.7% 11.0 25.8%
VH3–23 1,210 0.04 2.6% 9.4 2.5%
VH3–30 572 0.05 4.0% 9.9 1.9%
VH4–39 1,053 0.07 6.3% 10.1 7.9%
VK1–12 1,211 0.05 3.1% 10.0 8.7%
VK1–33 1,247 0.05 3.4% 9.9 6.4%
VK1–39 1,362 0.07 5.7% 9.9 9.2%
VK3–11 1,764 0.05 4.1% 9.7 5.3%
VK3–20 1,090 0.04 3.0% 9.5 3.2%
SCORE
ABOVE 0.27
THRESHOLD
MEAN HIC
RETENTION
TIME (MIN)
PERCENT
ABOVE 11.7
THRESHOLD
libraries is outside the scope of this chapter, it can be noted that the diversity is con‑
centrated almost exclusively on CDRs H3 and L3. Additional details may be found in
prior publication.
53
Table9.2 summarizes PSR binding and HIC data for the top ve VH and VL germ‑
lines by occurrence in the set. While the average PSR scores are very close to zero in all
cases, a trend for samples with antibodies using the VH4–39germline to have slightly
elevated PSR may be noticed. More than 6% of samples using VH4–39 exhibits PSR
scores above the 0.27 threshold, while for the other germlines, this percentage ranges
from about 2.5% to 3.9%. In the light‑chain analysis, the average PSR scores remain
very low, near zero, for all the germlines, but there is a slight trend for VK1–39 to have
a higher fraction of samples with PSR readouts above the 0.27 threshold: 5.7% vs. about
3%–4% for the others. We should add here that in many of the selections used to gener‑
ate the antibodies for this set, negative PSR pressures have been applied, as described
above and previously,24 so that the resulting samples will tend to have lower PSR scores
than what would be obtained in a selection process that lacked a PSR depletion step.
Table9.2 includes a similar analysis and breakdown for the HIC assay. We can see
that the VH germline appears to inuence the overall hydrophobicity, with VH1–69
showing a higher mean HIC retention time of 11minutes, while the mean HIC retention
time for the other germlines is in the approximate 9–10‑minute range. It is also notable
that IgGs using the VH1–69germline have a much larger proportion of cases with HIC
retention time above the 11.7‑minute threshold, over 25% compared to 0.3 to about 8%
for the other germlines. Finally, breaking the results down by light‑chain germline does
not reveal large differences in averages, but there is a tendency for VK1–39 and VK1–12
to have more antibodies above the 11.7‑minute threshold.
In Figure9.6, we present the PSR‑HIC bivariate analysis, with the data parsed
by the VH germline gene family. As could be expected from the prior analysis of
240 Biopharmaceutical Informatics
and HIC
21
70
VH3
16
14
12
10
HIC retention time (min)
8
0.0 0.2 0.4 0.6 0.8 1.0
16
14
12
10
HIC retention time (min)
8
0.00.2 0.40.6 0.8 1.0
PSR score
PSR score
VH1
VH4
16
14
12
10
HIC retention time (min)
8
0.00.2 0.40.6 0.81.0
16
14
12
10
HIC retention time (min)
8
0.00.2 0.40.6 0.81.0
PSR score
VH5
PSR score
Number of
VH Family
observations
VH1306
VH3369
VH4
2579 1.5%
VH5 297 0.0%
Percent with
PSR
outside
thresholds
.0%
.4%
FIGURE9.6 Bivariate plot of HIC retention time vs. PSR score for samples from primary
discovery; broken down by VH germline family. Thresholds at 0.27 for PSR and 11.7minutes
for HIC are shown as vertical and horizontal lines, respectively.
the individual assays, there is a slight trend for antibodies using genes from the VH1
and VH4 families to have larger percentages of cases with simultaneous high poly‑
specicity and hydrophobicity metrics, though, of course, these remain low overall
(under 2% in all cases).
The observation that certain germlines or even germline families tend to be asso‑
ciated with less favorable properties related to developability raises the question of
whether they should be avoided in diversity sources, be they libraries or even choices
to construct transgenic mice. We think this would be too radical a solution as, at
least for the metrics presented here, the overwhelming majority of molecules using
these “less desirable” germlines still can have favorable overall biophysical proles.
Removing or deprioritizing antibodies using, for example, VH4germlines will result
in the loss of unique canonical structures,54 which will impact overall structural diver‑
sity and may compromise the ability to nd binders with rare qualities in antibody
discovery projects.
To project how these metrics apply to subsequent stages of discovery, we next
focus down on a subset of molecules with collected PSR binding and HIC retention
time data emerging from a set of 15 discovery efforts that have resulted in lead mol‑
ecules currently in phase I trials or beyond (Figure9.7). In contrast with the cumu‑
lative campaign data presented earlier, which included only data from the earliest
discovery stages, the molecules presented here came from all stages of discovery,
including outputs from antibody optimization efforts as required by each project.
One can see in Figure 9.7 that the antibodies chosen for clinical development do
not populate the undesirable upper right quadrant (high PSR binding and high HIC
retention time), and in fact, with very few exceptions, these samples tend to exhibit
low PSR binding and low HIC retention times.