mAbs
`
`ISSN: 1942-0862 (Print) 1942-0870 (Online) Journal homepage: https://www.tandfonline.com/loi/kmab20
`
`Connecting the sequence dots: shedding light on
`the genesis of antibodies reported to be designed
`in silico
`
`Maximiliano Vásquez, Eric Krauland, Laura Walker, Dane Wittrup & Tillman
`Gerngross
`
`To cite this article: Maximiliano Vásquez, Eric Krauland, Laura Walker, Dane Wittrup & Tillman
`Gerngross (2019) Connecting the sequence dots: shedding light on the genesis of antibodies
`reported to be designed in silico, mAbs, 11:5, 803-808, DOI: 10.1080/19420862.2019.1611172
`To link to this article: https://doi.org/10.1080/19420862.2019.1611172
`
`© 2019 The Author(s). Published with
`license by Taylor & Francis Group, LLC.
`
`View supplementary material
`
`Published online: 20 May 2019.
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`Lassen - Exhibit 1062, p. 1
`
`

`

`MABS
`2019, VOL. 11, NO. 5, 803–808
`https://doi.org/10.1080/19420862.2019.1611172
`
`PERSPECTIVE
`
`Connecting the sequence dots: shedding light on the genesis of antibodies reported
`to be designed in silico
`Maximiliano Vásquez
`a, Eric Krauland a, Laura Walkera, Dane Wittrupa,b, and Tillman Gerngrossa,c
`
`aAdimab LLC, Lebanon, NH, USA; bMassachusetts Institute of Technology, Cambridge, MA, USA; cThayer School of Engineering, Dartmouth College,
`Hanover, NH, USA
`
`ABSTRACT
`Two recent publications out of the same research laboratory report on structure-based in silico design of
`antibodies against viral targets without sequence disclosure. Cross-referencing the published data to
`patent databases, we established the sequence identity of said computationally designed antibodies. In
`both cases, the antibodies align with high sequence identity to previously reported antibodies of the
`same specificity. This clear underlying sequence relationship, which is far closer than the antibody
`templates reported to seed the computational design, suggests an alternative origin of the computa-
`tionally designed antibodies. The lack of both reproducible computational algorithms and of output
`sequences in the initial publications obscures the relationship to previously reported antibodies, and
`sows doubt as to the genesis narrative described therein.
`
`ARTICLE HISTORY
`Received 16 April 2019
`Accepted 18 April 2019
`
`KEYWORDS
`Structure-based design; in
`silico antibody; peer review;
`sequence disclosure; virus
`neutralization
`
`Introduction
`
`Human antibodies are a major modality to treat human disease,
`and therefore the focus of significant technology development.1
`Historically, two main approaches have been used to discover
`antibodies against targets of therapeutic interest: 1) in vivo tech-
`nologies based on isolating B cell diversity from animals2-5 or
`humans,6–8 following immunization or exposure to infectious
`agents, respectively, and 2) in vitro technologies based on the
`display of synthetic or semi-synthetic human IgG diversity on
`the surface of phage or yeast.9–12
`More recently, a third approach of designing human anti-
`bodies in silico against specific epitopes has been fueled by
`two major trends: 1) ever-increasing structural information of
`antibodies and their potential targets, as well as 2) access to
`more powerful computational
`tools. Notwithstanding the
`potential impact, the general lack of published successes in
`this area has highlighted the extreme challenge of designing
`antibodies in silico,13,14 although progress in the area of affi-
`nity maturation has been made.15,16
`As such, two publications17,18 originating from the same
`research lab (based on the first and corresponding author), report-
`ing the in silico design of epitope-specific, broadly neutralizing,
`human antibodies against two infectious disease targets, garnered
`our attention. Strikingly, in both cases the extraordinary accom-
`plishments were not supported by a detailed description of meth-
`ods or intermediate results, nor were the end-products of these
`efforts, namely the amino acid sequences of the designed anti-
`bodies, disclosed, making it impossible to independently repro-
`duce the reported functional characterization. To understand how
`these results could have been achieved, we endeavored to better
`
`understand the identity and, potentially, the genesis of these
`antibodies. In this communication, we present evidence that in
`both cases, previously published antibody sequences and struc-
`tures are the basis for the in silico designed antibodies.
`
`Results
`
`Influenza antibody
`
`VIS410 is described17 as a broadly neutralizing antibody generated
`by a process that used “a database of nonredundant combinations
`of complementary determining region (CDR) canonical structures
`(antibody scaffolds), (to select) multiple antibody templates satisfy-
`ing shape complementarity criteria and systematically engineered
`energetically favorable, hotspot-like interactions between CDR resi-
`dues and these anchor residues on hemagglutinin (HA).” The
`authors then present experimental data on binding, neutraliza-
`tion, and protective efficacy in influenza animal models. However,
`the sequence of VIS410, the output of the design and engineering
`process, was neither provided in the publication, nor deposited
`into a public database. Using VIS410 as a search term in the
`USPTO database readily leads to a US patent application19 that
`also designates VIS410 as Ab044. This application further estab-
`lishes that the variable heavy- and light-chain (VH and VL)
`sequences correspond to sequence ID numbers 25 and 52,
`which are shown in Figure 1. Searching the patent database with
`the VIS410 sequences produces exact matches to an earlier US
`patent publication from 2013 describing Ab044 with a similar
`inventorship group.20 As shown in Figure S1, a comparison of
`tables from the two sources,17,20 confirms that VIS410 and Ab044
`are in fact the same antibody.
`
`CONTACT Tillman Gerngross
`Thayer School of Engineering, Dartmouth College, Hanover, NH03755, USA; Maximiliano Vásquez
`tillman@adimab.com
`Adimab LLC, 7 Lucent Drive, Lebanon, NH03766, USA.
`max.vasquez@adimab.com
`Supplemental data for this article can be accessed on the publisher’s website
`© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
`which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
`
`Lassen - Exhibit 1062, p. 2
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`804
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`M. VÁSQUEZ ET AL.
`
`Figure 1. VH and VL alignment of FI6v3 (PDB file 3ZTJ) and VIS410 (Ab 044) FI6. Non-conservative substitutions depicted in red font. CDRs are highlighted in gray
`according to Kabat’s23 definition.
`
`It is interesting that searching Genbank with the VH
`sequence, even today (April 2019, nearly four years after the
`original publication) does not yield a 100% match. However,
`the search does reveal FI6v3, a broadly neutralizing anti-
`influenza antibody first described in 2011 by Corti and et al.21
`An alignment of VIS410 and FI6v3 is shown in Figure 1. The
`overall percent identity values are 87% and 81%, respectively,
`for the VH and VL domains. This is achieved with no gaps in
`the alignment,
`indicating that all the corresponding CDR
`lengths are identical; this result is particularly significant for
`the CDRH3 and for the CDRL1, with the latter showing
`a relatively rare two amino acid deletion relative to the
`human germline of origin, as presented in the original pub-
`lication by Corti et al.21 Including conservative substitutions
`(“positives” as in the default settings for BLAST22) reveals an
`even closer relationship with a similarity of 96% and 88%,
`respectively, for VH and VL. The VIS410 publication includes
`in supplementary material a list of accession numbers to
`antibody variable regions as “top ranking templates” used
`for the design of anti-influenza antibodies.17 In Figure 2 we
`present the corresponding VH sequences, retrieved from the
`NCBI database using those accession numbers, and aligned
`with both VIS410 and FI6v3. Focusing on the CDRs, the
`portion of the antibody sequence expected to be most impor-
`tant to determine specificity and govern binding to antigen,23–
`25 the closest template CDRH3 to the output VIS410 CDRH3
`is 10% identical, while the FI6v3 CDRH3 to VIS410 CDRH3 is
`85% identical. Given the remarkable diversity of CDRH3
`sequences, it is hard to conceive how a computational method
`could have credibly and independently converged near the
`FI6v3 sequence. Further comparison of
`the other CDRs
`
`between VIS410 and FI6v3, shows just two non-conservative
`substitutions in VH CDRs, and three in CDRL2. According to
`the crystal structure, CDRL2 was deemed non-essential for
`antigen binding by FI6v3 and therefore an attractive location
`to introduce sequence alterations that are unlikely to compro-
`mise binding.21 Based on the structure of FI6v3 in complex
`with influenza HA, we mapped those differences, as shown in
`Figure 3. Only one of the non-conservatively substituted
`amino acid positions appears to be directly involved in anti-
`gen contact; this would be position 54 of the VH, which is Ala
`in FI6v3, but Gly in VIS410. As detailed in the original
`publication,21 the precursor of FI6v3, called FI6, does have
`Gly at the same position, and this change is among a few
`others demonstrated to have no impact on functionality.
`Given the remarkable degree of similarity, it is important to
`remember that FI6v3, including its sequence and structure,
`was described almost a year before the submission date of the
`first patent application describing the discovery of VIS410
`(Figure S2)
`
`Zika antibody
`
`More recently, the same research group with new collabora-
`tors reported to have “applied computational methods to engi-
`neer an antibody, ZAb_FLEP,” with broadly neutralizing
`activity against Zika virus.18 As in the earlier17 publication,
`no sequence information was provided for ZAb_FLEP.
`Following the same approach as in the previous section, we
`initially searched the patent
`literature with the
`term
`“ZAb_FLEP,” which proved unproductive. However, search-
`ing Zika together with some of the author’s names in a patent
`
`Figure 2. Design process for VH of VIS410: alignment of VH template sequences listed in table S1 of original publication17 with VIS410 and FI6v3. CDRs as defined in
`Kabat are highlighted in gray.
`
`Lassen - Exhibit 1062, p. 3
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`

`MABS
`
`805
`
`Figure 3. Depiction of non-conservative substitutions (red for the VH and magenta for the VL) in the context of the structure of FI6v3 complexed with influenza H1
`(gray, glycans shown as teal sticks). VH and VL are shown in orange and purple, respectively. PDB file 3ZTN.
`
`database led us to a published patent application26 titled
`“Antibodies that bind Zika virus envelope protein and uses
`thereof”. All six named inventors are also authors of the
`publication describing ZAb_FLEP.18
`Comparison of figures in the publication and patent appli-
`cation leaves little doubt that ZAb_FLEP corresponds to mAb
`8; see Figure S3.18,26 The sequences of mAb 8 are presented in
`the patent application26 (sequence IDs 6 and 15), and are
`shown in Figure 4.
`As in the influenza case, a Genbank search with mAb 8
`sequences fails to retrieve an exact match, but results in
`sequence hits to EDE1 C8,27–30 a previously reported den-
`gue/Zika cross-reactive antibody first described in 2015 by
`Dejnirattisai et al.29 An alignment of EDE1 C8 and mAb 8
`is shown in Figure 4.
`
`As in the influenza example, the similarity is remarkable, with
`89% and 90% identity, for VH and VL, respectively. This is
`obtained without any gaps in the alignment, and thus all CDR
`lengths are identical between ZAb_FLEP and EDE1 C8.
`Considering conservative substitutions, as before, yields simila-
`rities of 95% and 98%, respectively, for VH and VL. Within
`the CDRs of the VH, there is a single V to A non-conservative
`substitution (as defined by BLAST default settings). There
`are only three conservative substitutions in VL CDRs; in fact,
`one in each CDR, all
`involving S/T exchanges. The non-
`conservative substitutions in the context of the known EDE1
`C8 complex with Zika protein27 are depicted in Figure 5. Once
`again, given the remarkable sequence similarity, it is important
`to emphasize the prior publication of the EDE1 C8 sequences, in
`the context of both dengue and Zika (Figure S2).27,30
`
`Heavy Chain: 89% identity | 95% similarity
`
`EDE1 C8
`mAb8
`
`EDE1 C8
`mAb8
`
`Light Chain: 90% identity | 98% similarity
`
`EDE1 C8
`mAb8
`
`EDE1 C8
`mAb8
`
`Figure 4. VH and VL alignment of VH alignment of EDE1 C8 (PDB files 4UTA or 5LBS) and mAb 8 (likely ZAb_FLEP). Non-conservative substitutions depicted in red
`font. CDRs highlighted in gray according to Kabat’s definition.
`
`Lassen - Exhibit 1062, p. 4
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`806
`
`M. VÁSQUEZ ET AL.
`
`Figure 5. Depiction of non-conservative substitutions (red for the VH and magenta for the VL) in the context of the structure of EDE1 C8 complexed with ZIKV
`E (gray). VH and VL are shown in orange and purple, respectively. PDB 5LBS, chains AHL.
`
`It is interesting that EDE1 C8 is mentioned, among other
`antibodies, as a potential
`template for
`the design of
`ZAb_FLEP, as indicated in the supplementary material:18
`“Multiple antibody scaffolds (including mouse-derived pan-
`flavivirus 4G2, anti-EDE1 Dengue mAbs C8, C10 and anti-
`EDE2 Dengue mAb A11, anti-TDRD3 antibody and anti-HIV
`antibody PGT124) were used as starting templates for antibody
`engineering.”18 The lack of sequence disclosure for ZAb_FLEP
`and any direct data comparisons to EDE1 C8, however,
`obscures from readers, as well as peer reviewers, the remark-
`able similarity of ZAb_FLEP and EDE1 C8. Given the appar-
`ent origin of ZAb_FLEP from EDE1 C8, we wonder why
`a direct comparison between the two was not reported, espe-
`cially in light of the authors’ statement:18 “The in vitro neu-
`tralization potential of ZAb_FLEP approaches the potency of
`select Zika antibodies” (emphasis added).
`The narrative in the patent application,26 which is intended
`to teach the skilled artisan how to practice the invention, only
`provides sequences from an anti-TDRD3 antibody as input
`template, and it makes no explicit mention of EDE1 C8 as
`input. Moreover, sequences identical to EDE1 C8, represented
`as mAb 3 in the patent document, are said to have been
`“designed by computing the epitope-paratope connectivity net-
`work,” whereby “variable regions and CDRs (are) generated
`(and) shown in (…) Figure 1(a,b)” (see Figure S426). However,
`the designed mAb 3 has a non-traditional two amino acid
`addition (Arg-Ser) at
`the VL N-terminus. This sequence
`matches a non-coding cloning site present in the original
`EDE1 C8 VL expression vector.29 It is inconceivable that an
`unsupervised algorithm would produce vestigial vector
`sequence unrelated to antigen recognition.
`
`Discussion
`
`In this report, we examine two instances in which the same
`research group has made representations of structure-based
`computational design of anti-viral antibodies with exceptional
`
`neutralization breadth and potency.17,18 In neither case were
`the sequences of the designed antibodies disclosed, leading us
`to question the enforcement of editorial policies regarding
`reproducibility. Perhaps more concerning is the potential for
`contamination of the scientific literature with claims by inno-
`cent third parties. For example, in a commentary article31
`about the clinical evaluation of VIS410,32 it
`is said that
`“VIS410, however,
`is not
`just another HA-stem specific
`human monoclonal antibody. This human IgG1 monoclonal
`antibody is the result of man-made design and protein engi-
`neering and so is not derived from a natural source.” Clearly,
`the author of this comment was not in possession of the
`comparison presented in Figure 1.
`We present with a high degree of confidence the actual
`sequence identity of the designed antibodies, and a more
`plausible genesis narrative. Comparisons of these sequences
`to those of previously described human B cell-derived anti-
`bodies to the same targets show striking similarities. By con-
`trast, those designed sequences appear very dissimilar from
`the templates said to have been used to start the design
`process (Figure 2 and S4); we leave it to the reader to judge
`the likelihood of these highly homologous sequences being re-
`discovered coincidentally, or simply derived from existing
`antibodies targeting the same epitopes as those of the compu-
`tationally designed antibodies.
`In conclusion, the presented fact pattern calls into question
`the publications’ claimed genesis of VIS410 and ZAb_FLEP.
`Furthermore, the lack of sequence disclosure exposes a serious
`weakness in the peer review process in the emerging field of
`computational antibody design.13–16 (It is instructive to com-
`pare the level of transparency provided by some16 to the
`opaque disclosures in the publications examined here.17,18)
`Such obfuscation prevents independent confirmation, and is
`contrary to basic scientific norms. We find it difficult to view
`these authors’ approach17,18 in any light other than an intent
`to mislead as to the level of originality and significance of the
`published work.
`
`Lassen - Exhibit 1062, p. 5
`
`

`

`Disclosure of potential conflicts of interest
`
`All authors are equity stakeholders in, and or employed by, Adimab LLC.
`Adimab provides commercial antibody discovery and optimization ser-
`vices for the biotechnology industry, which includes infectious disease
`programs. Adimab also has research interests in aspects of computational
`antibody design. All authors are named inventors or co-inventors in
`multiple patent filing concerning antibody discovery and engineering.
`TG was a co-founder of Arsanis, a company with an infectious disease
`focus that recently merged with X4 Pharma.
`
`ORCID
`
`http://orcid.org/0000-0002-8838-2298
`Maximiliano Vásquez
`http://orcid.org/0000-0003-0657-8412
`Eric Krauland
`
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`Lassen - Exhibit 1062, p. 6
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`2016;5:147–55.
`A outbreaks.
`EBioMedicine.
`doi:10.1016/j.
`ebiom.2016.02.021.
`
`Lassen - Exhibit 1062, p. 7
`
`