ONCOIMMUNOLOGY
`2016, VOL. 5, NO. 10, e1220467 (14 pages)
`http://dx.doi.org/10.1080/2162402X.2016.1220467
`
`ORIGINAL RESEARCH
`
`Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against
`programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved
`tumor-growth inhibition
`
`Patricia Kleinpetera,*, Laetitia Fenda,b,*, Christine Thioudelleta, Michel Geista, Nathalie Sfrontatoa, Veronique Koerpera,
`Catherine Fahrnera, Doris Schmitta, Murielle Gantzera, Christelle Remy-Zillera, Renee Brandelya, Dominique Villevala,
`Karola Rittnera, Nathalie Silvestrea, Philippe Erbsa, Laurence Zitvogelb,c,d,e,f, Eric Quemeneura, Xavier Previllea,#, and
`Jean-Baptiste Marchanda
`
`aTransgene S.A., Illkirch-Graffenstaden, France; bInstitut Gustave Roussy Cancer Campus (GRCC), Villejuif, France; cINSERM U1015, GRCC, Villejuif, France;
`dCenter of Clinical Investigations in Biotherapies of Cancer (CICBT) 1418, GRCC, Villejuif, France; eUniversity of Paris Sud XI, Kremlin Bic^etre, France;
`fDepartment of Immuno-Oncology, GRCC, Villejuif, France
`
`ARTICLE HISTORY
`Received 23 November 2015
`Revised 18 July 2016
`Accepted 30 July 2016
`
`KEYWORDS
`Monoclonal antibody;
`oncolytic virotherapy; PD-1;
`vaccinia virus; vectorization
`
`ABSTRACT
`We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the
`murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of
`mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab)
`or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the
`expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1
`binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in
`tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in
`B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to
`1,900-fold higher than the level obtained after a subcutaneous (SC)
`injection (i.e., without tumor)
`confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral
`accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT
`administration of 10 mg of J43. The IT injection of viruses induced a massive infiltration of immune cells
`C
`C
`and CD4
`). Interestingly, in the MCA 205 tumor model, WR-mAb1
`including activated lymphocytes (CD8
`and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to
`systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the
`way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as
`mAbs.
`
`Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; APC, antigen presenting cells; CDC, complement
`directed cytotoxicity; CEF, chicken embryo fibroblast; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; DC, den-
`dritic cells; ECL, enhanced chemiluminescence; ELISA, Enzyme-linked immunosorbent assay; Fab, Fragment antigen-
`binding; GM-CSF, granulocyte/macrophage-colony stimulating factor; HC, heavy chain; HRP, horseradish peroxidase;
`ICI, immune checkpoint inhibitors; IgG, immunoglobulin G; IT, intratumorally; LC, light chain; mAb, monoclonal anti-
`body; MOI, multiplicity of infection; MVA, modified vaccinia Ankara virus; PD-1, programmed cell death- 1; PD-L1,
`programmed death-ligand-1; PFU, plaque forming unit; Q-PCR, quantitative polymerase chain reaction; RR, ribonu-
`cleotide reductase; SC, subcutaneously; scFv, single-chain variable Fragment; SEC, size exclusion chromatography;
`TAA, tumor-associated antigen; TK, thymidine kinase; TME, tumor microenvironment; VEGF, vascular endothelial
`growth factor; VGF, virus growth factor; VH, variable domain of heavy chain; VL, variable domain of light chain; WB,
`Western blot; WR, Western Reserve (strain of Vaccinia virus)
`
`Introduction
`
`Oncolytic viruses belong to different groups of viruses that
`share the properties to preferentially target and destroy tumoral
`cells. Some of those oncolytic viruses are currently evaluated for
`their safety and efficacy to treat several cancers in different
`
`clinical trials.1 The leading product: ImlygicTM (a modified her-
`pes simplex virus 1 expressing human granulocyte-macrophage
`colony-stimulating
`factor, GM-CSF) has been recently
`approved by the FDA for treatment of unresectable cutaneous,
`subcutaneous and nodal
`lesions in patients with melanoma
`
`jem@transgene.fr
`
`Transgene S.A., 400 Boulevard Gonthier, d’Andernach, Parc d’Innovation, CS80166, 67405 Illkirch-Graffen-
`
`CONTACT Jean-Baptiste Marchand
`staden Cedex, France.
`Supplemental data for this article can be accessed on the publisher’s website.
`*These authors contributed equally to this work.
`#Current address: Amoneta Diagnostics, 17 Rue du Fort, 68330 Huningue.
`Published with license by Taylor & Francis Group, LLC © Patricia Kleinpeter, Laetitia Fend, Christine Thioudellet, Michel Geist, Nathalie Sfrontato, Veronique Koerper, Catherine Fahrner, Doris
`Schmitt, Murielle Gantzer, Christelle Remy-Ziller, Renee Brandely, Dominique Villeval, Karola Rittner, Nathalie Silvestre, Philippe Erbs, Laurence Zitvogel, Eric Quemeneur, Xavier Preville, and
`Jean-Baptiste Marchand.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits
`unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
`
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`e1220467-2
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`P. KLEINPETER ET AL.
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`recurrent after initial surgery.2 Vaccinia viruses also have pro-
`vided several promising oncolytic candidates such as JX-594
`(SillaJen/Transgene), GL-ONC1 (Genelux), TG6002 (Trans-
`gene) and vvDD-CDSR (University of Pittsburgh). These onco-
`lytic vaccinia viruses originate from different strains and carry
`different genomic modifications (i.e., deletions with or without
`insertion of transgenes).
`Some deletions of viral genes are necessary to improve the
`safety profile of the virus, by restricting its amplification into
`actively dividing cells only, including tumor cells.3 Oncolytic
`vaccinia viruses can also be modified to express a transgene
`(armed virus) that either increases their safety profile or enhan-
`ces their oncolytic efficiency.4 For example, TG6002 is a double
`deleted thymidine kinase (TK-), ribonucleotide reductase (RR-)
`Copenhagen vaccinia virus strain encoding an enzyme (FCU1)
`that transform the anti-fungal prodrug 5-fluorocytosine (5-FC)
`into the cytotoxic 5-Fluorouracil (5-FU).5 The double deletion
`restricts the replication of the virus to cells containing a high
`pool of nucleotides (dividing cells). Therefore, TG6002 is
`unable to replicate in resting cells contrary to tumor cells that
`are permissive and destroyed by the virus.6 Another example of
`oncolytic vaccinia virus is JX-594 (Pexa-Vec) that is currently
`evaluated in several clinical trials for treatment of different solid
`tumors.4 JX-594 is a TK- Wyeth vaccinia virus that expresses
`both human GM-CSF and bacterial b-galactosidase. GM-CSF
`is produced in the tumor where it stimulates the immune sys-
`tem and b-galactosidase is used as marker to monitor the viral
`replication.
`In immuno-competent pre-clinical models, treatment of a
`tumor by an oncolytic vaccinia virus leads from partial to total
`regression depending of the host, the nature of the tumor, the
`dose and route of administration, the strain and the modifica-
`tions of the virus and the associated treatments. These anti-
`tumoral effects of oncolytic vaccinia virus are mainly due to a
`combination of at least three recognized activities: (i) direct
`lysis or triggered apoptosis of infected tumor cells; (ii) disrup-
`tion of tumor-associated vasculature by destruction of peri-
`tumoral endothelial cells and (iii) elicitation of an immune
`response against tumor cells.6,7,8,9 Concerning the latter point,
`virus replication stimulates the innate immune system by
`inducing an immunogenic cell death that is recognized by, and
`activates, neighboring professional antigen presenting cells
`(APC) such as dendritic cells (DC).10 The presentation of
`tumor-associated antigen (TAA) by these activated APC leads
`to an enhanced adaptive immune response against tumor cells
`that in turn participates in tumor destruction.11
`Moreover, oncolytic vaccinia virus has also been combined
`with successes in pre-clinical experiments with standard thera-
`peutic treatment of cancer such as chemotherapy, radiotherapy,
`thermotherapy and immunotherapy.4 Immunotherapies are
`particularly interesting because of the potential additive or syn-
`ergistic activities between an oncolytic virus that primes an
`immune response against the tumor cells, and immunomodula-
`tion molecules (such as mAbs) that sustain and/or amplify this
`response.
`Accordingly, John et al.12 reported the combination of a vac-
`cinia virus WR TK-, virus growth factor (VGF)- with an agonist
`mAb recognizing the T cell co-stimulation molecule 4-1BB
`(CD137). Crosslinking of CD137 with an agonist mAb induces
`
`C
`
`C
`
`and CD4
`proliferation, survival and activation of both CD8
`T cells. In a murine model of breast carcinoma, intratumoral
`(IT) injections of vaccinia virus combined with systemic
`administrations of agonist anti-CD137 had a better antitumoral
`effect than any of the treatment alone. Moreover, Rojas et al.13
`have demonstrated recently in different murine tumor models
`that combination of a vaccinia virus WR (TK-, B18R-) with an
`antagonist mAb recognizing the cytotoxic T-lymphocyte-asso-
`ciated protein 4 (CTLA-4), provided a better antitumoral
`response than either of the treatment alone. Antibodies against
`the checkpoint molecules PD-1 (Programmed cell death 1),
`CTLA-4 or PD-L1 (PD-1 ligand 1) are the most documented
`immune checkpoint inhibitors (ICI) both in pre-clinical and
`clinical studies.14 PD-1 or CTLA-4 ligations, by their respective
`ligands, on the surface of the activated T cells inhibit their acti-
`vation and proliferation in lymphoid organ and in tumor
`microenvironment. Therefore, mAbs that block the interaction
`of PD-1 or CTLA-4 with their ligands stimulate proliferation of
`T cells and then enhance the immune response.15
`These results paved the way to test more combinations of
`vaccinia oncolytic virus with other immunomodulatory anti-
`bodies or equivalent molecules (e.g., either antagonists of ICI,
`or agonists of co-stimulatory molecules). Even if these combi-
`nations prove to be pre-clinically advantageous, their medical
`implementations could be hampered by the development cost
`associated with the two drugs and would be limited to antibod-
`ies or molecules that are already on the market.
`One alternative to combination therapy could be the vectori-
`zation of mAbs (or equivalent molecules) into oncolytic vac-
`cinia viruses. Vectorization consists
`in the insertion of
`sequences coding for the two chains of a monoclonal antibody
`(or equivalent molecule) into the virus genome under the con-
`trol of vaccinia promoters. Therefore, the production of “bind-
`ers” (i.e., mAb, Fab, scFv, ligand…) would occur concomitantly
`with virus replication and mainly in the tumor. However, to
`validate this approach, several key questions remain to be
`addressed such as (i) the kind of molecule that can be vector-
`ized; (ii) the functionality of produced binders; (iii) the dura-
`tion and the amount of vectorized binder that can be produced
`in vivo in an immuno-competent host; and (iv) the putative
`competitive therapeutic advantage of this armed virus in com-
`parison to its parental counterpart.
`We present here experimental results providing answers to
`the above questions. This article focuses on the vectorization, of
`mAb, Fab and scFv forms of an anti mPD-1 antibody in a vac-
`cinia virus. These three forms of binders have been chosen as
`they offer different properties that could have an impact on the
`expected antitumoral effect. Mab are bivalent and therefore bind
`to target with an increased apparent affinity (avidity effect),
`whereas scFv and Fab are mainly monovalent. Mab have an Fc
`that is responsible for high circulating half-life but also for the
`engagement of complement and recruitment of killer cells (phe-
`nomenon known as, Complement directed cytotoxicity, CDC
`and Antibody-dependent cellular cytotoxicity, ADCC, respec-
`tively). Mab are much bigger than scFv or Fab (150 vs. 25 or
`50 kDa) and therefore their diffusion into the tumor could be
`limited by their size. Mab have also complex heterotetrameric
`structure that may impair their level of expression compared to
`scFv that are monomeric and Fab that are dimeric.
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`This article presents the vectorization in vaccinia virus of
`mAb, Fab, and scFv recognizing mPD-1. MAb, Fab, and scFv
`have been produced in vitro upon infection of permissive cells
`by the corresponding recombinant viruses. These molecules
`have been purified and characterized as functional (i.e., inhibit
`the PD-L1/PD-1 interaction). The kinetic of expression of the
`mAb in mice after IT injection of vaccinia virus carrying the
`sequences coding for the anti-PD-1 heavy and light chains was
`also investigated. Finally,
`in an immunocompetent murine
`model, the antitumoral efficacy of the unarmed virus, com-
`bined or not, with an anti-mPD-1 was compared with that of
`armed vaccinia viruses encoding for either mAb or scFv against
`mPD1. In this model, armed viruses were found as efficient as
`
`Figure 1. Schematic representation of the expression cassettes inserted in TK
`locus of the five WR vaccinia virus candidates constructed for this study. The
`insertion of cassettes disrupted the TK gene. The RR gene (not shown here)
`was also deleted in all the virus used in this study. For mAb and Fab each
`chain (heavy and light) is under the control of a different and independent
`promoter (namely p7.5K or pH5R) with their own strengths (i.e., level of pro-
`tein expression). Mab corresponds to the whole molecule with two heavy
`and two light chains assembled to form a bivalent molecule 2 £ (Light C
`Heavy). Fab corresponds to one light chain assembled with one heavy chain
`lacking their dimerization domains (i.e., hinge and Fc). Fab is a monovalent
`molecule. ScFv corresponds to the genetic fusion of VH to VL via a poly-GS
`linker. ScFv is monovalent molecule but a fraction of it can dimerize to form
`a divalent molecule. The variable and the constant domains of the light and
`heavy chains are represented with hatched and plain patterns, respectively.
`
`ONCOIMMUNOLOGY
`
`e1220467-3
`
`the combination of unarmed virus with anti-mPD-1 mAb, in
`term of effect on tumor growth and survival.
`
`Results
`Recombinant mAb, Fab and scFv, vectorized in WR
`vaccinia virus, are secreted and correctly assembled
`
`J43 mAb DNA sequence was designed using the publically
`available partially disclosed sequences of heavy and light chain
`(patent US 7,858,746 B2). The partial sequences were com-
`pleted by the constant heavy chain of anti-CD79b mAb and the
`signal sequence of the light chain of anti-CD79b mAb.
`Five WR recombinant vaccinia viruses were constructed by
`insertion at the TK locus of either the light and heavy chains
`(mAb and Fab) or the corresponding scFv (Fig. 1). In the case
`of mAb and Fab, two versions were constructed with the heavy
`and the light chain under the control of either pH5R or p7.5K
`promoters (i.e., WR-mAb1, WR-mAb2, WR-Fab1 and WR-
`Fab2). The WR strain was chosen for its ability to better propa-
`gate in murine cells in comparison to other vaccinia virus
`strains. All the WR virus presented in this article were also
`deleted of the ribonucleotide reductase gene (RR-).
`In order to select the combination chain/promoter that
`allows the best expression of the mAb and Fab, with a correct
`assembly, Chicken Embryo Fibroblasts (CEF) were infected
`with the two versions of recombinant virus and cell superna-
`tants analyzed by immunoblot, with a polyclonal anti-hamster
`IgG. Gel electrophoresis was performed in non-reducing condi-
`tions to preserve the assembly of light and heavy chains and to
`allow an optimal detection (i.e., the polyclonal antibody used
`for detection did not recognized reduced IgG and Fab chains).
`Fig. 2A demonstrates that WR-mAb1 and WR-mAb2 were
`equally able (in roughly same quantities) to generate a molecule
`with an assembly pattern comparable to that of commercial J43
`
`Figure 2. Expression of mAb, Fab and scFv by infected CEF. CEF in six wells plate were infected at MOI 0.2 by either WR (TK- RR-: negative Control: lanes 1 and 7),
`WR-mAb1 (lane 2), WR-mAb2 (lane 3), WR-Fab2 (lanes 4 and 9), WR-Fab1 (lanes 5 and 10) and WR-scFv (lane 8). After 24 h of infection the culture supernatants were
`collected and loaded on SDS-PAGE in non-reducing (A) or reducing conditions (B). Commercially available J43 was also loaded (lane 6) as a reference. After transfer onto
`PVDF membrane, mAb, Fab and scFv were detected using either an anti-hamster IgG (A) or an anti-Histidine tag (B). M: molecular markers. Arrow: putative dimeric light
`chain. Arrow head: correctly assembled Fab.
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`e1220467-4
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`P. KLEINPETER ET AL.
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`(i.e., an apparent size corresponding to two heavy and two light
`chains linked together). However, in case of WR-mAb2, an
`extra band between 43 and 55 kDa was clearly visible (lane 3).
`A band, in the same position, with the same intensity, was also
`clearly visible in case of WR-Fab2 (lane 4). WR-mAb2 and
`WR-Fab2 viruses have in common the same light chain under
`the same strong promoter (pH5R). Moreover, it is well known
`that antibody light chain can assemble in homodimers when
`overexpressed.16 This extra band, migrating between 43 and
`55 kDa, could correspond to the light chain disulfide cross-
`linked homodimers with a theoretical mass of 47 kDa.
`Fig. 2A also demonstrates that the WR-Fab1 produced the
`highest amount of correctly assembled Fab without any detect-
`able misassembled by-product (lane 5).
`In the case of WR-Fab1 & 2 and WR-scFv, cell supernatants
`were analyzed by immunoblot (gel electrophoresis in reducing
`conditions) with an anti-His tag to detect either the tagged scFv
`or the tagged heavy chain fragment of the Fab. Fig. 2B demon-
`strates that the scFv was expressed at the expected size (i.e
`27.5 kDa) and that infection by WR-Fab1 generated a larger
`amount of heavy chain fragment than infection by WR-Fab2.
`These expression tests were also performed using two mamma-
`lian cell lines (BHK-21 and A549) and provided similar results
`(data not shown). All
`together,
`these results showed that
`infected cells secreted mAb, Fab and scFv at detectable levels
`and, for some constructions, with the expected light and heavy
`chain assembly. WR-mAb1 and WR-Fab1 were selected for
`further experiments, as the expression pattern of their trans-
`genes was closer to the expected ones than those of WR-mAb2
`and WR-Fab2.
`The replicative and oncolytic abilities of WR-mAb1, WR-
`Fab1 and WR-scFv were compared to those of the parental
`virus (WR) to assess the impact of the different transgenes on
`the virus properties. Three cells lines were used: BHK-21 as
`permissive and production cell line, B16F10 and MCA 205 as
`murine
`tumor
`cell
`lines
`(melanoma
`and fibrosarcoma
`
`respectively). Figs. 3A–C demonstrates that on the three cell
`lines tested, none of the transgenes had a significant impact on
`the viral replication. It is noteworthy that the replication of WR
`viruses was similar in BHK-21 and MCA 205 but significantly
`slower in B16F10 (even if at 72H post-infection the same pla-
`teau of virus titer is reached for the three cell lines. Fig. 3D).
`The oncolytic activity of the different viruses was evaluated in
`the three cell lines at two MOI. Because of the intrinsic variabil-
`ity of the virus titration assay, two oncolytic activities are con-
`sidered different when at least one log difference is observed
`between them (i.e., same cell viability observed for the two
`viruses but at MOI different by at least 10-fold). According to
`this standard, neither the different transgenes nor the addition
`of J43 monoclonal antibody in culture medium had a signifi-
`cant impact on the oncolytic activity of the WR vaccinia virus
`tested (Figs. 3E–G; Fig. S1). Moreover, oncolytic sensitivity of
`the three tested cell lines follows the same pattern as the repli-
`cation results, with BHK-21 and MCA 205 cell lines being the
`most sensitive to oncolysis and B16F10 the most resistant. The
`relative resistance of B16F10 compared to MCA 205 and BHK-
`21 did not result in a reduced amount of produced mAb1, indi-
`cating that WR-mAb1 is clearly infecting and replicating in
`B16F10 (Fig. 3H).
`
`Purification and characterization of recombinant mAb1,
`Fab1 and scFv expressed by WR-infected cells
`
`Recombinant mAb1, Fab1 and scFv from pooled supernatants of
`WR-infected CEF were successfully purified to homogeneity by
`affinity chromatography followed by size exclusion chromatogra-
`phy (SEC). This expression/purification process was repeated once
`for mAb1 and Fab1 (batches 1 and 2). None of the three purified
`proteins contained significant amount of aggregated material. ScFv
`eluted in two separated peaks on gel filtration (Fig. 4A). The peak
`that eluted first (peak 1) had a surface 7-fold lower than the second
`one (peak 2). Peak 1 and peak 2 could correspond, respectively, to
`
`Figure 3. In vitro replication and oncolytic activities of the different viruses. Replication of WR-mAb1, WR-Fab1, WR-scFv and WR, and their effects on cell viability, have
`¡2 on BHK-21 (A),
`been assessed on MCA 205, B16F10 and BHK-21 cell lines. The virus replication was monitored over time by q-PCR after an initial infection at MOI 10
`B16F10 (B) and MCA 205 (C). The replication of WR-mAb1 on the three cell lines was compared and shown in panel D. Cell viability of BHK-21 (E), B16F10 (F) and MCA
`¡2 and 10¡3). MAb1 concentration in
`
`205 (G) was measured using trypan blue exclusion assay after 5 d post-infection with the different viruses and at two MOI (10
`supernatants collected 5 d post-infection was determined using a quantitative hamster IgG ELISA (H). Represented values are the mean (C/¡ standard deviation) of at
`least three measurements (see material and methods for details).
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`e1220467-5
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`Figure 4. Characterization and quantification of mAb, Fab and scFv purified from supernatants of infected CEF. Size exclusion chromatography profile of scFv after the
`affinity chromatography step (A). ScFv eluting from the Ni affinity column was pooled and loaded of Superdex 75 10/300 equilibrated in PBS. Absorbance at 280 nm and
`elution volume were recorded. Area of peak 2 was about 7-fold area of peak 1. V0 is the void volume of the column. SDS-PAGE profiles of purified recombinant mAb1
`and scFv in reducing and non-reducing conditions (B). 1 mg of purified recombinant mAb1 and scFv were loaded on SDS-PAGE in reducing (R) and non-reducing (NR)
`conditions. J43 (BioXcell) was loaded as reference in the case of mAb1. Quantification of mAb, Fab and scFv in supernatants of the infected cells (C). Supernatants of
`infected CEF were recovered 48 h after infection and loaded on stain-free SDS-PAGE together with corresponding purified and quantified molecules as standards. Fluores-
`cence intensity of the bands of interest was measured for each supernatant. Quantity of produced protein was determined using the fluorescence of standards as refer-
`ence. Represented values are the mean (C/¡ standard deviation) of three measurements.
`
`dimeric and monomeric scFv. Purified mAb and scFv loaded, on
`SDS-PAGE, displayed the expected band pattern in both non-
`reducing and reducing conditions without any visible contaminant
`(Fig. 4B). Recovered quantities and concentrations for each purifi-
`cation are summarized in Table 1. The purified molecules were
`used as standard on SDS-PAGE to quantify by fluorescence the
`corresponding protein in the supernatants of infected CEF (i.e.,
`starting material of the purifications). Results are summarized in
`Fig. 4C that shows that the scFv was expressed at the highest
`amount (»2 and 9-fold in mass or »4 and 54-fold in mole com-
`pared, respectively, with Fab and mAb).
`
`The glycosylation of the Fc part of a mAb plays an impor-
`tant role in its ADCC and CDC potencies. Therefore, glycosyla-
`tion of the purified mAb1, J43 and Rituximab (as reference)
`was analyzed by LC-MS/MS after trypsin digestion of the anti-
`bodies (mass of the glycosylated peptides). The results show
`that the glycosylation profiles of recombinant mAb1, commer-
`cial J43 and Rituximab were comparable (Fig. S2). This result
`indicates that in these conditions (i.e., hamster IgG vectorized
`in WR vaccinia virus and expressed by infected CEF) the
`glycosylation of Fc was similar to the one observed for a human
`IgG expressed by a stably transfected Chinese Hamster Ovary
`(CHO) cell line (i.e., Rituximab).17
`
`Table 1. Quantities and concentrations of purified recombinant mAb1, Fab1 and
`scFv recovered from supernatants of WR-infected CEF.
`
`Recombinant mAb, Fab and scFv are functional
`
`Virus/batch
`
`Conc (mg/mL)
`
`Total quantity (mg)
`
`WR-scFv peak 1
`WR-scFv peak 2
`WR-Fab1 batch1
`WR-Fab1 batch2
`WR-mAb1 batch1
`WR-mAb1 batch2
`
`22
`143
`51
`33
`33
`34
`
`28
`287
`30
`56
`13
`32
`
`In order to verify that the purified recombinant mAb1, Fab1
`and scFv were able to bind to mPD-1, these molecules were
`incubated with T lymphoma-derived EL4 cells that spontane-
`ously display mPD-1 at their surface. The binding of anti-PD-1
`molecules to cells was analyzed by flow cytometry. Fig. 5A
`shows that recombinant mAb1, Fab1 and scFv efficiently label
`the murine T lymphocyte cell line.
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`e1220467-6
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`P. KLEINPETER ET AL.
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`Figure 5. Binding of the purified recombinant mAb1, Fab1, scFv to mPD-1. Binding of purified mAb1, Fab1 and scFv to mPD-1-positive EL4 cells (A). Murine T lymphoma
`EL4 cells were incubated with commercially available J43 (positive control), hamster IgG (negative control), Fab1, monomeric scFv, mCD80-hFc-6xHis (His-tagged positive
`control, CD80 binds to PD-L1 expressed by EL4 cells) or hErbB2-hFc-6xHis (His-tagged negative control). Binding of mAbs and 6xHis-tagged proteins was detected by
`flow cytometry using either FITC-conjugated mouse anti-hamster IgG antibody or PE-conjugated mouse anti-His tag antibody. Competition between purified recombinant
`mAb1, Fab1, scFv (monomeric and dimeric fractions), J43 and mPD-L1 (B and C). Binding of biotinylated mPD-L1-hFc to immobilized mPD-1, or binding of unlabeled
`mPD-L1-hFc to EL4 cells, in presence of increasing concentrations of competitors (J43, mAb1, Fab1, scFv) or negative control (Hamster IgG) was measured in ELISA (B) or
`flow cytometry (C) assays. PD-L1 was detected using either streptavidin-HRP or anti-human-Fc-PE. The signal obtained with the lowest concentration of hamster IgG was
`set as 100%. Represented values are the mean of two normalized measurements.
`
`J43 has been selected for vectorization because it inhibits the
`interaction of mPD-1 with mPD-L1.18 Therefore, two competi-
`tive assays were designed to determine if the recombinant
`mAb1, Fab1 and scFv, expressed by recombinant WR vaccinia
`viruses infected CEF, were functional. The first assay was an
`ELISA in which the binding of labeled mPD-L1 to immobilized
`mPD-1-Fc was monitored. The second assay is a flow cytome-
`try assay in which the binding of unlabeled mPD-L1 to EL4
`cells was monitored. In both assays, the three forms of blocker
`tested were able to inhibit the interaction mPD-1/PD-L1 in a
`dose-dependent manner (Figs. 5B and C). Dimeric formats
`(either mAb1 or scFv peak1) were more potent competitor
`than their monomeric counterpart
`formats (Fab1 or scFv
`peak2) indicating the importance of avidity in the interaction.
`Interestingly, the purified recombinant mAb1 appeared in both
`assays to be more potent than the commercial mAb used as
`positive control indicating that the design of mAb1 from par-
`tial J43 sequence and anti-CD79b hamster IgG was correct.
`Note that Fab does not have any advantage over scFv (either
`in produced quantity or affinity for mPD-1). Therefore, the
`characterization of WR-Fab1 was not pursued.
`All together these data demonstrate that the vectorization in
`WR vaccinia virus of the three forms of anti-mPD-1 has yielded
`to functional molecules.
`
`Vectorization of mAb1 in WR allows its intratumoral
`expression
`
`In order to determine if the vectorized mAb could be produced in
`vivo, mice with and without subcutaneous B16F10 or MCA 205
`tumors were injected with WR-mAb1 (vehicle and WR were also
`injected as negative controls for B16F10 and MCA 205 models,
`respectively). Viruses were injected either SC (without tumor) or
`IT. In the case of B16F10 model, the virus was injected once
`whereas in the MCA 205 model it was administered twice (3 d
`apart). As a benchmark, 10 mg of commercial J43 was also injected
`IT. Virus intratumoral replication was assessed at different time
`points, by q-PCR on a dedicated experiment in the MCA 205
`model. Concentration of mAb was measured at different time
`points, both in serum and in tumor after a gentle homogenization
`to preserve cells integrity (interstitial fluid). All the negative control
`samples (vehicle in case of B16F10 or unarmed vector in case of
`MCA 205) did not yield any detectable hamster IgG (i.e., concen-
`tration below LOD). Fig. 6 shows that when WR-mAb1 was
`injected IT, mAb1 was detected in tumor from day 1 to day 11 with
`the highest concentrations reached at D3 (for MCA 205, before the
`second injection) or D5 (for B16F10). The concentrations of mAb1
`in serum followed the same trend as in tumor with peaks of accu-
`mulation at D3 or D5 depending on the model. However, in
`
`Replimune Limited Ex. 2020 - Page 6
`Transgene and Bioinvent International AB v. Replimune Limited
`PGR2022-00014 - U.S. Patent No. 10,947,513
`
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`ONCOIMMUNOLOGY
`
`e1220467-7
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`Figure 6. In vivo expression of mAb1 after injection of WR-mAb1. C57BL/6 mice were implanted SC with either 3 105 B16F10 (A, C) or 8 105 MCA 205 cells (B, D). When tumors reached
`100–200 mm2 (B16F10) or 40–60 mm2 (MCA 205), 107 pfu of WR-mAb1 or WR (negative control) or J43 (BioXcell, 10 mg) were injected IT. For mice without tumor, viruses were
`injected S.C. at the same time points. For MCA 205 tumors only, a second injection of the virus was performed 3 d after the first one. Blood, and tumors of three mice were collected
`at each time point i.e.: Days 1, 3 (MCA 205 only), 5, 7 (MCA 205 only) and 11 after virus or antibody injections. Concentrations of recombinant mAb or J43 were measured in tumor
`homogenates (A, B) or in sera (C, D) by sandwich ELISA using anti-hamster IgG antibodies and J43 as standard. The limit of quantification (LOQ D 2-fold the mean of blanks) of the
`ELISA was 2 ng/mL. The negative controls had no detectable hamster IgG (i.e., concentrations below the LOQ). The mean and the standard deviation of three measurements are
`represented.
`
`contrast to the situation in tumor, significant amounts of mAb1 in
`serum were no more detected after day 5. Interestingly, when the
`recombinant virus was injected by IT route, the concentration of
`mAb1 in serum was up to 68 (MCA 205) or 1,900 (for B16F10)
`times higher than the concentration measured when WR-mAb1
`was injected SC (i.e., without tumor). These results strongly sup-
`port the concept of preferential virus replication in tumor. The
`maximum concentration of mAb1 either in serum or in tumor was
`higher in the case of the B16F10 model after a single injection than
`in the case of MCA 205 model with two injections. This result
`could reflect the difference of antibody productivity observed in
`¡2), but could be also
`vitro with the two cell lines (Fig. 3H, MOI 10
`explained by a difference of tumor mass, and structure, at the first
`injection (»600 vs. »170 mg for, respectively, B16F10 and MCA
`205).
`The pharmacoki