Meeting Product Development Challenges in Manufacturing Clinical Grade Oncolytic Adenoviruses

Meeting product development challenges in manufacturing clinical grade oncolytic adenoviruses

Peter K Working*,1, Andy Lin1 and Flavia Borellini1

1Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, CA 94080, USA

Oncolytic adenoviruses have been considered for use as issue, is possible because the expression of the adeno- anticancer therapy for decades, and numerous means of virus genome is a tightly regulated cascade, which can be conferring tumor selectivity have been developed. As with considered as divided into early (E) and late (L) phases. any new therapy, the trip from the laboratory bench to the The E1 region gene products are critical for efficient clinic has revealed a number of significant development expression of essentially all the other regions of the hurdles. Viral therapies are subject to specific regulations adenovirus genome, such that controlling the expression and must meet a variety of well-defined criteria for purity, of the E1 region will effectively control adenoviral potency, stability, and product characterization prior to replication. Oncolytic adenoviruses that utilize this their use in the clinic. Published regulatory guidelines, approach include CG7870 (Yu et al., 2001), which although developed specifically for biotechnology-derived utilizes prostate-specific transcription regulatory ele- products, are applicable to the production of oncolytic ments to control E1 and target prostate cancer, and adenoviruses and other cell-based products, and they CG0070 (Bristol et al., 2003), which utilizes the human should be consulted early during development. Most E2F-1 promoter cloned in place of the endogenous E1a importantly, both the manufacturing process and the promoter to target the diverse group of cancers that development of characterization and release assays should have a defective retinoblastoma (Rb) pathway. CG0070 be science-driven, use the best available science and also encodes the cDNA for human granulocyte-macro- technology, and must consider the unique nature of the phage colony-stimulating factor (GM-CSF) in the E3 product: a living, and mutatable, virus. Potentially region. Both of these oncolytic adenoviruses are significant impacts on product quality and safety stem currently being evaluated in the clinic. from the possibility of genetic instability related to over- The second approach for engineering tumor specifi- engineering the viruses (as evidenced by their recombina- city entails the deletion of viral gene function(s) that are tion and/or occasional reversion to wild-type virus during critical for efficient viral replication in normal cells, but manufacturing). This report provides examples of some of dispensable in tumor cells. O’Shea and McCormick the critical components affecting the development and discuss this method further in this issue. Examples production of clinical grade material and summarizes the include Rb-inactivated E1a-mutated variants such as significant progress made in recent years. Addl922–947 and Ad5-D24RGD, and p53-defective Oncogene (2005) 24, 7792–7801. doi:10.1038/sj.onc.1209045 E1b-deleted adenovirus variants like Addl1520 (Onyx- 015) and Ad5-CD/Tkrep (Bischoff et al., 1996; Freytag Keywords: oncolytic adenovirus; product development; et al., 1998; Heise et al., 2000; Johnson, 2002). These manufacturing mutants have deletions of the E1a-CR2 and the E1b-55 kD gene regions that are responsible for binding and inactivating Rb family members and p53, respectively. Such viruses were originally believed to target cancer The idea that viruses could be useful as selective cells with genetic defects in the Rb and p53 pathways, anticancer agents is not a new one, dating back to which are defective in most human tumors (Biederer almost a century. In recent years, a variety of tumor- et al., 2002), but more recent work has shown that selective viruses, including over 50 tumor-selective differential late viral RNA export in tumor vs normal oncolytic adenovirus variants have been tested for cells, rather than p53 activation, appears to be the major antitumor activity in animals and humans. There are determinant of the selectivity of Onyx-015 (O’Shea three broad approaches to imbuing adenoviruses with et al., 2004). tumor selectivity. First, the viral genome can be The third approach is based on receptor-mediated engineered to limit the expression of essential viral targeting of tumor cells through genetic modification of genes to tumor tissues via the use of tumor- or tissue- the adenoviral capsid and can be termed transductional specific promoters, that is, by transcriptional targeting. targeting; Mathis et al. discuss it in detail in this issue. This approach, discussed in detail by Ko et al. in this To successfully create a virotherapy agent using this approach requires ablation of the natural viral uptake *Correspondence: PK Working; mechanism (deselection), identification of tumor-specific E-mail: [email protected] receptor targets on cancer cells, and the engineering of Product development of oncolytic adenoviruses PK Working et al 7793 new ligands into the viral coat (reselection), all without replication-competent and replication-deficient, and disrupting viral integrity (Douglas et al., 1999; Alemany the detection of wild-type adenovirus contaminants or and Curiel, 2001; Hemminki et al., 2001; Suzuki et al., wild-type virus produced during manufacturing. These 2001). unique components of product quality assessment of an The objective of this review is to discuss challenges in adenovirus can be evaluated using a bioamplification manufacturing selectively replicating oncolytic adeno- assay, that is, an assay that amplifies either pre-existing viruses for use in clinical trials and, eventually, recombinant replication-competent adenovirus (RCA) commercial use. It focuses on the developmental hurdles variants or wild-type adenovirus and that provides for transcriptionally targeted adenovirus variants made selective pressure to force the expression of genetic with the first strategy, such as CG7870 and CG0070, but instability. most of the hurdles discussed are relevant to the development of any type oncolytic adenovirus and, to Engineering a stable virus a lesser extent, any type of viral-based therapeutic. For more detailed discussions of the basis of adenoviral An unintended effect of engineering tumor-selective replication and strategies for its control, the interested adenoviruses is the potential introduction of genomic reader is directed to Ko et al. and O’Shea et al. (this instability. Wild-type adenoviruses have evolved over volume) and several other recently published reviews the millennia to do their job admirably well: they (Alemany et al., 1999; Biederer et al., 2002; Kirn, 2001; replicate efficiently in host cells and generate thousands Reid et al., 2002; Yu et al., 2002; Wakimoto et al., 2003). of progeny virions to maintain the infection. The adenoviral genome is stable, yet malleable enough to adapt to new circumstances, and changes that attenuate Development of oncolytic adenoviruses its replicative ability may result in genomic instability as the adenovirus attempts to revert to wild type. As with any drug or biologic product destined for use in Engineered oncolytic adenoviruses must retain a stable humans, oncolytic adenovirus products are subject to genome for consistent production and biological activ- broad, yet detailed regulations and must meet a variety ity, but engineering an adenovirus to direct its replica- of well-defined criteria for purity, potency, stability, and tion preferentially to tumor cells, effectively attenuating product characterization, among other things. Guidance its ability to replicate, may introduce significant genomic can be found in the US and EU regulatory documenta- instability reflecting the effort of the engineered virus to tion and policies for Good Manufacturing Practices ‘recover’ from that attenuation. (GMP) for making therapeutic products for human use. Overengineering, for example, the introduction of Particularly relevant are the guidelines for products large variations into the adenoviral genome, may result derived from biotechnology, the latest changes to which in significant genetic instability that prevents the clinical have been described in a recent review article (Jones and use of a virus and may lead to recombinants of differing Levine, 2004). Although developed specifically for specificity and/or potency, potentially including in- biotechnology-derived products, these guidelines are creased potency beyond that of wild-type adenovirus. applicable to the production of oncolytic adenoviruses Examples of genomic instability are reported in the and other cell-based products, and it is critical to consult literature, including dual-promoter-controlled viruses in them during product development, that is, they are which the same promoter is used twice in a single relevant and, perhaps more to the point, they are adenoviral backbone to improve tumor selectivity. In required. However, since oncolytic adenoviruses the Calydon/Cell Genesys, Inc. (CGI) virus CV716, E1a are living and, therefore, more variable, less stable and and E1b are independently controlled by separate PSE even mutatable, there are consequent unique and elements (Henderson et al., 2001; Henderson and Yu, significant challenges for their production, a major one 2002). Tumor selectivity was improved; compared to the being complying with regulatory guidelines for manu- single PSE-controlled oncolytic adenovirus CG7060, facturing and release while producing a living, poten- the prostate specificity of CV716 was increased over tially replicating product. The nature of the dilemma 100-fold. Southern blot analysis, however, revealed self- becomes clearer when one considers that the situation is inactivation of the virus due to homologous recombina- analogous to manufacturing a traditional pharmaceu- tion between the two identical inserted TREs. No effort tical or biotechnology-derived protein that hypotheti- to assess changes in viral potency was reported in this cally can reproduce itself, change itself to something else study. Similarly, the use of homologous controlling with decreased or increased potency or even with a elements in Onyx-411 results in recombination of the different biological target, and that either can happen viral backbone to self-inactivating recombinants as well during production or purification or both. This char- as to recombinants with undetermined changes in acteristic is perhaps the most significant development potency and specificity (Shen, 2003). In contrast, the challenge for oncolytic adenoviruses. With adeno- use of heterologous promoters to control the E1a and viruses, the biological and manufacturing consistency E1b transcription units results in a more genetically of the product depend in large part on the genetic stable virus, for example, CG7870, in which E1a is stability of the virus itself as well as the nature of the cell controlled by the rat probasin promoter and E1b by line used to produce the virus. Relevant issues include PSE (Yu et al., 1999; Henderson et al., 2001). However, the generation of recombinant adenoviruses, both OAS403, an oncolytic adenovirus developed by Genetic

Oncogene Product development of oncolytic adenoviruses PK Working et al 7794 Therapy, Inc. (GTI), uses heterologous promoters but is the potential for recombination during the manufactur- not genetically stable. In this variant, the E2F-1 ing process or to amplify any contaminating wild-type promoter controls expression of the E1a transcription virus to detectable levels. Nonpermissive cells are used unit and the hTERT promoter drives the expression of to assess the genetic stability of the engineered virus the E4 transcription unit, plus the packaging signal has since they provide selective pressure as described above. been relocated from the left to the right end of the Both pre-existing recombinants and those generated genome and a polyadenylation signal has been inserted during passaging can be detected by PCR and/or upstream of the E1a promoter (Ryan et al., 2004). restriction digestion if the structure of the potential Although highly tumor-selective, quality testing re- recombinant is known or can be predicted a priori. vealed a set of four or more recombinants detectable Altered potency in late-passage virus, which indicates after serial amplification through a nonpermissive cell that there has been a significant change in the viral line (see discussion of this type of assay below). The population, can be detected using a standard set of recombinants had altered selectivity and decreased in vitro cytotoxicity assays. potency. Sequence analysis revealed the presence of the packaging signal at both the right and left ends of Assessment of viral genetic stability and detection the genome, plus the relocation of an indeterminate set of wild-type adenovirus of adjacent E4 sequences to the left end, suggestive of rearrangement by either intermolecular recombination Wild-type adenovirus that contaminates a preparation or polymerase jumping (GTI and Cell Genesys, Inc., of engineered oncolytic adenovirus is typically detected unpublished data). Further work with other E4-altered using quantitative or real-time PCR techniques (Bernt adenoviruses that retain the packaging signals in its et al., 2002). The specificity of the assay for wild-type native left-end position suggested that E4 modifications adenovirus is conferred by the use of a forward primer alone were sufficient to induce an unacceptable degree of that hybridizes at the junction between the E1a genomic instability (Yu and Li, unpublished), and this promoter and E1a gene in the case of an E1-controlled engineering approach has been abandoned. engineered virus like CG0070 for example. In CG0070, The genomic instability of engineered adenoviruses this junction is eliminated by deletion of the endogenous will be most evident in vivo, where many of the infected E1a promoter and subsequent insertion of the E2F-1 cells will be nonpermissive for replication of tumor- promoter. Whereas both wild-type adenovirus and selective adenoviruses. The selective pressure imposed CG0070 can hybridize with the probe and the reverse may result in the emergence of more efficient or primer, only adenoviruses containing the wild-type E1a nonselective and possibly detrimental variants. It is promoter–E1a gene junction are expected to hybridize therefore critical to assess the genetic stability of the with the forward primer, initiate DNA polymerization, candidate adenovirus early in development using serial and produce a PCR signal, which is therefore indicative viral passaging in nonpermissive cells in vitro to assess of the presence of wild-type adenovirus. However, the genome stability in a bioamplification assay. The use of level of sensitivity of the PCR method, about one wild- nonpermissive cells mimics the situation in which a type adenovirus in 106–108 viral particles (the latter less tumor-selective oncolytic adenovirus infects a normal, common and only for shorter PCR products) is rather nontumor cell. low considering that clinical doses are in the range of 1012 viral particles per patient. Greater sensitivity can be achieved using a bioamplification assay in nonpermis- Bioamplification assays sive cells (i.e. cells that are minimally permissive for the engineered oncolytic adenovirus and maximally permis- Semiquantitative bioamplification systems are used to sive for wild-type). The selective pressure associated detect recombination that may occur during manufac- with passage in nonpermissive cells in vitro also provides turing. These assays are able both to detect contaminat- a sensitive method to monitor genetic stability of an ing wild-type adenovirus and evaluate the genomic engineered adenovirus. Nonpermissiveness for the tu- stability of an engineered adenovirus. The viral material mor-selective adenovirus is not absolute, however, with tested in such assays requires multiple rounds of varying degrees of selectivity among various cell lines. infection (passages). Amplification is initiated by infec- For example, the assay could be based on monitoring tion of permissive or nonpermissive cells (depending the preferential replication of wild type (or wild-type- upon the objective of the assay) with the purified like recombinants) in an oncolytic adenovirus prepara- working virus stock, which represents passage 0 (p0). tion in WI-38, a normal lung fibroblast cell line. WI-38 After several days of growth, the infected cells are cells provide a three-log selectivity factor between wild- harvested for viral purification using a CsCl gradient type and engineered adenoviruses in the case shown centrifugation method, or, alternatively, a clarified viral (Figure 1), but this factor is likely to vary among lysate (CVL) is prepared. An aliquot of purified virus or different oncolytic adenoviruses. As a result, the of the CVL is used to infect the cells for the next nonpermissive cell line must normally be selected passage. The process is repeated for several passages, specifically for each individual engineered virus to with four passages (p4) typically providing acceptable maximize both selective pressure and the degree of sensitivity. Permissive cells (preferably those that serve selectivity. This form of the assay maximizes the as producer cells during production) are used to estimate likelihood of detecting wild-type adenovirus in the

Oncogene Product development of oncolytic adenoviruses PK Working et al 7795 1.E+08 a 125 wild type adenovirus LD (ppc) 1.E+07 50 P0: 3.2 100 1.E+06 P4: 0.3

1.E+05 3 log selectivity 75

1.E+04 50

1.E+03 % of Control

pfu/mL on Day 3 25 1.E+02 Oncolytic adenovirus

1.E+01 0 -4 -3 -2 -1 0 1 2 3 4 5 1.E+00 PPC (log10) 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 Vector Particles per Cell b 125 LD50 (ppc) Figure 1 Relative selectivity of WI-38cells for wild-type and a 100 P0: 7.2 tumor-specific oncolytic adenovirus. Specific cell lines should be P4: 5.2 selected for each engineered adenovirus to maximize the selective advantage of wild-type adenovirus 75 oncolytic adenovirus product, with a typical bioampli- 50 fication assay able to detect the equivalent of one wild- % of Control 25 type adenovirus in 108—109 viral particles of oncolytic adenovirus, a two- to three-log improvement over the 0 PCR-based assay. -4-3 -2-1012345 Amplification in nonpermissive cells is also the PPC (log10) method of choice for detecting genomic instability, c 125 since multiple serial passages in nonpermissive cells LD (ppc) applies selective pressure similar to that in vivo, such that 50 100 P0: 17.7 unstable viruses may be subject to modifications that P4: 1.3 affect biological activity detectable using an in vitro 75 cytotoxicity assay. Other assays, including PCR and restriction digestion, can provide additional information 50

on the nature of the recombinant(s), if any, but the % of Control quickest, and arguably the more relevant way to detect 25 recombinants is the cytotoxicity assay. Figure 2 illustrates the outcome of cytotoxicity testing of three 0 different oncolytic adenoviruses. As shown in Figure 3, -4-3 -2-1012345 in all three variants the E2F-1 promoter drives the E1a PPC (log10) transcription unit. In two of them (Var1 and Var3) the Figure 2 Relative cytotoxicity in A549 cells of three different E4 transcription unit is controlled by the hTERT engineered adenoviruses serial-passaged in nonpermissive WI-38 cells, comparing passage 0 (p0; closed squares, ’) to passage 4 (p4; promoter; in the other (Var2) hTERT controls E1b. closed triangles, m) virus. Cell viability is expressed as a percentage The packaging signal is relocated next to the right-end of the uninfected control and processed for a sigmoidal dose– ITR in Var1, whereas it remains in its native left-end response curve fit. Left shift of a curve indicates increased potency. position in the other two. All three variants were Each data point is the mean of three replicates7s.d. Relative LD50 independently taken through four passages of amplifica- values are shown in each pane. (a) Var1; E2F-1 controlling E1a, hTERT controlling E4, packaging signal relocated to right end of tion in nonpermissive WI-38cells, which will exert the genome; (b) Var2; E2F-1 controlling E1a, hTERT controlling selective pressure on the attenuated virus. Virus from the E1b, packaging signal at left end of genome (native site); (c) Var3; final p4 passage was purified and used in cytotoxicity E2F-1 controlling E1a, hTERT controlling E4, left-end packaging tests in A549, SW780, or WI-38 cells. Data from the signal A549 cytotoxicity assay are shown in Figure 2. Viability curves were left-shifted in the Var1 and Var3 p4 virus result in a more genetically unstable adenovirus and preparations, indicating that a significant increase in should therefore be avoided if possible. potency for the virus had occurred and suggesting that a substantial fraction of the virus population represented Assessment of potential for recombination during recombinant forms. The potency of Var2, on the other production hand, was not increased, signifying that it had a relatively stable genome under the conditions of the Multiple serial passages in permissive cells such as A549 assay. Essentially identical results were seen with the or HeLa-S3 can be used to mimic the manufacturing other two cell lines with the three variants. These conditions as a means to evaluate viral stability under findings suggest that modification of the E4 region, or, these conditions. Cell lines selected for this test should more generally, the right-end of the viral genome will be identical to the producer cell line that will be used in

Oncogene Product development of oncolytic adenoviruses PK Working et al 7796 Endogenous promoters ψ

Wild-type E3 9K-2 E4 9K-1 Adenovirus E1A E1B Poly(A)

E2F -E1B

E1B 1 E1B E1A Prom-2 E1A 1 E1B E1B Prom. E1B E1A Prom-1 E1A 1kb+ E2F-E1B

Var1 E3 ψ E4 hTERT E2F Var2 E3 hTERT Poly(A)

Var3 E3 E2F E4 hTERT Figure 3 Schematic structures of three viral variants, including a wild-type adenovirus for reference. All three viral variants (Var1, Var2, and Var3) use the human E2F-1 promoter to control E1a. In two variants (Var1 and Var3), E4 is controlled by the hTERT promoter. The hTERT promoter controls E1b in Var2. The adenovirus packaging signal (C) is relocated to the right end of the genome in Var1. It remains in its native left end position in Var2 and Var3. Poly(A): SV40 early polyadenylation signal

the manufacturing process to get an accurate assessment of recombination potential during production. As described above, virus is purified after four serial passages, and the viral genome and viral biological activity are compared to the corresponding p0 virus by Figure 4 Detection of rearrangements in Var1 by PCR analysis after serial passaging in a nonpermissive cell line. Based on the various assays, including PCR, to detect the generation expected genomic structure of Var1, no PCR products should be of recombinant viruses and cytotoxicity assays to detected in the area delineated by the dashed box. The presence of a determine potency changes suggestive of a shift in viral band indicates recombination biological activity. In the PCR work, primer sets are chosen to detect likely recombinant viruses. In the case of the Var1 virus described above (Figure 3), multiple Master Viral Bank (MVB) of the virus; defined and primers to detect relocation of the right end of the viral optimized culture conditions for viral growth and DNA (including the packaging signal) to the left would harvest; an effective, efficient, and reproducible down- be selected, since such a relocation would amount to a stream purification process; and the identification of an potential restoration of the wild-type genome. The injectable final formulation in which the virus will be modified E1 region of this virus would be considered stable for long periods in storage. MCBs begin with a the most likely focus for potential recombination events. seed stock, or development bank, of well-characterized PCR analysis of Var1 using several different primers cells of the type selected for production, which is after bioamplification revealed unexpected bands repre- expanded to produce the MCB; selection of an appro- senting recombinants; a typical example is shown in priate producer cell is discussed further below. Similarly, Figure 4. Based on the known binding sites of the a well-characterized, genetically stable seed stock of primers in the Var1 genome, these likely represent engineered virus is expanded under controlled condi- relocations of the right-end packaging signal to the left tions to produce an MVB. side of the genome. DNA sequence analysis on several Once produced, an array of validated assays by which of the PCR products confirmed that the recombinants to qualify the MCB and MVB, as well as demonstrate contained a translocation of the right-end DNA the consistency of the manufacturing process (i.e. to fragment to the left end of the viral genome in the E1 show that the virus meets a predefined set of specifica- region. A caveat here is that one may not detect tions), is required so that the virus can be characterized recombinant that one cannot predict, since the appro- and released, as described in more detail below. priate PCR primers must be used to detect changes. However, the first critical decision in manufacturing a Once a producer cell line is selected and the viral viral product is the selection of an appropriate cell line genomic stability is deemed acceptable, one confronts to serve as the producer cell, which will not only serve as the necessity to consistently manufacture large quanti- the biological foundation of the manufacturing process, ties of the oncolytic adenovirus for use in clinical testing. but also be used to maintain a stable MVB.

Producer cells Manufacturing overview The producer cell line used to grow the virus must meet Manufacturing an adenovirus requires a Master Cell certain criteria. First, they obviously must be permissive Bank (MCB) of a producer or packaging cell line; a for viral replication. For a replication-defective adeno-

Oncogene Product development of oncolytic adenoviruses PK Working et al 7797 viral vector (e.g. adenoviruses rendered unable to exhibit greater potency or altered trophism or both. replicate via deletion of the E1 region), the producer Recombinants may be replication-competent or even cell line must provide in trans the complementary helper-dependent replication-defective viruses. Regard- function, that is, missing E1 functions. Since adeno- less of the type(s) of recombinants, their presence is viruses have a propensity to recombine, a key safety likely to have a negative impact on tumor selectivity, factor is the potential generation of recombinant RCA potency, and overall product safety and efficacy. The in the replication-defective population (Hehir et al., generation of RCA and recombinants is closely related 1996; Smith and Eck 1999; Murakami et al., 2002). For to both the basic genomic stability of the engineered a replication-defective adenovirus, the recombinant oncolytic adenovirus and the producer cell line used. RCA may, for example, be a wild-type virus that still Since multiple serial expansion passages in the permis- carries the inserted transgene of the replication-deficient sive producer cells are required to manufacture an variant and thereby raises safety concerns. FDA has set oncolytic adenovirus, each passage provides an oppor- a limit for the presence of RCA in replication-defective tunity for recombination to produce undesirable re- adenoviral vectors, and standard biological assays combinants, particularly if extensive sequence homology have been established to detect RCA. The selection of exists between the vector construct and the producer the proper producer cell line can go a long way to cells. address this concern. Thoughtful selection of the producer cell used for Among the most widely used cell lines are 293 cells manufacturing can decrease the potential for recombi- (Graham et al., 1977), derived from infecting human nation during the manufacturing process. Utilization of embryonic kidney cells with sheared adenovirus DNA. 293 cells, for example, with their extensive adenoviral These cells contain a long integrated copy of viral sequence homology, is likely to result in a high rate of sequences, including the E1a and E1b genes and their recombination. Designing a production plan that main- native promoters, the adenoviral inverted terminal tains the MVB and working viral banks in a tumor- repeat (ITR), and the packaging signal. The extensive specific cell line, for example, using the PSA-positive sequence homology between the engineered adeno- LNCaP cell line for production of the PSE-controlled, viruses and the integrated adenovirus sequences of the prostate-specific adenovirus CG7060 can significantly 293 cells gives rise to RCA recombinants. The PER.C6 reduce the potential for recombination and permit cell line (Fallaux et al., 1998), derived from human production of clinical material of acceptable quality. embryonic retinoblast cells and carrying a shorter In the case illustrated, to minimize wild-type virus adenoviral sequence containing the native E1a/E1b production, LNCaP cells were used to both expand the sequences with the E1a promoter replaced by a PGK seed virus and to produce the MVB itself; 293 cells were promoter, was developed to reduce the frequency of used for the final large-scale production of CG7060 RCA recombinants during production. PER.C6 cells are (Table 1). It is likely that the wild-type adenoviral titers reported to have produced no RCA in over 40 large- seen in this series of studies represent the expansion of scale batches of adenovirus (Lusky, 2005), although pre-existing wild-type contamination of the CG7060 there are reports of the generation of replication- seed stock or MVB (compare results using 293 vs deficient recombinants that cause cytopathic effects LNCaP as MVB cell line), not recombination during (CPE) (Murakami et al., 2002, 2004). More recently, a production. Nonetheless, this approach requires the novel cell line (C139) derived from A549 human lung characterization and maintenance of two cell banks: an cancer cells has been reported (Farson et al., 2004, LNCaP bank for supporting the viral banks and a 293 2005). In this cell line, the E1a and E1b coding regions producer cell bank for manufacturing, doubling both were reduced to their minimal sequences and their native the effort and characterization costs. Therefore, the promoters removed. The sequences were then intro- selection of production and MVB cell lines that do not duced in the A549 cells using separate MLV vectors to have integrated adenoviral sequences is more efficient ensure noncontinuous integration into the genome. and cost effective, and, more importantly, can further Although not yet utilized in large-scale production runs, extensive intermediate-scale laboratory studies using serum-free suspension culture of this E1A/E1B-comple- Table 1 Effect of cell line on frequency of wild-type virus menting cell line have demonstrated that neither RCA contamination during production of CG7060 nor CPE-inducing replication-deficient recombinants Seed virus cell MVB cell Production Wild-type are generated when replication-deficient adenoviruses linea line cell line frequencyb are produced, in contrast to both 293 and PER.C6 cells. Producer cells for replication-competent oncolytic LNCaP 293 293 1 in 1 Â 107 adenoviruses must meet other tests. Both the 293 and LNCaP LNCaP 293 1 in PER.C6 systems have been used for production of 1 Â 109 oncolytic adenoviruses, but since oncolytic adenoviruses are replication-competent, undesirable recombinants are aThree passages in the indicated cell line. bCount of wild-type not limited to wild-type virus, that is, the issue is not adenovirus in indicated titer of CG7060. Indicated cell lines were used to produce the seed virus prior to the MVB, to manufacture the MVB, strictly the generation of RCA but instead potential and in the production of the adenoviral product. LNCaP is a PSA- recombination to produce wild-type adenovirus or other positive prostate cancer cell line; CG7060 is a prostate-specific, PSE- replication-competent adenoviral variants that may controlled oncolytic adenovirus

Oncogene Product development of oncolytic adenoviruses PK Working et al 7798 decrease the potential for recombination during produc- producer cells and aliquots of the MVB. In the absence tion, so it is probably safer. of an MWVB, the use of a CVL extends the lifetime of Once selected and characterized, the producer cell line the MVB via an early expansion to an intermediate scale must be made into an acceptable MCB and MVB. for inoculation of the final production run. After sufficient culture time for viral amplification (generally 2–5 days), virus-containing producer cells are lysed by Master cell and virus banks detergent or freeze–thaw cycles, and the resulting lysate MCBs and MVBs are subject to demanding character- is clarified via filtration. The lysate is typically further ization and extensive quality control and must comply treated, for example, with Benzonase to digest free with established regulatory guidelines (FDA, ‘Points to unincapsidated RNA and DNA. Virus purification and Consider in the Characterization of Cell Lines Used to removal of process contaminants such as host cell Produce Biologicals’, CBER, 1993; ICH Q5D, ‘Deriva- proteins and medium components is typically carried tion and Characterisation of Cell Substrates Used for out using anion-exchange (AEX) capture and size- Production of BioTechnological/Biological Products’). exclusion chromatography (SEC); many processes A nonexhaustive list of characterization and release further utilize a diafiltration concentration and buffer criteria for MCBs and MVBs would include appearance, exchange step to concentrate the virus in the final identity, cell count and viability (MCB only), particle formulation buffer. The formulated virus is then sterile- and infectious titer (MVB only), sterility, mycoplasma, filtered and filled and finished into suitable pharmaceu- purity, absence of adventitious viral agents and replica- tical containers. A comprehensive description of tion competent virus, absence of specific human viral manufacturing, including a detailed treatment of vector pathogens (CMV, HIV-1, HIV-2, HBV, HCV, etc.), and quantification, quality control, and GMP regulations as more as detailed in the guidelines. Every product may they apply to producing replication-deficient adeno- also have Manufacturer’s Working Banks of producer viruses was recently published (Lusky, 2005) and should cells and virus (MWCB and MWVB) for production be consulted for more details. purposes, each of which will have a similar rigorous, Once the oncolytic adenovirus is produced, the although generally somewhat abbreviated, list of challenge becomes demonstrating the consistency of characterization criteria. the manufactured product using a set of appropriate and qualified analytical techniques. Production process Although the exact details will vary, oncolytic adeno- Characterization, testing, and release of oncolytic viruses are typically made and purified using the general adenoviruses scheme illustrated in Figure 5. In brief, a vial of the MCB or MWCB is expanded to sufficient cell number The regulatory guidelines provide a ‘generic’ list of the and infected with aliquots of the MVB, MWVB, or with release criteria that are useful for oncolytic adenoviruses a CVL produced in a preliminary incubation of and, where not specific, analogies to release criteria for

Measure virus Bioreactor concentration: in process Assay #1

UF/DF #1 4C Final O/N to target Fill Hold concentration

Triton lysed Buffer wash cells filters Sterile DF1 Filtration Clarification AEX Capture Wash/Elute In process filtration Assay #3 DF2 train

0.22um SEC load In process UF/DF #2 and collect Assay #2 to Add Mg++ final x2 formulation Add Benzonase 4C O/N Incubate Hold 60 minutes Pool Fractions at 37C Figure 5 Typical manufacturing scheme for an oncolytic adenovirus

Oncogene Product development of oncolytic adenoviruses PK Working et al 7799 Table 2 Typical release criteria for an oncolytic adenovirus particles is specified to be p30 : 1 in the US; details are End point Method provided in the regulatory documents referenced above. Although no specific guidance exists on the acceptable Appearance Organoleptic physical/infectious particle ratio for replicating vectors, Identity PCR or Southern blot the 30 : 1 ratio is generally adopted. Measurement of Vector titer Anion exchange HPLC physical titer is straightforward, typically accomplished Potency Physical particle to infectious titer ratio (o30 : 1) Purity Ratio OD260/280 using a chromatographic separation method with Safety Sterility detection of viral particles or absorbance at 260 nm in Mycoplasma the presence of detergent. Assessment of infectious titer Endotoxin is likely more virus-specific, since one must utilize a cell General safety Adventitious viruses (human, bovine, porcine) type or types that is permissive for the engineered virus. Host cell DNA Size Infectious titer is most commonly measured by deter- Quantity mining plaque forming units/cytopathic effects (CPE) in Process residuals Detergent, Benzonase, BSA, etc. permissive cells. In these assays, serial dilutions of virus Characterization Selectivity assays are used to infect permissive cells; CPE can be measured Empty particles by observing the formation of lytic plaques on a lawn of Vector aggregates infected cells (plaque assay) or by microscopic observa- tion of infected cell culture wells in a limiting-dilution format (TCID50). Other measurements of infectious titer include quantification of proteins or sequences derived biotechnology-derived product and even small mole- from either the vector (i.e. hexon) or the transgene in cules can be useful (Table 2). In general, the oncolytic infected cell cultures. A wild-type serotype 5 adenovirus virus product will be characterized for identity, purity, reference standard, produced via a collaboration of and the presence of contaminating adventitious agents industry, academia, and the FDA, is available from according to ICH Q5D, ‘Derivation and Characterisa- ATCC. It can be useful for normalizing interlab assay tion of Cell Substrates Used for Production of differences, as the exact methods for infectious titer BioTechnological/Biological Products’ and ICH Q5A, assay vary among labs. ‘Viral Safety Evaluation of Biotechnology Products Potency assessments must be expanded when the Derived from Cell Lines of Human or Animal Origin’. oncolytic adenovirus is armed. In the case of CG0070, However, more specific criteria that address the precise which carries the cDNA for human GM-CSF, resulting nature of the oncolytic adenovirus and its method of in production of active cytokine from transduced cells, production must be developed for individual viral assays were developed to measure both the amount and products. These additional criteria can range from bioactivity of the cytokine produced after transduction obvious criteria tailored to detect a known process of the target cells. GM-CSF secretion after transduction residual, to a requirement to develop expanded potency of target cells in vitro is measured using a quantitative measurements for an ‘armed’ virus. Two criteria may sandwich ELISA, and GM-CSF bioactivity is quantified pose special challenges and are addressed in more detail using a cell-based proliferation assay that utilizes the in the following subsections. The first, applicable to all TF-1 erythroleukemia cell line, which is known to be adenoviral vectors, is the development of an assay or dependent upon GM-CSF for growth (Kitamura et al., assays to define the potency of the virus, which will 1989). In the presence of bioactive GM-CSF, TF-1 cells include both the ratio of physical to infectious titer as become metabolically active and proliferate. The in- specified by the regulatory authorities and other assays creased metabolic activity of the proliferating cells can if the virus carries a transgene of some sort. Examples of be measured by the addition of a fluorogenic redox the latter include cases in which the oncolytic adenovirus indicator (Alamar Blue) to the cultures or by the more encodes a gene for a prodrug-activating enzyme, a traditional thymidine incorporation assay. GM-CSF chemosensitizer, a cytokine, or virtually any other bioactivity is expressed as a percent activity of a product biologically or chemically active molecule. The second, reference control, which must be established, typically which is more relevant to oncolytic adenoviruses, is using an early production lot that has been extensively selectivity, that is, how discriminating is the virus for characterized and is considered representative of the tumor cells compared to normal, nontumor cells? product, and which should be calibrated against the Selectivity is directly related to potency, as it is a World Health Organization (WHO) GM-CSF standard measurement of ‘relative’ potency. (GM-CSF First International Standard, OMS Report No. 840, 1994). As with any analytical assay, the GM- Potency assays CSF bioactivity assay should also be characterized in terms of intra- and interassay precision, accuracy, and A standard potency assessment for an oncolytic linearity as part of its qualification as a release adenovirus is the ratio of physical particles to infectious specification. It should be noted that assays of biological particles in the test material. In other words, how much activity are particularly prone to assay variability. For of what is in the product vial is actually living, infectious this reason, specification limits for potency of biologi- adenovirus? For replication-defective adenoviral vec- cals are often set for both the potency of the product tors, the ratio of noninfectious particles to infectious (acceptable limits around the stated potency are

Oncogene Product development of oncolytic adenoviruses PK Working et al 7800 80–125% for marketed recombinant GM-CSF, for infection between the normal and tumor cell lines are example) and the assay (confidence limits of the assay). taken into account. The Selectivity Ratio would then Since it is not uncommon for biological assays to have a take the following form: high relative standard deviation (e.g. 20–50%), the confidence limits of the assay tend to be wide. Normalized Selectivity Ratio Analogous bioactivity assays will be necessary for any TCID oncolytic virus=TCID wild-type Ad in normal cells ¼ 50 50 oncolytic adenovirus that results in the secretion of any TCID50 oncolytic virus=TCID wild-type Ad in tumor cells biologically active molecule, for example, an assay to 50 determine the specific activity of a prodrug-activating This reciprocal ratio is referred to as the Selectivity enzyme. In advanced stages of development, these Index (SI; Haviv and Curiel, 2003). assays may become part of the release criteria for the While the use of wild-type adenovirus in a laboratory oncolytic adenovirus product. producing or testing an oncolytic virus is certainly not desirable, the use of wild-type virus to identify a pair of Selectivity assays tumor/normal cell lines with comparable permissiveness during assay development and qualification is essential These assays are designed to demonstrate that the to the correct interpretation of selectivity results. Once manufactured oncolytic adenovirus consistently demon- an appropriate pair of cell lines is identified, the strates its intended selectivity for tumor cells relative to selectivity assay can become a relevant measurement nontumor cells. Selectivity is fundamentally a relative of the clinical safety of the oncolytic virus. expression of virus potency, as the biological activity of the virus in tumor cells is compared to its activity in normal, nontransformed cells (Figure 1 illustrates such preferential activity). As such, a measurement of Conclusions selectivity can take different forms, depending on the method chosen to measure virus potency. Selectivity can Engineered oncolytic adenoviruses can be produced in be expressed as a ratio. For example, if potency is large quantity and to accepted pharmaceutical stan- dards using the available regulatory documents as measured by a TCID50 assay, selectivity can be expressed as: general guidelines. These documents, although developed principally for biotechnology-derived products, TCID in normal cells provide a useful roadmap for the production and Selectivity Ratio ¼ 50 TCID in tumor cells characterization of oncolytic adenoviruses for human 50 use. Manufacturing processes and development of A Selectivity Ratio >1 indicates that the virus replicates appropriate characterization assays should be science- better (preferentially) in tumor cells, or, considered from driven and be designed to answer specific questions a different perspective, that virus replication is attenu- using the best available science and technology, keeping ated in normal cells compared to tumor cells. The higher in consideration the unique nature of the product: a the ratio, the more selective, and consequently safe, is living, and mutatable, virus. Particular attention should the virus. This interpretation is based on the important be directed towards understanding the associated assumption that the virus has equal opportunity to unusual challenges of product development in such a infect and enter both the tumor and normal cells in setting, most notably the potential instability of which viral replication is compared; in other words, the engineered constructs (as evidenced by their occasional pair of cells used for the comparison must express reversion to wild-type format) as well as the impact of similar levels of adenovirus receptors. Ideally, a matched the details of the manufacturing process, including the pair of normal and transformed cells of common origin producer cell line used, on final product quality. These should be used, although such reagents are unlikely to challenges notwithstanding, designing a rational and be readily available. An alternative approach is to science-based development program has the promise of normalize both measurements to wild-type adenovirus, delivering an ample supply of well-characterized, so that differences in permissiveness to adenovirus selective, and potentially potent oncolytic adenoviruses.

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