adenovirus boost

Int. J. Cancer: 120, 2290–2300 (2007) ' 2007 Wiley-Liss, Inc.

FAST TRACK Immunogenicity and safety of a DNA prime/adenovirus boost vaccine against rhesus CEA in nonhuman primates Luigi Aurisicchio, Carmela Mennuni, Patrizia Giannetti, Francesco Calvaruso, Maurizio Nuzzo, Barbara Cipriani, Fabio Palombo, Paolo Monaci, Gennaro Ciliberto and Nicola La Monica* Istituto di Ricerche di Biologia Molecolare (IRBM), Pomezia, Italy

Scaling up experimental protocols from rodents to humans is often not a straightforward procedure, and this particularly applies to cancer vaccines, where vaccination technology must be especially effective to overcome a variety of immune suppressive mechanisms. DNA electroporation (DNA-EP) and adenoviral vectors (Ad) have shown high potency and therapeutic efficacy for different antigens in several pre-clinical models. To evaluate the ability of DNA-EP and Ad to break tolerance to a self-antigen in large animals, we have cloned the CEA homologue (rhCEA) from rhesus monkeys (Macaca mulatta) colon tissue samples. rhCEA is a 705 aa protein and shares 78.9% homology to human CEA protein. Immunogenicity of rhCEA expressing vectors was tested in mice and subsequently in rhesus monkeys. To further increase the immunogenic potency of these vectors, a synthetic codon optimized rhCEA cDNA (rhCEAopt) was constructed. Genetic vaccination of rhesus monkeys was effective in breaking immune tolerance to rhCEA in all immunized animals, maintaining over time the elicited immune response, and most importantly, neither autoimmunity nor other side-effects were observed upon treatment. Our data confirm the efficacy of genetic cancer vaccines in large animals such as nonhuman primates and show that development of modified expression cassettes that result in increased potency of plasmid DNA and adenovirus may have a significant impact on vaccine development against malignancies expressing tumor associated antigens in patients. ' 2007 Wiley-Liss, Inc. Key words: CEA; cancer vaccine; DNA electroporation; adenovirus; self-antigen

Despite some recent failures in clinical trials, implementation of active specific immunotherapy for cancer still represents an attractive and promising idea.1 This wave of enthusiasm is rooted in recent advances in molecular engineering and a better understanding of tumor immunology2,3 bringing new paradigms, such as the dominant tolerance through T-regulatory cells and the instructive role of the innate immune system on the adaptive immune system. This ever increasing knowledge and the experience gained from earlier clinical studies indicate that efficient cancer vaccination could be feasible even without utilizing cancer neoantigens, provided that antigen expression and presentation are increased, and that regulatory circuits are controlled. In fact, several tumor associated antigens (TAA) have been identified and under appropriate conditions, are recognized by various components of the immune system.4,5 Carcinoembryonic antigen (CEACAM-5 or commonly CEA) was one of the first TAAs to be identified and has been well characterized.6 CEA is a membrane 180 kDa glycoprotein expressed at high levels in the fetal colon and at lower levels in the normal adult colonic epithelium.7 CEA is overexpressed in 90% of colorectal, 70% of gastric, pancreatic and nonsmall cell lung cancers and 50% of breast cancers.7 The biological role of CEA in cancer has not yet been fully clarified: it has been shown that higher CEA expression promotes an increase in intercellular adhesions, which may lead to metastasis.8 CEA is often used as a serological marker of malignancy because it is overexpressed in cancer as well as being present in serum.9 It has been recently reported, however, that human medullary thymic epithelial cells express CEA,10 and Publication of the International Union Against Cancer

protein members of the CEA family, which share regions of high homology are expressed in normal cells of haemopoietic origin (i.e. neutrophils).7 In view of these observations, central and peripheral tolerance is likely to interfere with the in vivo development of anti-CEA immunity. Thus, overcoming the immunological tolerance to CEA is considered a particularly challenging objective for the development of an effective vaccine. Genetic vaccines represent promising and efficient methods to elicit immune response against a wide variety of antigens.11 Among these, in vivo electroporation of plasmid DNA (DNAEP) has emerged as a safe method resulting in greater DNA uptake leading to enhanced protein expression in the treated muscle,12 and in a concomitant increase in immune responses to the target antigen in a variety of species.13–16 Similarly, recombinant viral vaccines based on replication-defective recombinant adenoviruses (Ad) have been proven safe and to induce strong antibody and cellular antigen-specific immune responses17,18 Combinations of heterologous modalities of immunization induce superior immune responses as compared with single modality vaccines.19–22 We have previously demonstrated that DNA-EP and Ad vectors coding for human CEA are capable of eliciting an immuneresponse against CEA in tolerant CEA transgenic (CEA.Tg) mice, and confer significant tumor protection.23,24 Furthermore, the expression level of the target antigen contributes considerably to antigen immunogenicity: we have shown that use of a codon-optimized cDNA for human CEA results in increased expression with concomitant induction of greater cell mediated immunity in CEA.Tg mice.23 Whilst studies in rodent pre-clinical models offer the advantages of testing the potency and therapeutic efficacy of cancer vaccines, they cannot fully predict efficacy when scaling-up doses in human patients, particularly when dealing with self-antigens and immune tolerance. This limitation can be overcome by testing vaccination regimens in large animals, such as nonhuman primates. To verify the efficacy of genetic cancer vaccines in nonhuman primates we have cloned the rhesus monkey homolog for CEA and analyzed the immunogenic properties of plasmid DNA and Ad vectors encoding rhesus CEA in rhesus monkeys. Our findings could have a significant impact on the implementation of DNA-EP and Ad vaccination in humans. Material and methods Rhesus monkey tissues and RNA preparation Colon samples from 2 different rhesus monkeys were used as RNA source for our experiments. Total RNA was prepared by Grant sponsor: MIUR (FIRB), Italy; Grant number: RBME017BC4. *Correspondence to: Istituto di Ricerche di Biologia Molecolare (IRBM), Pomezia, Italy. Fax: 139-6-91093-482. E-mail: [email protected] Received 25 August 2006; Accepted after revision 24 November 2006 DOI 10.1002/ijc.22555 Published online 15 February 2007 in Wiley InterScience (www.interscience. wiley.com).

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FIGURE 1 – Cloning strategy and expression of rhesus CEA. (a) Alignment of 50 and 30 ends of CEACAM family gene members. Upper Panel: 50 untranslated region of human CEACAM genes (1,3,4,5,6,7,8) and 2 partial nucleotide sequences found in Genbank (accession number is indicated). ATG codon is boxed and the designed degenerated primer is shown. Lower Panel: 30 untranslated region of human CEACAM genes (1,5,6,7,8). Stop codons (TAG) are boxed. For CEACAM1, stop codon is beyond the alignment. Designed primers for amplification and cloning are shown. Degenerated code: S 5 C or G; R 5 A or G; M 5 A or C; Y 5 C or T). (b) Expression of rhesus CEA clones. HeLa cells were transfected with phagemids obtained by screening the lambda-CEA library and a Western blot was performed using a rabbit polyclonal antibody raised against human CEA protein. Expression of 2 clones out of 15 is shown.

Ultraspec-II RNA isolation system (Biotecx) according to manufacturer’s instructions. Integrity of purified RNA was verified by formaldehyde-denaturing agarose gel and samples were aliquoted and stored at 280°C. Amplification of rhesus CEA cDNA On the basis of CEA gene family homologies among the different members and different species (see Fig. 1a), several primers were designed and PCR conditions were optimized to amplify rhCEA cDNA. The primers used to amplify the entire cDNA were 50 -rhCEA EcoRI 50 -CCGAATTCCGGACASAGCAGRCAGCAGRSACC-30 at 50 end of the gene and 30 -rhCEA A 50 GYCAGTCTTCCTGAAATGMAGAAACTACACCAGGGC-30 and 30 -rhCEA-8 XhoI 50 -CCGCTCGAGCGGCTGCTACATCAGAGCAACCCCAACC-30 . cDNA amplification was performed with SuperScript One-Step RT-PCR with Platinum Taq kit (Invitrogen). RNA (1 lg) and 200 pmol of both primers were used in 100 ll reaction volume and DMSO was added at 10% final concentration. After 30 min of incubation at 45°C, cycling conditions for the amplification were 2 min at 94°C followed by 40 cycles under the following conditions: 94°C for 15 sec, 52°C for 30 sec, and 68°C for 2 min and 20 sec. An amplified product of about 2,100 bp was obtained from both monkey RNA samples and purified from agarose gel. Generation and screening of a lambda rhCEA-specific library The amplified rhCEA PCR product was digested with EcoRI/ XhoI and ligated into the Lambda ZAP-CMV XR vector (Stratagene), according to manufacturer’s directions. The ligation product was incubated with Gigapack III gold packaging extract and the resulting phages were used to infect XL-1 Blue MRF’ cells. This primary library was then amplified, obtaining a titer of
1 3 106 pfu/ml. Screening of
5 3 103 plaques was performed

through lifting onto nylon filters and hybridization with 2 different DNA probes covering the 50 and the 30 of the molecule. Double positive plaques were excised in XL-1 Blue MRF’ cells and the derived filamentous phages were amplified in XL-OLR cells. The phagemids were then grown and analyzed with restriction enzymes. Sequence analysis and gene-bank comparisons revealed the highest homology with human CEACAM-5 cDNA. Construction of codon-optimized rhCEA On the basis of the predicted amino acid sequence of rhCEA protein, the rhesus CEA cDNA was designed and optimized according to the most frequent codon usage in human cells, by means of Vector NTI program algorithm (Informax). To increase the level of transcription, an optimized Kozak sequence was inserted at 50 of the ATG. Moreover, 2 consecutive stop codons were inserted downstream the coding sequence. The gene was synthesized by Bionexus (Oakland, Ca.) via PCR-mediated oligonucleotide assembly and cloned in the vector pCR-blunt (Invitrogen). To verify the sequence, both strands of the gene were sequenced by using ABI 377 automated sequencer. The autoassembler program was used to compare the sequence data of the synthesized gene with the expected sequence. Plasmid and adenovirus vectors rhCEA was excised with PstI/XhoI from pCMV-script EX phagemid vector and inserted in pBluescript II KS vector, obtaining pBS-rhCEA. The insert was entirely sequenced and then subcloned as SmaI/XhoI fragment in pV1JnsA vector,25 obtaining pV1J-rhCEA. The shuttle plasmid pMRK-rhCEA for Ad generation was obtained by subcloning the same fragment in polyMRK vector.18 rhCEAopt was excised as an EcoRI fragment from pCRblunt-rhCEAopt vector and inserted in pV1J-nsA vector, obtaining pV1J-rhCEAopt. pMRK-rhCEAopt was obtained by subcloning

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rhCEAopt as a HincII/XhoI fragment in SwaI/SalI sites of polyMRK vector. PacI/StuI fragments from pMRK-rhCEA or pMRK-rhCEAopt containing the expression cassettes and E1 flanking Ad5 regions were recombined to ClaI linearized pAd5 using BJ5183 E. Coli cells.26 The resulting plasmids were pAd5rhCEA and pAd5-RhCEAopt. These plasmids were cut with PacI to release the adenovirus ITRs and transfected in PerC-6 cells (Crucell) by Lipofectamine 2000 (Life Technologies). Vectors were amplified through serial passages. Viruses were purified through standard CsCl purification protocol and extensively dialyzed against A105 buffer (5 mM Tris pH 8.0, 1 mM MgCl2, 75 mM NaCl, 5% Sucrose, 0.005% Tween20). rhCEA expression and detection in vitro and in vivo Expression of rhCEA was verified by Western blot. Plasmids were transfected in HeLa cells with Lipofectamine 2000 (Invitrogen). Ad infections were performed in serum-free medium for 30 min at 37°C, and then fresh medium was added. After 48 hrs of incubation, whole cell lysates were analyzed by western blot using a rabbit polyclonal serum against human CEA (Fitzgerald, 1:1500 dilution). rhCEA was detected as a 180–200 KDa band. To measure rhCEA in blood, a semi-quantitative sandwich ELISA assay was set up. 96-well plates (Nunc maxisorp) were coated O/N at 4°C with a polyclonal rat anti-rhCEA serum (generated in house) in carbonate buffer (50 mM NaHCO3 pH 9.4) and21,22 were then blocked with PBS containing 5% BSA for 1 hr at 37°C. Sera were then diluted in PBS 5% BSA and incubated for 2 hr at RT. After washing 5 times with PBS/0.05% Tween 20, anti-CEA rabbit polyclonal antibody (Fitzgerald) was added at 1:2000 dilution and incubated for further 2 hr. Plates were then washed again and detecting antibody anti-rabbit IgG-AP conjugated was added at 1:2000 dilution for 1 hr at RT. Final detection was done with 100 ll/well p-nitrophenyl phosphate disodium, 1.0 mg/ ml in 10% diethanolamine buffer, pH 9.8 containing 0.5 mm MgCl2 and reading at OD405. Peptides Lyophilized rhCEA peptides were purchased by Bio-Synthesis (Lewisville, Texas) and re-suspended in DMSO at 40 mg/ml. Pool A (34 peptides), Pool B (45 peptides), Pool C (48 peptides) and Pool D (53 peptides) were assembled and final concentrations were the following: Pool A 5 1.176 mg/ml; Pool B 5 0.888 mg/ ml; Pool C 5 0.851 mg/ml; Pool D 5 0.769 mg/ml. Peptides and pools were stored at 280°C. Mice immunization and tumor challenge C57Bl/6 mice (H-2b) were purchased from Charles River (Lecco, Italy) and kept in standard conditions according to ethical committee approval. DNA-EP was performed in mice quadriceps injected with 50 lg pV1J-rhCEA or pV1J-rhCEAopt and electrically stimulated as previously described.27 Adenovirus injection in quadriceps was performed with 1 3 1010 vp of Ad5-rhCEA or Ad5-hCEAopt in a volume of 50 ll/mouse. Two weeks after the last injection antibody and cell mediated immune response were analyzed or tumor challenge was performed by s.c. injection of 5 3 105 MC38-CEA cells/mouse. At weekly intervals, mice were examined for tumor growth and tumor volume was measured with a caliper and calculated as previously described.28 Antibody detection and titration Sera for antibody titration were obtained by retro-orbital bleeding. Western blot was performed with extracts from HeLa cells transduced with Ad5-hCEA or Ad5-rhCEA run on SDS-page gel and transferred onto nitrocellulose filters. Sera from each group were pooled and diluted 1:50 for O/N incubation at 4°C. An antimouse IgG-AP conj. (Sigma, 1:2500) was used for the detection. Antibody titer was measured by ELISA assay as described.23

ELISPOT assay for IFNc ELISPOT for mouse IFNg was performed as previously described.23,24,29 A similar procedure was followed for rhesus IFNg ELISPOT. Briefly, rhesus monkey PBMC were purified as described.22 The coating antibody anti-rhesus IFNg (MD1 UCytech) was diluted 1:200 in sterile PBS (final conc. 10 ug/ml). PBMCs were plated at 4 3 105 and 2 3 105 cells/well, in duplicate. Detecting biotinylated anti-rhesus IFNg antibody (detector Ab U-Cytech) was diluted 1:100 in PBS. The next day, plates were washed and incubated for 2 hr at RT with Streptavidin-AP conjugate (Pharmingen) diluted 1:2500 in Assay buffer. After extensive washing, plates were developed by adding 50 ll/well NBT/B-CIP (Pierce) until development of spots is observed at the microscope. The reaction was stopped by washing plates thoroughly with distilled water. Plates were allowed to air-dry completely, and spots counted using an automated ELISPOT reader. Intracellular staining for IFNc Mouse assay was performed as described23,24,29 Macaques PBMCs (fresh or frozen) were resuspended at 2 3 106 cells/ml in R10 medium. Frozen samples were incubated at 37°C, in R10 for at least 5–6 hrs before proceeding with the assay. One ml of cells were aliquoted into round bottom polypropylene tubes (Falcon, 2059). Costimulatory monoclonal antibodies (CD28 and CD49d, Becton Dickinson) were added to each tube for a final concentration of 1 lg/ml. Individual peptides or peptides pools were added to samples for a final concentration of 2 lg/ml. Tubes were incubated at 37°C for 1 hr, then Brefeldin A (SIGMA) was added at final concentration of 10 lg/ml. After 15 hrs incubation at 37°C cells were washed with FACS buffer and incubated with the surface antibodies CD4 PE (clone L200, Pharmingen), CD8 PerCP (Becton Dickinson), CD3 Cy5, (rhesus clone FN-18 U-Cytech) diluted 1:10. Cells were incubated for 30 min at RT, washed and permeabilized with 750 ll of 2X FACS Perm buffer (Becton Dickinson) for 10 min at RT. Cells were washed with FACS buffer and incubated for 30 min at RT with IFNg FITC (clone MD-1 UCytech). Samples were resuspended in 1% Formaldheyde/PBS and analyzed with a FACS Calibur. Rhesus monkey immunizations Monkey immunization studies were performed at the Biomedical Primate Research Center (BPRC, Rijswijk, the Netherlands) using groups of rhesus macaques (Macaca mulatta). The DNA injection consisted of a 1 ml solution (split over two injection sites with 0.5 ml/site) containing 5 mg pV1J-rhCEA or pV1J-rhCEAopt DNA. Electrical conditions for electroporation consisted of 2 trains of 100 square bipolar pulses (1 sec each) delivered every other second for a total treatment time of 3 sec. The pulse length was 2 msec/phase with a pulse frequency and amplitude of 100 Hz and 100 mA, respectively (constant current mode). Rhesus monkeys were injected in deltoid muscle with a dose of 1011 Ad viral particles (vp). Blood was collected every 4 weeks and peripheral mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation. Fresh or frozen PBMC samples were analyzed for immunologic assays. Haematology, clinical chemistry and necropsy Haematological parameters were measured using a Goffin Meyis device with the programs Sysmex SF-3000 for flow cytometry and Sysmex R-500 for reticulocyte counting. Clinical chemistry parameters were determined by using the Cobas Integra 4001 machine (Roche Diagnostics). All animals were euthanized at the end of the observation period. A complete necropsy was carried out in which the abdominal and thoracic cavities were opened and internal organs examined in situ. Tissue samples of organs were preserved in neutral aqueous phosphate-buffered 4% solution of formaldehyde or frozen at 280°C.

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FIGURE 2 – Nucleotide and aminoacid sequence of rhesus CEACAM-5. (a) Comparison of rhesus and human CEACAM-5 ORFs. Nucleotide sequence identity is 88%. Sequence has been deposited in Genbank (bankit828720, DQ838491). (b) Alignment of rhesus and human CEACAM-5 polypeptide. Aminoacid identity is 78.9%.

Results Cloning and characterization of rhesus CEA cDNA CEA is a member of the superfamily of immunoglobulin genes, and currently 29 separate genes have been identified as coding

CEA-related gene products in humans.30 By comparing the 50 and 30 UTR of the mRNA of all known members of the human and nonhuman CEACAM gene-family, degenerated oligonucleotides were designed to amplify by RT-PCR rhesus monkey CEACAM5 cDNA (Fig. 1a). RNA extracted from rhesus colon tissue was

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FIGURE 3 – Immunogenicity of rhCEA in mice. (a) Groups of 6 C57BL/6 mice were vaccinated by DNA-EP (50 lg pV1J-rhCEA) and Ad injection (Ad5rhCEA, 109 vp) with 2 weeks interval. Fourteen days later mice were sacrificed and splenocytes cellular immune response was measured by IFNg ELISPOT using pools of 15 mer peptides encompassing rhCEA protein. Schematic representation of peptide pool distribution is shown on the right. (b) Antibody response. At Day 30 post-immunization mice were bled and sera were used to check the presence of antibodies anti-rhCEA. Extracts of HeLa cells transfected either with rhCEA or with hCEA were analyzed by Western blot. The results of 2 mice (no. 1 and no. 2) are shown.

used as template and a PCR product of about 2200 bp was amplified, the expected size for a CEACAM-5 cDNA homolog. An identical fragment was independently obtained from colon RNA, also isolated from a second monkey. Partial sequence analysis of the amplified DNA from both animals revealed a high degree of homology to human CEACAM-5 cDNA. Next, the RT-PCR product was cloned in a k vector, thus generating a CEA-specific phage-library and CEA positive clones were screened, sequenced, and analyzed for expression by Western blot using a polyclonal antibody against human CEA protein. All the selected clones expressed an 180 KDa protein when transfected in HeLa cells (Fig. 1b). rhCEA cDNA is an open reading frame of 2118 nucleotides encoding a 705 aa polypeptide. Comparison between human and rhesus homolog revealed 88% nucleotide identity (Fig. 2a) and 78.9% aminoacid identity (Fig. 2b). Interestingly, a 3 aminoacid insertion is present in the carboxyl-terminus of rhesus CEACAM-5, involving the presumed signal for GPI modification. However, the resulting polypeptide was as efficiently secreted as human CEA (data not shown).

rhCEA is immunogenic in mice With the final goal of immunizing macaques, plasmid DNA (pV1J-rhCEA) and Ad vectors expressing rhCEA under the control of human CMV promoter were generated. To test the efficacy and immunogenic potency of rhCEA vectors, C57BL/6 mice were vaccinated with DNA-EP followed by Ad5 injection. This heterologous combination had been previously demonstrated as highly efficient in generating an anti-CEA immune response.23 Two weeks later, cell mediated immune response was measured by IFNg ELISPOT using pools of 15 mer peptides, overlapping by 11 residues and encompassing the entire rhCEA protein. A significant immune response was detected against several epitopes distributed along the entire protein (Fig. 3a). To verify whether our genetic immunization was able to induce a humoral response, sera from

immunized mice were analyzed by Western blotting using extracts from HeLa cells transfected either with rhCEA or hCEA. rhCEA protein was efficiently recognized by mice sera, as well as human protein, thus indicating that similar epitopes were recognized by elicited antibodies (Fig. 3b). Genetic immunization of rhesus macaques with rhCEA vectors To test the efficacy of genetic vaccination and to break immune tolerance against rhCEA in rhesus macaques, a group of 4 monkeys was immunized with DNA-EP/Ad vectors. Animals were vaccinated intramuscularly with pV1J-rhCEA plasmid followed by EP at weeks 0, 4, 8, 12 and 16. The DNA injections consisted of a 1 ml solution (split over 2 sites with 0.5 ml/site) containing 5 mg plasmid DNA. No local reactions to the vaccine were noted in the following days. Cell-mediated response was measured over time by IFNg ELISPOT assay using pools of peptides covering the entire tumor antigen. Starting from the third DNA injection, all 4 monkeys developed a detectable immune response against epitopes distributed along the entire antigen (Fig. 4a). In particular, DNA-EP was effective in RI002, where specific T cell frequencies of 1/20,000 and 1/50,000 were measured against epitopes contained in Pool C and D, respectively. Upon Ad5-rhCEA injection, the immune response was boosted in all 4 monkeys. Above all, a very strong response directed against Pool C was measured in RI137 (up to 1/2,000 rhCEA specific effectors), but also at lower levels in RI311 against epitopes contained within the 4 pools. These data show that different genetic vectors expressing the same antigen can be used to maintain the amplitude of the immune response over time. To characterize the T cell subtypes elicited by the immunization regimen, an intracellular staining assay was performed on frozen PBMC from 2 monkeys at the time of their peak response, as measured by ELISPOT assay. In both animals, a consistent CD81

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FIGURE 4 – Genetic vaccination breaks tolerance in rhesus monkeys and is long-lasting. Four macaques (CO12, RI002, RI137, and RI311) were immunized with pV1J-rhCEA and Ad5-rhCEA vectors. Animals were vaccinated intramuscularly with 5 mg plasmid DNA at weeks 0, 4, 8, 12, and 16 by injection of DNA-EP in quadriceps. At Week 24–28, monkeys were boosted with intramuscular inoculation of 1011vp of Ad5rhCEA. (a) Cell-mediated response. Freshly isolated PBMC were collected every 4 weeks for a total duration of 9 months. Graphs show the numbers of spot forming cells (SFC) upon in vitro stimulation with rhCEA peptide pools (a–d) subtracted of the DMSO background (not shown). IFNg ELISPOT background was consistently <15% of the response in wells containing peptide. (b) Cell mediated immune response is CD81 specific. Intracellular staining for interferon gamma was performed on monkey PBMC frozen at week 40. Cells were stimulated with rhCEA Pool C (RI137) or Pool B (CO12). (c) Kinetics of humoral immune response in vaccinated monkeys. Humoral response was measured by ELISA assay at the indicated time points using human CEA protein as substrate (see Material and Methods).

T cell-specific IFNg secretion was measured (Fig. 4b). A CD41 cell response of lower amplitude was also detectable (not shown). To determine whether genetic immunization was capable of eliciting antibodies against the tumor antigen, the titer of antibodies specific for rhCEA was measured by ELISA throughout the entire course of the experiment (Fig. 4c). Anti-rhCEA antibody titers were detected over time for 2 monkeys (CO12 and RI311). Notably, the animal which showed the higher specific cell-mediated immune response (RI137, see Figs. 4a–4c), did not show measurable anti-CEA antibodies.

Construction, expression, and immunogenicity of codon optimized rhesus CEA The increased immunogenicity of codon-optimized genes coding for a variety of antigens including human CEA have been recently shown.23,31 The optimized cDNA for rhesus CEA was designed using a computer algorithm by back-translating the protein amino acid sequence according to the most common human codon usage and synthesized by PCR-mediated oligonucleotideassembly. Transfection with an expression plasmid (pV1J-rhCEAopt) carrying the optimized cDNA of rhesus CEA (rhCEAopt) at different

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FIGURE 5 – Gene optimization of rhCEA. (a) In vitro expression. HeLa cells were either transfected with plasmids at the indicated doses or infected with Ad at the indicated MOI (multiplicity of infection). Forty-eight hours later, whole cell lysates were analyzed by Western blot. Rhesus CEA was detected as a 180–200 KDa band. (b) In vivo expression. C57BL/6 mice were injected intramuscularly with Ad5-rhCEAopt and compared with similar vectors carrying wild type CEA. Four days later, mice were bled and circulating levels of rhCEA were measured by ELISA. Each symbol represents the measure for a single mouse and bars indicates the geometric mean of the group (T-student test at 107vp, p 5 3.6 3 1025). (c) Immunogenicity of codon optimized rhCEA in mice. Groups of 6 C57BL/6 mice were vaccinated by DNA-EP or Ad injection 2 weeks apart. Fourteen days later mice were sacrificed and splenocytes cellular immune response was measured by ELISPOT using rhCEA pool D as antigenic stimulus. (d) Antitumor effect of rhCEAopt expressing vectors. Groups of 8 C57BL/6 mice were vaccinated by DNA-EP (50 lg) and Ad (1 3 1010vp) injection 2 weeks apart. Fourteen days later mice were challenged subcutaneously with 5 3 105 MC38-CEA colon adenocarcinoma cells. Left Panel: tumor free mice. Vaccinated mice (solid line) and control mice (dashed line) are compared. Right panels: tumor growth. Tumors were measured weekly with a caliper. Each line indicates the tumor size per each single mouse. The black thick line is the average tumor size per group. Tumor growth of vaccinated mice was significantly different at all analyzed time points (T-student test, p < 0.003).

doses showed about 100-fold greater protein levels than a similar vector carrying the native cDNA (pV1J-rhCEA) as detected by Western blot (Fig. 5a). Similarly, infection of HeLa cells with Ad5-rhCEAopt showed an increased expression of rhCEA of at least 10-fold. Thus, the optimization of rhesus CEA coding sequence efficiently enhances the level of expression of rhCEA in vitro. To determine whether a higher level of expression could be measured also in vivo, C57BL/6 mice were injected i.m. with different doses of pV1J-rhCEAopt or Ad5-rhCEAopt and compared with the respective vectors carrying the wt cDNA. Four days later, mice were bled and circulating levels of rhCEA measured by ELISA: higher protein levels were obtained in Ad5-rhCEAopt injected mice than those detected upon injection of vectors encod-

ing the wild type rhCEA (Fig. 5b). No significant expression was measured upon DNA-EP treatment (data not shown). To verify whether this higher expression was connected with higher immunogenicity, cellular immune response in injected mice was measured by IFNg ELISPOT. As shown in Figure 5c, significant enhancement of immune response was measured using rhCEAopt expressing vectors. Antitumor effect of rhCEAopt genetic vaccine To assess if genetic vaccination with rhCEAopt vectors could protect mice from tumor development, C57BL/6 mice were immunized with DNA-EP/Ad vectors and challenged subcutaneously with mouse colon adenocarcinoma cells expressing human CEA

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strong cell-mediated response upon Ad5 injection (533 and 870 SFC/106 PBMC, respectively, Fig. 6a). Both for RI497 and RI512, as observed for RI137 in the previous vaccination, the immune response was probably directed against one or more immunodominant epitopes present in Pool C and D. rhCEA-specific antibody titers were also measured upon DNA-EP and were maintained over time by Ad boosting (Fig. 6b). Evaluation of clinical parameters and necropsy Body weight and temperature were measured throughout the entire course of the vaccination protocols without significant variations. To monitor potential signs of toxicity connected with the vaccination protocol and/or to detect indications of autoimmunity, vaccinated animals were constantly monitored for abnormal values in haematological and clinical chemistry blood parameters. The parameters measured before (PRE, Week 6) and after (POST, Week 52) vaccination are listed in Table I. Only minor deviations from normal values were detected: monkey CO12 and RI497 showed slightly elevated percentages of eosinophilic granulocytes; monkey RI002 and RI311 had elevated liver enzyme ALT. These variations were, however, also noted before initiation of the vaccination study. RI512 also showed a transient increase of ALT transaminases. Thus, no significant haematological side effects connected with the immunizations were detectable. All animals were euthanized at the end of the observation period and internal organs were examined in situ. For all animals, gross examination of the organs at necropsy did not reveal any abnormalities. In conclusion, neither significant side effects nor phenomena of autoimmunity were observed in relation with the genetic vaccine and the immune response against rhCEA. Discussion

FIGURE 6 – Genetic vaccination with optimized rhesus CEA. Two macaques (RI497 and RI512) were immunized with pV1J-rhCEAopt and Ad5-rhCEAopt vectors. Animals were vaccinated in quadriceps with 5 mg plasmid DNA at weeks 0, 4, 8, 12 and 16 by DNA-EP. At Week 27–31 monkeys were injected with 1011vp of Ad5-rhCEAopt. (a) Cell mediated response. Graphs show the numbers of spot forming cells (SFC) upon stimulation with rhCEA peptide pools (a–d) subtracted of the DMSO background. (b) Humoral response. Antibody titer was measured by ELISA assay at the indicated time points using human CEA protein as substrate (see Material and Methods).

(MC38-CEA). As shown in Figure 5d, all control mice developed palpable tumors by Day 9 after implantation, while 57% of vaccinated mice remained tumor free for the entire course of the experiment. Additionally, adenocarcinoma growth rate in vaccinated tumor-bearing mice was noticeably slower than in control mice (Fig. 5d), thus indicating that the immune response elicited by rhCEAopt vectors was able to interfere with tumor expansion. Vaccination with rhCEAopt expressing vectors in rhesus macaques An immunization protocol similar to that described above was used to immunize 2 macaques (RI497 and RI512) with vectors expressing the optimized version of rhCEA. Monkeys were immunized by DNA-EP with pV1J-rhCEAopt at weeks 0, 4, 8, 12 and 16, and then were inoculated with Ad5-rhCEAopt at week 27-31. Only RI497 developed a detectable cell mediated immune response following DNA-EP, but both animals showed a very

Nonhuman primates such as macaques are valid models to determine the safety and immunogenicity of candidate vaccines that are being developed for implementation in humans.32 In fact, the immune response is similar to that expected in humans, and in the last 2 decades numerous immunogenicity studies have been performed in nonhuman primates utilizing preclinical candidate vaccines, most of them utilizing recombinant proteins of bacterial or viral origin as immunogen33,34 or genetic vectors coding for viral antigens.18,20,35,36 This is a reasonable approach when dealing with bacterial or viral diseases, where the organism recognizes the antigen as an exogenous protein and consequently the elicited immune-response is generally strong and effective against the target pathogen.18 Similarly, other studies involving the use of TAAs have been conducted with human proteins or vectors encoding for human TAAs including CEA.37–39 In these reports, the elicited immune response is not expected to be fully predictive of the possible outcome in human patients, since the antigen is recognized as a non-self protein. For cancer vaccines the most widely used preclinical models for immunologic and antitumoral studies are transgenic rodents expressing the human TAA,28,40 which show central and/or peripheral tolerance to the antigen of interest. The objective of our study was the evaluation of DNA-EP/Ad vaccination regimen in nonhuman primates using CEA as target antigen. In view of the important role of tolerance in evaluating the immunogenic potency of a cancer vaccine targeting self-antigens, the rhesus monkey homolog of CEA was cloned and used as immunogen in the rhesus monkeys vaccination studies. The human and rhesus proteins were similar, but different enough to consider use of the human antigen inappropriate for vaccination studies in nonhuman primates (Fig. 2b). In humans, CEA messenger RNA translates to a protein that has a molecular weight of 70 kD: the additional weight of this protein derives from an extensive level of glycosylation, leading to a final size of 180 kD. Moreover, CEA protein includes an N-terminal sequence followed by 3 disulphide-linked repeats of 178 amino acids, and a hydrophobic C-terminal domain

Test Anaemta Iron Diabetes Glucose Proteins Total Albumin Electrolytes Sodium Potassium Chloride Bicarbonate Calcium Phosphate Liver/heart, enzymes Alk. phosphatase Bilirubin Gamma-GT AST ALT Lac. dehydrogenase Kidney function Urea Creathinine Fat metabollam Cholesterol Hematology Analysis White blood cells Leukocytes Neutrophils Lymphocytes Monocytes Ensinophil granolocytes Basophil granolocytes Neutrophils Lymphocytes Monocytes Eosinophil granolocytes (%) Basophil granolocytes (%) Red blood cells Erythrocytes Haemoglobin Haematocrit Mean corpusc. volume Mean cell haemoglobin Mean cell haemoglobin conc. Thrombocytes Platelets Platelets width distribution Mean platelet volume

Monkey #

2.76

5.82 2.13 3.39 0.08 0.2 0.02 36.7 58.2 1.4 3.4 0.3 4.59 7.4 36.9 80.4 1612 20.1 234 15.4 13.1

2.47

5.72 3.24 2.2 0.06 0.2 0.02 56.7 38.5 1 3.5 0.3 4.59 7.3 35.8 78 1590 20.4 199 13.5 11.9

146.5 4.7 70.6 31.1 63.9 360

135 5 79.5 25.3 45.5 188 6.13 82

144.9 3.13 105.3 24.5 2.24 1.42

144.3 3.59 106.6 27.9 2.28 1.31

4.93 76

71.3 47.9

2.59

3.26 71.5 47.2

27.93

POST

242 9.1 9

4.81 7.7 39.5 82.1 1601 19.5

6.07 2.72 3.16 0.11 0.06 0.02 44.8 52.1 1.8 1 0.3

3.73

4.79 63

234.7 5.2 73.2 43.9 90.2 289

144.7 3.15 105 29.1 2.29 1.42

68.9 46.2

144.7

6.97 2.96 3.86 0.06 0.07 0.02 42.4 55.4 0.9 1 0.3

3.16

6.18 65

257.4 4.6 80.2 39.2 67.5 457

144.1 3 102.5 26.6 2.31 1.54

65.7 46

144.1

25.29

POST

303 9 9.1

4.71 7.4 38.1 80.9 1571 19.4

RI137

30.69

PRE

333 9.4 9.6

4.11 7.2 35.8 87.1 1752 20.1

61 2.85 3.09 0.07 0.07 0.02 46.8 50.7 1.1 1.1 0.3

3.09

4.73 76

146.3 4.6 81.3 29.6 96.6 379

142.7 3.17 108.1 24.7 2.28 0.92

70 45.4

3.09

6.52 2.19 4.11 0.09 0.1 0.03 33.6 63 1.4 1.5 0.5

3.19

6.49 79

149.4 3.8 76.8 59 403.7 733

144.9 3.01 108 22 2.24 1.07

67.5 46.8

2.5

18.44

POST

358 10.1 10.7

3.91 7 34.4 88 1790 20.3

RI002

25.98

PRE

283 10.3 9.9

4.53 71 36.2 79.9 1567 19.6

7.12 2.92 4.07 0.07 0.03 0.03 41 57.2 1 0.4 0.4

3.07

4.58 57

128 6.7 65.2 94.9 385.8 234

144.5 3.41 107.4 26.5 2.2 1.44

69.2 45.8

2.65

8.3 2.36 5.75 0.08 0.07 0.04 28.4 69.3 1 0.8 0.5

3.83

6.71 69

93.8 7 50.9 42.2 152.7 384

143.8 3.44 105.5 23.4 2.22 1.11

71.2 46.6

2.56

18.6

POST

311 11.4 10.7

4.7 7.2 27 78.7 1532 19.5

RI3l1

22.46

PRE

300 12.6 11.6

5.17 7.8 41.8 80.9 1509 18.7

6.14 3.44 2.35 0.06 0.24 0.05 56 38.3 1 3.9 0.8

3.16

3.75 74

402.5 5.3 78.1 23.3 16.1 214

143.8 3.3 105.6 26.4 2.33 1.43

66.7 48.3

3.6

4.85 1.7 2.83 0.05 0.22 0.05 58.4 58.4 1 4.5 1

3.01

4.35 67

207.5 3.6 94.3 28.7 47 237

145.3 3.23 106.1 20.8 2.33 1.52

67.8 46.6

3.87

23.18

POST

310 13.1 12.1

4.83 7.4 40.3 83.4 1532 18.4

RI497

21.29

PRE

TABLE I – EVALUATION OF CLINICAL PARAMETERS OF VACCINATED RHESUS MONKEYS

24.09

PRE

col2

334 11.6 11

4.98 7.8 0.4 80.3 1566 19.5

6.96 3.37 3.4 0.13 0.04 0.02 48.3 48.9 1.9 0.6 0.3

3.63

4.98 64

94.8 5.3 60.5 13 27.3 159

141.4 3.15 107.5 17.4 2.28 1.39

66.4 47.8

4.33

3.69 1.14 2.39 0.07 0.08 0.01 30.8 64.8 1.9 2.2 0.3

2.8

4.33 65

60.2 3.5 54.8 49.1 165.8 209

144.6 3.84 109.6 19.8 2.38 1.2

67.6 47.6

4.22

27.09

POST

361 12.3 11.6

4.88 7.6 0.4 81.6 1557 19.1

RI512

17.75

PRE

192–529 3 109/L 7.7–14.4 fL 8.5–12.7 fL

4.36–6.02 3 1012/L 6.7–9 mmol/L 34.5–46.9% 72.6–84.4 fL 1371–1653 amol 17.4–21.2 mmol/L

2.37–12.98 3 109/L 0.51–11.18 3 109/L 0.25–9.00 3 109/L 0.0–1.30 3 109/L 0.0–0.21 3 109/L 0.0–0.11 3 109/L 21.5–86.1% 10.7–69.3% 0.0–10.0% 0.0–1.6% 0.0–0.81%

1.85–4.83 mmol/L

2.94–9.5 mmol/L 44–l12 mol/L

53.2–672.9 U/L 1.4–6.4 mol/L 24.1–122.6 U/L 10.5–52.1 U/L 4.3–80.5 U/L 71–2006 U/L

138.9–150.7 mmol/L 2.94–4.26 mmol/L 100.8–111.2 mmol/L 18.6–33.8 mmol/L 2.05–2.52 mmol/L 0.94–2.28 mmol/L

61.8–78.1 g/L 39.6–53.7 g/L

1.68–4.81 mmol/L

13.8–37.22 mol/L

Normal values units

2298 AURISICCHIO ET AL.

GENETIC VACCINE AGAINST CEA IN NON-HUMAN PRIMATES

which is linked via lipid into the membrane, through a glycosylphosphatidylinositol moiety. The polypeptide is also shed in the blood, and for this reason considered a suitable marker for cancer progression.9 All these features are common to rhCEA, as shown by protein sequence and observed by Western blot and ELISA (Figs. 1, 3 and 5b), So far, only a few studies have shown that members of the CEA family are expressed in nonhuman primates.41,42 It would be interesting to verify whether colon adenocarcinomas in macaques overexpress rhCEA antigen similarly to humans. To increase the level of antigen expression we have codon-optimized the cDNA of rhesus CEA and generated genetic vectors which displayed enhanced immunogenicity. Rhesus CEA was immunogenic in mice (Figs. 3 and 5c), conferred a significant antitumor effect against adenocarcinoma cells overexpressing human CEA (Fig. 5d) and, most importantly, combining plasmid DNA-EP and Ad vectors resulted in disrupting immune tolerance in all vaccinated monkeys. Significant cell-mediated and humoral immune responses against the antigen were detected in immunized animals, albeit with a qualitatively and quantitatively different elicited response among monkeys, since it was directed against several epitopes contained in various peptide pools encompassing the antigen. Interestingly, the type of elicited immune response was mainly CD81 T cell specific, which may be crucial for effective antitumoral action against CEA-expressing carcinomas. Some of the animals developed a consistent immune response against putative specific immunodominant determinants: for instance, monkeys RI137 and RI497 showed strong IFNg secretion upon stimulation with rhCEA Pool C of peptides, whereas monkey RI512 specificity was directed mainly toward rhCEA Pool D (Figs. 4 and 6). This observation is not surprising since, contrary to inbred laboratory animals, the monkeys included in our study were not selected in advance for the same Mamu HLA Class I haplotype. Two monkeys vaccinated with vectors bearing wild type rhCEA and both monkeys immunized with optimized vectors developed significant antiCEA antibody titers (Figs. 4c and 6b). Due to the small number of animals, we cannot conclude that rhCEAopt expressing vectors are more immunogenic than vectors carrying the native cDNA in rhesus monkeys. Nonetheless, the increased efficacy of codon-optimized human CEA in transgenic mice,23 the data obtained with mice vaccinated with rhCEAopt and the observation that cell-mediated and antibody response in monkeys RI497 and RI512 were significantly higher than that in the other monkeys all indirectly support the notion that codon usage optimization of cDNA enhances the immunogenicity of genetic vaccines in large animal models. In the past years several Phase I clinical trials have demonstrated that patients vaccinated with vaccinia and avipox vectors

2299

expressing CEA were able to mount anti-CEA specific immune response with no significant adverse effects,43–47 although combining the antigen with costimulatory molecules and cytokine administrations had a significant impact on the efficacy of vaccination with these vectors. The feasibility of implementing these strategies in cancer derived from preclinical data obtained with transgenic mouse models. By contrast, our genetic vaccination protocol breaks tolerance and exerts antitumor effects in the absence of concurrent expression of costimulatory molecules and exogenous recombinant cytokines. In vivo electroporation of plasmid DNA resulted in increased antigen expression in the treated muscle, although the enhanced immunogenicity may also derive from undefined adjuvant effects connected with transfection of other cells, muscle damage, and/or release of danger signals.48 Similarly, the strong immunogenicity of replication-defective recombinant Ad may be in part due to the induction of proinflammatory cytokines and chemokines with immune cell infiltrates in the transduced tissue.49 Recently, different genetic vaccination strategies and protocols have been directly compared in nonhuman primates.21 In particular, DNA vectors, modified vaccinia virus Ankara vectors (MVA), and Ad vectors have been evaluated for immunogenicity, each of them expressing the same codon-optimized HIV-1 gag gene. Immunization with the MVA vector as a single modality elicited only minimal cellular immune responses. Similar observations were obtained using another avipox vector (ALVAC).50 The best overall immune response was provided by DNA/Ad5 combination, but a major drawback to using Ad vectors is the r influence exerted by pre-existing anti-Ad immunity, which may attenuate immunogenic potency.21 We are presently evaluating novel Ad serotypes to induce and maintain over time the elicited immune response against specific TAAs. The results of this study imply that a cancer vaccine immunization strategy centered on the use of DNA-EP and Ad vectors could be valid in humans. These findings may confer a significant advantage over the ongoing genetic vaccine strategies, and have important implications for the design of clinical trials for patients with colorectal cancer and other tumor types overexpressing CEA. Acknowledgements We thank Dr. Armin Lahm for help in designing degenerated oligonucleotides for rhCEA cloning. We thank Prof. B. Rosenwirth for coordinating monkey studies and BPRC personnel for technical support in conducting animal experiments. We also thank Mrs. J. Clench for editorial assistance.

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