> 1 DEPARTMENT OF HEALTH AND HUMAN SERVICES

FOOD AND DRUG ADMINISTRATION

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

> BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

MEETING #31 - VOLUME III

Friday, October 26, 2001

8:14 a.m.

> Holiday Inn Gaithersburg Two Montgomery Village Avenue
>
> Gaithersburg, Maryland

2

PARTICIPANTS

Daniel R. Salomon, M.D., Chairperson

Gail Dapolito, Executive Secretary

MEMBERS

> Richard E. Champlin, M.D. Katherine A. High, M.D.

Alison F. Lawton (Industry Representative) Richard C. Mulligan, Ph.D.

Mahendra S. Rao, M.D., Ph.D.

Edward A. Sausville, M.D., Ph.D.

CONSULTANTS

Jonathan S. Allan, D.V.M.

> Kenneth Cornetta, M.D. (by telephone) Michael Emerman, Ph.D. (by
> telephone) David W. Gaylor, Ph.D.

Katherine E. Knowles (Consumer Representative)

Bruce E. Torbett, Ph.D.

John A. Zaia, M.D.

GUESTS/GUEST SPEAKERS

Boro Dropulic, Ph.D.

Yvette Delph, M.D. (Patient Representative)

Marina O\'Reilly, Ph.D.

> Amy Patterson, M.D. Marvin Reitz, Ph.D.

FDA PARTICIPANTS

> Philip D. Noguchi, M.D. Daniel Takefman, Ph.D. Carolyn Wilson, Ph.D.

3

C O N T E N T S

Opening Remarks: Dr. Daniel Salomon

Topic 2: Lentivirus Vector Gene Transfer Product

for Treatment of People with HIV

Autologous E-Cells Transduced with VRX496: HIV-1-Based

Lentiviral Vector Treatment for Patient-Subjects Infected with HIV-1:

Boro Dropulic, Ph.D.

VIRxSYS

Open Public Hearing

Susan Kingsman, Ph.D.

Committee Discussion of Questions

> 4 1 P R O C E E D I N G S

2 Opening Remarks

3 DR. SALOMON: I apologize. I did say we

4 would start at 8:00. Today is the second day of

5 deliberations on the lentiviral class of vectors.

6 I think yesterday set the ground rules for sort of,

7 in general, things about assays, about safety,

8 about mobilization, about transient versus stable

9 production.

10 I think it is a wonderful opportunity as a

11 follow up this morning to begin with Dr. Boro

12 Dropulic from VIRxSYS presenting now what has been

13 presented to the RAC and has been presented to the

14 FDA as what could be the first lentiviral gene-delivery

15 vector trial in patients with HIV entitled

16 Autologous T-cells Transduced with VRX496, HIV-1-Based

17 Lentiviral Vector Treatment of Patient-Subjects Infected

18 with HIV-1.

19 Boro? TOPIC 2: LENTIVIRAL VIRUS GENE

TRANSFER PRODUCT FOR

20 TREATMENT OF PEOPLE WITH HIV

21 Autologous T-Cells Transduced with VRX496

22 HIV-1-Based Lentiviral Vector Treatment of

23 Patient-Subjects with HIV-1

24 DR. DROPULIC: First of all, I would like

25 to thank you, the committee members, for the

> 5 1 opportunity to present our work.

2 \[Slide.\]

3 First of all, I would like to say that I

4 really appreciated the discussion that went on

5 yesterday regarding HIV vectors, regarding issues

6 of using HIV vectors in the clinic. I wanted to

7 say that the issues that were discussed yesterday

8 were really the same issues that we have been

9 grappling with for the last several years, both

10 when I was at Hopkins and now with my team VIRxSYS.

11 The point that I want to make as a

12 backdrop for my presentation is that we believe

13 that this vector system that we are proposing is

14 the safest vector to use in the first instance of

15 clinical trial in humans using an HIV vector. That

16 is with two important considerations in mind; one,

17 that the vector works, that it can actually inhibit

18 HIV in our case and, second of all, that the

19 payload gene is regulatable. And so, in our case,

20 it is tat- and rev-dependent regulation.

21 With that, I will start with the first

22 slide.

23 \[Slide.\]

24 Just one slide on the company, really,

25 just to tell you who we are. We are established

> 6 1 for three years. We have 40 employees. We are

2 located about three blocks from here. Our mission

3 is to develop gene therapies for serious disease

4 states and our present entire focus is the

5 development of a gene therapy for the treatment of

6 individuals with AIDS. That is all the company is

7 doing at the moment.

8 \[Slide.\]

9 So target disease AIDS. This is my slide

10 where, every morning, the reason I come to do what

11 we are doing. There are 40 million people infected

12 with the virus worldwide, 1 million in the United

13 States.

14 The drug therapy, HAART therapy, can

15 suppress HIV infection but is not a cure. HAART

16 therapy is toxic. There is a cumulative failure to

17 therapy and, also, resistance to HAART is on the

18 increase. So there is a definite need for new

19 approaches for the treatment of HIV infection.

20 Our approach is to turn the virus against

21 itself, develop and use an HIV vector with anti-HIV

22 payloads to interfere with wild-type HIV

23 replication. We are not saying that this is a

24 cure. Our goal here is not to remove the virus

25 from the patient but basically to interfere with

> 7 1 HIV replication in order to decrease viral loads

2 and postpone the development of AIDS. That is our

3 goal.

4 \[Slide.\]

5 We believe that HIV infection is the

6 appropriate disease target to first test HIV-based

7 vectors. The reasons for that are as follows:

8 testing of HIV vectors in non-HIV-infected

9 individuals could result in their seroconversion.

10 There is a risk that you could give that patient

11 AIDS. There is that risk.

12 If non-HIV-infected individuals became

13 infected with HIV, there will be ambiguity as to

14 the source of infection. Our target patient

15 population is already laden with wild-type HIV so

16 that is why we really believe that this is the

17 right candidate patient-subject population to

18 deliver an HIV vector and also we are selecting a

19 patient population that has no good treatment

20 options left. They are failing HAART therapy; they

21 have a viral load of greater than 5,000; and they

22 show the X4 strain of HIV.

23 The presence of X4 strain, the T-cell-tropic virus

24 means that the patient is in more

25 advanced disease, they are in the later stages of

> 8 1 disease, rather than treating somebody that is

2 early.

3 \[Slide.\]

4 We believe that HIV vectors are the

5 appropriate lentiviral vector for clinical testing

6 as the first vector. The biology and pathogenesis

7 of HIV in humans is well understood in contrast to

8 other lentiviruses. We are selecting an HIV

9 population with no good treatment options and this

10 population can be identified. They exist.

11 The introduction of non-HIV vectors into

12 humans, particularly those infected or at risk of

13 being infected with HIV, could result in

14 unpredictable consequences. So that is why we

15 think HIV vectors are appropriate.

16 \[Slide.\]

17 So this is our proposed clinical protocol.

18 It is an ex vivo. We are not directly injecting

19 the vector so, basically, it is a process where we

20 are taking out cells from the patient, transducing

21 the cells with vector and then expanding the cells

22 and then reintroducing them back into the patient.

23 VRX, the acronym that we are using for our

24 vector is an HIV vector that contains an anti-HIV

25 antisense sequence. The patient populations I have

> 9 1 already described, failing HAART, viral load of

2 greater than 5,000, CD4 counts of 200 to 600

3 because you need at least a certain number of CD4

4 T-cells to be present in order to be able to

5 isolate and amplify them so that you can

6 reintroduce them back into the patient, and the X4

7 strain of HIV.

8 T-cells are isolated, hit with the vector,

9 expanded and then reintroduced back into the

10 patient. That is our proposed protocol.

11 \[Slide.\]

12 So, a schematic representation of our

13 vector is as follows. Basically, it is derived

14 from pNL4-3, one of the best-studied molecular

15 clones of HIV. These are the fragments in which we

16 have derived our vector. It contains a region from

17 the 5\' that contains the packaging sequence. It

18 also contains a region from pol that incorporates a

19 central polypurine track region.

20 The antisense payload is also derived from

21 wild-type HIV. We are not placing in a protein or

22 something that could be potentially antigenic. It

23 is an antisense RNA and it is derived from the

24 envelope region of the wild-type HIV. You can see,

25 it is just flipped and inserted in here.

> 10 1 Then what we have is a fragment from the

2 RRE which regulates the messenger RNA expression

3 from the vector. The only heterologous sequence

4 that we have here is a small, non-coding disposable

5 marker sequence for GFP. We have inserted this in

6 there so we can basically track the vector

7 uniquely.

8 What is important is that the antisense

9 payload is tat and rev regulatable. There is a

10 splice, a sector site located just downstream of

11 the RRE region, that basically makes this antisense

12 payload both tat and rev dependent. Tat and rev

13 are needed for genomic messenger-RNA expression

14 from the vector. So it is a highly regulatable

15 system for the expression of the payload sequence.

16 The reason why I have outlined here VRX496

17 and 494 is because, for our laboratory studies, we

18 actually have a vector that expresses GFP. So we

19 can look at marking. We can look at transduction.

20 For the clinical-grade vector, there are no coding

21 regions, no protein-coding regions in this vector.

22 It is a completely gutted vector.

23 \[Slide.\]

24 So some of the safety features of our

25 vector for our proposed gene transfer in HIV-infected

> 11 1 patients; we believe it is the safest

2 approach for gene transfer in HIV-infected

3 individuals because the vector is entirely derived

4 from wild-type HIV and a well-studied wild-type

5 HIV.

6 No sequences other than HIV are introduced

7 into the patient. The patient subjects are laden

8 with the virus and the vector backbone is actually

9 constructed from highly conserved regions of the

10 virus. And NL4-3, the backbone, is derived from

11 strains common to North America.

12 As evidence for safety from nonhuman

13 primates, that also may add weight to the safety of

14 this vector system. Attenuated SIVs that are

15 deleted in accessory genes do not cause disease in

16 nonhuman primates. There are no accessory genes in

17 either our vector or VIRPAC.

18 Now, although attenuated SIVs containing

19 genes such as IL2 can exacerbate the disease, no

20 such reports for attenuated SIVs without genes have

21 been reported and, importantly, our vector does not

22 encode for a gene, any gene, like IL2, for

23 instance.

24 \[Slide.\]

25 Additional safety features for our vector

> 12 1 are that we have targeted expression of the anti-HIV

2 antisense payload. The antisense payload would

3 only become expressed in cells transduced with the

4 vector that then become infected with wild-type

5 HIV. So it is a highly regulatable way of

6 expression your anti-HIV gene.

7 Another safety is that the antisense

8 payload appears to decrease mobilization of the

9 vector to cells. I am going to show you data for

10 that in a few slides. Expression of the

11 antienvelope antisense results in decreased

12 mobilization of packaged VRX496 genomes, so

13 mobilization is the term where the vector comes out

14 and then goes into another cell.

15 Also, our vector contains a stop codon in

16 gag so that recombination with the helper of the

17 wild type would result in a nonfunctional gag/pol

18 open reading frame if that event should occur.

19 \[Slide.\]

20 Let me tell you a little bit about this

21 stop codon. The stop codon is located just

22 downstream of the packaging signal. In the helper,

23 what we have engineered is we have basically gotten

24 rid of the packaging signal and degenerated this

25 first region of gag.

> 13 1 So if a recombination event would occur

2 where, for example, the reverse transcriptase comes

3 and jumps over in this region that is homologous

4 between the vector and the helper, it would

5 incorporate this gag/stop signal so the resulting

6 recombinant would not be able to produce gag/pol.

7 \[Slide.\]

8 So there was a little bit of a confusion

9 yesterday as a result of the comments that I have

10 made in the document regarding that the result of

11 recombination between the vector and the wild-type

12 HIV would result in either a noninfectious

13 recombinant or wild-type HIV.

14 I wasn\'t referring to sequence-specific

15 effects. What I was referring to is that if the

16 vector does recombine with wild-type HIV, you can

17 only get either a noninfectious event, a

18 noninfectious recombinant or a wild-type HIV type

19 of virus. It is not meant to be sequence specific,

20 per se.

21 So let me go through some of these

22 possible recombination events that we have modeled

23 here. I have already mentioned the gag stop for

24 the helper. A similar thing would happen if wild-type HIV

25 would be recombining. Let\'s just look at

> 14 1 this event. If the reverse transcriptase comes

2 along here and crosses over in the common region of

3 the cPPT, between the vector and the wild type, you

4 would get the crossover event but the result would

5 be is that you would get a truncated gag/pol.

6 Another event is that if the reverse

7 transcriptase here would cross over in the RRE

8 region, you would have a truncated envelope. This

9 event would probably take two events to occur but

10 you could imagine that if, basically, the reverse

11 transcriptase picks up this antisense payload and

12 then puts it back into the virus, you would still

13 get a wild type. Yes; its phenotype would be

14 changed because now it would contain envelope

15 sequences that could possibly confer an X4

16 phenotype strain to this virus but, nevertheless,

17 it would be a wild-type HIV.

18 \[Slide.\]

19 But, in order to address the sequence

20 issue of increasing the pathogenicity of the virus

21 through recombination between the vector and the

22 wild type, I just want to make one point\--a few

23 points, but one point here. The backbone of the

24 vector contains regions of HIV that are highly

25 conserved; the LTR, this packaging gag, cPPT and

> 15 1 RRE, are all highly conserved regions. I would

2 imagine that a patient infected with HIV at the

3 late stages of disease would contain these

4 sequences.

5 The only region that is actually not

6 highly conserved is a region in the payload

7 sequence, the antisense payload sequence. That

8 confers an X4 tropism. It is in the V3 loop that

9 gives rise to X4 tropism of the GP120 sequences.

10 The way that we address that issue is that we are

11 going to restrict our patient-subject population

12 only to those that demonstrate the X4 strain of

13 HIV. So, if recombination should occur between the

14 vector and the wild type, that patient already has

15 X4.

16 One point that is very important is that

17 we are not claiming that mobilization of our vector

18 is required for anti-HIV efficacy. We have vectors

19 that mobilize well. We have vectors that don\'t

20 mobilize virtually at all, which is VRX496. We

21 have specifically chosen VRX496 because of its

22 extremely poor mobilization characteristics because

23 of the issue of mobilization for the first

24 lentiviral-vector clinical trial.

25 In the future, mobilization may provide

> 16 1 some additive effect but I doubt very much that

2 VRX, its anti-HIV efficacy is at all contributable

3 to any mobilization effect because we don\'t really

4 see any significant mobilization. I will show you

5 the data.

6 VRX496 is a maximally gutting vector with

7 poor mobilization characteristics but yet it

8 maintains its high transduction and anti-HIV

9 efficiencies and it can still regulate its payload

10 expression through tat and rev dependencies making

11 expression of the payload only occurring in cells

12 containing vector and wild-type HIV.

13 \[Slide.\]

14 There was also discussion about using

15 potential SIN vectors. But we believe that these

16 are not optimal for AIDS gene therapy. First of

17 all, you would lose the target of expression of tat

18 and rev because, by activating the 3\' LTR, all the

19 LTRs, you would have to substitute some other

20 promoter.

21 That other promoter cannot be HIV-LTR

22 because you would create direct repeat sequences

23 making the vector unstable. You would have to use

24 some sort of other promoter. So, likely, you would

25 use a constitutive expressing promoter and, in that

> 17 1 case, you would have constitutive expression of the

2 antisense payload and that may be detrimental to

3 the host cell.

4 We have avoided constitutive promoter

5 elements in our vectors because we wanted to make

6 it highly regulatable. Also, by replacing or

7 modifying the HIV sequences 5\' and 3\' to the

8 antisense, we would have a detrimental effect on

9 HIV efficacy because we believe that these 5\' and

10 3\' sequences allow trafficking of the vector RNA to

11 its target wild-type HIV-RNA. So disrupting this

12 may affect trafficking and the ability of the

13 antisense to accumulate at the epicyte where wild-type HIV

14 is accumulating.

15 Also, we don\'t believe that the antisense

16 is the only thing having its effect. We believe

17 that, through competition for packaging, and that

18 can be at the level of all these elements that are

19 in the vector, that the anti-HIV effect is

20 cumulative, is due to the vector and the antisense

21 payload because we can see, and others have

22 reported, that just the basic vector alone can have

23 some antisense anti-HIV activity.

24 A SIN vector, by definition, would not be

25 able to compete with wild-type HIV for packaging.

> 18 1 Still, you cannot guarantee that a SIN vector will

2 not be able to mobilize.

3 \[Slide.\]

4 So let me tell you a little bit about the

5 features of our VIRPAC helper construct. It is a

6 two-plasmid system. One of the reasons that we are

7 using a two-plasmid system is because we do get

8 higher levels of production. That may not be

9 important for academic-scale production of the

10 vector but it is very important for scale-up

11 manufacturing of the vector.

12 Every complication, every additional thing

13 that you add to the manufacturing process, results

14 in a decreasing yield. So that is one of the

15 reasons. But what we have done is we have some

16 features in our VIRPAC construct that we believe

17 make it safe.

18 There are no accessory genes in VIRPAC.

19 We have done degeneration of several regions of the

20 helper in order to decrease the likelihood of

21 recombination with the vector, as I have already

22 mentioned to you, the gag region. Also, we have

23 four strong poly-As and two transcriptional pause

24 sites to partition the structural envelope genes.

25 So, let me tell you, we have here a CMV

> 19 1 promoter that drives gag/pol. And we have an RRE

2 element that is degenerated that basically then

3 allows tat and rev here to be\--tat and rev is first

4 expressed. When there is an accumulation of tat

5 and rev, then that allows the gag/pol to be

6 expressed.

7 Then what we have between this open

8 reading frame and the VSV-G here are two poly-A

9 sites, the bovine growth-hormone poly-A site and

10 then, tandem to it, is the alphaglobin poly-A site.

11 Then, in addition to that, there is a pause site to

12 decrease the chance of transcriptional read through

13 between the gag/pol tat rev and the VSV-G.

14 In addition, there is a poly-A, SE40 poly-A here.

15 There is a synthetic poly-A and another

16 pause site located here to prevent the

17 transcriptional read-through going in the other

18 direction. So we have taken a different strategy

19 to make our helper safe.

20 \[Slide.\]

21 Some of the safety features of our VIRPAC.

22 First of all, I want to say because we are using

23 the VSV-G and HIV vectors, there is always a

24 theoretical possibility that they can recombine.

25 But what we can do is minimize the potential for

> 20 1 vector and helper recombination by intelligent

2 construct design. What that means is that we try

3 to force the putative RCL to go through multiple

4 events. Multiple events would be needed to obtain

5 a putative RCL. That is how you minimize the

6 potential for recombination.

7 You have to do that, minimize the

8 potential for homologous DNA recombination and also

9 reverse-transcriptase-mediated RNA recombination.

10 So there are strategies other than simply splitting

11 plasmids that are available to minimize the

12 potential for recombination.

13 \[Slide.\]

14 Let\'s look at DNA recombination and the

15 events. So this is a depiction of the vector here.

16 And I am showing the plasmid backbone here. This

17 is a depiction of the helper. So let me go through

18 it again. CMV promotor driving the gag, which is

19 degenerate. There is no packaging sequence here.

20 pol. Degenerate RRE.

21 Then we have rev IRES tat. Then we have

22 two poly-A sites, a transcriptional pause site.

23 Then we have the promoter that drives the VSV-G,

24 the poly-A and then the backbone of the plasmid.

25 These are the regions of homology. There is

> 21 1 homology between the backbones of the plasmid.

2 There is a region of homology between the cPPT

3 regions that are common in both the vector and the

4 helper, and there is a very small region here in

5 gag, just before the stop site, that is common to

6 both the gag and vector\--the helper and the vector.

7 \[Slide.\]

8 So can homologous recombination produce a

9 putative RCL in one step? No. The answer is no.

10 So let\'s look at this. Two classes of events. The

11 first event here is that you get recombination,

12 DNA, homologous DNA recombination, occurring

13 between two sites; here the pol and this small gag

14 region.

15 So what would be the resulting virus, the

16 resulting recombinant? It would contain the

17 vector. It would contain gag/pol and it also would

18 contain the LTR here. The other class of event is

19 that you get recombination in the backbone and,

20 say, in one of these two sites, for instance. I

21 haven\'t shown all the three sites, just to make it

22 simple.

23 So you get recombination here and

24 recombination here. What would be the resultant

25 recombinant? Well, you would have the backbone.

> 22 1 You would have the poly-A, the VSV-G. You go

2 through all these elements, and then you pick out

3 the gag and that is what you have. You would have

4 the LTR.

5 So the first event is not an RCL because

6 it still doesn\'t contain any envelope. There is no

7 VSV-G. The second event basically contains no 3\'

8 LTR that is necessary for reverse transcription.

9 So one event does not produce an RCL.

10 \[Slide.\]

11 So do two events produce an RCL? Let\'s

12 look at that. We have now this gag/pol recombinant

13 that is formed. Let\'s look at, then, DNA

14 recombination between the gag/pol and the backbone

15 of the plasmid. What is the recombinant? The

16 recombinant has to go this way. It then picks up

17 the VSV-G, the rev and the tat and then recombines

18 that way.

19 So it now does contain VSV-G, tat, rev,

20 gag/pol, but it doesn\'t contain and 3\' LTR.

21 \[Slide.\]

22 So what does this mean? This is what you

23 would have. This thing would still have a

24 difficult time in being replication competent.

25 First, in the center of its genome, there are two

> 23 1 strong polyadenylation sites, a bovine growth-hormone poly-A

2 and an alphaglobin poly-A. There

3 is, in addition, a transcriptional pause site

4 located between the gag/pol and the VSV-G.

5 To get rid of this event, the virus would

6 have to be able to synthesize this read-through

7 transcript and then delete the pause site, the two

8 poly-As, without removing the promoter element

9 because they would need the promoter element to

10 express the VSV.

11 In addition, it still has this stop site.

12 So if this transcript is made, you cannot produce

13 gag/pol because you have the gag stop here. Quite

14 frankly, it is difficult to see how additional

15 events would produce an RCL beyond that which would

16 be common to any production system, and what I mean

17 is nonhomologous-type events. And, in addition,

18 there is no 3\' LTR.

19 We are not trying to say this is better.

20 We are saying this is comparable to the other types

21 of production systems that are available.

22 \[Slide.\]

23 Now let\'s look at RNA recombination. RNA

24 recombination requires RNA not DNA, so let\'s look

25 first at the transcripts that are produced. There

> 24 1 are three transcripts that are produced from the

2 helper. The first transcript is the gag/pol open-reading

3 frame. And then, also, the second

4 transcript is the rev IRES tat that is needed to

5 further express the gag/pol.

6 The third transcript is the VSV-G. Now,

7 all these transcripts don\'t contain a packaging

8 site and it must be remembered that these RNAs

9 would have to be copackaged with the vector RNA in

10 order to mediate an RNA recombination event. So

11 already the event is fairly low because you would

12 require copackaging of these RNAs with the vector

13 RNA in order for reverse transcriptase to mediate

14 the crossover event.

15 So let\'s look at some of these events.

16 \[Slide.\]

17 Basically here is the reverse-transcriptase

18 molecules using the poly-A\--first,

19 let me answer the question. RNA recombination does

20 not produce an RCL in a single event. So let\'s

21 look at this. This is that the reverse

22 transcriptase takes the poly-A, binds to the helper

23 and then basically crosses over again in order to

24 pick up the packaging sequence.

25 This results in no envelope. There is no

> 25 1 VSV envelope incorporated into this RNA. So it

2 does not produce and RCL in a single event.

3 \[Slide.\]

4 Does the RNA recombination produce an RCL

5 in two events? Well, in contrast to DNA, the next

6 event\--so you have this gag/pol now. The next

7 event would mean that it would have to come up here

8 with the poly-A, bind to the poly-A VSV-G and then

9 pick up the VSV-G sequence. There is no other

10 homologous region to cross over so the next step

11 would have to occur by nonhomologous recombination.

12 I will address that event in another

13 slide, but you can see, in two events, through

14 homology, you cannot get beyond an RCL in two

15 events.

16 \[Slide.\]

17 What would happen if the read-through

18 transcript would get packaged? Again, I want to

19 caution you that the read-through transcript does

20 not contain any packaging sequence so, again, it

21 has lowered the chance for it to be copackaged.

22 Then it would have to read through these two poly-A

23 sites and the pause site in order to create this

24 read-through message.

25 Let\'s say that that does occur. If this

> 26 1 occurs and then gets packaged, and then

2 recombination occurs, this is the event that would

3 occur. The reverse transcriptase would take the

4 poly-A, here, pick up the VSV-G, pick up all these

5 sequences and then come back here.

6 So the next slide shows the event.

7 \[Slide.\]

8 This is what you would have. But this

9 thing still has problems\--it is not an RCL. It

10 still would have problems to replicate. Again,

11 there are two polyadenylation sites that would be

12 located within its genome and a pause site. A stop

13 site would be located to prevent gag/pol

14 translation and, again, it would be difficult to

15 see how an RCL would be produced beyond that which

16 would be common to any production system.

17 \[Slide.\]

18 So, in summary, comparison of VIRPAC

19 between other production systems, including a

20 stable producer cell line, currently there is no

21 producer cell line that has been described, at

22 least to my knowledge, that can produce vector

23 titers to at least 10

7 transducing units of cGMP-grade HIV

24 vector in what we think for the proposed

25 clinical trials in order to get a sufficient dose

> 27 1 to get the transduction that you need to get the

2 vector in.

3 VIRPAC offers advantages. You can produce

4 sufficient amounts of clinical-grade vector from a

5 scale-up manufacturing process. Multiple

6 recombination events would be required to generate

7 a putative RCL. And we believe that VIRPAC

8 contains safety features that are comparable to

9 other transient production systems and produce a

10 cell line.

11 \[Slide.\]

12 So let\'s move on from the constructs to

13 the data. These vectors can transduce primary

14 human T-cells and many other primary human cells

15 with very high efficiency. This is just to show

16 you what we can robustly produce with the VRX494

17 vector that expressions GFP.

18 This is done in the multiplicity infection

19 of 20. Control cells. And essentially greater

20 than 99 percent transduction with the vector. Very

21 high transduction efficiency.

22 \[Slide.\]

23 One of the things that we looked at was

24 how stable was this transduction. We looked at it

25 by a number of means and we also wanted to look at

> 28 1 whether the vector was toxic. The way that we are

2 measuring vector toxicity is by the effects of the

3 vector on the cells during expansion of the cells

4 in vitro.

5 So what we have here is an arbitrary

6 scale, depending upon what you are really looking

7 at. This is the data culture of the transduction.

8 So we are looking at EGFP. You can see that it is

9 very high transduction and it remains stable during

10 the course of this experiment.

11 Actually, we have shown that these

12 vectors, because the GFP is expressed from the LTR

13 through the spliced message, it is stable for

14 months. If you look at the copy number of the

15 vector per cell by TaqMan PCR, it is also very

16 stable during the course of the experiment.

17 This is all occurring, and this is what is

18 very interesting, during over 1000-fold expansion

19 of the cells. So, if you look at the fold

20 expansion of the untransduced cells in red and when

21 you compare them to the transduced cells, you can

22 see that there is no real significant difference

23 between the level of expansion. This level of

24 variation you would see in any two particular

25 cultures of expanded T-cells.

> 29 1 So we get very high and stable

2 transduction efficiency with this class of vector.

3 \[Slide.\]

4 And the vectors inhibit wild-type HIV

5 extremely well. The cells were transduced. We

6 don\'t select the cells. We just directly then

7 challenge them with wild-type HIV. This is the

8 p24. This is a log scale. This is the date and

9 culture and infection.

10 In this particular case, we are using a

11 multiplicity infection of 0.001 really because we

12 are trying to mimic low amounts of virus that could

13 be seen in a patient, but I am going to show you

14 data that we have done it for higher MOIs, 0.01,

15 0.1, and we get similar effects.

16 Basically, you can see that, while control

17 cells replicate wild-type HIV extremely well, there

18 is three logs of inhibition of wild-type-HIV

19 replication by vector-containing cells. This is

20 really extraordinary.

21 \[Slide.\]

22 Showing it at higher MOIs. So what we are

23 doing here is we are varying the dose of challenge

24 virus that is inputted into the challenge culture.

25 So these are transduced cells that were challenged

> 30 1 either with an MOI of 0.001, 0.01 and 0.1. You can

2 still see that, when you compare them to the

3 untransduced cells, that you still are getting very

4 effective inhibition of wild-type HIV replication.

5 \[Slide.\]

6 What is also interesting is that it seems

7 that the T-cells are also resistant to productive

8 HIV infection. How do we look at productive HIV

9 infection? Well, when HIV infects a cell and

10 productively infects the cell, it expresses GP120

11 VPU amongst other proteins. These proteins,

12 besides their other effects, can also downregulate

13 CD4 expression. They bind for CD4 and downregulate

14 the expression.

15 So we are using downregulation of CDR as a

16 means of looking into whether the cells are

17 productively infected with wild-type HIV. As you

18 can see, while control cells decrease their

19 frequency of CD4-expressing cells during the period

20 of the culture, transduced cells do not.

21 So it suggests that the cells have a

22 resistance, a selective resistance, to productive

23 HIV infection.

24 \[Slide.\]

25 Also, what we have looked at is inhibition

> 31 1 of vector-containing cells with different strains

2 of HIV. We have chosen two viruses that are X4-tropic HIVs

3 and two strains of virus that are R4.

4 NL4-3 is virus derived from the prototypic

5 molecular clone. BK132 is a primary isolate that

6 has X4 tropism that is the only passage through

7 once in tissue culture. This was derived from an

8 HIV-infected patient from an associate of Carl June

9 at the University of Pennsylvania.

10 Ba-L and US1 are R5 tropic strains of HIV.

11 As you can see here\--maybe you can\'t see this

12 obviously at the back; I can hardly see it here\--but this is

13 Day 23 out after an infection. You can

14 see that both X4 and R5 strains of HIV are

15 controlled fairly well by vector-containing cells,

16 by the vector.

17 You can make an argument that perhaps the

18 antisense payload is having an additional

19 inhibitory effect on the X4 rather than the R5 but

20 with our later data showing that it is really

21 inhibiting well at later stages, I would say the

22 conclusions of this data are that the payload is

23 effectively inhibiting both X4 and R5 strains of

24 HIV.

25 \[Slide.\]

> 32 1 What is also interesting is that cells

2 containing the VRX494 show, again, selective

3 resistance to CD4 downregulation in a mixed culture

4 that contains transduced cells and untransduced

5 cells. So what we are trying to do here is move to

6 situations that more mimic what it would be like in

7 the body or in an HIV-infected individual.

8 So what we did is we transduced the cells

9 so that we would get roughly half of them to

10 basically contain the vector, and that means that

11 they are EGFP positive, and half the cells don\'t

12 contain the vector. And then we challenged them

13 with different strains of HIV in the MOI, as you

14 can see right there.

15 This is actually data from 36 days after

16 infection, but you can see that, while cells that

17 don\'t contain the vector downregulate CD4, cells

18 that contain the vector have a selective resistance

19 to this CD4 downregulation indicating that they

20 have a selective resistance to productive HIV

21 infection.

22 \[Slide.\]

23 So, a summary of the in vitro transduction

24 and challenge data is as follows. We can see high

25 transduction efficiencies in primary human T-cells

> 33 1 with this class of vector. When you challenge

2 these cells with wild-type HIV, over 99 percent of

3 the wild-type HIV can be inhibited upon challenge

4 with a variety of MOIs. Both X4 and R5 strains of

5 HIV can be effectively inhibited by the antisense

6 payload which is almost 1 kb in length targeted to

7 HIV envelope.

8 Cells transduced with the vector show a

9 selective resistance to CD4 downregulation and,

10 hence, to productive wild-type HIV replication.

11 \[Slide.\]

12 As we are moving now towards the clinic,

13 we wanted to do a comparative study between our

14 laboratory-grade vector which expresses EGFP and

15 our candidate clinical vector which only has that

16 GFP marker fragment, no proteins expressed. So we

17 did a transduction in Sup-T1 cells at various

18 transduction MOIs and then challenged with wild-type HIV at

19 an MOI of 0.01.

20 As you can see, while control cells

21 replicated the virus very well, the cells

22 containing the vector inhibited extraordinarily the

23 replication of wild-type HIV, no matter, really,

24 the transducing MOI here. So this shows that our

25 laboratory-grade vector and our clinical candidate

> 34 1 vector are comparable.

2 \[Slide.\]

3 So the next stage is that we wanted to see

4 if would can transduce CD4 T-cells at the patient

5 scale. So we basically asked Bruce, who is sitting

6 in the audience here\--we sent him up some vector.

7 We made it at the right amounts. He got a whole

8 leukopheresis product and transduced it with our

9 vector.

10 Then he looked at some interesting

11 toxicity endpoints to see whether the vector is

12 toxic to cells during our mock transduction that

13 would be very, very similar to the transduction

14 procedure that would go on in a clinical trial. So

15 the toxicity parameters, we looked at the doubling

16 level, the population doubling level during the

17 culture.

18 In blue, these are the transduced cells

19 and, in red, these are the mock cells. As you can

20 see, there is no appreciable difference between

21 mock and transduced cells. If you look at the cell

22 size during the expansion period, it is eleven days

23 in culture, no significant difference between

24 vector transduced and mock cells.

25 If you look at the viability of the cells

> 35 1 during the expansion period, again, no significant

2 difference between mock and transduced cells. If

3 you look at a cell-surface profile between mock and

4 transduced cells, you will see no significant

5 difference. The way that you read this is that the

6 first two blue and red bars are blue is transduced

7 and red is mock at Day 7 while blue transduced and

8 mock at Day 11.

9 But if you look at these doublets\--and you

10 can look at that in the handouts; we have submitted

11 this data before\--there is no significant

12 difference between the surface expression of these

13 markers on these cells.

14 DR. SALOMON: Dr. Dropulic, I will resist

15 the temptation to interrupt you too often, but I am

16 a little stuck here. What exactly did you do here?

17 DR. DROPULIC: What we did here was we

18 made a preparation of vector\--

19 DR. SALOMON: What does a \"preparation of

20 vector\" mean?

21 DR. DROPULIC: We made a pilot lot of

22 vector using our manufacturing procedure. So that

23 was, then, put into bags, sent up to the University

24 of Pennsylvania. The whole leukopheresed product

25 was isolated and then transduced\--the cells were

> 36 1 isolated, the T-cells were isolated and then

2 transduced with the vector.

3 DR. SALOMON: So it was all T-cells.

4 There was no CD4 purification.

5 DR. DROPULIC: Not in this particular

6 case.

7 DR. SALOMON: There was a monocyte

8 depletion?

9 DR. DROPULIC: There was a monocyte

10 depletion. Bruce, could you comment on that?

11 DR. LEVINE: The pheresis unit that we

12 obtain is first washed in a Code 2991 cell

13 processor and some of that was removed. It is

14 washed to remove the platelets. And then we do a

15 monocyte depletion by adherence that takes about an

16 hour. The cells that we\--

17 DR. SALOMON: Can I ask you a question?

18 How many cells did you eventually take\--when you

19 say a monocyte depletion by adherence. To what?

20 To large plastic bags? Or did you just take a

21 couple hundred million cells and put them down on

22 plastic Petri dishes?

23 DR. LEVINE: They adhere to magnetic

24 beads. It provides a much larger surface area in a

25 smaller bag than you could do with a T175 flask.

> 37 1 Basically, the entire pheresis unit can be

2 monocyte-depleted in two 150 ml bags. From that

3 preparation, we take approximately a billion cells,

4 stimulate them with other magnetic beads that have

5 conjugated to them anti-CD3 and anti-CD28

6 antibodies that we have used in all our trials.

7 The vector is added pretty much as a media

8 supplement just as you would add glutamine to the

9 media, we just add vector at the appropriate

10 dilution. Then those cells are grown in gas-permeable

11 culture bags for the duration of the

12 culture that you see up there.

13 DR. SALOMON: No interleukin 2.

14 DR. LEVINE: Well, we don\'t have to add

15 interleukin 2 but we add a low level of interleukin

16 2, approximately 100 units per ml.

17 DR. SALOMON: That is low? If you gave

18 100 units per ml of interleukin 2 to a human being,

19 they would die.

20 DR. LEVINE: 100 units of\--

21 DR. SALOMON: If you gave 100 units to the

22 whole patient, no. But I mean if you tried to

23 achieve a level of 100 units per ml in a patient,

24 that would be very high.

25 DR. LEVINE: What I mean by low is when

> 38 1 previous investigators have cultured what have been

2 called the lac cells, they use very high amounts of

3 IL2, 800 units per ml or higher. What that tends

4 to do is make the CD8 cells grow out in the

5 culture.

6 What we have found is if we add 20 to 100

7 units per ml, that we maintain the CD4 to CD8

8 ratio. The second point is that that is low enough

9 that you are not conditioning the cells to be

10 cytokine dependent. We believe that when you

11 culture the cells with very high amounts of

12 interleukin 2, 800 units, 1000 units per ml, when

13 you would infuse those cells, they would be

14 dependent on that high level of interleukin 2 in

15 vivo. That is obviously not present.

16 DR. SALOMON: Your evidence that 100 units

17 per ml of IL2 does not condition the cells is\--

18 DR. LEVINE: Well, I would say in vivo we

19 don\'t have evidence of that. But if we do grow the

20 cells without IL2, for most of the normal donors we

21 are growing, they grow just as well. We think of

22 it with some of the HIV cells that we get, it helps

23 maintain an adequate level of expansion.

24 DR. TORBETT: Can I ask a question. Can

25 you tell me, just a little bit, do you add the

> 39 1 virus during the full stimulation period? Do you

2 add your vector preparation to the bag during the

3 full preparation?

4 DR. LEVINE: Yes.

5 DR. TORBETT: Could you go over a little

6 bit how you actually transduce the cells during

7 this time on volume?

8 DR. LEVINE: As I said, you just add it as

9 a media supplement depending on the MOI that you

10 would like to achieve and the titer of the virus,

11 so it is a very small amount of the vector added to

12 the culture media. It is added on Day 0 and then

13 basically diluted out as you add media to expand

14 the cells. Then, at the time of harvest, the cells

15 are washed, completely washed, three or four times

16 with volumes of Plasmalyte prior freezing.

17 DR. SALOMON: Last question. What was the

18 MOI in these experiments?

19 DR. LEVINE: Was it 40, I believe?

20 Vladimir?

21 DR. SLEPUSHKIN: I think, in this

22 experiment, this was a clinical experiment and it

23 was an MOI of 200.

24 DR. SALOMON: 100 did you say?

25 DR. SLEPUSHKIN: 200.

> 40 1 DR. SALOMON: 200 MOI?

2 DR. DROPULIC: Right; by copy number,

3 because you don\'t have GFP.

4 DR. SALOMON: Okay; I\'m sorry for

5 interrupting.

6 DR. DROPULIC: No; my pleasure.

7 DR. SALOMON: I was just\--there are some

8 details here.

9 DR. DROPULIC: Please interrupt.

10 \[Slide.\]

11 So then we took this preparation and

12 basically they are normal human\--patients that are

13 not infected with HIV\--and basically challenged

14 them with NL4-3, and basically looked at the

15 inhibitory effects of the vector-containing cells.

16 As you can see, it inhibited about 2 logs of virus

17 here when you compare the controls to the vector.

18 \[Slide.\]

19 Now we have moved on with additional

20 studies to take CD4 T-cells from HIV-infected

21 donors. Basically, this was a patient that had a

22 viral load of about 92,000 and a CD4 count in about

23 the 600 range. What we are looking for here is are

24 there any effects of the vector specifically on

25 cells derived from HIV-infected individuals.

> 41 1 So what we are looking for here is for

2 cumulative cell expansion. In red are the mock

3 cells. In blue are the transduced cells. You can

4 see no appreciable difference. If we look at copy

5 number per cells with this preparation, we can find

6 that normal human T-cells can be transduced at

7 about three copies per cell while HIV-infected CD4

8 T-cells are in the same range, about two copies per

9 cell.

10 \[Slide.\]

11 So these cells were first expanded, then

12 frozen down and then we thawed them so that they

13 would be frozen down as they would have been done

14 in a clinical trial. And then we thawed them and

15 then grew them out to look at whether the virus

16 would come back and replicate. So this is virus

17 that is endogenous to the patient. We are not now

18 infecting with another virus.

19 So when we cultured them\--so we expanded

20 for eight days, froze the cells down, thawed them

21 and then grew them up again and then looked for p24

22 after expansion. So what we have here is that the

23 mock-containing cells, you can see that the virus

24 were all back and replicated while, in the vector-containing

25 cells, it controlled HIV replication for

> 42 1 about two logs for about ten days and then you see

2 this bump or breakthrough effect.

3 \[Slide.\]

4 I will show you what the breakthrough

5 effect is\-- we have done analysis on that\--in just

6 a moment. But, at the same time, what we had done

7 is we looked at the presence of CD4 on these cells.

8 So this is looking at downregulation of CD4 from

9 cells that are transduced from an HIV-infected

10 donor.

11 So here are the non-treated cells. You

12 can see the amount of CD4-expressing cells is about

13 40 percent while almost twice as many cells were

14 CD4-positive when treated with the vector.

15 DR. SALOMON: Marvin?

16 DR. REITZ: Excuse me. The question I had

17 was this an HIV-infected donor that was under

18 treatment or a treatment-naive patient?

19 DR. DROPULIC: No; I think that patient

20 was on treatment but failing. I can\'t exactly

21 remember. I have to go back to the people at

22 Hopkins, but I believe it is a patient that was

23 failing therapy.

24 So that is that. Twice as many cells

25 appear to be less\--twice as many cells are CD4-positive in

> 43 1 cells treated with the vector compared

2 to nontreated controls.

3 \[Slide.\]

4 So, getting back to this issue of what is

5 this bump, we did RT PCR analysis where we looked

6 at the types of viral RNAs that were present in

7 this culture. So what we have here is we do RT PCR

8 where we have two sets of primers. One is for wild-type HIV.

9 That is this band that goes across here\--and one that

10 specifically detects the vector.

11 Each of these is days after infection, so

12 what we are doing is we are comparing mock\--this is

13 the transduced cells. As you can see, M is mock.

14 V, here, is the vector-containing cells. You can

15 see early in an infection that the wild-type virus,

16 we can detect the wild-type virus. In the

17 transduced cells, you don\'t see that packaged

18 vector until very late in infection, in this

19 infection process.

20 And then you can see that the vector is

21 coming up. So the vector is copackaging, or being

22 packaged. In this case, it is being selectively

23 packaged into progeny virions because there is more

24 of the vector being present. So this bump is not

25 entirely wild-type HIV. It is some wild-type HIV

> 44 1 but the vector is being packaged.

2 Now packaged vector doesn\'t mean it is

3 mobilized. It is going out there. Mobilization

4 means that it has to go in to a neighboring cell.

5 So we looked at that question by taking samples and

6 then infecting them on naive CD4 cells. That is

7 represented by the next slide.

8 \[Slide.\]

9 So here you have mock HIV. These are the

10 two primers here. These are mock HIV cells. These

11 are just mock controls. This is a vector and no

12 HIV. And these are the cells, vector, that are

13 infected with HIV.

14 Although you can see the wild-type HIV

15 band, you don\'t see the vector, suggesting that,

16 actually, the copackaged virus is having a very

17 difficult time mobilizing, getting to the

18 neighboring cell. In fact, you can\'t really detect

19 the band and we have to resort to TaqMan PCR to see

20 whether there was any mobilized vector at all.

21 What we found, by TaqMan PCR, was that, in

22 these samples, 30 copies of vectors were mobilized

23 into CD4 T-cells per 10,000 cells analyzed. So an

24 extremely low frequency.

25 DR. MULLIGAN: May I interrupt for just a

> 45 1 second. First of all, I would dispute the\--you can

2 call it mobilization of whatever you want to call

3 it, but I would say that what you are demonstrating

4 would be what I would call mobilization. That is

5 mobilization out. There is a component. And

6 mobilization into cells.

7 DR. DROPULIC: I call it packaging,

8 actually. I call it co-packaging and mobilization

9 is as it is actually going into the cell.

10 DR. MULLIGAN: Okay. I wouldn\'t call it

11 that. But if you quantify the mobilization event,

12 or packaging event, if you look at the efficiency

13 of that event relative to the packaging in wild

14 type in this experiment here, can you give us a

15 sense of how\--well, not this one, but the one you

16 just showed\--how efficient is that?

17 DR. DROPULIC: How efficient is what?

18 DR. MULLIGAN: How efficient is the

19 generation of packagable vector in this system?

20 DR. DROPULIC: It only occurs at low copy

21 numbers of cells and, on the packagable vector, it

22 is not very efficient.

23 DR. MULLIGAN: Maybe go back a couple to

24 where\--

25 DR. DROPULIC: Sure. That is a problem

> 46 1 with this. Let me just try that. There we go.

2 \[Slide.\]

3 DR. MULLIGAN: So it is a little busy for

4 me to tell what bands we are looking at here, but\--

5 DR. DROPULIC: These are the bands that

6 you are thinking about.

7 DR. MULLIGAN: So you are looking at the

8 ratio of those versus what represents the\--

9 DR. DROPULIC: So this is the vector here

10 and this is the wild type here. All I am saying is

11 that we can see this. This is happening. We

12 didn\'t do any studies in terms of ratios or

13 anything. But when you take this type of soup and

14 passage it, it doesn\'t go into the cells very

15 efficiently.

16 DR. MULLIGAN: I just want to make sure I

17 have got this right, though. So if you look at the

18 ratio of those two, you are saying, of the virus

19 particles that you are looking at, there is

20 significantly more of the vector than there is of

21 the \"helper,\" the wild type; is that right?

22 DR. DROPULIC: I wouldn\'t say

23 significantly more. I would say marginally more;

24 yes.

25 DR. MULLIGAN: You would say that that is

> 47 1 a marginal difference between the\--

2 DR. DROPULIC: I don\'t know what is

3 significant? What is it, maybe a five-fold effect?

4 DR. MULLIGAN: That is a marginal

5 difference?

6 DR. DROPULIC: A five-fold effect.

7 DR. MULLIGAN: I would not call that a

8 marginal difference. But I guess my point is that

9 is suggesting that is a very, very efficient

10 packaging of a vector. So you call it packaging.

11 I will call it mobilization. But there is a very

12 significant amount of vector that has been out in

13 the soup.

14 DR. DROPULIC: Okay. I can tell you that\--do you

15 want to say? Go ahead.

16 DR. LI: My name is Yuexia Li. I work for

17 VIRxSYS. First of all, I want you to know there is

18 a duplex RT PCR. The lower band is a smaller piece

19 so you have a more efficient\--when you do the PCR,

20 it is more efficient. So you may have more signal

21 here amplified. Also, this is a qualitative assay.

22 It is not a quantitation so you can\'t just see the

23 intensity of the band and say, okay, you have much

24 more vector here than the wild type.

25 If you repeat it exactly, you may get a

> 48 1 slightly different result. What we want to show

2 here is in the p24 peak, we want to characterize

3 just by the nature of that peak, that the p24 value

4 was contributed by the wild type and some vectors.

5 It is not a qualitative assay. You can\'t just

6 stick in that band and say, okay, you have much

7 more vector than the wild type. We can\'t draw that

8 conclusion.

9 DR. MULLIGAN: I think I was just trying

10 to raise is that this is am important clue. This

11 is what we have been looking for to see whether

12 there is, indeed, packaging. I think the next step

13 is, of course, why you don\'t detect it as being

14 capable of infecting.

15 DR. DROPULIC: Right.

16 DR. MULLIGAN: And, in fact, what,

17 exactly, is that species.

18 DR. DROPULIC: Right. We haven\'t looked

19 into what is the event. But what is likely is

20 happening is that you are getting copackaging of

21 wild-type HIV and vector. You have got two

22 different genomes. If that occurs, you have got

23 the antisense binding by hybridization and that

24 would make it very difficult for that vector, for

25 that packaged recombinant, in order to reverse

> 49 1 transcribe and integrate because there is 1 kb of

2 antisense that is binding to the envelope region of

3 the wild-type virus.

4 So that is our explanation of why we are

5 seeing it.

6 DR. SALOMON: Have you characterized this

7 at all, then? That was actually in your clinical

8 protocol, I believe so, if this was found during

9 the trial.

10 DR. DROPULIC: Yes.

11 DR. SALOMON: You say you will stop and

12 characterize this.

13 DR. DROPULIC: No; if we see an RCL, we

14 will stop and characterize it or if we see

15 packaging of a VSV signal, we will stop and

16 characterize it. But I don\'t think if we see this

17 event, we would stop and characterize it. We will

18 monitor for whether VRX is mobilized or not, but it

19 is not a stopping event presently.

20 DR. SALOMON: It would just be interesting

21 after all the very elegant sort of molecular

22 strategy you showed us earlier about this

23 recombination could occur and this recombination

24 could occur to actually look at what got packaged

25 here.

> 50 1 DR. DROPULIC: I agree.

2 DR. SALOMON: Just to finish this. Then

3 the idea here is\--I guess the problem I am having

4 just a little bit here is with adjectives; low,

5 nothing, minimal. So 30 copies in 10,000 cells, I

6 calculate to 3,000 copies in a million cells or

7 about 1 million PBL per ml. So we are talking

8 about 3,000 copies of packaged vector per ml of

9 blood, basically.

10 DR. LI: Actually, that 30 copies is the

11 30,000 cells because when you run the TaqMan, you

12 run triplicate in all three wells together, they

13 only find 30 copies. Each well, you have it.

14 DR. SALOMON: Oh; okay. It says 30 copies

15 per 10,000. So, 1000 copies per ml of blood.

16 Okay.

17 DR. DROPULIC: Next slide.

18 \[Slide.\]

19 So we looked at mobilization, what I

20 define to be mobilization, by taking basically

21 cells, either primary human CD4 T-cells or Sup-T1

22 cells, and then transducing them with vector and

23 then taking the supernatants, transducing the cells

24 with the vector and then basically challenging them

25 with a very high MOI of 0.2 with wild-type virus,

> 51 1 and then taking the supernatants of those cells and

2 then infecting MT4 cells, which is a very sensitive

3 cell for HIV infection.

4 So what we have here is we have our

5 untransduced controls. We have here VRX430 which

6 does not contain the antisense payload. It is

7 analogous to VRX494 that does contain the antisense

8 payload. So when we look at the MT4 cells, we can

9 see that, with the vector that contains no

10 antisense payload, you see a very small amount of

11 mobilization. I am just going to use my adjective.

12 You can correct me later\--because I like them.

13 While, when we looked at the cells that

14 were infected with the supernatants from VRX494-challenged

15 cells, we saw no mobilization events.

16 In Sup-T1 cells, which are more permissive

17 to HIV infection, we saw low levels of mobilization

18 with the vector that did not contain the antisense

19 payload. But, again, it decreased, significantly

20 decreased, when the vector contained the antisense

21 payload.

22 So the conclusions are that there is a

23 very low level of mobilization that is occurring

24 and, in addition, the antisense payload decreases

25 mobilization. So it is an additional safety

> 52 1 feature of the vector system.

2 \[Slide.\]

3 So we did this in vitro and now we want to

4 do it in an animal model. This is very difficult

5 to do in an animal model so we kind of made our

6 best stab at it. This is what we came up with

7 looking at in vivo mobilization in SCID mice. SCID

8 mice are nice because, as you all know, you can

9 transplant them with human cells. What we are

10 doing here is we are injecting human CD4 T-cells IP

11 into the animal so you have a small local

12 environment to look for mobilization events.

13 So you isolate human CD4 T-cells. Then we

14 divided the T-cells into two lots. One lot of

15 cells received a vector that expresses EGFP. The

16 other lot of cells were transduced with a vector

17 that expressed EYFP. The next thing about EGFP and

18 EYFP is you can discriminate by FACS. So you can

19 look for dual events. That is what we are trying

20 to look for here.

21 So we have CD4 cells transduced with

22 either vector, mixed together, challenged with

23 wild-type HIV. Then we mix that back with CD4-negative

24 PBMCs and then we injected those cells IP

25 back into the animal, you know, just into the

> 53 1 animal because they are human cells.

2 The types of events that we were looking

3 for is whether the vector mobilized authentically

4 from CD4 cells to CD4 cells. The way that you

5 would see that event is by looking for double-positive

6 cells. If this green vector mobilized to

7 a yellow vector-containing cell, that would be

8 authentic mobilization.

9 It is mobilization that is restricted to

10 the target tissue. While an adverse, if you like,

11 mobilization event would be if the vector, either

12 green or yellow, would mobilize to a marker CD4-negative

13 cell. We have used B-cells because B-cells are lymphocytes

14 closely related and they are

15 definitely CD4-negative.

16 \[Slide.\]

17 So VRX mobilizes poorly between primary

18 CD4 cells in vivo. So what we have here is these

19 are the cells that were inserted into the animal.

20 The only difference between this and this is that

21 these cells were not challenged with wild-type HIV

22 and these cells were challenged with wild-type HIV

23 at an MOI of 0.2.

24 This is the background of the events, so

25 you can see here that this is the background of

> 54 1 double-positive events. You can see some

2 mobilization occurring above the background. So

3 some low level of mobilization is occurring between

4 CD4 T-cells because you are seeing these double-positive

5 events.

6 \[Slide.\]

7 However, the vector does not mobilize

8 adversely. It does not mobilize to CD19 cells.

9 Again, now we are looking for either GFP or YFP

10 expression and then looking for whether we can see

11 that expression on CD19 cells. These are the

12 noninfected background controls and these are the

13 cells from animals that were infected, the cells

14 that were infected with wild-type HIV.

15 You can see no significant events over the

16 background events.

17 \[Slide.\]

18 So, a summary of the in vitro and in vivo

19 mobilization data is as follows. Mobilization, we

20 believe, is only a safety concern when the vector

21 spreads beyond the intended target tissue. That

22 is, in our case, CD4 T-cells. Our in vitro and in

23 vivo data show that VRX496 mobilizes poorly between

24 primary CD4 T-cells and our in vivo data shows that

25 our vector does not mobilize beyond CD4 T-cells,

> 55 1 the intended target tissue. No mobilization was

2 seen into CD19 cells, B cells, which is our CD4-negative

3 marker cell.

4 \[Slide.\]

5 So we also wanted to look at when

6 mobilization occurs between CD4 and CD4 T-cells, is

7 the vector structure or sequence somehow affected.

8 So we did this analysis where we basically produced

9 our vector in our 293 cells by cotransfection and

10 then transduced primary CD4 cells with the vector

11 preparation.

12 Then what we did is we did PCR sequencing.

13 We PCRed out the vector and then looked for were

14 there any deletions or mutations present in the

15 vector genome. We found none, no deletions, no

16 mutations, no insertions. We sequenced the PCR

17 product. That is what I want you to understand.

18 Then what we did is we challenged with

19 wild-type HIV and then infected\--and this was very

20 difficult to do because there wasn\'t a lot of

21 vector sequence in these cells, but we were able to

22 get out a signal. Again, we PCRed out a product

23 and that product, again, when mobilized with wild-type HIV,

24 again showed no deletions, no mutations,

25 no insertions. It didn\'t pick anything up. So

> 56 1 those are the studies that we have just completed,

2 actually.

3 DR. ALLAN: Can I just ask a question

4 right there?

5 DR. DROPULIC: Sure.

6 DR. ALLAN: Did you look at any for

7 recombination? I mean, you are just looking at

8 deletions, mutations?

9 DR. DROPULIC: Anything. Absolutely. I

10 should have added recombination. What we did is we

11 PCRed out the vector sequence. So we used primary

12 in the LTR and one primary in G-tag, the G-tag

13 sequence, PCRed that one fragment out, sequenced

14 it.

15 And then the other side, PCRed that

16 sequence. And there were no changes from the

17 original.

18 DR. ALLAN: But it depends on which

19 primers you are using because you can get

20 recombination and the one set of primers may not

21 function because you do have recombination between

22 wild type virus.

23 DR. DROPULIC: We tried to choose a primer

24 set that would uniquely pull out our vector.

25 DR. ZAIA: I have a question, also. Are

> 57 1 you saying that\--you are looking at virus now two

2 weeks into the infection. This is the virus that

3 is growing out in the presence of transduced cells.

4 DR. DROPULIC: What are you specifically

5 talking about, this last slide?

6 DR. ALLAN: That last slide. When you

7 looked at the\--

8 DR. DROPULIC: This was short-term. This

9 wasn\'t long-term. I am not making a statement

10 about it being long-term. I am just simply saying

11 that when you did this experiment\--

12 DR. ALLAN: I guess my question is are you

13 characterizing the virus that is being selected by

14 the transduced\--

15 DR. DROPULIC: No; this is not selection.

16 This is just simply transduction, challenge, hit as

17 many cells as you can with that supernatant and try

18 to PCR it out. When you PCR it out, that is what

19 we got. This is no long-term selection. But we

20 have done selection experiments. I am going to go

21 into that in just a moment.

22 DR. ALLAN: But you showed us that it

23 takes about two weeks to see a virus grow out in

24 the system.

25 DR. DROPULIC: This is a separate

> 58 1 experiment. Xiaobin?

2 DR. LU: This is Xiaobin Lu from VIRxSYS.

3 This experiment actually characterized the vector

4 cells, not the escape. The escape comes later.

5 DR. ALLAN: I see. Okay.

6 DR. DROPULIC: Next slide.

7 \[Slide.\]

8 We have shown this data before to the FDA.

9 What we have seen is that when you transduce Sup-T1

10 cells\--this particular event doesn\'t occur in

11 primary human CD4 cells. When you transduce at a

12 relatively low MOI of 5 and then only when you

13 challenge with a high dose of wild-type HIV that

14 you see these effects.

15 So this is p24. This is days after

16 infection. You can see that the control cells

17 replicate wild-type HIV very nicely. Now, cells

18 that contain the vector that does not contain the

19 antisense has an effect. It delays it but it still

20 comes up and goes back down.

21 However, when you challenge the cells

22 containing the vector with the antisense payload,

23 you see strong inhibition early but then you see

24 this breakthrough effect. What we wanted to do is

25 really look into this, what this breakthrough virus

> 59 1 could be and try to understand these events more

2 clearly.

3 \[Slide.\]

4 So what we did is we did an experiment

5 where we did high-dose challenge of wild-type HIV

6 at 0.1 in Sup-T1 cells and we did a range of

7 transduction doses with the vector. So these cells

8 were transduced with the vector. Now, this is GFP

9 transduction at an MOI of 20.

10 This is with a transduction MOI of 10.

11 This is a transduction MOI of 5. So, clearly,

12 suboptimal doses of vector can give rise to a

13 breakthrough. But it is related to dose. It is

14 not an escape, per se, in this experiment. It is

15 dose-related.

16 However, that still begs the question can

17 you get an escaped variant that is resistant to the

18 vector.

19 DR. SALOMON: Can I just interpose again?

20 It is this adjective thing. So high MOIs, low

21 doses of vector. So the MOI is\--

22 DR. DROPULIC: 0.1 is considered a pretty

23 high challenge MOI. I don\'t know if anybody would

24 disagree with that.

25 DR. SALOMON: So that is high.

> 60 1 DR. DROPULIC: Yes.

2 DR. SALOMON: And that would be high

3 relative to what is going on in the patient.

4 DR. DROPULIC: No; it is convention in the

5 field, I think, pretty much, an in vitro challenge

6 dose. I don\'t think anybody has really looked at

7 MOIs in a patient, per se. I think that is very

8 difficult to study. Low transduction is just by

9 experience, that the MOI of 5 is relatively low

10 compared to an MOI of 20 where we get very good

11 levels of transduction with the vector.

12 So we wanted to look at whether, really, a

13 resistant variant to the payload can occur.

14 \[Slide.\]

15 So what we did was we took these VRX Sup-T1 cells

16 that were transduced with a lot MOI and we

17 took control cells, and then we passaged the

18 breakthrough virus, as shown here. If you passage

19 this any time on a transduced cell that is higher

20 than an MOI of 5, a transduced MOI of 5, you don\'t

21 see breakthrough.

22 You have to keep on reinfecting at this

23 low suboptimal dose of vector transduction in the

24 cells in order to carry this infection forward.

25 After three passages, we basically took that,

> 61 1 basically the sample, and then did PCR sequencing.

2 So the question is do variants develop

3 that, escape the antisense action.

4 \[Slide.\]

5 So this is the original passaged\--this is

6 the original breakthrough. Then, after three

7 passages, what we found is that it seems like there

8 is something that basically is more resistant to

9 the antisense payload effect because the peak of

10 titer here is at Day 12 compared to Day 20.

11 However, it appears that its fitness, compared to

12 wild type, is significantly impaired.

13 So that is what we found. We found a

14 shift but the peak was significantly lower.

15 \[Slide.\]

16 So then what we wanted to do is take\--

17 DR. TORBETT: Excuse me for just a second.

18 Could you back up to the slide. I didn\'t quite

19 understand what you were doing. I apologize.

20 DR. DROPULIC: All right.

21 \[Previous slide.\]

22 DR. TORBETT: Could you go over in detail

23 what you did, explain it to us?

24 DR. DROPULIC: Okay. This is the Sup-T1

25 cells containing the vector. We challenged with

> 62 1 wild-type HIV. We got the breakthrough. We

2 passaged once, twice and I believe at this time we

3 took the cells out and then did PCR sequence

4 analysis on these cells, I believe.

5 DR. TORBETT: So if spread was occurring

6 during these successive passages\--

7 DR. DROPULIC: Yes; we could see that\--

8 DR. TORBETT: Is that why it is suppressed

9 then at the end because what happened was the low

10 number of copies that you got into the original in

11 the MOI-5 gradually was increasing because of

12 mobilization of the vector, so that, by the time

13 you got to the third or fourth passage, it was

14 equivalent to a starting T-cell population that had

15 an MOI of 10 or higher?

16 DR. DROPULIC: No; we didn\'t look into

17 that. All we were looking for in this experiment

18 was purely the resistance issue. That is all I can

19 tell you.

20 Xiaobin, did you want to mention

21 something?

22 DR. LU: I think what we have done is,

23 after the third passage, we take the soup and

24 transduce, infect Sup-t1 cells. Then we extract

25 the DNA and do the PCR and clone the corresponding

> 63 1 sequence in the plasmid. And we sequence.

2 DR. DROPULIC: Right. That is the next

3 slide, actually.

4 DR. SALOMON: How many clones did you

5 sequence?

6 DR. DROPULIC: I will show you that right

7 now.

8 \[Slide.\]

9 So here we are basically. We took wild-type HIV-

10 specific primers here. And then we looked

11 at a comparison between the number of deletions in

12 the antisense. We compared the wild-type cultures,

13 and what that means is wild-type HIV just passaged

14 on Sup-T1 cells, compared to the breakthrough virus

15 being passaged on the vector-containing cells.

16 The frequency of deletions in the

17 breakthrough virus was extraordinarily high, 91

18 percent. We did 290 clones, to your question, and

19 264 of those contained some form of deletion. We

20 did 40 control clones and 11 of those were deleted

21 so a frequency of 27 percent.

22 Then, also, we looked at the mutation rate

23 and compared it to the wild-type passage cultures

24 compared to the breakthrough passage cultures. We

25 saw there are mutations here. But what was

> 64 1 particularly interesting was this one clone here

2 that had 109 mutations which reflected 12 percent

3 of this antisense binding region contained

4 mutations that would try to convert resistance

5 against the antisense payload.

6 That is the next slide. So I hope

7 everybody understands that. Basically, it is PCR

8 out to just wild-type-specific primers and then we

9 do subcloning into plasmids and then we PCR

10 sequence that as plasmids.

11 DR. ALLAN: What is wild-type virus that

12 you are using?

13 DR. DROPULIC: NL4-3.

14 DR. ALLAN: It is?

15 DR. DROPULIC: Yes.

16 \[Slide.\]

17 So this one mutant I am calling BTP1

18 mutant displayed a mutation frequency of 12 percent

19 in the envelope region that binds to antisense. It

20 is really interesting because this is the region

21 that binds to the antisense. What we did is when

22 we PCRed our cloning, we PCRed a 2 kb fragment.

23 So 1 kb would be reflective inside the

24 region that the vector antisense would bind and the

25 other 1 kb reflects outside that region. It was

> 65 1 interesting that this 12 percent mutation frequency

2 occurred exclusively in this region and not outside

3 the region, if you can sort of think of it as a

4 control.

5 What also was interesting was that

6 essentially all the changes were A to G base-pair

7 substitutions that we saw in this mutant clone, all

8 along the fragment here. This is just a very small

9 region of it. You can just see an example right

10 there.

11 \[Slide.\]

12 This is consistent with the known action

13 of antisense. So the deletions and the mutation

14 data strongly suggests that our antisense payload

15 is behaving by the mechanisms of known antisense

16 action.

17 So let me tell you about how antisense

18 works, what are the mechanisms. Adosine deaminases

19 act to convert adenosines to inosines in double-stranded

20 RNA. This conversion leads to an unstable

21 base pair which leads to partial or complete

22 unwinding of the region and degradation or nuclear

23 retention of the RNA.

24 The mutations that we are seeing would

25 hamper ADA conversion of adenosines and then make

> 66 1 resistant to the effects of the antisense sequence.

2 \[Slide.\]

3 So what we did now was is we took this

4 fragment and we cloned it back into wild-type HIV

5 to see whether it could replicate. So this is the

6 data. What we did is we made both of the plasmids,

7 the mutant and the wild type, transfected it, I

8 believe, into 293 cells\--I think it was 293 cells

9 because we are just looking at one round here for

10 the first round.

11 Then, basically, we find that, in the

12 first round, you could produce p24 from both the

13 wild-type HIV and the mutant. However, when you

14 take that supernatant and passage it on Sup-T1

15 cells, CD4 cell line, to look for replication,

16 while the wild-type HIV can replicate\--this is the

17 second round\--the mutant does not appear to

18 replicate. It has very low fitness, if anything,

19 below detection.

20 When you do a TCRD50, you can see that the

21 wild-type HIV can replicate nicely while the

22 mutant, it was below detection. So this suggests

23 that the virus is trying to create mutations

24 against the antisense payload but it pays a price

25 in terms of its own replicated fitness.

> 67 1 DR. SALOMON: That was the one mutant.

2 DR. DROPULIC: Just the one mutant, but a

3 deletion would decrease the replicative fitness as

4 well, if you know you haven\'t got N.

5 DR. MULLIGAN: Have you ever associated

6 that mutation with the rest of the proviral

7 sequences? Often, there are compensatory mutations

8 at other locations.

9 DR. DROPULIC: We haven\'t looked. I mean,

10 the only thing we have done is what I have shown.

11 We did 1 kb downstream of the site. We didn\'t look

12 at the whole genome, if that is the question. No.

13 DR. MULLIGAN: The question is whether or

14 not that, indeed, this is a fair test in the sense

15 that you have never asked the question whether the

16 entire sequence\--

17 DR. DROPULIC: True. We have not done

18 that. But this is what we have done.

19 DR. TORBETT: I have a question real

20 quickly. You flashed by pretty quickly on your

21 envelope sequence.

22 DR. DROPULIC: Oh; I\'m sorry. Do you want

23 me to go back?

24 DR. TORBETT: No; that\'s okay. I just

25 have a quick question. You are going back in to

> 68 1 Sup-T1s. Your area you are targeting I believe is

2 the V3 area; is that correct, on your antisense?

3 DR. DROPULIC: It is not just specifically

4 V3. It is a 1 kb stretch that is basically most of

5 the 5\' end of the envelope.

6 DR. TORBETT: Is there any chance, and I

7 am sure you have done that, looked on CCR5 using

8 cell line to find out if the tropism is changing.

9 You are going back into Sup-T1s which restricted to

10 X4 using viruses. Is there any chance that it

11 switched over to an R5 during these kinds of tests

12 and you would miss it in your biological assays?

13 DR. DROPULIC: We haven\'t done that. We

14 haven\'t done that experiment. We certainly can do

15 that, but we haven\'t done it.

16 DR. TORBETT: Have you run your sequence

17 through any type of blast search to see homologies

18 with other types of envelopes?

19 DR. DROPULIC: No; we have not. We can do

20 that.

21 DR. TORBETT: Thank you.

22 DR. DROPULIC: We can go back. I wanted

23 to make one last point. It is a speculative point.

24 The mutation severely restricts virus replication.

25 And we believe that, and this is speculation, that

> 69 1 this mutant may persist by pseudotyping wild-type

2 HIV. It can persist and survive. It can\'t

3 replicate on its own but it can survive by having

4 some wild type around to complement it and drive it

5 forward, but it cannot replicate on its own. Mere

6 speculation, but that is our hypothesis of why we

7 think that this thing can be picked up after

8 multiple passages.

9 \[Slide.\]

10 So a summary of the breakthrough data.

11 The initial breakthrough virus is due to high MOI

12 of wild-type HIV overcoming suboptimal transduction

13 levels in Sup-T1 cells. No breakthrough is seen

14 when sufficient doses of vector are used.

15 A variant HIV can be selected that shows

16 increased resistance to vector inhibition.

17 However, the consequence of this resistance appears

18 to be a decreased fitness for replication.

19 We have seen a very high deletion and

20 mutation frequencies in the target env region of

21 wild-type HIV which strongly indicates that the

22 vector is acting upon wild-type HIV by the known

23 antisense base-pairing mechanism.

24 \[Slide.\]

25 Now we move on to in vivo by distribution

> 70 1 and safety studies. There has been a lot of talk

2 about animal models to test the safety of these

3 vectors. We believe that, given everything, this

4 is the best model that we can have. It may not be

5 an ideal model, but it at least has features that

6 basically uses human cells in a xenotransplantation

7 model.

8 So let me tell you a little bit about how

9 these studies were performed. What we have here is

10 we have human cells that are then transduced with

11 the vector and then we inject the cells IV into the

12 mice, and then they distribute throughout the

13 animal. The human cells distribute throughout the

14 organs of the animal.

15 Then, at various time points, we are

16 harvesting the organs and then undergoing PCR to

17 detect for the presence of vector. So the days

18 that we are looking at here is Day 2, immediately

19 after infusion where that is your positive control,

20 if you like, where you would see a lot of vector.

21 You would see the distribution of your vector-containing

22 cells in the animal.

23 Then we looked at Day 30, Day 90 and Day

24 131. Over this period of time, the human cells are

25 dying in the animal. That is the useful nature of

> 71 1 this model is because, as the cells die, you can

2 look for events of autonomous vector mobilization

3 into mouse tissue.

4 So what we are doing here\--

5 DR. SALOMON: Boro, may I just ask one

6 quick question.

7 DR. DROPULIC: Please.

8 DR. SALOMON: These cells, now; were these

9 activated with\--

10 DR. DROPULIC: This are from the pilot

11 lot. These are exactly those cells.

12 DR. SALOMON: So these got the 100 units

13 per ml of IL2 and the whole shtick.

14 DR. DROPULIC: These are exactly that lot.

15 DR. SALOMON: Okay. Fine.

16 DR. DROPULIC: So what we are trying to

17 analyze is for the presence of vector in murine

18 tissue by DNA PCR. So the murine tissue contains

19 human cells and then the vector signal will either

20 be due to the vector being present in the human

21 cells or, if some adverse mobilization event is

22 occurring, the vector would mobilize and then go

23 into the mouse tissue. That is the adverse event.

24 We are only looking for a single event.

25 We know that HIVs cannot replicate in mouse tissue.

> 72 1 The application of this model system occurs totally

2 within the human cells, the infused cell product.

3 What we are looking for is a single event of

4 mobilization, adverse mobilization, into the mouse

5 tissue.

6 So, if an RCL-like event of the vector is

7 detected, that means if you detect vector in the

8 absence of a signal to a human marker gene, that

9 would be an adverse event. Now, the reason why we

10 used human CART is because\--you can\'t use actin

11 because there is 100 percent homology between human

12 and mouse actin. You have to use something where

13 you can find some primers to be able to

14 distinguish.

15 Hu CART has these regions of this homology

16 so you can specifically amplify whether you have

17 got human cells left in the mouse tissue.

18 \[Slide.\]

19 So what are the advantages of using this

20 mouse for biodistribution and toxicity studies?

21 First of all, we are introducing human cells which

22 is what we will be doing in the clinic containing

23 HIV vector into an animal model. It is difficult

24 to conceive of other animal models. This is nice

25 because it is immunodeficient and allows for the

> 73 1 persistence of these human cells in an animal

2 setting.

3 The injected human cells\--this is a nice

4 feature. The injected human cells survive for long

5 periods of time in the animal permitting

6 amplification of an adverse event in the human cell

7 population that is resident in the animal.

8 We have got two windows of amplification

9 of an RCR event here. One window is when the cells

10 are amplified ex vivo in Bruce\'s facility during

11 the ex vivo expansion process. Once you inject the

12 cells, the cells persist. Again, if there is an

13 RCR event that allows for that amplification step

14 to take place within the human cells.

15 Then our final readout is if there is any

16 one single event that just integrates into the

17 mouse tissue. That is what we are looking for.

18 Another feature is that the human cells do

19 eventually die, permitting visualization of adverse

20 events in whole tissues by PCR. So if the human

21 cells would survive indefinitely, you would never

22 be able to discriminate between a signal that was

23 in the human cell compared to a signal that was

24 then mobilized into the mouse tissue.

25 The fact that they die means that you can

> 74 1 look for those events. The HIV vectors can

2 transduce murine cells efficiently, one event,

3 which is likely sufficient for the detection of an

4 overt adverse event. Sensitivity of this assay may

5 be an issue. We acknowledge that. What we are

6 looking for is an overt adverse event in mouse

7 tissues despite the lack of productive HIV

8 infection in the animal cells per se.

9 \[Slide.\]

10 This is just to show you that murine

11 hemopoietic cells are efficiently transduced by HIV

12 vectors. These are human bone-marrow cells

13 transduced with an HIV vector with a very low MOI

14 of 2 and than analyzed 13 days later. We are

15 getting a 73 percent transduction efficiency.

16 So it validates the fact that, if that one

17 event would occur, you could pick it up. It does

18 transduce the cell, murine cell.

19 \[Slide.\]

20 This shows you a little bit about our

21 study design. Basically, these are the animal

22 groups. These are the days that we killed the

23 animals and isolated the organs. The first group

24 is just an infusion media control. The second

25 group is a mock transduced control cells. These

75

1 are cells that do not contain the vector.

2 The third group is vectors, transduced

3 cells, at low dose, 3 by 10

5 cells per mouse. Then

4 the fourth is vector-transduced T-cells at high

5 dose, 2 by 10 7

cells per mouse. These are the

6 number of mice that we used.

7 \[Slide.\]

8 So what I would like to do is just to show

9 you example PCR data and then a summary slide of

10 the data. What we are looking at here first is two

11 days post-injection of the control cells. So these

12 are cells that don\'t contain the vector. What we

13 are looking for here specifically is the G-tag

14 sequence, this unique sequence that is present in

15 our vector by PCR.

16 This is the G-tag sequence, this unique

17 sequence that is present in our vector by PCR. So

18 this is the G-tag. This is the positive controls

19 here. Then what we have is this is an example of a

20 spleen, DNA from the spleen from two animals. Then

21 what we have here is we have two test articles

22 without a spike, and then the third one is spiked

23 with 50 copies of DNA.

24 As you can see, we can detect, by the

25 spiked control, validating the sensitivity of the

> 76 1 assay and, in these control cells, we don\'t see any

2 signal, specific signal for the G-tag sequence.

3 Obviously, there is no vector in the cells so you

4 would not expect to see the G-tag sequence in these

5 cells.

6 \[Slide.\]

7 Then this is an example of data of murine

8 tissues two days post-injection of cells transduced

9 with our vector. Again, this is a specific band

10 that we are looking for. You can see here that it

11 is positive, the spiked control and the no-spike

12 test articles were positive, showing that the

13 tissues contained vector-containing cells.

14 We know that the tissues from this data\--we know

15 the tissues contain vector. What we now

16 have to do is PCR for a human specific sequence to

17 see whether that signal is due to human cells or an

18 adverse mobilization event.

19 \[Slide.\]

20 This is to show you when you do hu-CART

21 analysis of these samples at Day 2, transduced

22 cells, that they are positive. So this is the hu-CART band,

23 positive control, negative control. And

24 these are those two tissues that you saw that are

25 positive for hu-CART showing that the bands that

> 77 1 you saw of VRX496, the vector, are due to human

2 cells. That is what you would expect at Day 2,

3 right after injection.

4 DR. MULLIGAN: One question. I am missing

5 how you link this to the\--why couldn\'t both things

6 be occurring? I don\'t get that.

7 DR. DROPULIC: I\'m sorry; say that again.

8 DR. MULLIGAN: Why couldn\'t both events be

9 occurring; that is, you are getting mobilization in

10 your human cells.

11 DR. DROPULIC: You can\'t detect that. You

12 can\'t detect that in this animal. What you are

13 looking for is animals, that when the cells have

14 died off, the human cells have died off, if that

15 event has occurred, it would have integrated into

16 the human cells and then that is when you are

17 looking for it.

18 DR. SALOMON: At Day 2, you cannot make

19 the conclusion you just made. At Day 2, you have

20 surviving human cells in a compartment and you get

21 a PCR signal for the hu-CART. So that tells you

22 you have surviving human cells.

23 DR. DROPULIC: Correct.

24 DR. SALOMON: At Day 2, you also find the

25 VRX496.

> 78 1 DR. DROPULIC: Right.

2 DR. SALOMON: That doesn\'t mean it is all

3 in the human cells in that compartment. It could

4 be in mouse cells in that compartment.

5 DR. DROPULIC: All right.

6 DR. LI: In order to answer you and Dr.

7 Mulligan\'s question, you have to use a specific

8 technology called in situ amplification. So you

9 have to see your human signal coincide with the

10 vector sequence. But that technology has not been

11 developed. In situ PCR does exist, but it is a

12 tremendous problem.

13 DR. SALOMON: There are other ways to do

14 it. I mean, one of the ways to do it is to use

15 ratios like we published and GTI, our collaborators

16 at GTI published, when we did this because we had

17 the same concerns in xenotransplantation infection

18 where you could have a situation where you had pig

19 cells chimeric in compartments that were also

20 expressing porcine endogenous retrovirus. That is

21 all published. We don\'t want to spend ten minutes

22 talking about that. You can find that in papers in

23 Science and Nature. It would be another strategy

24 here.

25 DR. LI: We know such a problem will be

> 79 1 existing because this is not in situ technology.

2 You disrupt the tissue. You can see both signals.

3 You cannot say\--in vivo location they are together.

4 But, at the current technology we can do, this is

5 the best.

6 DR. DROPULIC: The way the assay is

7 designed is to look for those events later when the

8 human cells die off. Point taken.

9 \[Slide.\]

10 So this is a summary of the Day 2 data.

11 Basically, you can see that these are the groupings

12 here. These are the tissues that we analyzed,

13 heart, testes, ovary, liver, lymph node, blood,

14 tail, spleen, lung, bone marrow. These are the

15 groupings. These are the animal groupings that

16 received the cells, either low dose or high dose.

17 As you can see, most of the animals

18 contain both the vector signal. Whenever we saw a

19 vector signal, we always saw a concomitant human

20 signal as well. Blood, in certain cases, was

21 difficult. These assays generally fail because of

22 sampling size but we had no problems in terms of

23 all the other organs.

24 \[Slide.\]

25 SO now we go on to Day 30, thirty days

> 80 1 post-injection. What we see here is that the human

2 cells\--what we see here is that certain tissues are

3 lighting up positive for vector and certain tissues

4 are not. are not.

5 This is an animal here. This is the

6 spiked control. You can see that, in this

7 particular sample, you can detect the vector

8 sequence. In this particular sample of DNA, the

9 spiked control validates the sensitivity of the

10 assay and there is no presence of vector-containing

11 sequence in this particular sample.

12 \[Slide.\]

13 So the summary of the data. We took those

14 samples that were positive for the vector and then

15 did Hu-CART analysis on these samples. In every

16 case, whenever there was a positive signal for the

17 vector, we also saw a concomitant positive signal

18 for Hu-CART.

19 \[Slide.\]

20 This is the summary of the Day-30 data.

21 You can see that basically the numbers of samples

22 that are lighting up positive for the vector is

23 decreasing. But whenever you saw a sample that was

24 positive for the vector, you always saw a

25 concomitant Hu-CART-positive signal as well.

> 81 1 \[Slide.\]

2 When we look at the Day-91 data, basically

3 most of the tissues are negative for the vector.

4 This is the spike controls. These are the test

5 articles.

6 \[Slide.\]

7 This is a summary of the data. You can

8 see that everything except four independent tissues

9 from four independent mice were negative for the

10 vector. However, in the case that, again, the

11 tissue was positive for the vector, you saw, again,

12 a concomitant positive Hu-CART signal.

13 \[Slide.\]

14 This is not showing up well, but this our

15 latest data, Day 123, post-injection. All the

16 tissues are now negative for the vector.

17 \[Slide.\]

18 Again, that is just the complete data.

19 Everything now has turned negative.

20 \[Slide.\]

21 So, a summary of the animal by

22 distribution. Toxicity data is that infused human

23 T-cells containing vector could survive for long

24 periods, up to 91 days, in these SCID mice. All

25 the tissues studied with the G-tag vector signal

> 82 1 was associated with a concomitant Hu-CART signal

2 for human DNA.

3 So the interpretation is, and this was

4 just discussed, I suppose, that the G-tag signal is

5 due to vector-containing cells. That is the

6 extrapolation I would make.

7 A total of six animals from the study

8 displayed clinical manifestations that were not

9 treatment related. There were some clinical

10 manifestations but they were all, by a certified

11 pathologist, shown to be not as a result of the

12 test article. And no adverse RCL-like events were

13 observed.

14 \[Slide.\]

15 Now a little bit about the manufacturing

16 process for the vector. We used certified 293

17 cells, transfected in NUNC-cell factories with

18 VRX496 and VIRPAC plasmid DNA constructs using a

19 calcium-phosphate precipitation method. The vector

20 is then purified using ultrafiltration,

21 diafiltration and column chromatography.

22 The plasmid raw materials and the purified

23 vector preparation is prepared at VIRxSYS\'s

24 manufacturing facility using GMP conditions. We

25 both have BL3 labs and Class 10,000 Labs. The

> 83 1 Class 10,000 Labs are used for manufacturing of the

2 vector.

3 Then the cell processing will be performed

4 at the University of Pennsylvania Hospital\'s

5 Clinical Cell Production Facility using GMP

6 conditions with Carl June and Bruce Levine

7 \[Slide.\]

8 So there are two steps; making the raw

9 material, the plasmids, and then using those

10 plasmids, then, to transfect into cells to produce

11 the vector. This is routine. Basically the

12 plasmid manufacturing process; you culture the

13 bacteria. You centrifuge them down.

14 \[Slide.\]

15 I am not going to belabor on this too

16 long. I just want to give you a feel for it. The

17 cells are lysed. They undergo filtration.

18 \[Slide.\]

19 Then the plasmid is purified, centrifuged

20 and then filtered, stored and then QC testing is

21 performed on the plasmid.

22 \[Slide.\]

23 When it passes QC testing, then it can be

24 released for use in vector manufacturing. The

25 plasmids are one of the raw materials for the

> 84 1 vector-manufacturing process.

2 \[Slide.\]

3 The vector-manufacturing process; cells

4 from a 293 master cell bank are thawed and

5 expanded, five passages to 16 cell factories. The

6 293 cells, then, are transfected with vector and

7 helper plasmid DNAs using calcium phosphate

8 precipitation. The bulk harvest, the viral vector

9 harvest, is the medium and they are collected at

10 24, 36 and 48 hours.

11 The vector-containing medium is stored at

12 2 to 8 degrees until 60 hours post-transfection.

13 The vector then undergoes filtration and

14 concentration. Then the product is subsequently

15 concentrated via ultrafiltration.

16 \[Slide.\]

17 Then the vector undergoes diafiltration

18 and benzonase treatment to remove cellular host DNA

19 and also the plasmid DNA from the transfection

20 process. The vector is then purified using size-exclusion

21 chromatography and stored at -20 pending

22 the results of in-process tests.

23 \[Slide.\]

24 Then, finally, the vector is formulated to

25 storage. We can store this vector at -20 degrees

> 85 1 for over six months and then basically it undergoes

2 a lot of QC testing before it is released by QA for

3 use in the cell-processing part of that process.

4 \[Slide.\]

5 Just a couple of data slides. Our vector

6 purification; we can purify from our bulk harvest

7 to our chromatography step 1000-fold. It is 1000-fold

8 purification.

9 \[Slide.\]

10 This is just to show you a gel here. So

11 this is what the vector looks like after the bulk

12 harvest. We are validated by Western blot, but

13 this is a VSV-G band. This is p24. This is p17.

14 This is what it looks like after bulk harvest,

15 after diafiltration. And this is the final

16 product, so it cleans it up very nicely.

17 \[Slide.\]

18 After the product is made and there are a

19 bunch of QC tests. There is a whole battery of QC

20 tests that really need to be performed but I think,

21 for the purposes of here, I just really want to

22 talk about the RCT assays and the detection of an

23 RCL because I think that is very important. I

24 think that is the critical thing in this protocol,

25 the assays and the detection of a putative RCL.

> 86 1 What we will do for the final\--after

2 production of the vector from the 293 cells, we

3 will take both the bulk harvest and the end-of-production

4 293 cells and run them through and RCL

5 assay.

6 So let\'s go through the bulk harvest. The

7 bulk harvest will take out, and basically will

8 infect the bulk harvest on H9 cells using the

9 specifications, the guidelines, recommended by the

10 FDA and then passage it for six times to amplify

11 any potential RCL.

12 Then use TaqMan RT PCR on the supernatant

13 to detect HIV gag and VSV-G in that final sixth

14 passaged supernatant. So we will also take the

15 end-of-production 293 cells, co-cultivate them with

16 H9 cells, the correct amounts, passage that for six

17 times and, again, perform RT PCR, TaqMan RT PCR

18 using both gag and VSV-G primers.

19 Negative results will mean that the vector

20 can be released for use pending other QC tests. If

21 it is positive\--we haven\'t seen this to date\--but

22 if a lot would become positive, we would obviously

23 not release it and fully characterize it.

24 Our sensitivity of our RT TaqMan PCR is

25 HIV gag, is 10 copies per input volume. For VSV-G,

> 87 1 it is 10 copies per input volume. The overall

2 assay sensitivity is that we believe or have

3 extrapolated that we can detect the HIV with a

4 fitness of 1 percent of NL4-3.

5 The next slide will show you how we came

6 to that calculation.

7 \[Slide.\]

8 This tells you a little bit about the

9 sensitivity of our assay. What we have done here

10 is we take the cells and then we start either

11 cocultivating or incubating the supernatant in H9

12 cells and then we are passaging, and we are

13 splitting the cells as we are passaging.

14 What we have found out is that if we have

15 taken one infectious unit of wild-type HIV and

16 passaged it, that, after three passages, one

17 particle comes up positive by p24. What we will do

18 is, in addition to that, we are going to

19 additionally passage the culture for another six

20 times.

21 Given that this is 100-fold expansion of

22 the H9 cells, we are extrapolating that this is

23 giving us 100-fold sensitivity over our positive

24 control which, in this case, is wild-type HIV.

25 The problem with positive controls is that

> 88 1 nobody wants to make a VSV-G recombinant HIV. Even

2 if you would, you don\'t know whether that would be

3 the event that would ultimately be seen. So our

4 approach has been just to use wild-type HIV and

5 then increase the sensitivity of the assay by

6 passaging for longer to try to pick up the event.

7 And p24 and RT PCR will be used for the virus

8 detection. In this case, we just used p24.

9 \[Slide.\]

10 Another thing, one of the questions,

11 should an in vitro for the detection of functional

12 gag/pol LTR be used as a lot-release assay. First

13 of all, I would like to say that this assay may

14 have utility for HIV vectors in non-HIV disease

15 applications. However, HIV disease, the final

16 product, already contains these types of events in

17 abundance. There is wild-type HIV there. So we

18 don\'t necessarily see the relevance for our

19 particular case although it certainly may have

20 relevance for other situations.

21 Also, another thing to consider is that

22 those events that you have seen, we have found that

23 when we construct vectors with those types of

24 events, they are actually more efficient in

25 inhibiting wild-type HIV than our fully gutting

> 89 1 vector. So the presence of such an event in a

2 final product may not be a detrimental one in AIDS

3 gene transfer.

4 \[Slide.\]

5 So what is our cell-processing procedure?

6 Again, Bruce and Carl are doing that. We are

7 providing our vector to their facility. They run

8 the cell processing at U. Penn. The patient

9 undergoes leukophereses and T-cell selection. I

10 think Bruce has already described the process but I

11 will just briefly go through here. The cells are

12 transduced with the vector in presence of

13 immobilized CD3, CD28, antibodies. The beads are

14 removed. The cells are washed and concentrated.

15 \[Slide.\]

16 Then the cells are formulated and then the

17 cells will undergo QT testing. The cells are

18 frozen. That is the nice thing about this whole

19 procedure is that we can freeze the cells, perform

20 the QC testing before releasing the cells to use in

21 the clinic.

22 \[Slide.\]

23 So, again, what are the important assays

24 for an RCL detection. Obviously, this is now the

25 final product. These are the cells transduced with

> 90 1 the vector and this is what we really have to

2 examine very carefully for a putative RCL. So,

3 other than all the other assay, these, we believe,

4 are the critical ones.

5 We look at it at two levels. One, we will

6 perform a biological type RCL assay. The second is

7 a molecular detection type RCL assay to detect for

8 any residue of VSV-G DNA that may be present in the

9 final cell product.

10 So we have our ex vivo transduced and

11 expanded T-cells and we will take both the

12 supernatant and the transduced cells and undergo

13 these tests. So let\'s look at the transduced cells

14 first.

15 We take the transduced cells and we will

16 run through the biological assay. The biological

17 assay is now we will take the cells, cocultivate

18 them on 293 T-cells because H9 cells would be

19 permissive to wild-type HIV and kill the culture,

20 so we want to use a CD4-negative cell line in this

21 case. We will take the correct amounts, passage it

22 for six times and then look, by TaqMan PCR, on the

23 amplified supernatant using particularly VSV-G

24 primers.

25 The transduced cells we will also take

> 91 1 directly and undergo DNA PRC directly on those

2 cells to look for any residue VSV-G that may be

3 present. If the results are negative, then we will

4 release the cells. However, if the results are

5 positive, we will not release it.

6 Obviously, if there is anything going on

7 here, we will fully characterize the event. We

8 will also take the supernatant from these ex vivo

9 transduced cells. We will infect them onto 293 T-cells,

10 passage them for six times and again, the

11 passage supernatant will undergo TaqMan PCR to

12 detect for any potential event using VSV-G-specific

13 primers.

14 The supernatant we will also directly take

15 and perform RT PCR to look for any VSV-G signal

16 that may be present in that final supernatant.

17 Again, if it is negative, we will release it. If

18 it is positive, we will not release and we will

19 characterize.

20 Our sensitivity assays for VSV-G DNA

21 detection; we can see our assay down to 1 copy

22 sensitivity. But because of the issue of false

23 positives over sampling size, we are now saying

24 that we can definitely detect our detection limit

25 to be 10 copies per 10,000 cells. And I have

> 92 1 already mentioned that.

2 \[Slide.\]

3 Just to show you that we can get rid of

4 the VSV-G DNA in our final cell product, what we

5 have taken is basically the cells of the

6 transduction. Before the wash, we do have a very

7 low residue of VSV-G DNA in the cells. However,

8 when the cells are washed, we can get rid of this

9 DNA. At least, it is below the detection limit. I

10 don\'t want to say that there may not be absolutely

11 no VSV-G DNA present in that preparation, but it is

12 below our detection limit by TaqMan PCR.

13 So this is two independent experiments.

14 Before wash, we do see some residual VSV-G DNA.

15 After wash, it is below the detection limit.

16 \[Slide.\]

17 So a summary of our proposed clinical

18 trial. We are taking patients that have no good

19 therapeutic options left. They are failing or

20 discontinued therapy. They do not have

21 opportunistic infections. They have a CD4 count of

22 between 200 and 600 and a viral load of greater

23 than 5000 and they demonstrate X4 strain of HIV.

24 That means, they are more advanced in their

25 progress to disease.

> 93 1 The patient comes in. Twelve patients.

2 Basically the T-cells are isolated by a

3 leukopheresis procedure. The vector is produced.

4 When it is released by QC, then it can be

5 transduced onto the cells. It undergoes an ex vivo

6 transduction and expansion process. Then the cells

7 are frozen down.

8 After the frozen-down cells are tested for

9 QC and then released, then the cells can be

10 released for infusion into the patient. Our dose-escalation

11 scheme starts off with a very low dose

12 of cells. The first dose, there will be only one

13 patient at that dose, the first dose escalation.

14 Then the next escalation will be the other

15 two patients at this first 10

9 dose. Then we will

16 dose escalate in patients of three up to 3 by 10

10

17 T-cells infused into the patient.

18 DR. SALOMON: Can you make one thing clear

19 to me. You started out\--what you called patient

20 scale was 1 times 10

9 which is about a unit of

21 blood. Then you activated and these cells

22 proliferate.

23 DR. DROPULIC: 50-fold or so expansion.

24 DR. SALOMON: But you are only giving 1

25 times 10 9. I guess what

confused me is in these

> 94 1 early ones, are you not going to do any activation

2 and proliferation or every time they are going to

3 be activated and proliferated just like they were

4 described and cultured for X number of days? How

5 long? Is it going to be longer if you want more

6 cells?

7 One of the ways to interpret something in

8 the protocol is that you would count every other

9 day or something and stop it when you got to the

10 right number of cells which means that patients

11 later in the trial at higher doses would get cells

12 that had been in culture for longer. That seemed

13 like a very awkward trial.

14 DR. LEVINE: I can tell you the cells will

15 be stimulated and then expanded in the same way for

16 every subject at every dose. From an average

17 leukopheresis, we have 50 to 100-fold times more

18 cells than we would need for the given dose. So we

19 freeze the excess cells at two points; after the

20 monocyte depletion and then, if we have excess

21 cells after the expansion, we freeze those cells.

22 That is useful if something were to go

23 wrong in the culture or the transduction. Then we

24 have these cells frozen. After the monocyte

25 depletion, we can do a second transduction, expand

> 95 1 the cells frozen from post-monocyte depletion.

2 They behave in exactly the same way and then that

3 could be used for infusion as well.

4 DR. SALOMON: So, just as a bottom line,

5 how many days will these cells be in culture with

6 activating antibodies and interleukin 2 before you\--

7 DR. LEVINE: Eight to ten days.

8 DR. TORBETT: I have a naive question.

9 These are from HIV-infected patients. What is

10 just, on the average, the number of T-cells that

11 are infected and, if you activate the cells and it

12 is a low-level infection, would that virus spread?

13 Are you going to include antiretrovirals during the

14 cultures situation?

15 DR. LEVINE: Again, it depends on the

16 viral load. I think on the order of 1 out of

17 10,000, 1 out of 50,000, cells would be infected.

18 In someone with a CCR5 virus, we have shown that

19 CD28 simulation downregulates CCR5 and upregulates

20 the beta chemokines and that there is a diminution

21 to below detection of HIV in CCR5 patient cells.

22 CXCR4; that is not the case. What we have

23 been able to do is to demonstrate that in the

24 CXCR4-positive patient, if we add antiretrovirals

> 96 1 to the culture after the transduction that we can

2 suppress the virus.

3 DR. TORBETT: Thank you.

4 \[Slide.\]

5 Next slide.

6 DR. DROPULIC: So patient monitoring; we

7 are going to perform patient monitoring both early

8 and late. Let me just take you through. There

9 will be patient monitoring, samples taken at 24

10 hours, 48 hours, 72 hours, one week, two weeks, 28

11 days, three months, six months, a year and then

12 yearly for life.

13 I am just giving you one example here, the

14 28 day, because that is where we are proposing our

15 dose escalation to occur after the 28-day period

16 sample is processed.

17 I will just go through the ones that are

18 important. Basically, we will perform, obviously,

19 CD4 counts. Basically, we will also perform this

20 differential viral-load assay. This is kind of

21 important because what we will be doing here is

22 looking for mobilization of the vector. We will

23 have a PCR assay where we will be comparing the

24 amount of wild-type HIV RNA compared to, if there

25 are, any vector RNA present in the serum of the

> 97 1 patient.

2 That is what we mean by a differential

3 viral load. We will also perform some

4 immunological assays. This is very important.

5 Basically, we will also have a TaqMan PCR assay too

6 to look for VSV-G RNA in the plasma. If we start

7 detecting that guy, and it is a consistent result,

8 then we will immediately end the trial because this

9 would be like a red flag to say, hey, there is some

10 sort of VSV-containing RNA that is replicating in

11 this patient.

12 In that case, the patient will undergo

13 leukopheresis and then we will fully characterize

14 the event. So that is a red flag there.

15 We will look for the VSV antibody

16 response. We will look for the repertoire, of the

17 T-cell repertoire. We will also monitor for the

18 phenotype and genotype using the drug-resistant

19 profile assay kits that are available to see

20 whether there is any change in the virus phenotype

21 with respect to drug resistance.

22 Then there are other chemical type assays

23 that we will also perform.

24 \[Slide.\]

25 So, I again reiterate\--I should have put

> 98 1 this slide before the other slide\--patients will be

2 monitored for the short term, 24, 48, 72 hours; 7,

3 14, 28 days and long-term; 3, 6, 12 months and

4 yearly for life.

5 The dose escalation would proceed after 28

6 days. This is our rationale, because most of the

7 short-lived activated T-cells would have died

8 within a few weeks. These are the cells that are

9 most capable to support wild-type HIV replication

10 or the replication of the putative HIV recombinant.

11 Long-lived cells, on the other hand, are

12 normally quiescent and, during their quiescent

13 state, they are not capable of supporting HIV

14 replication. However, they could support HIV

15 replication upon their sporadic activation with

16 antigen. That is why we have a long-term follow-up

17 scheme as well.

18 Since activated T-cells are most abundant

19 immediately after infusion because we are using an

20 immobilized CD3-CD28 approach to activate and

21 expand the cells, the greatest risk for an adverse

22 event, we believe, is short-term. So that is why

23 we have done the dose-escalation scheme that we

24 have proposed.

25 \[Slide.\]

> 99 1 So, in summary, HIV vectors can transduce

2 at greater than 90 percent transduction

3 efficiencies. We can inhibit wild-type HIV

4 replication by over 99 percent and provide CD4 T-cells with

5 what appears to be a selective

6 resistance to productive HIV infection.

7 Our vector, we believe, is the safest

8 vector for this type of trial. It is a fully

9 gutted vector. There are no novel sequences. We

10 are not putting in a CMV promotor or something else

11 into the vector. Even the antisense payload is

12 entirely derived from wild-type HIV. The backbone

13 of the vector is derived from highly conserved

14 sequences.

15 We have shown VRX does not mobilize beyond

16 its target tissue. In vitro and in vivo studies

17 showed poor mobilization occurs only between CD4 T-cells.

18 Our vector-production methods use vector-packaging

19 systems that we believe are comparable to

20 those used in other gene-transfer studies. Our

21 animal by-distribution toxicity studies show the

22 vector to be safe, we believe.

23 \[Slide.\]

24 Our final cell-product release-testing

> 100 1 criteria are highly stringent; no detection of VSV-G DNA in

2 the final cell product, no detection of

3 VSV-G RNA in the final cell supernatant, no

4 detection of an RCL after biological amplification

5 in a highly permissive human cell line, and

6 detection by TaqMan PCR.

7 Treating HIV individuals with advanced

8 disease, we believe, that have no good treatment

9 options left affords the lowest risk for testing an

10 HIV vector and the highest chance\--I am not saying

11 at the low doses anything will happen, but the

12 highest chance for benefit, particularly at the

13 high doses.

14 Drug-therapy failure due to toxicity is

15 common and viral resistance to these drugs is

16 increasing. So there is a real need for new

17 approaches for the treatment of HIV infection. We

18 believe that biological control using HIV against

19 itself may offer new treatment opportunities for

20 individuals with AIDS.

21 \[Slide.\]

22 Would like to thank particularly the

23 VIRxSYS team. They are really a great bunch of

24 people. The reason why we have been able to

25 progress so rapidly is because of their talent and

> 101 1 critical thinking. So critical thinking they are,

2 sometimes I think that I founded VIRxSYS, I thought

3 I founded the first debating society because we are

4 always discussing things.

5 I think that makes for good science so I

6 really am honored to have each and every one of you

7 a member of our team.

8 I would also like to thank Rob MacGregor

9 and Carl June and Bruce Levine and all the team up

10 at the University at Pennsylvania for their

11 collaborative efforts. I would also like to thank

12 Tony Pascorelli, our CEO. He has done a great job

13 in running the company.

14 That\'s it. I will leave it now open for

15 discussion. Thank you.

16 DR. SALOMON: Thank you very much, Dr.

17 Dropulic.

18 \[Applause.\]

19 DR. SALOMON: What I would suggest we do

20 is\--I have a couple of announcements. I would like

21 to introduce a new person who joined us on the

22 committee and then take a break, come back and

23 start to discuss this.

24 So I guess first I would like to note the

25 arrival of Dr. Marvin Reitz. Welcome. Dr. Reitz

> 102 1 is from the Medical Biotechnology Center Institute

2 of Human Virology. Do you want to give us just a

3 quick two cents for what you are doing there?

4 DR. REITZ: Actually, it is the University

5 of Maryland Biotechnology Institute, Institute of

6 Human Virology. We are an institute that is headed

7 by Bob Gallo and mostly do work related to HIV. I

8 am a molecular virologist. I have been fiddling

9 around with retroviruses for about thirty years or

10 so.

11 DR. SALOMON: Good. I am glad you are

12 still healthy.

13 Ten minute break?

14 \[Break.\]

15 DR. SALOMON: I have one additional

16 administrative duty at the beginning of this next

17 session, and that is to introduce Marina O\'Reilly

18 from OBA covering for Amy Patterson again this

19 morning. Welcome, Marina.

20 What I have decided to do here is,

21 basically, go right to the questions, the specific

22 questions from the FDA. I think that what we will

23 do is we will begin a discussion of the questions

24 which were posed by FDA staff specifically about

25 the protocol.

> 103 1 The discussion that will develop is,

2 obviously, specific enough to what you have

3 presented that you will be more than encouraged to

4 make your responses and comments.

5 I was trying to think of how to do this.

6 You either go through all the different phases of

7 this very complex system that you presented\--I am

8 afraid that it would degenerate into sort of an NIH

9 study section which is not what we are here to do.

10 I think, rather, what I would like to do,

11 in terms of being responsive to the very, very

12 important issues that are on the table here would

13 be to go to the questions and, as the conversation

14 evolves, I think individuals on the committee can

15 go to the details. I think, in this way, we will

16 stay grounded in the specifics of the clinical

17 trial and the major questions that were developed

18 yesterday rather than getting lost as can always

19 happen to a group of academics when they get

20 presented a lot of interesting data.

21 So that is kind of a chairman\'s executive

22 decision. If there is anybody who absolutely

23 believes that I have gone inappropriately, I would

24 like to hear it because I don\'t want to be

25 tyrannical on this.

> 104 1 Is that okay with the FDA staff if I take

2 that strategy. Okay.

3 Open Public Hearing

4 But, before we begin, this is also a time

5 where we would normally allow public comment.

6 Again, I don\'t change what we said yesterday, but

7 let me repeat it for the record. Anyone in the

8 audience is welcome to step up to the mike. You

9 were comfortable doing that yesterday. If you were

10 not here yesterday, then please note that you

11 should be encouraged to do so and contribute.

12 But there is also a time for just a formal

13 public comment before we begin any discussion and

14 it is my understand that Dr. Susan Kingsman would

15 like to start.

16 DR. KINGSMAN: Thank you very much. I

17 think most of the discussion is going to relate to

18 the very specific questions that have been posed.

19 The only comment I would like to make is whether

20 the safety of the trans gene has been adequately

21 considered because I think there is a lot of focus

22 on the vector system.

23 I just want to pose the question as to are

24 we delivering, in this therapeutic strategy, a

25 potent and specific mutagen of the HIV envelope

> 105 1 sequence, something that will change its sequence

2 at a higher rate than the normal mutation frequency

3 and that this mutant coding sequence, albeit

4 partial or with low reproductive fitness, in an

5 assay developed in the lab can actually be

6 disseminated in patients with a very high viremia

7 and it is in a region that is functionally

8 important for the envelope.

9 Even if it doesn\'t change tropism, it

10 could create new immune determinants. So I am not

11 entirely convinced that there has been

12 consideration of the trans gene, itself.

13 That is the only comment I would like to

14 make.

15 DR. SALOMON: Thank you, Dr. Kingsman.

16 Committee Discussion Of Questions

17 DR. SALOMON: I am certainly not trying to

18 restrict the scope of the discussions that now

19 follow, but I want to try, before lunch\--how is

20 that for optimism\--to answer the questions and then

21 we will just see where this goes.

22 The first question is, \"Is the VRX496

23 vector proposed for use in the clinical trial by

24 VIRxSYS designed and manufactured in a manner to

25 sufficiently address safety concerns relevant to

> 106 1 generation of replication-competent lentivirus?\"

2 That certainly is something that we discussed at

3 some length yesterday; what is an RCL assay, what

4 are alternative assays, what is the sensitivity?

5 \"Please consider that the vector will be

6 used in HIV-positive subjects. How does this use

7 of a transient transfection system versus a stable

8 packaging line for vector production affect the

9 rate of recombination in a manner that would be

10 sufficiently compensate for the use of one plasmid

11 to encode all helper functions?\"

12 So there are a lot of different pieces we

13 could start off with, but let\'s start with Question

14 1 in general. Does anyone want to jump in and I

15 will try and guide it.

16 DR. EMERMAN: This is Mike Emerman.

17 DR. SALOMON: Excellent. I didn\'t know

18 you were on the phone, Dr. Emerman. But you are

19 more than welcome to jump in.

20 DR. EMERMAN: Some of these questions are

21 related with the RCL. We talked a lot about RCLs

22 which are weird recombinants with VSV-G and things

23 like that. The RCL that they\--well, the lentivirus

24 that they don\'t test for is actually the wild type

25 HIV from their protocol, so they expanding HIV-infected

> 107 1 cells. There will be some HIV that comes

2 out of that.

3 The previous person who spoke spoke to the

4 real question here. What is coming out at the end

5 of the transduction, at the end of the expansion,

6 that is. So I think there has to be some kind of

7 test for how much HIV is going back into the

8 patient and what is the nature of that HIV. Is it

9 a variant or is it what they started out with?

10 DR. SALOMON: Thank you. That came across

11 very clear, by the way. I am just reminded, Dr.

12 Cornetta\--Ken, are you there as well?

13 DR. CORNETTA: Yes; I am.

14 DR. SALOMON: Excellent. You are also

15 coming through very clear. So both of you, please

16 feel free to participate.

17 Dr. Emerman picked up a theme that Dr.

18 Torbett brought up during the presentation. Dr.

19 Torbett, do you want to just sort of amplify?

20 DR. TORBETT: I do have a concern with

21 that, given the length of time that the assay goes

22 one. Furthermore, the studies earlier didn\'t

23 indicate they were looking\--in their preliminary

24 work, it didn\'t look like they were looking for a

25 CCR5 using variant.

> 108 1 I guess I have a little concern. Maybe it

2 is out of scope right now, but it comes back to

3 actually the trans gene, itself, and its ability to

4 derive a different species. So the question I am

5 posing is whether something that would come out,

6 get back into the cells, they would be infused back

7 into the patients, would create additional problems

8 above and beyond what the patient has as his viral

9 load, or her viral load.

10 DR. MULLIGAN: Back to Sue\'s point. I

11 think that I would like to see more

12 characterization of what that virus is, as we were

13 discussing during the talk. We really need to look

14 at the entire profile genome, not just the

15 mutations, because it may well be that, as an

16 entire package, the virus has replication

17 competence and may have some unique biological

18 properties.

19 I think that is very, very key and would

20 help us further assess whether or not more kinds of

21 tests are necessary.

22 DR. SALOMON: Just for my clarity, Dr.

23 Mulligan, what virus did you mean just now needs to

24 be characterized?

25 DR. MULLIGAN: The genome that had various

> 109 1 envelope mutations. I think what I heard was that

2 the proviral sequences were never tested in

3 totality. The mutant was taken and moved into an

4 otherwise wild-type background and that mutant

5 seemed to severely retard the characteristics of

6 the virus.

7 The question is can we go from that data

8 that the background of that particular set of

9 mutant viruses is the same as wild type or have

10 you, in fact, generated something, evolved

11 something, that now has different biological

12 properties.

13 DR. EMERMAN: Hello. This is Mike Emerman

14 again.

15 DR. SALOMON: Yes; go ahead, Dr. Emerman.

16 DR. EMERMAN: I am not so worried about

17 the virus that came out of that Sup-T1 experiment

18 as much as I am concerned about the virus that

19 comes out of the T-cell expansion from the patient.

20 The Sup-T1 is obviously biased because they are

21 challenging with homologous virus to the antisense.

22 So they are going to come up with something there

23 that has to evolve, has to change a lot more.

24 What comes out of the patient is not going

25 to have to evolve as much because its envelope is

> 110 1 different than the envelope that is used in the

2 antisense which was used from NL4-3. So actually

3 that virus, the break-through virus in that one

4 patient, that is the one I would like to see

5 characterized.

6 In a sense, they have to know, for each

7 patient, how much is coming out; that is, how much

8 virus are they going to be putting back into that

9 person as part of the protocol.

10 DR. SALOMON: Yes; I think that is very

11 clear, Dr. Emerman.

12 DR. ALLAN: This is in the same line which

13 is you have done a lot of work on looking at the

14 escape mutants but only in terms of mutations

15 within the vector. The issue about, well, it is

16 NL4-3 so it shouldn\'t be any worse than the virus

17 even if you got a recombinant than the wild-type

18 virus. It also goes to what Susan was saying which

19 is I don\'t think you can predict whether it is

20 going to be better or worse or just as bad or

21 whatever.

22 The thing is you could get gag/pol

23 recombinants and then the virus that is coming out

24 of those patients\' T-cells could actually be

25 replication-competent recombinants because you have

> 111 1 intact LTRs. You could have a gag/pol recombinant.

2 The issue with that also is, and this was

3 alluded to earlier, too, which is the immune

4 system. CTLs are supposed to very important for

5 limiting viral replication in HIV-infected

6 patients. If you introduce a different gag

7 sequence, a different pol sequence, you may be

8 generating viruses that are no longer recognized by

9 those CTLs and so you may actually get an increase

10 in viral replication in those patients.

11 So I think that is something else you have

12 to consider, too. So I think, again, which is the

13 same issue which is you really need to characterize

14 what is coming out of those T-cells and those HIV-infected

15 cells.

16 DR. REITZ: One thing that might be done

17 is to do the Sup-T1 experiment but with cells that

18 would support the replication of an R5-tropic virus

19 like PM1 and then look for conversion, and do the

20 experiment with an R5 input virus and see if you

21 got conversion to any X4 phenotypes because the

22 NL4-3 is a X4-tropic virus.

23 I think that would give you an indication

24 of whether you could possibly change the envelope

25 tropism of the patients\' viruses.

> 112 1 DR. SALOMON: I think the other point I

2 would make directly on that is I am uncomfortable

3 with an experimental result in which there is some

4 evidence for a breakthrough in in vitro that, then,

5 goes and clones 200-and-some clones, which is very

6 appropriate, picks the absolutely most changed

7 clone and shows that it seems to be packaged but it

8 is not very infectious.

9 So my response is okay. That is one. Now

10 you have got 260 more to go. So not crossing that

11 fine line that I realized of being ridiculous, I

12 just am saying that results on a single clone, what

13 it means to me, thinking about what is going to be

14 going on in this patient, is that there is going to

15 be literally hundreds of opportunities in a

16 relatively short time and then extrapolate that to

17 potentially months and years for all kinds of

18 variants to be developed.

19 DR. DROPULIC: Most of the variants that

20 we saw were deletions in envelope.

21 DR. SALOMON: Right. But I think we would

22 all agree that the larger amounts of deletions,

23 you picked the one that had almost 12 percent of

24 the genome replaced, or deleted.

25 DR. DROPULIC: That wasn\'t a deletion.

> 113 1 That was a mutation, base substitution. That is

2 the only one I picked because the other ones had

3 deletions in the envelope.

4 DR. EMERMAN: I really think it is not

5 that virus that really needs to be characterized.

6 It is the one from the other experiment where you

7 actually expanded T-cells from the HIV-infected

8 person.

9 DR. SALOMON: Yes, Dr. Emerman. I agree.

10 There are kind of two threads going on. We will

11 get back to that one in a second. You are

12 absolutely right. We kind of skirted that for a

13 moment. I agree.

14 DR. TORBETT: I think there are two parts

15 to this. One is replication fitness and the other

16 is resistance. I think Dr. Mulligan made a very

17 good point that when one inhibits, for example,

18 envelope, they can pick up other mutations which

19 compensate and that is very true in the protease

20 situation. There have been other examples as well,

21 CTLs, whatever.

22 I think, since this is one of the first

23 demonstrations, it behooves the investigators to go

24 that little extra mile and do full sequencing and

25 find out some of this information. I think that is

> 114 1 fairly critical here.

2 DR. SAUSVILLE: So, along the lines of

3 what seems to have emerged here is issues

4 ultimately with what we might call characterization

5 of what comes out of the T-cell passage process.

6 Is there any evidence or entertainment of the issue

7 that obviously patients come in very many different

8 flavors. So CD4 cells are going to be quite

9 different from patient to patient ranging on

10 everything from what medications they are on to

11 what coinfections they may have.

12 So does that enter into some of the

13 variants that might actually come and be generated

14 here and should this discussion also consider that

15 issue. I raise that as a question. It would seem

16 to me it would be relevant in the clinical sense.

17 DR. SALOMON: Certainly, there is some

18 expertise around the table to address that

19 question. Of the population that is being proposed

20 for this study, it is defined as HAART\--I guess the

21 beginnings of a HAART failure. These are not total

22 HAART failures because, at that point, I would

23 think that their CD4 counts would basically

24 plummet. I guess one of the difficulties here is

25 defining exactly what is a HAART failure. We will

> 115 1 get to that maybe later.

2 So for those of you who are real HIV

3 experts, what will this patient group be like? Are

4 there going to be any commonalities for this

5 particular subset of patients?

6 DR. ZAIA: If you look at the definition

7 of who is included, it is everyone above 5000. So,

8 assuming that let\'s say failure is some other

9 level, or any level, while on HAART, you can have

10 anyone from 5000 to 1 million. So the question is

11 is the person at a million going to be different

12 than a person at 5000. I would guess they are

13 different in some ways. Maybe they are different

14 because you will have a harder time isolating their

15 T-cells or expanding them.

16 But at least it introduces the idea of

17 heterogeneity in the population. So when you are

18 looking at toxicity, you may have a difficult time

19 comparing strata. If, in one stratum, you had

20 enrolled three people who had had a million and, in

21 the other stratum, you enrolled three people who

22 had had 5000\--I mean, that is a design problem.

23 But it is still important.

24 DR. SALOMON: The specific question, not

25 withstanding the quality\--I mean, those are

> 116 1 important comments. I didn\'t mean to trivialize

2 them, but the specific question I was asking was do

3 you think, then, that there would be a similarity?

4 Would you come down at all the kind of viral

5 populations that would be present in these patients

6 because you are defining them as being relative

7 HAART failures?

8 I guess I could interpret from what you

9 said that the answer is no, that these would really

10 be still very heterogenous groups from the point of

11 the viral species.

12 DR. EMERMAN: There is one point which is

13 important here which is whether or not they have

14 drug resistance at the time you are staring the

15 therapy. So, if they fail HAART because they have

16 resistance, that is one thing.

17 DR. SALOMON: Wouldn\'t that be the reason

18 they would be failing HAART would be drug

19 resistance of some sort?

20 DR. EMERMAN: They have failed HAART

21 because their virus is resistant to an antiviral.

22 That is one different kind of criteria. I think it

23 was alluded to the fact that one could suppress the

24 HIV activation when you are expanding if you do it

25 in the presence of antiviral.

> 117 1 That is a way, actually, they can get

2 around having new variants come up during the

3 expansion if they include antiviral during the

4 expansion. But that predicates knowing whether or

5 not there are resistant viruses present already.

6 DR. SALOMON: I thought about that, too,

7 when that came up. Exactly.

8 DR. EMERMAN: So that is a strategy to get

9 around these concerns we have of generating new

10 viruses is if they can show, out to the expansion,

11 that there are no viruses because they have

12 expanded in the presence of antiviral.

13 DR. SALOMON: Dr. Sausville and then Dr.

14 Allan.

15 DR. SAUSVILLE: So then, to expand on

16 that, I don\'t think that we came to a\--at least I

17 didn\'t think we came to a clear notion of whether

18 or not there would be a relative advantage to

19 include the drugs while the T-cells were being

20 expanded. I think that was alluded to before.

21 But, as I listen to the discussion, if the

22 goal is to minimize the risk of coming up with yet

23 a worse flavor of HIV during this process, it would

24 seem that the preclinical data is a little thin on

25 considering the merits or demerits of that.

> 118 1 DR. ALLAN: I can see individual variation

2 from patient to patient in terms of when you expand

3 the T-cells, what percentage of the cells are going

4 to be expressing HIV. If you have got somebody who

5 has really failed on HAART and they have a high

6 viral load or a high viral burden, then you have

7 got essentially a high multiplicity of infection

8 circulating into the cells that you are getting the

9 vector in which may increase your chances of

10 recombination.

11 The other issue that follows with that is

12 I didn\'t see anything where they filed CXCR4 versus

13 CCR5 expression in those activated T-cells. I

14 think that might be an indication as to how much

15 virus breakthrough you are going get, maybe

16 variability between patients that you have looked

17 at but you are taking out and how many CCR5-positive CD4

18 cells, versus CXCR4.

19 DR. SALOMON: Dr. Zaia?

20 DR. ZAIA: I think one question we have to

21 ask ourselves is why would you analyze the virus

22 coming out of the transduction in these patients.

23 Is it because you want to exclude that lot of cells

24 or do you want to prove the implication that Dr.

25 Kingsman started with, that the design of the study

> 119 1 is flawed; that is, if you found one patient that

2 had that, you would accept that premise that you

3 are selecting for a resistance that may change the

4 phenotype. So it is a slightly

5 different question. I don\'t think we are asking

6 for release testing. I think we are asking for

7 proof from the sponsor that this is not a poorly

8 designed study from its inception; that is, the

9 target is asking for selection in vivo.

10 DR. REITZ: In addition to the question of

11 resistance, I think, since this is targeted to

12 antisense envelope, you also have the question of

13 the cell tropism or envelope phenotype of the virus

14 as well because one thing that would be concerning

15 is if you converted a CCR5 to a X4 tropic virus,

16 you would generate something that is at least

17 seemingly more virulent than the R5 populations of

18 virus.

19 So you might be generating a fitter virus in some

20 sense in that way.

21 One way I think that you could look at

22 proof of concept is what I had mentioned before,

23 which is to see if you do get phenotypes in a

24 system that allows in vitro testing or growth of

25 both R5 and X4 viruses. I think you could look at

> 120 1 it using that kind of system.

2 DR. SALOMON: So, trying to capture this.

3 What I hear here is a couple of threads. One

4 thread is that if we assume that you are doing your

5 transductions on patient T-cells that are HIV-infected,

6 which is, of course correct\--that is what

7 is going to happen\--then I think Dr. Torbett, Dr.

8 Emerman, made a very concrete point that I think we

9 need to start with because that is just the

10 simplest point.

11 Dr. Zaia makes it more complicated and we

12 need to go there, too. But the first point is I

13 think the committee was saying that you have to

14 look at the amount of\--the number of CD4 T-cells

15 that are infected in the input and the virus that

16 those cells carry. And we should go back and say

17 what does characterizing that virus mean, but

18 characterize it in some way and, after eight to ten

19 days of activation with the vector, characterize

20 the number of CD4 T-cells that are infected and

21 characterize that population of virus.

22 There is a lot more to go there. But, how

23 about starting there? What do you guys think of

24 that statement?

25 DR. ALLAN: I don\'t know about days. You

> 121 1 are saying eight to ten days. It may not be

2 enough.

3 DR. SALOMON: Eight to ten days was the

4 period of time that this procedure will be done

5 after which the cells are taken, washed and frozen

6 and assays done.

7 DR. MULLIGAN: I think the timing may not

8 be all that important. It is important relative to

9 what the clinical protocol will be. But if you

10 want to see the events that occur, I think what you

11 are saying is you go further and you try to really

12 see what happens when you have these cells infected

13 with vector.

14 DR. SALOMON: Okay. Now, there what I am

15 doing is I am following what I think Dr. Zaia

16 articulated beautifully. The first point is what

17 is a safety request for the protocol as proposed.

18 The second is getting at the science behind the

19 strategy which I am not saying, in any way, shape

20 or form, isn\'t equally important, but I am, right

21 now, just trying to start with one point and make a

22 step forward.

23 So this is just, they do the protocol\--at

24 the end of nine days when they freeze these cells

25 down, should we do this? Then we will go exactly

> 122 1 where you are going, and that is longer culture and

2 more characterization. But you wouldn\'t do that

3 for every single lot; right? If that is what you

4 are suggesting, then\--

5 DR. MULLIGAN: I think we are saying the

6 same thing, that you would, before you ever do

7 this, collect much more information on the kinds of

8 viruses that come. And you would do that for

9 longer periods of time. Whether it would be ever

10 meaningful to test at nine days or ten days, once

11 you decide you are going to do the protocol, I am

12 not sure.

13 DR. EMERMAN: Can I make a suggestion,

14 perhaps. You would want to know what percentage of

15 cells are infected at the beginning of the

16 expansion and then you want to know what percentage

17 of cells are infected at the end of the expansion

18 and had it increased. Then that would be a

19 criteria for not continuing with that particular

20 lot.

21 DR. SALOMON: Yes. That is what I wanted

22 to hear someone say. That is what I was

23 suggesting, that that would be the minimum.

24 Dr. Sausville and Dr. Rao.

25 DR. SAUSVILLE: At one level, this is

> 123 1 maybe a more exotic form of quality control and

2 release specifications than we are used to

3 considering but is brought by the nature of this in

4 that\--I would like to see not only what, in

5 essence, goes in which is easy to define but also

6 what comes out on several levels, but across a

7 reasonable spectrum of T-cell populations that

8 might be expected to emerge from a representative

9 set of patients because, to me, in terms of

10 interpreting the outcome of any clinical trial, I

11 don\'t have any context in which to judge what comes

12 out of the clinical trial in terms of what the

13 product is going in at this point.

14 DR. SALOMON: So we can have the sponsor

15 determine the percent of infected CD4 in and the

16 percent CD4-infected out at the end of a nine-day

17 period. Then the question is what would we request

18 in terms of characterization of the virus in and

19 virus out?

20 DR. ALLAN: You are talking about the

21 virus out\--by saying virus out, what you are saying

22 is\--

23 DR. SALOMON: Whatever combination of

24 wild-type HIV and vector comes out.

25 DR. ALLAN: You are talking about the

> 124 1 breakthrough kind of scenario that was shown with

2 the patient\'s T-cells.

3 DR. SALOMON: No, no. I think that what

4 we have heard is that two possible things could

5 happen. Dr. Emerman and Dr. Torbett, correct me if

6 I didn\'t get you guys right, and also Dr. Reitz.

7 The idea here is that in vitro, during CD3, CD28,

8 activation in high concentrations of IL2, and you

9 can play adjective wars here, but in IL2, there

10 would be a change in the wild-type virus, a

11 selection, perhaps, of an R5 variant or something.

12 Or there could be recombination with a

13 vector that could create a different species.

14 DR. REITZ: I think that something like

15 that could happen in vivo, also. But you can

16 probably get some kind of indication on the

17 likelihood of that by looking at it in vitro and

18 then characterizing the virus that comes out in

19 vitro more fully.

20 DR. SALOMON: So can we be a little more

21 specific just because the word \"characterize\" does

22 not mean anything to a sponsor or to the FDA.

23 DR. REITZ: In the case I am thinking of,

24 it would be a relatively simple readout of

25 recombination with the NL3-4 envelope sequences of

> 125 1 just looking to see whether you generate X4

2 viruses, whether you can show them coming out of

3 the gemish that you have after doing some kind of

4 relatively extensive culturing.

5 DR. SAUSVILLE: But, isn\'t that choosing

6 one particular marker situation? I am not a

7 virologist, but when one looks at the efforts to

8 describe populations of viruses and different

9 clades, different epidemiologic scenarios, et

10 cetera, one could potentially imagine a more

11 detailed characterization, at least at this initial

12 get-go.

13 DR. REITZ: What this would answer, and I

14 think is using a relatively simple readout, is it

15 does this occur.

16 DR. SAUSVILLE: But would that be

17 sufficient if it were to potentially detect other

18 types of changes and then that gets back to the

19 whole issue of\--

20 DR. EMERMAN: I think there are other

21 techniques that could be used. For example, there

22 is the technique HMA, heteroduplex mobility assay,

23 which can look at the variability within that

24 region of envelope before expansion and after

25 expansion. You can ask, are there different

> 126 1 species there after expansion than before

2 expansion.

3 DR. MULLIGAN: I think there are two types

4 of things. There are, obviously, these more

5 important biological characterizations but then

6 there is also the vector-specific question which

7 really requires a detailed molecular

8 characterization, pro-viral DNA, PRC sequence,

9 whatever you want to do, but to see what variations

10 you have, what pieces of the vector you pick and

11 incorporate, not that particularly that is going to

12 be worse, in any means, than picking up a new

13 envelope.

14 DR. SALOMON: Dr. Torbett?

15 DR. TORBETT: I guess, for a minimum, I

16 think what I am hearing is that we would like to

17 have the same populations going back into the

18 patient and started and the same number of cells

19 infected so the individual is not worse at the go.

20 Otherwise, it will be very difficult to analyze

21 efficacy of the trial.

22 So, at the minimum, I would think that the

23 envelope sequences, if they are breakthroughs, and

24 this is very important because we are using cells

25 from patients that are presumably resistant to the

> 127 1 current protease regime. So there will be high\--there

2 possibly will be replication.

3 So I think, at the minimum, the question

4 of what is changed in the envelope region by

5 sequencing and, at the minimum, by heteroduplex

6 analysis, is something that should be considered as

7 well as the number of cells going in and out in

8 terms of bookkeeping infectivity are infected.

9 DR. DELPH: Would it be important to look

10 as well at viral fitness or replication capacity of

11 what is coming out?

12 DR. SALOMON: I think that what Dr. Reitz

13 was saying is that. He is talking about biological

14 assays of viral fitness. That was picked up as a

15 theme by others. So, yes.

16 DR. REITZ: Also using that as an index

17 for the possibility of recombination.

18 DR. ALLAN: I would go another step,

19 though, because it is not necessary to have a

20 replication-competent virus coming out that is a

21 recombinant. I think that all you need to have is

22 a gag/pol recombinant because, if you are

23 introducing that into the patient, at some point

24 later, you may get the recombination occurring. So

25 it is not even just looking for viruses that have

> 128 1 the ability to replicate but you have to start

2 looking at large sequences. So you have to do

3 sequencing across a genome.

4 It is not just envelope that you are going

5 to be worried about. I agree; that is a major

6 concern. But, also, LTR gag and pol.

7 DR. SALOMON: Okay. So, trying to keep

8 this in focus. We definitely hear that we need to

9 know percent CD4 going in, percent CD4 infected

10 going out. We also appear to be in consensus that

11 we need some sense of the biological activity of

12 the virus going in, as a baseline, but more

13 importantly, of course, the virus coming out to

14 make sure that just the procedure of transduction

15 activation and in vitro culture doesn\'t alter the

16 characteristics of these viral species that get

17 returned.

18 But then, as we go to where Dr. Allan have

19 taken us, and Dr. Mulligan, we need more background

20 information is what I hear the committee saying, as

21 well. I think that is where Dr. Zaia began.

22 I think, Dr. Mulligan, why don\'t you pick

23 up on that. What additional information does the

24 committee want that we wouldn\'t necessarily want

25 them to do every time they did a patient but that

> 129 1 we need now to even think about it as an issue for

2 the protocol.

3 DR. MULLIGAN: The simple, I think, would

4 be to, even in vitro, to go through culturing the

5 cells for a longer period of time because I am not

6 sure we would ever be able to assess exactly what

7 the test would be during the actual clinical

8 procedure until we knew what we were looking at.

9 We didn\'t really talk about this but my

10 conclusion is that there is really not a very good

11 in vivo mobilization assay, from what we have heard

12 and, therefore, we may be limited to these in vitro

13 assays. So, carrying on the cells in culture and

14 just looking for the kinds of things that occur

15 won\'t necessarily tell you what is going to occur

16 in the patient.

17 But things may well go on after the nine

18 or ten days of culture. Different things might go

19 on\--and just seeing the kinds of variants that you

20 would get I think would be very important.

21 DR. SALOMON: I agree with that. I was

22 thinking about the experiment that could be done

23 here. There is always a flaw in thinking of

24 experiments on the fly. For that, I apologize to

25 you because I am sitting here for a day. You guys

> 130 1 have done this for a year, probably.

2 But the experiment I was thinking about

3 was if you took a group, X number of fresh CD4

4 cells from an HIV-infected individual, activated

5 them in your protocol, go about nine days, wash

6 them, then add in new fresh CD4 cells from a

7 noninfected individual, and sort of do serial

8 passages each time bringing in some fresh CD4 cells

9 from an individual and begin to look at\--sort of

10 use that as a real-life sentinel cell, at the same

11 time doing parallel experiments in a very highly

12 permissive cell line for R5 and X4 variants, just

13 to really push the system through a couple of times

14 to see what kind of viral variants are going to be

15 produced and selected.

16 I don\'t think you are going to do those

17 kinds of experiments in a SCID-mouse model. I

18 would like to point out that my lab does NOD/SCID-mouse

19 work. I am not in the anti-SCID-mouse

20 population. I think it is an extremely useful

21 model. But I am saying to you my feeling is I

22 don\'t think these are experiments that could be

23 done in that system.

24 DR. ALLAN: The monkey studies. There was

25 a monkey study that was shown yesterday, and it was

> 131 1 nice, a lentiviral vector and it was targeting to

2 the brain. You ramp up the concern when you

3 introduce the wild-type virus into the person who

4 is getting the vector.

5 So my question is, in this particular

6 protocol, I am not certain that it is\--designing a

7 situation in a monkey-model system is strictly a

8 basic academic endeavor because the thing you don\'t

9 want to do is you don\'t want to kill an AIDS

10 patient with the protocol.

11 I am not certain, at this point, whether

12 or not that might happen. From what I have seen, I

13 don\'t know that you are going to make that patient

14 worse by introducing this vector. So the issue

15 then is how do you define\--we don\'t have a model

16 system. The SCID mice aren\'t so good, while

17 monkeys you have got to do all this stuff to.

18 But, yes; you do have to finagle a few

19 things, not huge, but I am just saying if you want

20 a model system, that is what you are going to have

21 to do. I am not saying you have to do it. I am

22 just saying that it is available.

23 DR. SALOMON: Dr. Torbett.

24 DR. TORBETT: I think what we are asking,

25 I thought we were asking, is whether we could at

> 132 1 least look for the minimum number of changes seen

2 over a certain period of time in culture without\--and I can

3 think of many studies in some of our

4 mouse models and our other models that are probably

5 beyond the scope here.

6 But I think, at the minimum, we need a

7 little bit more information, at least in classic

8 cultures that people have done in vitro. I think,

9 at least myself, that would give me a little bit

10 more reassurance.

11 DR. SALOMON: To Dr. Allan, I think the

12 point here, to put this in context, is we are

13 trying to focus on the protocol and we are trying

14 to do it in the context of the principles we

15 articulated yesterday. We talked about the

16 relative value of the monkey model yesterday. I

17 don\'t think that, unless the committee wants to

18 take me otherwise\--I don\'t think that the message

19 to the FDA or the sponsor now is that these guys

20 should go and generate a SHIV-modified monkey model

21 before we would allow a protocol like this to go

22 forward.

23 Do we agree on that? I mean, there are

24 problems in the field. We acknowledged those

25 yesterday. I thought your ideas were really

> 133 1 interesting.

2 DR. ALLAN: This is a Chicken Little

3 thing, obviously. It is not going to create a

4 public-health nightmare. But still the issue is

5 the AIDS patient is at risk. The patient may have

6 600 CD4 counts, may only have 5000 copies per ml of

7 plasma, so it is not that they are in bad shape at

8 that point.

9 So they are actually, I would think,

10 relatively healthy. And you are going to treat

11 them with this and you could kill them depending on

12 what happens. I don\'t know what the risk is. But

13 it is the same sort of situation when you say,

14 well, gee; anthrax, we are not going to test postal

15 workers or make them do this stuff because we don\'t

16 believe that it is\--and then you go back\--you just

17 don\'t want to be on a committee where you say,

18 well, gee, a year ago, we didn\'t think that was

19 going to happen and it happened.

20 DR. SALOMON: Okay.

21 DR. ALLAN: I am not saying that you

22 should do a monkey model. I am just saying that

23 there is a level of concern and the question is

24 whether or not you want to take it to the next

25 level.

> 134 1 DR. SALOMON: And I was saying that we

2 have articulated that concern yesterday, and I am

3 testing you now to see whether or not you want to

4 elevate the level of concern higher than we

5 elevated it yesterday to say that you would put

6 that as a barrier, basically, before going forward.

7 I am saying that I haven\'t heard anything

8 from the rest of the committee that suggests that

9 this is a requirement to go forward in this area,

10 but I certainly am not arguing with you about what

11 you have articulated as an issue for the field.

12 DR. ALLAN: I am not saying that you would

13 use a monkey model to test a lentiviral vector that

14 was used for some other reason. But, in this

15 particular case, you are using a lentiviral vector

16 in the context of an AIDS patient who has a wild-type HIV.

17 To me, that is different.

18 DR. SALOMON: Dr. Zaia?

19 DR. ZAIA: We are being unfair to the

20 sponsor if we don\'t set the bar, or at least advise

21 the FDA on how to set the bar. So let\'s talk about

22 two bars. A high bar may be evidence in the monkey

23 that you cannot change the biotype of the virus in

24 such a way to make the monkey worse.

25 So let\'s suppose that were done and you

> 135 1 actually constructed the vector with antisense to

2 SIV envelope. You then went through the experiment

3 and you found that the monkeys did make more virus

4 and they grew faster and they got AIDS quicker.

5 You failed the bar. You did not pass the bar. The

6 bar was set high and now you probably would never

7 bring this to the clinical trial.

8 That is one bar. That is not an

9 unreasonable bar if you are concerned about what

10 let\'s call it the anthrax level of concern. But

11 the other bar, then, would be looking at\--let\'s

12 call it the Torbett bar.

13 DR. TORBETT: It is always a lower bar.

14 DR. ZAIA: You look at what goes in and

15 ask the question, is there some change in that

16 virus. If we look at, let\'s say, 200 clones and we

17 characterize the mutations. We look at some

18 fitness and we have this experience. We don\'t see

19 any more increased fitness from these three

20 experiments we did, let\'s say. So that is the low

21 bar that you pass.

22 I guess there is no real way to know what

23 is best. If we are very concerned that the design

24 of the experiment may lead to worse virus, then I

25 think it is reasonable to ask for the higher bar.

> 136 1 It is not an impossible task to accomplish.

2 Once accomplished, it may give us a lot

3 more\--lessen the anxiety, let\'s say, of the

4 scientific community that is arguing in defense of

5 this study to go forward to advance the field.

6 DR. EMERMAN: A way to get around all

7 these questions of a worse virus coming out of the

8 transduction is to\--I think they proposed was to do

9 an expansion in the presence of antiviral drug, or

10 antiviral drugs, assuming they pretest virus from

11 the patients knowing that\--

12 DR. SALOMON: Dr. Emerman, there is

13 nothing in the protocol at the moment suggesting

14 these expansions will be done in the presence of

15 antiviral drugs, number one. Number two, we have

16 pointed out that if, indeed, these are patients who

17 are, at some level, failing HAART, then the

18 significance of the antiviral drugs is less clear

19 yet.

20 DR. EMERMAN: But that could be tested

21 because, if, at the end of your expansion, you

22 don\'t see any virus, then that is fine. That means

23 that they didn\'t expand a drug-resistant virus.

24 People fail HAART for a lot of reasons only some of

25 which are resistance to the drug.

> 137 1 DR. SALOMON: That\'s fair.

2 DR. ALLAN: The thing is, it doesn\'t fail\--it

3 doesn\'t end when you put the cells into the

4 patient. You put the cells in the patient, you

5 could continue to get recombinants. You could

6 continue to get things that happen later on. So it

7 is not just, this is safe now and we can put it in.

8 DR. CORNETTA: Dan, this is Ken Cornetta.

9 One of the things that has been disturbing, I

10 think, for me over yesterday and today is not only\--I think

11 most of the assays that have been looking

12 for recombinants have been geared for common

13 recombinants. But even looking back at the old MLV

14 data, various cells that can be tested may or may

15 not express various recombinants very well.

16 It seems most of the people that have

17 presented have looked at one assay using one or two

18 cell lines. I think, in just general, I am not

19 sure folks have spent enough time looking at

20 assays for trying to detect recombinants that

21 really make me feel very comfortable that people

22 have a good handle on being able to analyze both

23 the product and then the transduced cells.

24 DR. SALOMON: Good.

25 Dr. Sausville and then Dr. Reitz.

> 138 1 DR. SAUSVILLE: I just wanted to\--with all

2 due respect to the monkey models, I would counsel

3 against making that a bar because I think that is

4 investing what is a biologically very informative

5 model with a level of ultimate access in the

6 clinical-trial sense that I just feel very

7 uncomfortable with. I think that I would rather

8 characterize well the product that comes out and

9 address some of these issues of variability in the

10 transduced cell population rather than the monkey

11 situation.

12 DR. ALLAN: I understand what you are

13 saying, too. But what happens is that you don\'t

14 know what is going to happen when you put it into a

15 biological system. You can do tissue culture and

16 you can passage it, but that is not what happens in

17 a person that is infected. It is not what happens

18 in a monkey that gets SHIV or whatever. There are

19 studies that are done which, if you take a deletion

20 in nef and you put SIV into a monkey, then you get

21 nef back.

22 You can put a defective virus in one leg

23 and another defective virus in another leg, and you

24 get replication-competent virus.

25 So I don\'t know that an in vitro tissue

> 139 1 culture system is going to be able to tell you

2 that. I am still wondering whether\--because the

3 monkey-model system is not that difficult to

4 engineer, really, because you can still use the

5 env, because if you use the SHIV system, you don\'t

6 have to redesign the antisense env. You can use

7 that.

8 The only thing you have to redesign is

9 really the gag/pol. So it is not that big a deal.

10 I am not doing it so I can say that.

11 DR. MULLIGAN: I was just going to say

12 that we may not want to equate the level of concern

13 with whether we do the monkey\--I think that is

14 maybe what Ed is saying. I would look at it that

15 we have the level of concern, I think. the issue

16 is if we were really to analyze the data that we

17 would get from the monkey, would it really satisfy,

18 for sure, our concern one way or the other; that

19 is, this data could be very, very helpful.

20 I am sure everyone would love someone to

21 do this and test it. But if you really ask this

22 question which we always ask is how relevant is the

23 model system, how meaningful is it, I am not sure,

24 in this particular case, we would get any clear

25 consensus, even though we might get a clear

> 140 1 consensus that the level of concern we have is very

2 high.

3 DR. ALLAN: It wouldn\'t tell you yes or

4 no, it is safe or it is not safe. What it does do

5 is it give you a lot more information in terms of

6 safety in a patient. It gives you a lot more

7 information.

8 DR. SAUSVILLE: But, on the other hand, if

9 you are going to pursue the point, if the animal

10 model were to faithfully replicate the clinical

11 scenario, you would have to have some sort of

12 control where the animal is observed for the same

13 period but with a non-changed virus.

14 You are talking about a scope of an

15 experiment that we just usually don\'t require, I

16 don\'t think, in any therapeutic area. I am

17 concerned that that could ultimately block progress

18 if that were set as a bar for every type of therapy

19 of this sort.

20 DR. ALLAN: I\'m only suggesting it in the

21 sense of treating an AIDS patient with an HIV

22 vector. I am not suggesting that in other types of

23 therapeutic modalities but only in this particular

24 case.

25 DR. DELPH: I am obviously not competent

> 141 1 to make any determination as to whether in vitro

2 testing is going to give you better information

3 than monkey models, how much better the information

4 from monkey models is going to be. But I do think

5 that there is a level of concern there, which I

6 share. And I also think that this is a new area.

7 This is completely new territory as far as we are

8 concerned in terms of putting lentiviruses into

9 HIV-positive patients, an HIV-positive patient.

10 I think, for that reason, we need to err

11 on the side of caution. So I would urge that the

12 bar be set higher rather than lower. I don\'t know

13 what the tests are that you need to get to that bar

14 and whether they are worth doing. But I would err

15 on the side of caution.

16 DR. SALOMON: As someone who is

17 representing the AIDS community for us, my

18 interactions with the AIDS community in the past

19 wouldn\'t have prepared me for that statement. I

20 just wanted\--if you could kind of deal with that.

21 Do you think that what you have just said would

22 represent, obviously not every person in the AIDS

23 community. No one is trying to be that absurd, but

24 it seems to me there are groups in the AIDS

25 community that feel that a patient failing HAART is

> 142 1 a young person, innocent person, dying.

2 The relative risk here of generating a new

3 virus and spreading it into the community, which is

4 what you should be concerned about, would be very

5 low providing you had patients who adhered to

6 appropriate behavior during the trial not to bring

7 this whatever was happening inside them, if

8 anything bad was happening, to another patient.

9 So I guess I am a little uncomfortable. I

10 just hope that we think about that for a minute.

11 The best way to kill an idea is a committee. At

12 some point here\--I don\'t want to go too\--I just

13 want to make sure that I raise the issue for

14 discussion here. I think I am being clear.

15 DR. DELPH: Obviously, I cannot speak for

16 the HIV community and, obviously, there are going

17 to be very different opinions from mine. But I do

18 think that if you look at\--there are people who

19 really have virtually no options at the moment and

20 who are in dire straights.

21 But I have no idea what the relative risk

22 that you are talking about, how quantifiable that

23 is. So it is very difficult for me to say at this

24 point is the relative risk worth it for those

25 people or for the rest of the HIV community. I

> 143 1 guess what I am saying is that, yes, we need to

2 balance the risks and benefits but this is

3 completely new territory and whereas, I think, we

4 have familiarity with antiviral drugs, it is a lot

5 easier to say what you need to do and to assess

6 those relative risks.

7 Right now, I have no way of assessing

8 those risks. And, from what I have heard, I don\'t

9 know that many people here can quantify that risk

10 for me.

11 DR. SALOMON: Just to share with you kind

12 of where I was from, just to finish the thought. I

13 went through this over the last five, seven, years

14 with xenotransplantation. Today, we still don\'t

15 have a very specific measure of relative risk and

16 so this equation of risk and benefit has been as

17 much a part of those debates as we have now been

18 having here.

19 I think that one has to think about\--one

20 of the things that came out in our

21 xenotransplantation considerations was the idea of

22 individual risk and public risk. Individual risk

23 can be handled very well by a consent form. The

24 risk that a more rapidly, more fit, HIV species

25 could come out in an individual and lead to a more

> 144 1 accelerated death can be explained in an informed

2 consent and put in the context of, we have done our

3 best to select a patient population with HIV that

4 really has no option.

5 If I wasn\'t convinced of that, if we were

6 talking about relatively healthy people being

7 pulled off the street, that would be a different

8 issue. But providing that we would assure

9 ourselves later that the clinical-trial design

10 chose an appropriate population, that is individual

11 risk.

12 So, the only thing that goes to public

13 risk is that then there would be transmission from

14 that patient in the trial to the public, anyone

15 that they would be in contact with in any context.

16 That is manageable and small if the patient

17 population is mature and compliant. That is where

18 things get gray.

19 DR. ALLAN: I am concerned about the

20 patient. I guess, in contrast to

21 xenotransplantation, I am little bit more concerned

22 about the patient here. With xenotransplantation,

23 the patient is going to die within a week, month,

24 or whatever. An HIV-infected patient who has got a

25 600 cell count and 5000 viruses, I don\'t know how

> 145 1 long they are going to be able to live for. Do you

2 know? A couple of years?

3 DR. DELPH: Longer than that.

4 DR. ALLAN: Maybe longer than that.

5 DR. DELPH: Somebody with a viral load of

6 5000 and CD4 cells of 600 is really not in dire

7 straights.

8 DR. ALLAN: No. So you are jeopardizing

9 the individual health. The other point about

10 xenotransplantation, just to take the other aspect,

11 is we do require, in xenotransplantation, that they

12 do an animal-model study, that they put pig hearts

13 into baboons. We even set a bar in terms of

14 primates in xenotransplantation.

15 DR. SALOMON: Okay; but just remember that

16 was a benefit bar, not a risk bar, in the primate.

17 DR. ALLAN: We are doing the same thing if

18 you require a macaque model. You would be looking

19 at efficacy as well.

20 DR. SALOMON: Okay; just the discussion

21 was on risk.

22 Dr. Zaia and then Dr. Reitz.

23 DR. ZAIA: I think that I am persuaded by

24 Dr. Sausville\'s point that a monkey model as the

25 bar may not be adequate for two reasons; one, it

> 146 1 may not answer the question and it may be too

2 impractical for other studies as they come along in

3 the future.

4 But I think that there still is an

5 intermediary situation, that the sponsor carries a

6 burden to show, and that is the fitness of the

7 virus. So if there were, say, 100 isolates

8 analyzed and 100 of them were unfit, or whatever

9 the word is for that, and there were no better-fit

10 viruses in that, at least you have assured the

11 patient who is undergoing the consenting procedure

12 as well as the FDA that it is a highly unlikely

13 event to occur.

14 I think maybe that is all we can say.

15 DR. SALOMON: And that was the consensus

16 earlier

17 DR. SAUSVILLE: I further make the point

18 that, even if you had a very successful monkey

19 experiment, you would still have to do that for the

20 product that you are going to put into the person.

21 So that is why I see this is a product-characterization

22 issue at one level.

23 DR. SALOMON: Dr. Reitz?

24 DR. REITZ: I would just like to agree for

25 a slightly different reason with Dr. Allan about

> 147 1 there being\--I think you have got to think about

2 risk to the patient more than the population

3 because it seems to me the likeliest phenotypic

4 conversion in this particular situation would be

5 the an X4-tropic virus which could be fitter within

6 the patient but it is also a virus that is less

7 easily transmitted, almost never transmitted from

8 one person to another. So I would be more

9 concerned about the patient than the population.

10 DR. DROPULIC: Could I just answer that?

11 DR. SALOMON: Okay; she has been standing

12 there, too. Go ahead.

13 DR. DROPULIC: I just want to answer that

14 comment by Dr. Reitz. We will restrict the patient

15 study population to X4 strains. So we will

16 demonstrate that the patient has X4.

17 DR. SALZMAN: This is Rachel Salzman, STOP

18 ALD Foundation. I want to comment about the bar

19 setting. We talked about the high bar, the low

20 bar, the in-between bar. When you start talking

21 about a high bar for just the HIV subject patient

22 population, that causes a little bit of problem

23 because there has been so much discussion here that

24 the non-HIV population may become and HIV-positive

25 population.

> 148 1 So when you start saying, well, the bar

2 for HIV studies is this and the bar for non-HIV

3 studies is that, then there is kind of a logic

4 flaw. So, of course, I am kind of coming more from

5 the perspective of you invest all this time and

6 effort into accomplishing and meeting the standards

7 of the high bar and what you have learned,

8 ultimately, just doesn\'t seem like it is of the

9 value for what you have invested into it.

10 So just remember that the non-HIV

11 population, technically speaking, could become

12 potentially HIV and we wouldn\'t want to exclude

13 them and make them have to meet these higher bar

14 standards.

15 DR. SALOMON: I don\'t necessarily see the

16 logical flaw, to be honest.

17 DR. SALZMAN: The logical flow is every

18 time that it has come up saying, well, let\'s put

19 the HIV patients in this category, and then the HIV

20 specialists say, well, just remember that the non-HIV people

21 can become HIV-positive. So suddenly

22 you have done\--

23 DR. SALOMON: There is nothing illogical

24 about that. That is real.

25 DR. SALZMAN: Right. Exactly. So my

> 149 1 point is that if you are going to say, okay, the

2 high bar is only for studies that you are going to

3 treat HIV patients, well, then, by definition, the

4 high bar has to be for non-HIV patients because

5 they could become HIV patients.

6 DR. SALOMON: Exactly. I don\'t think

7 anyone suggested an illogic on that. That\'s all.

8 DR. ALLAN: But what I think she is saying

9 is that she is afraid that what we are going to do

10 is\--

11 DR. EMERMAN: \--we are not doing that in

12 the non-HIV people. If they get infected later,

13 that is a totally different story. We are talking

14 about what goes into those people.

15 DR. MULLIGAN: Another point that may make

16 you feel a little more comfortable is that the

17 mobilization sorts of things we are talking about

18 are dependent upon what HIV infects, too. So, in

19 many of the cases, you may have a particular

20 interest in. Although it is true that you could be

21 infected by HIV, you may be doing some cells,

22 trying to transduce cells, that are not susceptible

23 to HIV.

24 So it wouldn\'t necessarily mean that it

25 would completely cover all kinds of activities. So

> 150 1 if you were doing muscle gene transfer, for

2 instance, even if you were infected by HIV, in

3 principle, you wouldn\'t be infecting muscle cells.

4 DR. SALZMAN: Right. I am coming at it

5 from the other end, just saying\--I mean, I think

6 you see my point. I understand what you are

7 saying, also. I am just saying I have concern when

8 you are saying, well, we have these standards for

9 the HIV population that are strict. What I am

10 saying I like having the more middle bar and that

11 then you are saying everyone could potentially be

12 HIV positive, so potentially all patients should

13 have to meet those higher standards. That is all.

14 It just seems to me the value is not necessarily

15 weighing what you are investing into it.

16 DR. SALOMON: Dr. Wilson?

17 DR. WILSON: I wanted to make one point to

18 clarify regarding the last comment which is that if

19 I had been following the train of discussion, I

20 think Dr. Allan\'s proposal to raise the bar is

21 specific to this particular protocol because of the

22 use of the antisense envelope where there is data

23 suggesting that that would drive changes in the HIV

24 envelope.

25 As Dr. Mulligan was also saying, not only

> 151 1 is it an issue of different target cells, but

2 presumably that issue wouldn\'t arise if you are not

3 treating HIV with that type of\--you wouldn\'t be

4 putting into a non-HIV-positive patient population

5 that type of a vector.

6 MS. KNOWLES: I am hearing the scientific

7 concerns from others on the committee and I agree

8 with those concerns. But I have some other safety

9 concerns in terms of the proposed trial. I think

10 that the proposed tests in terms of in-process and

11 QC during manufacturing are vague except for the

12 RCL assays and that those other tests ought to be

13 identified and detailed.

14 Also, I am concerned, and this is a little

15 further down the line, but there is very little

16 information regarding the dosage schedule after the

17 initial dose and what parameters are in place for

18 continued treatment, et cetera.

19 DR. DROPULIC: Could I just answer that?

20 DR. SALOMON: No. Don\'t answer that

21 because we will get to that in a second. Those are

22 very important points so don\'t let us forget that.

23 We are getting close to what I think could be a

24 consensus of this, and this is so important to the

25 whole field, not just to your protocol.

> 152 1 So, trying to capture this here, I think

2 we have all agreed that we need to know the percent

3 of CD4 cells going in that are infected, the

4 percent that are coming out and some

5 characterization of the virus before and after to

6 look at what is happening during activation and in

7 vitro expansion.

8 I think that we also have all agreed\--stop

9 me if I am wrong, here\--but we have all agreed that

10 before you really even go forward in new clinical

11 protocol, we want to see more data for longer in

12 vitro culture, naive T-cells, T-cell lines selected

13 to reflect different sorts of biological tropism,

14 X4, R5.

15 I think the point came that we should be

16 more careful about different kinds of cell lines,

17 not just one kind of cell line. So I think we all

18 sort of follow that. As for the monkey studies and

19 where we set the bar, I think that in the final

20 discussion here, and again I would encourage a

21 vigorously defended and articulated minority

22 opinion, Dr. Allan, but I think that the final

23 sense here is that a monkey study, even for this

24 protocol, is too high a bar, particularly based on

25 the fact that I, too, am not convinced, as Dr.

> 153 1 Sausville said, that it is clear that the monkey

2 study will answer the question.

3 I, at the same time, as a scientist, would

4 look very favorably on such a project had it come

5 across to me in a different role as a study

6 section. But I would have to see that one and

7 really be sure that it would work before I could

8 come back on a regulatory advisory committee and

9 suggest that that is where the bar should be set.

10 I think that that was the sense of the

11 majority of the committee. Did I capture it right?

12 DR. CHAMPLIN: I believe so. I was just

13 going to make one little point. We talked about

14 longer-term culture with toxicities in mind, but

15 efficacy is also dependent upon the lack of

16 emergence of recombinants that would not be

17 affected by the antisense. So, if this proved to

18 something that readily occurred in a high frequency

19 of samples, that that would suggest that the

20 strategy was unlikely to be successful in vivo.

21 I am not sure, as a nonvirologist, how

22 predictive those types of assays could be, but one

23 would like to see some assessment of long-term

24 sensitivity to the antisense approach and the lack

25 of emergency of emergence of resistant virus.

> 154 1 DR. SAUSVILLE: I would heartily endorse

2 that position because I think it gets to the

3 essence of knowing more about what you have got

4 coming after the T-cell incubation procedure before

5 going on.

6 DR. ALLAN: The only thing I am going to

7 say about the monkey model at this point is that it

8 is not an academic exercise. It actually goes

9 right to the heart of efficacy. It goes right to

10 the heart of safety. It is a straightforward

11 experiment and, without that, I think you really

12 have to\--and this is something we will probably get

13 to later which is patient selection.

14 So if you are not going to do the efficacy

15 or the safety issue in the monkeys, then do we want

16 to only select patients that we know have a short

17 life span. In other words, maybe their CD4 counts

18 are 50 or 100 or something that\--or their viral

19 loads are a million, something that would give you

20 some sense that they may be in crisis, because you

21 are actually treating someone is relatively

22 healthy.

23 So that is my concern here.

24 DR. SALOMON: I think that that was

25 corroborated by everybody, that patient-selection

> 155 1 issues are relevant here.

2 DR. SAUSVILLE: I just had a question for

3 Dr. Noguchi and the FDA staff in relation to this

4 issue of efficacy because, again, while efficacy is

5 certainly a criterion for success\--i.e., marketing\--my

6 impression was that efficacy, as any type of

7 bar to enter a phase I, is not defined as a

8 regulatory issue. Would you care to address that?

9 DR. NOGUCHI: We would put it slightly

10 differently, that in many cases the question of

11 efficacy is certainly not the primary consideration

12 that we have when evaluating first time in man.

13 There unusual circumstances such as this one and is

14 the reason for the extended discussion where the

15 question of safety in this particular case and the

16 question of efficacy does become one of those real,

17 almost beyond FDA balance, because part of the

18 issue that has been discussed here is is this

19 experiment just wrong at this time or is it right

20 at this time, or are there other things that can be

21 done.

22 Part of that must be in the context of

23 potential benefit. If there is no chance for

24 benefit, because of the nature of the vector coming

25 from a disease-causing agent, one might really want

> 156 1 to pose the question, maybe this should not be done

2 at this time because there is no chance of

3 efficacy.

4 In the absence of being able to say that,

5 then I think we need to consider to do these very

6 careful discussions of the nuances of risks,

7 possibly future benefits, potentially no benefits

8 at this time.

9 So the long, roundabout way, is it is not

10 the primary determinant of whether to go forward at

11 this time by FDA in terms of that and, for these

12 early trials, first time in man, it is always

13 primarily based upon safety. But the questions of

14 efficacy do enter in the discussion. They may or

15 may not influence the final decision, but they do

16 play a part.

17 DR. SALOMON: There are some details in

18 Question 1 that I would like to go to try and

19 organize it but not, of course, restrict anyone

20 from bringing up anything else. I think one issue

21 we need to deal with is the vector. Implicit here,

22 Dr. Rao reminded me a moment ago, is we have got to

23 go back now and say how about this vector.

24 It is a very interesting strategy that has

25 been proposed and described, I think, very nicely

> 157 1 by Dr. Dropulic this morning, to use a single

2 helper plasmid, obviously, a very distinct strategy

3 from that employed and described in detail

4 yesterday by Dr. Verma and Didier Trono and Luigi

5 Naldini, et cetera, where each generation actually

6 broke it into more and more so now we are into

7 four-plasmid systems.

8 That\'s fine. It is two different

9 strategies. So one of the things we have got to

10 talk about is the vector and what safety issues are

11 there.

12 A second of three things that I want to

13 talk about is to use a transient-transfection

14 system versus a stable packaging cell line. We

15 have heard discussions of that yesterday and we

16 should talk about whether or not that has major

17 safety implications and put that in the context of

18 where the field is today.

19 The third thing we need to talk about, I

20 think, is we need to come back, at the end here, to

21 deal with Dr. Cornetta\'s comments which I take very

22 seriously, and that is concerns with the RCL assays

23 because that is something we grappled with

24 yesterday and I think we all admit that that is far

25 from perfect.

> 158 1 I think Dr. Cornetta could maybe start us

2 with that in a minute or two. So can we start with

3 vector, transient versus stable, and then RCL

4 assays. I think that will kind of get us through

5 Question 1.

6 Vector; one plasmid helper versus four-plasmid or

7 three-plasmid systems, transient.

8 DR. MULLIGAN: After the heavy

9 conversation we just had, I am not sure that this

10 is all that big an issue since we talked about it

11 yesterday in great detail. I would weigh in that

12 this certainly, I would consider, the safest vector

13 for this clinical trial, as we have mentioned

14 several times. I think that could be possibly the

15 case.

16 I think, on the other hand, it is not very

17 different than the transient transfection approach

18 that others talked about. So, whether you use

19 three separated pieces that all go back together

20 after you transfect them or use one that is already

21 together I don\'t think makes a big difference.

22 I guess I would say that looking at the

23 packaging construct in some detail, it has these

24 fancy bells and whistles but I didn\'t see a

25 characterization that the features that it has

> 159 1 actually do what they are intended to do. I am not

2 sure, in this group, we really want to harp on this

3 but psychologically the fact that investigators put

4 three or four poly-A sites and pauses between the

5 gag/pol and the VSV-G makes me think that they

6 think that it is important that there isn\'t

7 transcriptional read-through.

8 They may want to comment on that, but\--

9 DR. DROPULIC: Could I comment on that?

10 DR. MULLIGAN: I am not finished. Let me

11 just finish. I saw no evidence from any of the

12 information that anyone ever looked, for instance,

13 at the RNA species after transient transfection

14 being a dinosaur in the vector field, and I have

15 seen everything that can go wrong, not according to

16 theoretical principle.

17 Certainly, in this case, when you have all

18 those bells and whistles, all those sequences, you

19 can have cryptic donors and acceptors, you can

20 cross poly-A sites so they are not functional.

21 Transcriptional pauses don\'t always work when you

22 put them where they ought to be put.

23 So many things can happen. I just think

24 it would be nice to actually, for the FDA at some

25 time\--maybe we don\'t need to discuss it now\--to see

> 160 1 the information that validates the fanciness of the

2 vector system. All that being put, it is probably

3 comparable to the other transient-transfection

4 systems.

5 Just on the vector side, other than this

6 issue of whether you want a mobilizable vector or

7 not, it is a garden-grade variety vector like the

8 other vectors people have. So I don\'t think there

9 is a big issue there.

10 DR. SALOMON: Dr. Dropulic.

11 DR. DROPULIC: I think we could those

12 studies if you wanted us to do them to validate the

13 bells and whistles, if you like. But we didn\'t

14 have any concern\--I mean, actually we got the

15 vector from a commercial supplier. It came with

16 those bells and whistles already there.

17 DR. SALOMON: I guess, from my point of

18 view, I agree with Dr. Mulligan\'s concerns. I had

19 the same concerns. I like the idea of this high-level

20 molecular discussion and we put a pause site

21 here, we put a poly-A site here, we put an ATG stop

22 code on.

23 Okay, but, I guess I grew up in the

24 Midwest. It is sort of like, show me and I will

25 believe you. I think that the problem is that\--I

> 161 1 am not quite a dinosaur in that I have come to it

2 more recently, and what bothers me continually is

3 that there are all these sort of glib statements

4 of, this will work, or, this will work.

5 I am not accusing you of being any more

6 guilty of it than all of us. It is all fine as

7 long as we are doing stuff in vitro and we are

8 arguing with each other about whether you like my

9 work and will publish it. But it is a real issue

10 when you start talking about doing a human clinical

11 trial.

12 I just think, as a principle, what I am

13 defending as a chair, here, is yeah; I think if you

14 are going to tell us you have got all these bells

15 and whistles in the vector, then all I want is some

16 reasonable evidence that those bells and whistles

17 do what they say. Otherwise, they are irrelevant

18 to me and I don\'t need to know about them.

19 DR. MULLIGAN: The other thing is just on

20 the fancy models and pictures of all the

21 recombinations that have to occur which, again, I

22 have lived a life of this, that is all well and

23 good as long as the original materials are as

24 advertised.

25 DR. DROPULIC: It is validated, the

> 162 1 plasmids.

2 DR. MULLIGAN: I don\'t exactly mean that.

3 I mean after you do a transfection and you have

4 these two constructs, those two constructs will

5 become one construct. Therefore, your DNA

6 substrates for the events you are talking about are

7 very different.

8 So, again, I think I am just echoing your

9 point that, at the end of the day, it probably

10 doesn\'t make much difference and it is probably

11 just as complicated with three- or four-part

12 things. But it doesn\'t give you confidence,

13 basically, when you don\'t see the backup for the

14 features of the vectors and it a difficulty in the

15 vector field over the years.

16 We probably started this off the worst by

17 making very fancy vectors and then realizing it is

18 far better to get rid of things than to add things,

19 and it is far better to have simplicity. But it is

20 important that we get a sense of confidence that

21 people are looking at these issues in a general way

22 in a critical way.

23 DR. DROPULIC: We have always wanted to do

24 the right thing. So, if that is what the committee

25 requests of it, we will be happy to do to satisfy

> 163 1 the committee.

2 DR. SALOMON: I think one of the things,

3 again, that maybe we could note here is that when

4 the RAC reviewed it, one of their statements was,

5 wow, this is just a first-generation vector and

6 then you come back and say, well this is\--that is

7 where I was getting with Dr. Verma yesterday in

8 trying to articulate the fact that this use of the

9 word generation\--again, it is word problems.

10 So what I am saying is that I agree with

11 Dr. Mulligan, also. I don\'t think there is

12 anything inherently wrong with your strategy. You

13 guys have got to deal with this. I don\'t think

14 that a four-plasmid vector system and a transient

15 system is necessarily safer than the strategy you

16 took, necessarily.

17 You got up and said, well, we did this and

18 we did this because\--and that is your argument to

19 me that it is as safe, or safer, maybe. Dr. Verma

20 and Didier Trono, these guys did their four-plasmid

21 system. They did one. And then they did two, and

22 they did three and four, all based on the premise

23 that that would be safer.

24 All we are asking is that that all be

25 backed up scientifically. It is not a

> 164 1 responsibility of your company more than anyone

2 else, is all I am saying..

3 DR. ALLAN: Can I address the other issue

4 about the vector is that you have an intact LTR. I

5 know the reasons why you want to use an intact LTR

6 so that you can get differential expression, so

7 that you don\'t get the expression of antisense

8 unless the cell gets infected with wild type. I

9 like that. That is pretty cool. I like that.

10 But, on the other hand, if you have an

11 intact LTR, it gives you a great chance for

12 recombination because you have got LTRs there. So

13 it is like you are between a rock and a hard place

14 because you have increased your chance that you are

15 going to get recombination, copackaging

16 recombination, all these things with your vector.

17 So you are trying to balance these two

18 things and I am a little uncomfortable with that.

19 DR. MULLIGAN: The recombination issue, I

20 thought this would be dealt with in the

21 mobilization; that is, if you drew the conclusion,

22 as we kind of did yesterday, that we didn\'t want

23 mobilization, then you are, obviously, in a

24 quandary relative to the whole concept here.

25 Tracing the logic, it appears that some of

> 165 1 this may depend upon how much of a safety feature

2 it is to have inducible antisense. I know that was

3 emphasized in the talk. One of the conclusions

4 that that is important is that you have the LTR

5 present.

6 There are some other ways you could do it,

7 but you certainly have to, otherwise, make a brand-new

8 vector system. I didn\'t even want to get into

9 that but I think that is an issue\--I don\'t see

10 particularly making antisense in an uninfected

11 cell. I haven\'t seen, again, any data of how that

12 would be dangerous. I may be interested to hear

13 that. But I agree with you, that is the feature

14 that is totally related to the mobilization

15 question.

16 DR. DROPULIC: The efficacy of our vector

17 is not only relative to the antisense payload. As

18 I mentioned, it is a combination effect. Having

19 the UTRs helps the tracking, helps competition for

20 packaging. These are all attributes that help

21 inhibit HIV.

22 DR. MULLIGAN: As Ronald Reagan would say,

23 \"There you go again.\" This is the same issue of

24 there is\--and I read the protocol very carefully\--the issue

25 of whether or not antisense worked better

> 166 1 when the RNA is localized. The data was not there

2 for that.

3 Again, if that is the case, we need to see

4 data that is rigorous and supports that.

5 DR. DROPULIC: I can tell you one example.

6 We have a collaborator in the Boston area who has

7 done some challenge experiments with a SHIV. It is

8 the D12. Now, the SHIV D12 is very interesting

9 because it contains both the tat and the rev and

10 the envelope from pNL4-3, and yet it is in an SIV-type

11 backbone.

12 So what our collaborator had done is

13 transduced cells with our vector and then

14 challenged both pNL4-3 and this SHIV onto the

15 cells. He found three logs inhibition of NL4-3

16 while two-fold or so inhibition of the SHIV.

17 Now, the SHIV is very interesting because

18 it has got tat and rev that would also stimulate

19 the vector LTR; right? There is not much

20 difference there. When you think about it though,

21 the level of expression is similar. It should

22 affect and express from the vector.

23 The only thing that you can think about

24 that is different between the two is

25 colocalization. I suppose I am not articulating

> 167 1 that very well, but the differences in inhibition

2 between SHIV and NL4-3 in transduced cells with a

3 vector is indicative of some sort of differential

4 effect.

5 DR. SALOMON: What I don\'t want to do now

6 is\--this is the path into a study section

7 discussion. I would love to have it, to be honest

8 with you, but that is not the job today. That

9 doesn\'t trivialize the value of the science you

10 just described or the issues that Dr. Mulligan has

11 brought up.

12 Transient transfection versus a stable

13 line; I have not heard anything in the last two

14 days that makes me more or less comfortable with

15 one or the other, provided the same sort of safety

16 characterizations are done with both. In fact, my

17 sense from everything I have heard from everyone is

18 that we are probably not quite\--the field is

19 probably not quite ready for stable lines yet, that

20 everybody sounds like they are working on them but

21 that there are issues of titer and truncated LTRs

22 rather than self-inactivating LTRs and different

23 sorts of strategies.

24 Is there anyone who disagrees, basically,

25 with the statement that\--I don\'t think that is a

> 168 1 go/no-go criteria to use a transient. I think that

2 the field could be moved forward now relatively

3 safely with a transient expression system.

4 DR. MULLIGAN: I think so but, in fact, I

5 think there was a consensus by everyone that the

6 stable packaging cells did offer theoretical\--

7 DR. TORBETT: I tend to agree with that,

8 but in the absence of data, I think it is an even

9 call all the way around. I see no data either way,

10 again, with the one-plasmid versus four. Again,

11 without the data, it is a personal judgment.

12 DR. SALOMON: All I was trying to get at

13 was I think the advice that we can give the FDA on

14 this point is that we wouldn\'t tell you to refuse a

15 transient at this point. I just don\'t think there

16 is anything here that suggests that is less safe

17 than a stable.

18 So that brings us to Dr. Cornetta\'s

19 comments and our discussion yesterday on RCL. Ken,

20 do you want to just start us off, sort of restate

21 your issues and add whatever you want to the RCL

22 issue so we can get through that?

23 DR. CORNETTA: I just was looking at what

24 has been presented and what is out there. I am

25 just not very confident that we have something that

> 169 1 has been anywhere close to being validated for

2 detection. Some of the things that are presented

3 may not address two issues; one, when you are

4 talking about amplification, you need to consider

5 the effects and efficiency of the amplification

6 cells.

7 Since we are not sure exactly what these

8 recombinants might be in regard to both what their

9 LTRs may be and also what the envelope may be, we

10 need to be thinking about how efficiently might

11 this recombinant infect the amplification cell and

12 then, also, if it does infect the amplification

13 cell, will it really be amplified.

14 That came to some issues, for example,

15 yesterday talking about the PERT assay and using

16 MLV as the amplification control. MLV probably

17 would be amplified very efficiently in 293 cells

18 and so you may well be setting the bar too high in

19 trying to figure out what is your positive control.

20 I am not sure I have an answer for these,

21 but I just don\'t have a lot of confidence right now

22 that we can feel that the material that is being

23 tested has been in a very rigorous way to date.

24 DR. SALOMON: Thank you. I think that

25 that nicely states, also, the kinds of discussions

> 170 1 that we ended up with yesterday where I felt that

2 the consensus we had reached at the end of

3 yesterday was that, right now, still the RCL

4 assays, and I am not contradicting what Dr.

5 Cornetta just said, still the RCL assays are what

6 we have.

7 So we have to think about maybe ways to

8 enhance the quality of the RCL assays, and I think

9 Dr. Cornetta gives us a couple of suggestions along

10 that line. The fact that there are alternative

11 assays, the PERT assay, looking for the gag/pol

12 recombinants, I think we all agreed that that ought

13 to be pursued and, yet, the fact that neither had

14 really been that well validated made us all a

15 little uncomfortable advising the FDA that that

16 should be a requirement.

17 Are we sort of all agreeing on that sort

18 of thing? Ken, are you comfortable with how I said

19 that?

20 DR. CORNETTA: I think so. Just talking

21 about your prior point about whether we use

22 packaging cell lines or using transient

23 transfection, the way we produce virus in the

24 vector system is going to change very rapidly. But

25 one thing that may well be the determinant of where

> 171 1 to go with these new systems is what are we really

2 going to say qualifies that end product for our

3 clinical use.

4 That, I think, needs to be a major focus

5 and I am not sure we are there yet.

6 DR. SALOMON: One issue, just to be

7 specific, is choice of cell lines. Just given that

8 we are dealing with the VIRxSYS protocol, what

9 process did you do to decide that the H9 cell line

10 was your cell line of choice for RCL assays? That

11 was the only one you tested and you liked it, or

12 did you test ten others? You didn\'t show us any

13 data for validating the use of the H9.

14 DR. LI: NL4-3 is a very classic T-cell-topic

15 virus. You can infect H9, Sup-T1, whatever

16 you can choose with all similar sensitivity.

17 However, the reason we chose H9, FDA requires that

18 all the reagents that we are using has a CFA. So

19 we can\'t just make a virus stock.

20 So we bought a commercially available

21 pNL4-3 virus stock with identified PCR50. It was

22 provided by the manufacturer and where the titer

23 was sensing the H9 cells. So we want to verify

24 this one virion particle and go back to the

25 parental cell line where the virus was produced.

> 172 1 DR. SALOMON: So that would tell you that

2 the wild-type virus from which you made the

3 antisense envelope sequence would be caught if it

4 flipped, if it reinserted in the right direction,

5 recombined. Then the H9 would be the ideal. But

6 that, to me, isn\'t a very good rationale for its

7 use for detecting replication-competent lentivirus

8 emerging from this very complex recombinant-prone

9 transduction system.

10 So I might argue that you should probably

11 test a number of cell lines and, in addition,

12 consider the use of target cell lines that, let\'s

13 say, express tat. What was the one, CC\--it was one

14 of the cell lines there. Someone help with me. Is

15 it rev that is expressed at low levels? Carolyn?

16 DR. WILSON: C8166 that has HTLV1 tax.

17 DR. LI: Can they have a clarification

18 here? We are talking about two places where we do

19 the RCL test. One is for viral production. In

20 there, there is no HIV. There is nothing, except

21 the\--

22 DR. SALOMON: No; I am talking about the

23 RCL testing of the T-cell-transduced product at the

24 end of the nine- to ten-day period before infusing

25 it into a patient.

> 173 1 DR. LI: I mean, there we do not use H9

2 because you can\'t the H9. The cell already has

3 HIV. The cell is from an HIV-positive patient.

4 DR. SALOMON: Right; so what do you use to

5 detect\--

6 DR. LI: We use 293-T because\--

7 DR. SALOMON: Because that won\'t be

8 infected. Okay. I admit to a confusion on my part

9 on that one.

10 DR. LI: Okay. So we know VSV-G can, if

11 any recombination happens between the gag and pol

12 and VSV-G, it can be very productive, in fact, for

13 the 293T-cells.

14 DR. SALOMON: Any comments from the

15 committee? Is that going to be sensitive enough,

16 the way they are going at this? I am still not

17 totally clear here, because I kind of blew it,

18 didn\'t I?

19 DR. KAPPES: I see what that approach does

20 is it takes us back to the question of you may have

21 generated a recombinant. We don\'t know what it is.

22 We haven\'t characterized it, but we know it likely

23 does not contain VSV-G environment. My point is

24 there still is a void of information as to what

25 recombination may have generated.

> 174 1 DR. ALLAN: Are you on Question 2 and, if

2 you are on Question 2, where are you on Question 2?

3 DR. SALOMON: You don\'t have to make it

4 worse.

5 DR. ALLAN: You are talking about assays

6 for RCL, and there is the virus production and then

7 there is the stuff that goes into the patient;

8 right? If you are talking about, like this one

9 says LTR gag/pol recombination of media\--

10 DR. SALOMON: No; let me clarify. That is

11 where I blew it. I should be talking about, right

12 now, virus production in which case my earlier

13 comments were okay. When I blew it was when I went

14 and talked about what was coming out of the viral

15 product which we need to get back to. But you are

16 right. That is Question 2. I apologize to

17 everybody. I am just human.

18 DR. BORELLINI: I have another question on

19 the assay. What is the effect of the vector that

20 is in large excess and carries the antisense for

21 HIV on the amplification of the RCL?

22 DR. LI: We did a spiking experiment, used

23 the bulk harvest which has lots of virus, our viral

24 production. And the spike positive HIV. Actually,

25 that is required by FDA. You have to prove your

> 175 1 viral production does not inhibit your positive

2 control. And that was characterized. There is no

3 difference with the viral production bulk harvest

4 liquid, solution, whatever you want to call it, or

5 just simply the H9 cells media. There is no

6 difference, no comparable difference.

7 DR. SALOMON: So I think we are at the end

8 of Question 1. I still believe\--again before I

9 made the misstep\--I still believe that you should

10 probably go back and test several cell lines, not

11 just the H9.

12 DR. MULLIGAN: I think the question was if

13 you are using a therapeutic vector that is supposed

14 to suppress the emergence, how does that affect the

15 detection of HIV. So I am not sure how to

16 interpret the results you just presented. That is

17 it doesn\'t work in this case, or what? Do you get

18 my point?

19 What HIV you are putting in; could you

20 maybe go back?

21 DR. LI: For the spike?

22 DR. MULLIGAN: Yes.

23 DR. LI: It is the bulk harvest. The

24 vector we use for the cell transduction is

25 processed and concentrated. For the bulk harvest,

> 176 1 we just use that as a culture\--it is very diluted.

2 We are not using that for transduction purposes.

3 It is the very raw first harvest from the producer

4 cells after DNA transfection. We just want to

5 demonstrate, because that is the step. FDA

6 requires you to do the RCL test.

7 DR. SLEPUSHKIN: I am Vladimir Slepushkin.

8 I am responsible for production so maybe I can

9 explain better. In the first step of the

10 production, one of the first steps of the vector

11 production is collection of the supernatant that

12 contains vector. That is what is tested in this QC

13 test for RCL.

14 In this supernatant, vector is in a very

15 diluted concentration compared to the final

16 product. Therefore, I think it doesn\'t impede

17 sufficiently wild-type replication.

18 DR. SALOMON: Then I believe we are

19 through Question 1. Do we agree with that?

20 DR. TORBETT: I guess I am a little

21 confused because if it was expressing, wouldn\'t you

22 knock down the number of possible recombinants in

23 your readout assay? I think that is what Dr.

24 Mulligan was getting at. The answer probably is

25 yes? I think I just heard that is an MOI-dependent

> 177 1 phenomenon. I think that is what he was saying.

2 Is that correct?

3 DR. HIGH: But the answer we got is that

4 it is okay because it is done on the bulk harvest

5 and the concentration is dilute. But it must be

6 done at the end, also; right? It is not just done

7 on the bulk harvest, is it?

8 DR. LI: That is not a requirement.

9 DR. SAUSVILLE: That is the issue, the

10 characterization of the final product is, as we

11 started all this out, problematic.

12 DR. SALOMON: Right. So our job isn\'t,

13 however, to express our angst. Our job is to give

14 them some sort of specific guidance on what we

15 should do. I am being a little quiet because this

16 is getting close to the edges of my experience and

17 expertise. So I am looking to my colleagues here.

18 You have got to know when you know and when you

19 don\'t.

20 DR. SAUSVILLE: Correct me if I am wrong,

21 but the point that\--the specific issue in the

22 question was for the manufacture of virus. There,

23 they are doing what the FDA requires. What was

24 just articulated though was that the concentrated

25 material at the end of the day, after it has been

> 178 1 through T-cells, et cetera, what I, and I think a

2 number of us had in different ways, have issue with

3 is that that entity remains somewhat undefined.

4 There, I think your point is quite well

5 taken, that one would like to know, and one could

6 imagine infecting different cell types as part of

7 that. Again, whether this fits into the

8 requirement guidance, et cetera, I think it would

9 convey confidence that the product is going to

10 perform in the way we think it is.

11 There, it would be a dilution issue among

12 others to ferret out the different variants that

13 might be present.

14 DR. CONDE: Could I just make a comment?

15 I am Betty Conde, also with VIRxSYS. I work on QC.

16 The reason why we test the end-of-production cells

17 and the bulk harvest for the RCL assay is because

18 that is what the FDA requires. So we are trying to

19 meet the requirements as stated by the FDA.

20 The reason why, as Xuexia and Vladimir

21 mentioned before, it looked like the bulk harvest

22 wasn\'t affected is because it is about 100-fold

23 less concentrated than our final product.

24 DR. SALOMON: I suppose if I understand

25 it, then you know that you have come to the lowest

> 179 1 rung in this particular area. What I get is you

2 have a viral containing supernatant that you want

3 to test if you have replication-competent

4 lentivirus. So you put it on H9 and then you say

5 the supernatant; right? Very low MOI at this point

6 which is a big problem because we have already

7 seen, with your vector, at least, and I am sure it

8 is really true with everyone\'s, that the lower the

9 MOI, you begin to get grayer and grayer.

10 So you do it at a very low MOI and you do

11 six passages now. So if replication-competent

12 lentivirus is happening, then, theoretically, it

13 should be coming out passage to passage and

14 anything left over from the early inoculum is long

15 gone by the sixth passage. I am okay with that.

16 And six passages to amplify it\--well, I would again

17 defer to my colleagues, but some of the stuff we do

18 with endogenous retrovirus, six passages is pretty

19 reasonable.

20 So you get amplification. And then you go

21 back in and you do PCR for VSV-G and for\--that is

22 end. And also gag.

23 DR. CONDE: And also gag; right.

24 DR. SALOMON: You don\'t detect anything;

25 right? You get zero signal.

> 180 1 DR. CONDE: Right.

2 DR. SALOMON: That is replication-competent

3 lentivirus-negative.

4 DR. CONDE: Right.

5 DR. SALOMON: So now these guys are going\--well,

6 wait a minute. Later you are going to take

7 200 MOI and go to T-cell transduction, do the same

8 assay at 100 MOI. I think that is what Dr. High

9 was suggesting.

10 DR. CONDE: Yes. But we also test the

11 transduced cells at the end, the same way, using an

12 RCL assay.

13 DR. SALOMON: That\'s okay. The question

14 here is knowing that you don\'t have any detectable

15 RCL at an MOI that really doesn\'t represent the MOI

16 that you are going to go on to your T-cells with an

17 issue to anybody. It is a test. It is a crude

18 test. It doesn\'t convince any of us absolutely

19 that there is no RCL in that prep, is all I am

20 saying.

21 Then the question is, okay, but leave us

22 alone because we do it again after the

23 transduction. Is everybody okay with that?

24 Because, after the transduction, of course, life is

25 different. You are not going on to H9. You are

> 181 1 going on to 293. You have got a background of

2 wild-type HIV. There could have been

3 recommendation. So a premise you are trying to say

4 here is that your assays are relatively equivalent

5 so it is okay because we were going to reassure you

6 after the transduction.

7 I guess my concern here is that this 293

8 assay after the transduction is more problematic

9 and less sensitive, but maybe that is wrong. That

10 is what we need to discuss.

11 DR. ALLAN: The 293 assay is just to

12 detect whether you have got VSV-G; right? It is

13 not to detect whether you have got any HIV-related

14 sequences. So, again, why are they testing bulk

15 rather than the purified virus prep?

16 DR. WILSON: Perhaps I could clarify that

17 because I am not exactly sure where that impression

18 that we only require testing of the bulk harvest

19 came from. So, just to clarify, that is not

20 something that we require, recommend. I think that

21 we would determine that on a case-by-case basis,

22 what the appropriate stage for testing would be,

23 whether it be a bulk harvest or a final product.

24 DR. LI: That was the document based on

25 the murine leukemia virus.

> 182 1 DR. BO: Mark Bo from GTI. I may have

2 missed this. What is the positive control, then,

3 in the 293 test of the cell?

4 DR. LI: That question has been answered

5 many, many times because we do not want to create

6 an HIV VSV-G virus. I don\'t think anybody wants

7 that around.

8 DR. BO: So there is no positive control?

9 DR. LI: We do not have RCR-positive\--you

10 can\'t create a positive control to predict an

11 unpredictable event.

12 DR. EMERMAN: Hi. This is Mike Emerman.

13 DR. SALOMON: Go ahead, Mike.

14 DR. EMERMAN: One suggestion is, instead

15 of H9 to use primary CD4 cells which would also

16 amplify the HIV but then you come back with your

17 PCR assay for VSV-G. So what you are really

18 worried about is recombination between I guess

19 anything that is VSV-G and HIV. Even though you

20 would be getting replication of HIV in that

21 culture, your test would be is there any VSV-G

22 still around.

23 By using 293 cells, you are getting rid of

24 all of the HIV. That is certainly a fair test, I

25 think.

> 183 1 DR. SALOMON: Dr. Emerman, you are saying

2 as long as they do the 293 test and maybe do a CD4

3 test at the end of the transduction that you are

4 okay with the current strategy?

5 DR. EMERMAN: Yes; I am not sure what the

6 293 test is actually going to\--yes; I am okay with

7 that.

8 DR. ALLAN: I think the reason for the 293

9 is because, if you have got 10

6 HIV particles and

10 you have got one particle of potentially

11 recombinant VSV-G, and you just threw it on CD4

12 primary cells, you are just going to get\--you may

13 not see VSV.

14 DR. EMERMAN: I think if you had 10

6 HIV,

15 you wouldn\'t want to be putting that into a person,

16 anyway.

17 DR. ALLAN: Well, no; this is what is

18 coming off the transduced cells, so you are going

19 to get some\--you may not get a lot of HIV,

20 actually, but you would get some probably because

21 of the breakthrough studies we saw earlier. So I

22 would expect you would see some HIV.

23 DR. EMERMAN: Right. But, again, that is

24 what you are worried about, is there something

25 really, really bad in that stuff. One thing that

> 184 1 would be really bad in that stuff is something that

2 had VSV-G in it.

3 So you are amplifying something that is

4 actually more realistic. And you are also

5 amplifying in the same cells which it is going into

6 in the body, which is primary CD4 cells. Whether

7 or not something grows in 293 cells is somewhat

8 irrelevant. Those are not the relevant cell types.

9 DR. MULLIGAN: He is getting at the issue

10 of how you preamplify before you test for the VSV-G.

11 DR. EMERMAN: Yes.

12 DR. SALOMON: Right. I heard that. I

13 guess, trying to synthesize it here, I am still

14 uncomfortable with the idea of testing the low-MOI

15 bulk supernatant because I am not convinced by

16 anything that the 293 VSV-G assay that you are

17 employing to test the final T-cell-transduced

18 product and, even accepting Dr. Emerman\'s excellent

19 suggestion that you do it with fresh human CD4\--I

20 like to do that and that is something you should be

21 doing.

22 But I still am not convinced, because of

23 the sensitivity of the VSV-G assay and some of the

24 unknowns like the absence of a good positive

> 185 1 control, which is now anticipating a little bit

2 more the discussion in this second question\--so I

3 would still like to know, coming into the high MOI,

4 200 MOI, bulk, ready-to-go into the T-cell mixture,

5 that there is no RCL.

6 I would like to know that before it goes

7 into the human T-cells.

8 DR. SALOMON: Dr. Torbett?

9 DR. TORBETT: I guess it is a question of

10 the percent they are testing of the sample, whether

11 it is a low or high MOI. I kind of thought they

12 were testing 1 percent of the sample of 3 percent,

13 it shouldn\'t make a difference. But maybe I am

14 wrong here.

15 DR. LI: We do test the 5 percent of the

16 total harvest when the total harvest is 6 liters.

17 But, above 6 liters, we test 300 ml.

18 DR. WILSON: I need to explain. That 300

19 ml is really specific for MLV vectors using an RCR

20 standard that was developed for MLV vectors with an

21 amphitropic envelope and qualifying your RCR assay

22 for the sensitivity of detection of that particular

23 standard.

24 We have not addressed that issue for

25 lentivirus vectors.

> 186 1 DR. LI: So what should we do? That is

2 the only thing out there we can follow.

3 DR. WILSON: For now, I would go with the

4 5 percent rule because we really don\'t have the

5 tools and we haven\'t had extensive discussion to go

6 with a model like we have for the MLV-based vectors

7 with the 300 ml rule.

8 DR. SALOMON: That 5 percent, I believe,

9 again looking for confirmation or discussion, that

10 5 percent should be the concentrated vector to be a

11 model for what you are going to put on the T-cells

12 a few minutes later.

13 DR. LI: Let\'s clarify one more point. I

14 think that the rationale we do on the bulk harvest,

15 because it is a transient system, you have a chance

16 of DNA contamination. Most likely, RCR will happen

17 in that step than later. You go through

18 purification. You get rid of all your DNA\--most of

19 your DNA by benzonase treatment. So the cleaner

20 product.

21 So if RCR will happen, RCL, if you like,

22 it will happen in the more dirty, like you say,

23 step. That will be the first step. That is the

24 rationale behind it.

25 DR. SALOMON: That is an interesting

> 187 1 point. Again, I think we have to be very cognizant

2 of production issues in the kind of advice we give.

3 So I think that is very good. What I am sitting

4 here thinking is, okay, fine. Then take the 5

5 percent, benzonase treat it, purify it and test it.

6 DR. LI: You mean after it goes through

7 all the purification and test it again, 5 percent.

8 DR. SALOMON: I say that. But everybody

9 here who knows me knows I have never made a 40-liter batch

10 of anything and benzonse treated it and

11 purified it, so it is perfectly okay for someone to

12 say, easy for you to say but it is hard for me to

13 do.

14 But I need to hear you tell me that.

15 DR. LI: The other thing is I think we

16 have to really remember the vector is not our

17 product. Our product is vector-transduced cells.

18 That; we are vigorously testing that with DNA PCR,

19 RT PCR, and biological assay.

20 DR. SALOMON: Dr. Cornetta, you are

21 someone here that does this all the time. I know

22 Dr. Mulligan, also, and maybe others. I don\'t mean

23 to exclude anyone. Ken, do you have a comment?

24 How do you come down on this?

25 DR. CORNETTA: Again, I would like to see

> 188 1 more data. You are talking about two potentially

2 different products if you are talking about the

3 undiluted or unconcentrated material versus the

4 concentrated material. I would suspect\--I guess I

5 would tend to like to look downstream. It may be

6 that the initial product, which is not

7 concentrated, may have inhibitors to transduction

8 or something else in there, too, that you may also

9 have some more interfering particles, or something

10 else, that might change how your readout is for

11 detecting of RCL.

12 So, again, that can be done fairly quickly

13 once you are confident of what your RCL assay is.

14 But, again, those are the kinds of thing that you

15 would like to look at to see what kind of level of

16 detection you are getting.

17 It is a very difficult issue since we

18 don\'t really have a good positive control to be

19 able to follow through there. Again, there seems

20 to be a concentration or a tendency to try to look

21 at one cell line for detection. I am not sure that

22 is good. I think, from what Mike Emerman was

23 saying, looking at CD4-positive cells are probably

24 good at looking for HIV recombinants that may have

25 the envelope for CD4.

> 189 1 But then again, if we are looking for VSV-G, it

2 may be that other cell lines are better. So

3 I think people need to be expanding what kinds of

4 cells they are eventually testing but, again,

5 looking through the process of where you are

6 testing, you may get different results depending on

7 where you test that and at least some initial work

8 should be to justify where your testing is going to

9 be.

10 DR. SALOMON: Dr. Mulligan, Dr. Zaia. I

11 know you guys also have experience.

12 DR. MULLIGAN: I would just say the

13 concentrated product, with all the other caveats of

14 how to do the amplification, but the concentrated

15 product. You should copurify\--whatever you had in

16 the unconcentrated should coconcentrate, I would

17 think. That is the source of the virus you are

18 using for the infection.

19 DR. SALOMON: Dr. Zaia, do you have any

20 comments on that?

21 DR. ZAIA: No comments.

22 DR. LI: Excuse me, because after you guys

23 say it, I have to go home and do it. So I would

24 ask you a question. For the concentrate, remember

25 our product is an anti-HIV product, whatever the

> 190 1 positive control will be. After they concentrate

2 it, if we do on the HIV-permissive cell line, the

3 positive control won\'t grow because it will have

4 anti-HIV activity. What am I going to use? How

5 will I even validate my assay without creating an

6 RCR, which nobody wants to see HIV VSV-G RCR.

7 DR. MULLIGAN: I don\'t know. I think this

8 is probably being nonproductive to go through this

9 in any detail, but it may depend upon the eventual

10 multiplicity of infection at which you test the

11 product. That could be prohibitive if you have to

12 test a lot of product and you can\'t use a high

13 multiplicity infection.

14 But that would alter, I assume, the

15 efficacy of the effect of your construct. So,

16 depending on how you did the infection, you might

17 have to use more and more cells to do the

18 infection. But that might be some way to go.

19 DR. LI: You are saying to do very low MOI

20 transduction to\--

21 DR. NOGUCHI: Yes, but these are the kinds

22 of discussions\--

23 DR. LI: So then we go back to the bulk

24 harvest diluted

25 DR. NOGUCHI: Rather than get into details

> 191 1 here, these are the precise kinds of interactions

2 that we invite you to share with our regulatory

3 staff who will, in fact, work with you to determine

4 the proper conditions.

5 I will just say that, even if your

6 anticipated product is going to be the transduced

7 cells, we require quality control of all components

8 of that which includes the vectors and

9 characterization and so forth. But we really would

10 welcome further interaction directly with our

11 staff.

12 I think it is unfair and counterproductive

13 to try to work out the details here.

14 DR. SALOMON: I agree and I think Dr.

15 Mulligan was starting to get that, too. I guess

16 the only part that I don\'t totally understand yet

17 is at that point all I want to know is if there is

18 replication-competent lentivirus. I don\'t care

19 whether wild-type HIV grows in these or not. There

20 hasn\'t been any wild-type HIV yet. This is the

21 concentrated supernatant. So tested on a number of

22 different cells would work for me.

23 Let\'s talk about the VSV-G assay since

24 that is part of this RCL assay. So now we are

25 switching\--oh; I\'m sorry. Please.

> 192 1 DR. KHAN: Arifa Khan, FDA. I just wanted

2 to mention something about the considerations that

3 were taken when coming up with the 1 percent and

4 the 5 percent. That really was specific to the

5 therapeutic vectors that were generated using the

6 MULV-based vectors.

7 A lot of things were taken into

8 consideration. Basically, the bottom line was that

9 the 1 percent really must at least reflect the

10 volume of the vector virus that is being used in

11 your transduction. Everything does have to

12 correlate eventually with the actual dose, with the

13 volume that is really going to be affecting the

14 transduced cells going back into the patient.

15 The 5 percent also takes into

16 consideration the number of cells used to generate

17 the volume that is going to be used, at least in

18 one human dose equivalent, used in the

19 transduction.

20 So the numbers just didn\'t come out of

21 thin air. For the MULV-based system, all those

22 factors were taken into consideration including the

23 total volume of the lots and the volume used in the

24 transduction. So I think we will have to take

25 those things into consideration for these

> 193 1 particular situations.

2 DR. SALOMON: Thank you. I think we will

3 take Dr. Noguchi\'s and Dr. Wilson\'s guide here very

4 literally and that is these are details. This is

5 not what the committee is really supposed to be

6 doing. I think we stay on a higher ground. I

7 appreciate that.

8 So I am going through Question 2 here,

9 whether we like it or not. By the way, I skipped

10 a) because I think we answered that yesterday, and

11 we have answered it earlier today, \"Should an in

12 vitro assay for detection of functional LTR-gag/pol-LTR

13 recombinant be used as a lot-release

14 assay?\" I think we all agreed that that has not

15 been a validated assay and shouldn\'t be a lot

16 release, unless someone wants to disagree strongly

17 with that.

18 Nonetheless, I think that we all recognize

19 the scientific value and encourage that it be done.

20 DR. LAWTON: The only comment I would have

21 on that if, in this particular case, it is a

22 sensible assay to develop and validate, then it may

23 be appropriate.

24 DR. SALOMON: So it brings me to b) which

25 is the RCL infectivity assay of sufficient

> 194 1 sensitivity and is the positive control for the

2 assay adequate for determining the sensitivity

3 which then gets back to the comment we got also

4 from the audience and sort of where I was going a

5 minute ago.

6 So let me go back there; ten copies in

7 10,000 cells, by TaqMan PCR\--you will have to

8 correct me here. We do TaqMan PCR all the time in

9 our lab and we can detect ten copies in a million

10 cells. So I don\'t understand an assay\--there has

11 got to be something wrong here. Either I am being

12 dumb, which is very likely, or you guys need to go

13 back and work on this assay.

14 DR. LI: The TaqMan assay, each well you

15 can put 500 nanograms of DNA. More than that, and

16 the sensitivity gets affected. This is when they

17 sell the instrument, what it can do, unless you can

18 do it differently or you have a way to control your

19 background signal. For ten copies, actually we can

20 claim one copy. But anybody knows, one copy, you

21 run into the statistical problem because you have

22 to run nine, ten, twelve replicates in order to

23 catch that one, and also you do have a false-positive

24 possibility.

25 If we use that as a lot release, that will

> 195 1 run into a lot of problems. In terms of cell

2 number, I would like to know how you do the million

3 cells for one copy in the TaqMan in that one well.

4 How do you run that? May you have a custom-made

5 plate or something.

6 DR. SALOMON: No. We use the TaqMan

7 technology in a somewhat different cycler where we

8 can go do multiple dilutions going down from 1

9 microgram of DNA as cells. But I will have to\--for

10 this point, I will just say, fine. If you don\'t

11 think you can do it on technical grounds, I am not 12 a TaqMan, such
an expert that I am going to argue 13 it in this. I will do some of my
own homework on 14 that one as well.

15 DR. WILSON: Perhaps a different way of

16 framing the question is if the qualification assay

17 is done on 10,000 cells and the total dose is 10

9

18 cells, is that of adequate sensitivity.

19 DR. SALOMON: No. I am not comfortable

20 with that at all. So I think that, unless someone 21 disagrees, you
either have to have a more sensitive 22 TaqMan assay, so I need to go
back and figure out 23 why there is such a disparity between your

24 sensitivity and the one I am used to, in a 25 different system,
though.

> 196 1 Or you need to have a different assay

2 because\--see, that is kind of what scares me

3 because if it is an insensitive assay, then the

4 risk of moving RCL into the patient is all that

5 more great.

6 DR. LI: No. The thing is we also go

7 through six passages, remember, for the culture.

8 So, after that, we do TaqMan again. So six

9 passages of amplification.

10 DR. SALOMON: In this system, now, you are

11 amplifying HIV, wild-type HIV, very likely, in

12 addition to your RCL. Therefore, I am concerned

13 now that there may be issues of competition, for

14 example.

15 DR. LI: No; 293T is nonpermissive for HIV

16 replication. We specifically try to catch VSV-G

17 pseudotype.

18 DR. SALOMON: That\'s right. You are using

19 293 in one assay and 293 in another. Our

20 collaborator and guru on TaqMan just stepped up.

21 DR. LONG: Zhifeng Long from GTI. I just

22 wanted to clarify one thing. I mean, in TaqMan

23 PCR, you can easily do 3.2 micrograms DNA per 96

24 well reaction. We do that all the time. We

25 actually can do up to 6.6 micrograms. So nanograms

> 197 1 is not a limit that you can put into a 96-well PCR.

2 But that is not the point. I think I want

3 to be fair is that, while in the assay system, they

4 do a six-passage amplification, so they are not

5 required to test 10

6 cells because you know, if

6 they can verify or qualify their system in a way

7 that they can show the original input one virus

8 somehow a positive control in 10

9, they will six

9 passage it. At the end, even using 10,000 cells,

10 they can detect that positive control virus, that

11 would be an elegant way of showing that the assay

12 is sensitive enough

13 I don\'t really see that 1 or 10 copies in

14 10,000 cells is the limit because they amplify six

15 passages. What I see is whether your PCR assay is

16 an adequate assay. Actually, the PERT assay is

17 more sensitive because it detects all spectrum of

18 retrovirus. Or you may want to think of other

19 assays like P24. But, in your case, if you mix

20 with the HIV virus, you probably don\'t want to use

21 it. So I think you want to think of some other way

22 to verify your system separately with a positive

23 control virus that is distinguished from your wild

24 type.

25 DR. SALOMON: Okay. There are a bunch of

> 198 1 different things there. Thank you, Dr. Long. One

2 of the issues is to use a control virus. I think

3 that all the sponsors, not just VIRxSYS, have

4 stated that they are not comfortable making a VSV-G

5 control virus. I am okay with that. I don\'t think

6 we need to go there unless someone on the committee

7 feels really strongly about it.

8 DR. ZAIA: I don\'t feel strongly, but we

9 saw an example yesterday that it can be done with

10 the VSV, with the mutated accessory protein virus.

11 If it is good enough for a cell genesis, why isn\'t

12 it good enough\--

13 DR. VERES: I have to qualify that that

14 was pseudotyped. That was not HIV with a VSV

15 envelope cloned into the virus. That was a

16 chimeric attenuated HIV which was pseudotyped. I

17 had both the HIV envelope and the VSV envelope on

18 it. So it is only for a first round of infection.

19 DR. ZAIA: But it is still a virus that

20 has VSV-G envelope. It may not be exactly what the

21 virus would be like\--

22 DR. SALOMON: But it doesn\'t have VSV-G

23 DNA or RNA.

24 DR. ZAIA: No, but\--

25 DR. SALOMON: That is what they would need

> 199 1 as the positive control for this assay.

2 DR. ZAIA: To further spreading. If you

3 don\'t have an amplification step, you don\'t need to

4 have it spread.

5 DR. SALOMON: I guess I would be okay

6 with, at this point, using VSV-G RNA and VSV-G DNA.

7 I guess the idea in the amplification is they can

8 do it all with DNA. You don\'t need RNA. That

9 probably is reasonable. So the question, then,

10 would be Dr. Long\'s comments\--I didn\'t totally

11 follow this part, Dr. Long, is that you felt,

12 because of the amplification, the sensitivity of 10

13 copies in 10,000 was okay because, after six rounds

14 of amplification, you really have dramatically

15 gone\--you could have detected 1 in a million, is

16 what you were trying to say; right?

17 DR. LONG: Yes; that\'s right. I think we

18 have experienced six amplifications is ample to

19 amplify a single virus from the beginning. The

20 fact is that here it is a different system, that

21 you need to validate whether the cell line, like

22 Dr. Khan, I think, mentioned about whether you use

23 appropriate cell lines to show, demonstrate, your

24 assay system is sensitive enough to detect RCL. I

25 think that is the key question.

> 200 1 The PCR part, you are right. You can put

2 more than 10,000 cells in a well. But no limit

3 here. You can put 1 million there. But I think

4 the assay is okay in terms of using the final

5 endpoint detection after six passages. More key is

6 whether the assay is capable of detecting RCL

7 without a positive control.

8 DR. SALOMON: Okay. I think that is

9 definitely some progress on this.

10 Dr. Rao?

11 DR RAO: I just had an additional question

12 here. All of this is only detecting RCL which has

13 VSV-G in it; right? None of the other possible

14 recombinants which might be because of the wild-type HIV

15 where there is stress, where you are

16 selecting the envelope, none of those will be

17 deleted at all so you have no detection for what

18 else might be happening other than the VSV-G.

19 DR. SALOMON: I think that is the point

20 that Dr. Long made, Dr. Kingsman, Dr. Kappes and

21 Dr. Cornetta. So the question to the committee is

22 that is a general area of discomfort. I think we

23 all share that. VIRxSYS shares it. They have come

24 up with their strategy. CellGenesis, Oxford

25 Biomedica, GTI, for that matter.

> 201 1 Can we do more substantively? We have

2 identified the issue. I don\'t think that any of us

3 have got any more of an answer yet, though. I

4 think Dr. Cornetta put it as well as anyone could.

5 DR. WILSON: I think you are right that

6 this is a difficult issue which we probably won\'t

7 be able to come to a definitive answer on today,

8 and a number of important points have been raised

9 for our own internal consideration and discussion.

10 Probably, for the sake of time, we should go ahead

11 and move on for now.

12 DR. SALOMON: Dr. Cornetta, I don\'t get

13 any physical input because I can\'t see you. Are

14 you okay with that? Again, I want to respect the

15 fact that you brought it up.

16 DR. CORNETTA: No; I don\'t think it is a

17 thing that you are going to be able to answer today

18 and I think the overall point, and I think that was

19 probably Carolyn, was that I think it is something

20 the FDA is going to have to think hard about and,

21 just as they went through with the retroviruses and

22 coming up with guidelines for testing, this is

23 going to be something that they are going to have

24 to struggle with.

25 I think all the appropriate comments have

> 202 1 been made.

2 DR. SALOMON: Then we will move on. I

3 think it would be fair to say, since I have been

4 sort of giving you guys a hard time about this

5 particular one, that I do think that you have\--as

6 all the other sponsors appropriately acknowledged,

7 that this is an area of difficulty and you have

8 used what are available guidances to try and help

9 you to figure it out.

10 So I do commend you for what I think was a

11 good-faith effort, very much so, in this and it is

12 not your fault that we haven\'t solved it yet.

13 Are there additional in vivo studies that

14 need to be performed is c), regarding now the

15 safety testing of VRX. I think we have gone over

16 that, too.

17 DR. WILSON: I think we have really

18 covered c) and d) already.

19 DR. SALOMON: Good. I was going to try

20 and agree with that.

21 Question 3. \"Please discuss whether

22 vector mobilization is considered an advantage or a

23 safety concern for the proposed clinical trial and

24 consider. Please consider the following,

25 specifically.\" Now we clearly had a discussion of

> 203 1 mobilization yesterday. I know Dr. Mulligan had to

2 leave to the airport, so we will try and remember

3 and be faithful to some of the comments that he

4 made yesterday.

5 \"Are the data available from the assays to

6 assess vector mobilization by wild-type HIV

7 sufficient? Are there additional preclinical

8 studies to assess vector mobilization that should

9 be performed and, if some, discuss the optimal

10 study design.\"

11 Dr. Allan, we have discussed the monkey

12 study; right? So that is on the record. I make a

13 joke of it just not to take myself too seriously

14 but not to trivialize it. That was a good

15 discussion.

16 So, are there additional studies? Do we

17 think that the data here was sufficient? So I will

18 jump in there and say, yes; it was sufficient to

19 demonstrate to me that there was a lot of

20 mobilization. Again, now, we are doing adjectives;

21 low mobilization, high mobilization.

22 To me, what I calculated out in the in

23 vitro culture system, which is reasonably what has

24 been studied, was about 1000 copies per ml. I

25 wrote that down. Yes; 1000 copies of

> 204 1 packaged/mobilized, depending on which terminology

2 you want to use, per ml is occurring.

3 To me, that is a lot when one copy of HIV

4 theoretically can infect a cell and set off an

5 infection of a patient.

6 The studies in the mouse show that there

7 is mobilization to CD4 cells. The studies of

8 failure to mobilize to the B-cells, I would

9 suggest, are uninterpretable because there were not

10 enough CD19-positive cells, when you are looking at

11 1 or 2 percent, to find it. So I think those, you

12 would have to go back, if you want to sell those

13 studies, and do much higher injury levels of CD19

14 cells.

15 DR. ALLAN: Do other target cells need to

16 be considered there? Is there tropism,

17 necessarily, uniformly to all non-CD4-positive

18 cells so should macrophages or some other cell type

19 be considered?

20 DR. TORBETT: And that issue is relevant

21 because, as was mentioned before, you are changing

22 or stressing the envelope nature of this. If you

23 would think about it in antibody terms where an

24 antibody has to be qualified against as panel of

25 cell types, it would seem reasonable, since we are

> 205 1 trying to detect the nonprobable but bad thing to

2 happen, that that issue be explored a bit further.

3 DR. SALOMON: Yes; it suggests

4 experiments. Again, I don\'t want to get down into

5 the detail, but there are a lot of other things

6 that could be relatively simply done; for example,

7 putting EGFP into H9 and putting H9 into the\--or

8 Jerkit or Mold4, different cell lines into the SCID

9 mice to see whether it was mobilization to

10 noninfected T-cells, for example.

11 Again, the details of that; all I am

12 saying is that, from my point of view, just to

13 start this, I think that you have demonstrated that

14 there is mobilization. I think the question now

15 is, unless someone says no, I disagree with you and

16 that is what I am waiting for, but if you agree

17 that there is mobilization, whether it is a little

18 mobilization or a lot of mobilization, I don\'t know

19 what that means in terms of the biological

20 significance of it, and therefore is that an issue

21 now?

22 Is that a problem? Are we going to accept

23 mobilization in this system.

24 DR. ALLAN: What struck me as the fact

25 that it wasn\'t clear to me what was being mobilized

> 206 1 and whether they are actually recombinants or not,

2 because the data was presented, which was good,

3 which was it directly looked at either the

4 antisense vector or\--let me see; what else was

5 looked at. My consideration is whether you have

6 got some sort of recombinant in there, not that it

7 is replication-competent, but which could be, at

8 some point later, which is like a gag/pol

9 intermediate or something else, that has been

10 mobilized.

11 So not just that the vector has been

12 mobilized but anything else.

13 DR. TORBETT: I think the question is is

14 the vector getting mobilized and you are asking is

15 the vector now becoming infectious; is that right?

16 DR. ALLAN: Not necessarily infectious but

17 that, beyond the vector being mobilized, which they

18 have demonstrated, are there other things being

19 mobilized. It is just a question of terms. I

20 think it is terminology.

21 DR. TORBETT: I guess it is two different

22 questions, really. I mean, the question of vector

23 mobilization was very hard to assess in any of

24 their animal models. All I can say is it occurred.

25 To what degree, how much, is unclear to me and what

> 207 1 is coming out is unclear.

2 DR. SALOMON: So I think the suggestion

3 would be to better characterize\--I think, as an

4 overall safety concern, is the idea that what is

5 happening is mixing, matching and evolution of the

6 species in the system as a model for what might

7 happen in humans.

8 I think that, to understand better the

9 mixing and matching, we go back to a discussion we

10 have already had, that we are going to need to see

11 more data in longer-term in vitro cultures with

12 both characterization\--I don\'t want to restate what

13 we have already agreed on and restated.

14 I don\'t know whether I want to go into

15 saying\--get into the details now because I think we

16 are then going into the study-section mode again,

17 whether you should do it in the NOD, or the NOD-SCID, or how

18 you should do it. Again, I would love

19 to have those conversations, but I don\'t think that

20 is appropriate for today.

21 DR. NOGUCHI: Some of this is sounding, to

22 get back into that, \"We would like to see something

23 else like a nonreplicative recombinant, and an

24 assay for that.\" Yet there has been a discussion

25 as well, maybe that is too much to ask at this

> 208 1 time.

2 DR. SALOMON: I don\'t think that is too

3 much to ask. I don\'t think anybody told you that

4 was too much to ask. I think everybody here is

5 saying that we want evidence that there is not a

6 real evolution, if it is non-replicative, of viral

7 sequences in this kind of a system and, if there

8 is, I think we need to know about it and try and

9 regulate on that and consider its safety

10 intelligently.

11 DR. TORBETT: I think this is an important

12 point because we are using not a self-enacting

13 vector but something that has an active LTR which

14 can make a full-length transcript which increases

15 the chance for recombination.

16 So I think these are serious

17 considerations. I don\'t know which vector is

18 better, whether it is a SIN or an active, but I

19 think if you are using it as a full-length

20 transcript, then some of this information needs to

21 come out for safety concerns.

22 DR. EMERMAN: This is Mike Emerman, again.

23 It was alluded to that SIN vector wouldn\'t work.

24 It would be interesting to note that was actually

25 tested. I guess my sense of this is that the

> 209 1 antiviral effect depends on copackaging. If it

2 does depend on copackaging, it is hard for us to

3 ban vector mobilization except to have it

4 characterized.

5 DR. SALOMON: I think, in terms of an

6 assay design for assessing mobilization, I think

7 what we all agree on is that it should be done in

8 long-term, more complex, multicellular in vitro

9 cultures.

10 Now, whether or not you should also

11 include animal studies, sort of building on\--you do

12 have some expertise and have been pretty successful

13 with your SCID studies. I am, again, trying to

14 articulate a decision here, or a recommendation. I

15 think that would be a positive.

16 I am not certain, though, as I said, that

17 I would insist that you do that because I think

18 that\--I am not sure how that\--that is science.

19 That is not necessarily simple testing.

20 DR. DROPULIC: It is an extreme amount of

21 work to do that.

22 DR. SALOMON: As I said, as my laboratory

23 does it, I absolutely agree. It is a lot of work.

24 Are we done with Question 3?

25 DR. WILSON: Part b) of Question 3 is more

> 210 1 specific to the study subjects.

2 DR. SALOMON: Yes; I missed that right

3 now. But you are right. So what should we do\--we

4 have gotten so focussed on this first part that we

5 haven\'t talked enough about what is happening in

6 the study subjects which is what we are being

7 reminded of now.

8 So what should be done on the study

9 subjects in terms of looking for vector

10 mobilization?

11 DR. TORBETT: Many of the things that we

12 discussed, the technology is very similar, whether

13 it is before you put the cells in, do it in culture

14 or coming out of the patients. That is very

15 similar.

16 So I think that the same kinds of studies

17 we have discussed in the past need to be applied to

18 the patients as well. I am opening that up for

19 discussion, I guess.

20 DR. SAUSVILLE: I would echo that. I

21 think that is the logical follow-through, the issue

22 about the evolution of this virus or viruses into

23 something that\--we don\'t know exactly what we are

24 going to see in the clinical situation.

25 I would just note that, in the long list

> 211 1 of things that are going to be looked for in the

2 clinical trial, I didn\'t go back and look, but this

3 issue of variants that emerge, I think, is very

4 important and should be captured. That would

5 include whether you mobilize some aspect of the

6 vector or whether you change the population of the

7 HIV that is running around.

8 DR. SALOMON: Dr. Zaia?

9 DR. ZAIA: I just want to say that within

10 the clinical-trial groups, AIDS clinical-trial

11 groups, that are looking at fitness of virus

12 relative to new drug additions, that technology is

13 well worked out so you are going to very rapidly

14 detect changes in fitness in this population of

15 patients who clearly will have virus in their

16 blood.

17 DR. REITZ: I think, also, you want to

18 follow the virus populations with some sort of

19 genotype analysis over a period of time. You will

20 learn stuff from it.

21 DR. SALOMON: I think that there is

22 nothing but logical follow-through here in terms of

23 saying that the same concerns we have of mixing and

24 matching viral parts leading to evolution of the

25 species is as big a concern after the treatment as

> 212 1 before. I think everyone has said that.

2 Therefore, if you agree, the advice would be that

3 it should be characterized biologically, on

4 multiple cell lines with different tropisms but,

5 also, some of it has to be done molecularly either

6 through genotyping or through specific\--I think

7 what Dr. Zaia is saying is interesting because it

8 also implies that some of the things could be just

9 looking at changes in drug resistance which could

10 be done more rapidly than full sequencing and

11 trying to imply changes on that basis.

12 Is that right, Dr. Zaia?

13 DR. ZAIA: Yes, but I was just thinking

14 about fitness of the virus, per se. If we are most

15 concerned about that, we could easily do that.

16 DR. SALOMON: So fitness would be like a

17 quantitative assay that demonstrated just an

18 increase in the rate of spread through a given

19 indicator cell line. Okay; I stand corrected.

20 Dr. Long?

21 DR. LONG: I would just like the ask the

22 panel to consider what kind of assay would be

23 appropriate given the fact that they only detect

24 one variant when the screen 240-some clones

25 sequencing.

> 213 1 DR. SALOMON: No; you didn\'t get that

2 right. There were hundreds of variants. About 80

3 percent of them were changed. That is a rough

4 calculation. It was 264 clones. 240 had various

5 mutations.

6 DR. DROPULIC: No. They had all

7 deletions. Most of them were deletions. There

8 were relatively few that had the base substitutions

9 that we are seeing. 91 percent were deletions.

10 DR. COHEN: I am Ruben Cohen. I manage

11 the clean room for VIRxSYS. I would like to just

12 offer kind of a global perspective in terms of

13 patient safety. I have graduated from GTI so I

14 know a little about the patient trials there having

15 been involved in training the medical centers that

16 were involved in collaboration.

17 I also would like to say that one of the

18 reasons I am happy with the way VIRxSYS is handling

19 this is because I have also come through the

20 agricultural world. The fact that I consider this

21 vector is under a sentinel control, its expression

22 is limited to the cells that basically have some

23 activity going on with relationship to what the

24 problem is.

25 One of the lessons I would like the FDA to

> 214 1 be thinking about is that, having come out of the

2 agricultural world and working in molecular biology

3 there, the antisense tomato is not something we buy

4 in the market. The reason, I think, for its

5 failure is because it was under a constitutive

6 promotor, it was always expressed. It was

7 everywhere and everybody was afraid of that.

8 The fact that this has a kind of a

9 sentinel function, it only works where it is

10 needed, I think is both expressing something

11 towards both the safety and the appeal of the way

12 this product is being handled.

13 DR. NOGUCHI: I think the point is well

14 taken. However, I think that the reason that we

15 are actually having this extensive discussion is

16 that the absence of evidence is not evidence of

17 absence. The fact that this is to be a sentinel-only

18 function is, in fact, the question.

19 We agree that the scientific basis for

20 this is to have it work only in that which is

21 affected, but what biology teaches us, and, from

22 GTI, you know this as well, what we believe today

23 is not what we know to be true tomorrow and it is

24 very likely not to be what will be approved.

25 So we need to be able to go through this

> 215 1 in an open a fashion as we possibly can. I think

2 the fact that we have spent such a grueling set of

3 time here really going over all the pluses and

4 minuses and the pros and cons really does

5 illustrate the intense interest in being able to

6 develop something that does have this type of

7 specificity.

8 As is often said, now we are getting down

9 to the details and both god and the devil are

10 always in that. So your point it absolutely well

11 taken, but we need to be assured that, in fact, the

12 sentinel remains specific for only one particular

13 cell.

14 DR. DELPH: I have a question. If

15 mobilization occurs into other tissues, is it

16 necessary to look at viral reservoirs and

17 characterize HIV species there. I am asking that

18 because the species that you find in viral

19 reservoirs may be very different from what you find

20 in blood.

21 DR. SALOMON: I think that is fair. One

22 of the difficulties, of course, is sampling and

23 characterizing viral reservoirs. So, again, I

24 would defer to my colleagues who do HIV as their

25 primary business.

> 216 1 Dr. Zaia?

2 DR. ZAIA: I would suggest that that is a

3 question for the phase II study.

4 DR. ALLAN: You could actually look at

5 macrophages and see if you get mobilization of the

6 macrophages and, if you do, what effect that might

7 have on function. So I think there are some things

8 you can do even within the blood compartment.

9 DR. DELPH: You can look at semen.

10 DR. SALOMON: Yes. You can. That is a

11 point. I think we should let it stand and I think

12 Dr. Zaia\'s comment about whether that should come

13 in in a phase I or a phase II is also well taken.

14 I think we are done in the sense that,

15 what I was going to say is Question 4, we have

16 really covered. I think we all agree on the basic

17 principles here. I think we have also articulated

18 for you up to the edge how far we can go without it

19 getting gray.

20 Yes? Dr. Zaia?

21 DR. ZAIA: There is one aspect, though, of

22 Question 4 that I think needs to be discussed and

23 that is the safety assessment is linked to the

24 rules of escalation. The rules say that we will

25 look at 28 days and look for toxicity. If the

> 217 1 toxicity is not there, we will escalate.

2 I just have a problem with that. I don\'t

3 think 28 days convinces me that you can see some of

4 the things that we have been talking about. But,

5 more importantly, I don\'t even think this study

6 needs to be a dose escalation, unless you want to

7 dose escalate the transducing agent. But that is

8 not the research agent, or the investigational

9 agent.

10 So I would really encourage the sponsors

11 to rethink the design of this study because they

12 are not asking how to get T-cells expanded and

13 infused safely. I mean, there are several other

14 studies I am sure the FDA knows more about than I

15 do where they are infusing cells around the same

16 level, 3 times 10

10, and we all know how safe that

17 is.

18 I don\'t think there is anything about

19 these cells that make them more dangerous in

20 regards to infusion-related toxicity. What is

21 different about these cells is these other

22 toxicities that we are talking about which are

23 more, I guess, virological or less easy to

24 elucidate within the normal observation periods

25 that we look at in the standard trial.

> 218 1 So I think that is a critical question

2 that the sponsor has to contend with, I think, is

3 how can they use this dose-escalation rule for the

4 kinds of toxicities that we are most concerned

5 with.

6 DR. SALOMON: Actually, that is perfect.

7 What I was going to say is I think we are kind of

8 done with the questions. What I would like to do

9 now, in coming to a close, is for people to weigh

10 in with other things here because I have got a

11 list, too, and I wonder if some of you don\'t.

12 We have addressed the questions. I would

13 say that there were a couple of things that

14 bothered me. That was one. I don\'t think it is

15 very clear to me whether the dose escalation\--and

16 the way I get it is the dose escalation is in

17 different patients. Part of me is going, well, why

18 not\--you could do a dose escalation in the same

19 patient.

20 Certainly, I would echo Dr. Zaia\'s

21 comments that a 28-day\--the things that are really

22 safety concerns to me; 28 days? No; I don\'t think

23 so. I would say more like three to six months.

24 DR. CHAMPLIN: Although these are not

25 dose-related issues so much.

> 219 1 DR. SALOMON: No.

2 DR. CHAMPLIN: So the issue of infusion

3 toxicity is sort of all you are looking for

4 realistically in the short term toxicity analysis

5 and the emergence of resistant virus, of course,

6 over a longer period of time and is probably going

7 to be dose independent. So you could sort of

8 minimize the importance of the dose escalation

9 because that is just to establish the tolerance to

10 the infusions. But, really, you are looking at the

11 sort of long-term phase-I aspects of the biology

12 of the whole approach.

13 DR. SALOMON: That is good point. But,

14 then, I might say, that you don\'t need 28 days to

15 find out. I guess I am a little confused on the

16 premises here. There is toxicity from infusion.

17 You know that in two or three days.

18 DR. HIGH: But if what we are talking

19 about is doing better characterization of what is

20 happening to HIV variants, you would like to at

21 least wait 28 days, probably, before you even drew

22 that blood. Then, once you have got it, it is

23 going to take a little while to characterize it.

24 So you might not want to enroll the second

25 patient before you have analyzed at least, at 28

> 220 1 days, what is going on with the first one.

2 DR. SALOMON: Yes. I guess what I am

3 saying is I agree with you, Dr. High, and that is

4 the conundrum. I think the way Dr. Zaia and I were

5 coming at it was 28 days is not long enough to see

6 the safety issues that we are concerned. But Dr.

7 Champlin said, but the safety issues concerned

8 with the dose escalation is simply a dose effect of

9 the infusion.

10 My response to that is, okay, good point,

11 but that is three to five days. We have got to be

12 consistent here, logically. If the concern is an

13 evolution of the viral species present, then I

14 think a couple of months is probably appropriate.

15 But that should be discussed.

16 DR. SAUSVILLE: Recognizing that that gets

17 into the issue of why the dose escalation. It does

18 lead to the protocol-design issue as to what are

19 you going to consider your endpoint here.

20 Certainly, matters related to the infusion, while

21 we don\'t think they are going to be a big issue,

22 they are formally something that does have to be

23 captured and scoped out.

24 Certainly, if we are looking at the

25 incidence of variants mobilization et cetera, a

> 221 1 dose response relationship to input would be

2 certainly of interest to also capture. So I guess

3 I sort of come down in the middle, but I agree 28

4 days is probably a little soon. I do believe there

5 is a role for dose escalation here and I do believe

6 that there is a role for longer periods of

7 observation before doing the dose escalation.

8 Let\'s put it that way.

9 DR. SALOMON: Dr. High, did that capture

10 kind of where you were going?

11 DR. HIGH: Yes.

12 DR. SALOMON: Dr. Allan and then Dr.

13 Torbett.

14 DR. ALLAN: Safety is also\--you are hoping

15 to get some efficacy with this or you wouldn\'t be

16 doing it. But you know there is always the

17 possibility that it is not going to have a good

18 outcome. That outcome might not happen for six

19 months or longer.

20 So my question, then, is do you need to

21 rush doing dose escalation anyway. What is the

22 need to have to do it after 28 days. If this is

23 the first patient or the first three patients who

24 get this, do we want to sit back and go, okay, what

25 is going to happen, let\'s see what happens before

> 222 1 we do anymore and let\'s see if we get a\--not to

2 look at efficacy but to look at is there an outside

3 possibility that it may not be a good outcome.

4 So I would say you want to wait.

5 DR. CHAMPLIN: You need well defined early

6 stopping criteria, what events would make you not

7 enter another patient. You need to define exactly

8 what those things are and then work out your

9 accrual of patients accordingly. So, obviously,

10 the faster you do the initial process that allows

11 additional accrual, the faster they can complete

12 the trial.

13 If you ask for a year follow up for every

14 patient before you enter another one, it is going

15 to take forever. So there has to be some sort of

16 middle ground where you have an accrual of a

17 reasonable number of patients that you can observe

18 and then make it a go/no-go kind of determination

19 should you continue the trial.

20 DR. SALOMON: Dr. Torbett and then Dr.

21 Sausville.

22 DR. TORBETT: I guess I have a little bit

23 more fundamental question. In some of the earlier

24 data, it was shown that, depending on the number of

25 integrins per cell\--that is, the number of hits per

> 223 1 cell\--you can or can\'t select quickly the variants

2 coming out. Am I correct?

3 DR. DROPULIC: Can you repeat that?

4 DR. TORBETT: The question is, I think you

5 showed us originally that, if you had one integrin

6 per cell, or an MOI of 10 or 5, 10 and 20, the

7 breakthrough came slower or faster.

8 DR. DROPULIC: Right. So, as you dose

9 down the vector\--I am not so sure faster.

10 DR. TORBETT: I am sorry about that word.

11 DR. DROPULIC: We saw breakthrough at a

12 transduction MOI of 5 and a challenge MOI of 0.1 in

13 that Sup-T1-cell experiment.

14 DR. TORBETT: The reason I am bringing

15 this up, it comes to the whole question\--and it

16 wasn\'t clear from the document that I had what the

17 MOI is per number of cells. But depending on the

18 number MOI per cells, even if you can get 100

19 percent and you increase the number of integrins,

20 then the time period to look for variants coming

21 out would vary. Is that logical?

22 DR. DROPULIC: At the optimally transduced

23 cells, we haven\'t see any.

24 DR. TORBETT: Maybe it is the way I am\--it

25 is late in the day and I haven\'t had any more

> 224 1 sugar, so maybe that is part of the problem here.

2 But if you had one integrin per cell and, at a

3 certain time, you had a turnover virus, say you had

4 more integrins, less, and you had 10 integrins per

5 cell\--I am just making these numbers up\--and you

6 had pretty good control but it took a longer time,

7 the length of time that you would want to look

8 could very well be longer and the time that you

9 would want to sample each time would vary.

10 DR. SLEPUSHKIN: I just would like to

11 answer it. In any case, we have specification for

12 copy number per cell. That won\'t change during

13 escalation. So there will be about the same amount

14 of integrins on all steps of escalation. So you

15 are just changing the amount of cell injury.

16 DR. TORBETT: So the MOI is going to be

17 constant.

18 DR. SLEPUSHKIN: Yes.

19 DR. TORBETT: Just, out of curiosity, what

20 is it?

21 DR. SLEPUSHKIN: I probably will be about

22 200 as it was in the animal or the clinical

23 experiment.

24 DR. TORBETT: So it is going to be an MOI

25 of 200.

> 225 1 DR. DROPULIC: That is not copy number.

2 DR. SLEPUSHKIN: Not copy number. It is

3 MOI; yes.

4 DR. TORBETT: I understand, per X number

5 of cells. Okay.

6 DR. SLEPUSHKIN: Copy number

7 specifications; copy number per cell should be

8 between 1 and 10. And, in the clinical animal

9 experiments, it was 6.

10 DR. TORBETT: So is there any data

11 suggesting\--well, I guess you can\'t control with

12 the variability. Maybe that is something that

13 shouldn\'t be brought up here.

14 DR. SALOMON: Unless you guys want me to,

15 I am not going to try and come to consensus on each

16 of these points. I think we have done our job.

17 But I would like to, in this kind of concluding

18 process, have everyone share with you, and with the

19 FDA staff, additional things that were not in your

20 question list.

21 So I believe my job as chair has suddenly

22 now to just making sure that everybody gets a

23 comment and everyone gets heard. So unless someone

24 is insisting that I come to consensus, I am not

25 going to.

> 226 1 Dr. Torbett?

2 DR. TORBETT: This is a first trial and I

3 would like to commend the company for going

4 forward. It was a pretty brave step and I think,

5 without too many boundaries, they done a fairly

6 admirable job of starting up the stairway.

7 DR. DELPH: I would like to echo that. I

8 would also like to express some concern about the

9 patient population that has been selected for this

10 trial. I really think a CD4 count of 600 as an

11 upper limit is way to high. right now, even the

12 DHHS guidelines for treatment are really not coming

13 out in favor of recommending treatment until CD4

14 counts drop below 350.

15 In the European guidelines, they are even

16 dropping them as low as 200. Generally speaking

17 and, of course, there are exceptions, patients with

18 CD4 counts of over 200 are not that ill. So I

19 would certainly\--I understand the difficulties that

20 the company says that they may have in getting T-cells from

21 patients who have CD4 counts of under

22 200, but I think we need to also need to balance

23 that with the need for the patient population and

24 what is safe for them.

25 The other thing, and I don\'t know what

> 227 1 your actual inclusion criteria are for \"failing or

2 discontinued HAART therapy.\" But I think that

3 needs to be far more clearly defined and I would

4 recommend that you define it in terms of the number

5 of drug classes that someone is resistant to. I

6 would recommend that someone be resistant to at

7 least two of the drug classes currently on the

8 market and possibly even three, but at least two

9 protease inhibitors.

10 You are going to look at people who really

11 have few, if any, drug options left. So those are

12 my major comments.

13 DR. DROPULIC: I appreciate those comments

14 and I just want to assure you that we want to work

15 with the FDA to finalize these patient criteria.

16 It is not set in stone now and your comments are

17 well taken.

18 DR. DELPH: I also suggest that you work

19 with the HIV community on that.

20 DR. DROPULIC: Okay. Yes. Thank you.

21 Yes.

22 DR. LEVINE: Let me address the issue of

23 the cell number, if I could. Once you get below

24 200 cells, there are increasing difficulties with

25 the transduction and the culture. I think if we

> 228 1 could make an analogy about immunologic treatment

2 in a different setting, with Cliff Lane\'s studies,

3 where he has given IL2 to the patients, there was a

4 real dichotomy with the effectiveness of raising

5 the CD4 count with patients who came in with a

6 count above 200 versus those that came in with a

7 count below 200; that is, there was not an

8 effective of the IL2 once you had patients coming

9 in below 200.

10 So I think, at that point, the immune

11 system has suffered what may be irreparable injury

12 and would make any trial safety and feasibility

13 more difficult.

14 DR. NOGUCHI: The question is not simply

15 one of difficulty. I will point out that

16 interleukin 2 has been actually approved for the

17 treatment of renal-cell carcinoma so that the

18 parameters of experience are far different than

19 what we are talking about here. But we do

20 understand the technical aspects of this particular

21 approach which is trying to take cells and expand

22 them. There very well may be areas that we will

23 need to really fine tune.

24 DR. CHAMPLIN: They are few and far

25 between, but the availability of identical twins

> 229 1 sometimes can give you sort of an opportunity to

2 treat a seriously ill patient who now has a normal

3 twin that you can get unlimited numbers of

4 leukocytes from. So, for proof of principle kinds

5 of things, that offers, sometimes, unique

6 opportunities.

7 DR. SALOMON: Dr. Golding.

8 DR. GOLDING: I just to bring a little

9 perspective from the Office of Vaccines. Our group

10 is responsible for a lot of HIV vaccine including

11 therapeutic vaccines. Most of the therapeutic

12 vaccines, as you know, are done in the context of

13 antiviral therapies. So what are sort of

14 guidelines that we are using in terms of the safety

15 monitoring of the patient.

16 Of course, just like in this case, when an

17 outcome can be either no effect or worse disease

18 progression, is the same thing we have to deal with

19 when we deal with when you deal with therapeutic

20 vaccine, that even though you gave something that

21 is supposed to help to control viremia, you

22 actually have a negative effect.

23 I think that can be seen relatively

24 quickly by measuring viral-load changes in the

25 patient. So I think it is important, once the

> 230 1 cells have been transduced into the individuals is

2 to really take multiple measurements of viral loads

3 over this first period, whether it is one month or

4 six weeks, and to actually have a sense of the

5 slope of viral load measurement as well, of course,

6 of CD4 counts because you are talking about all

7 kinds of toxicities.

8 There is the infusion toxicity. There is

9 potential emergence of more fit viruses that, down

10 the road, can dominate the patients. But I think

11 if you have a really adverse reaction, you have

12 something really bad in your product, what you are

13 going to see is much more rapid increase in viral

14 load.

15 For that, you have to have patients that

16 are not in the millions of viral loads to start

17 with. You have to have a window that would really

18 allow you to see some really enhanced increased.

19 DR. SALOMON: Right. The logic to what

20 you have said, Dr. Golding, though, is that you are

21 probably going to want to choose a patient

22 population that is not all that far gone and wildly

23 rising HIV viral loads on the day that treatment is

24 initiated.

25 DR. GOLDING: I wouldn\'t say total failure

> 231 1 of anti-HAART with a million copies because I

2 really don\'t think you will be able to see this

3 type of adverse reaction that you want to use to

4 allow you to go to the next patient.

5 DR. SALOMON: So we have to put that in

6 the context with what Dr. Delph shared with us in

7 terms of the patient selection.

8 I had some other concerns I wanted to put

9 on the table. One would be, in terms of the CD3,

10 CD28, beads, you have less than 100 beads per 3

11 million cells which means you could literally be

12 putting in thousands of beads into someone. I do

13 not buy that one at all. I think you would cause a

14 pulmonary embolus. But maybe you have experience

15 to answer that.

16 DR. LEVINE: That number was developed in

17 consultation with the FDA. There have been

18 toxicity studies done by what used to be Baxter

19 Immunotherapy infusing a large number of beads into

20 rats looking for that sort of thing. There were no

21 toxic effects at levels of beads very much higher

22 than what we are infusing.

23 We are also very much below that number of

24 100 beads per 3 million cells. What we are able to

25 achieve currently is a depletion of greater than

> 232 1 six logs to what we start with. So if we were to

2 stimulate 50 million cells, 100 million cells, a

3 billion cells, and have, say, a 3-bead-to-cell

4 ratio of, let\'s say, up to 2 to 3 billion, we would

5 anticipate easily being able to deplete six logs of

6 those beads.

7 DR. SALOMON: Another issue; I think I

8 understand why you want to activate these cells is

9 because you think you are going to have very low

10 numbers and you want to get up to these higher

11 numbers of 10 10 and

higher in your infusion.

12 Pheresis, even\--I am not getting into the

13 state of the T-cells when you are really getting

14 down to 150, 200 CD4 cells. Your comments stand on

15 that. But I don\'t understand that. I don\'t

16 understand why everyone wants to ignore the biology

17 of T-cells and activate them and culture them in

18 nonphysiological concentrations of interleukin 2,

19 inject them back in the patients.

20 I mean, the whole purpose of a lentiviral

21 vector is it is incorporated into non-replicating

22 cells. So my murine Moloney leukemia virus

23 backbone, I have to activate my T-cells and I am

24 not happy about it. But you don\'t. For studies

25 that you are trying to maintain a normal immune

> 233 1 repertoire yet you are doing these things that I

2 don\'t think there is any data here demonstrating

3 what the immune repertoire is that is left in these

4 cells after you do this. I just don\'t understand

5 why you want you to go there.

6 DR. LEVINE: I can tell you that the

7 maintenance of the repertoire after 60 days in

8 culture is published in 1996 in the Journal of

9 Immunology showing that we do maintain the entire

10 repertoire.

11 DR. SALOMON: Defining repertoire as the

12 CD4/CD8 ratio?

13 DR. LEVINE: As 24V beta families as

14 analyzed by the CDR3.

15 DR. SALOMON: Okay. I guess, again, this

16 is not a comment coming from the chair. We are not

17 going to try and get consensus, so just a comment

18 to you. I just don\'t believe that these assays

19 maintain the normal T-cell phenotype. To go into

20 these initial studies at the early low-dose effects

21 where you don\'t have to activate and you don\'t have

22 to treat with interleukin 2 just seems to me you

23 are adding another variable to prepare yourself for

24 a later thing based on an assumption that you have

25 maintained your repertoire.

> 234 1 DR. LEVINE: I would say that we have

2 experience with these T-cell infusions, with CD4-cell

3 infusions and with bulk T-cell infusions in

4 HIV patients and in cancer patients. We,

5 ourselves, have done 51 infusions in HIV patients.

6 CellGenesis has done with CD3-28-stimulated cells I

7 am guessing 60 or 80 infusions and, in cancer

8 patients, even more.

9 So I think we just have to agree to

10 disagree.

11 DR. SALOMON: Right. That\'s fine. That

12 is perfectly fine.

13 MS. KNOWLES: I would like to take Dr.

14 Delph\'s comments one step further and caution\--in

15 terms of her comments about the other

16 pharmacological agents in the research pipeline

17 because she is right. There are more things coming

18 down the pipeline. As such, I would like to

19 caution the sponsor to not put the message forward

20 that your proposed clinical trial is going to be a

21 last-ditch treatment effort for people with HIV

22 because it is one potential of the armamentarium.

23 DR. DELPH: I have another question

24 because it wasn\'t clear from what you have given us

25 about the protocol. Are these subjects going to be

> 235 1 on antiretrovirals or not?

2 DR. DROPULIC: They will be failing HAART

3 and, if they are not on a therapy, then they are

4 not on. But we are not going to require them to

5 come off therapy. We think that that is unethical.

6 So, if they are on one or two drugs and they are

7 failing therapy, then they can enroll in the study.

8 That is how we have defined it so far but, again,

9 we can negotiate this with the FDA to see how we

10 approach this. That is how we have characterized

11 it presently.

12 MS. KNOWLES: If they go on study drugs,

13 are you going to pay for them? Who is going to pay

14 for the drugs?

15 DR. DROPULIC: I hadn\'t thought of that.

16 We will think about that one.

17 DR. TORBETT: You propose in your set of

18 criteria that you follow these individuals for

19 life. Who would pay for those, assuming that the

20 company had problems?

21 DR. DROPULIC: We plan to be around a long

22 time.

23 DR. TORBETT: In any event that you don\'t,

24 is there going to be\--I am just curious. Would you

25 take out insurance to make sure that that is done?

> 236 1 This is a serious consideration. It has been

2 discussed before. I am just curious.

3 DR. DROPULIC: If that is a requirement,

4 we can do that.

5 DR. TORBETT: I just wanted your thoughts

6 on that.

7 DR. DROPULIC: Haven\'t thought about it,

8 quite frankly, because we expect to be around a

9 long time.

10 DR. SALOMON: I think one of the comments

11 I have, and this is not specific to VIRxSYS, but

12 that I think the focus of these discussions in the

13 last two days have, and perhaps very appropriately,

14 focused on the biggest risk, the low-hanging fruit,

15 if you will, of the replication-competent

16 lentivirus and shuffling of the DNA species, et

17 cetera, which is fine.

18 I guess I still feel like, as part of this

19 sort of last number of comments here\--it continues

20 to bother me what is happening also to the trans

21 gene that is being delivered, the payload, if you

22 will. That, to me, is as much a part of the

23 product as the issues of safety.

24 Here, you get close to this gray area of

25 \"okay.\" But remember this is phase I and we want

> 237 1 safety not efficacy. But, as Dr. Noguchi said,

2 when there is significant risk and unclear benefit,

3 it is very hard to construct risk/benefit ratios

4 and I think the rules change.

5 We have been through that with

6 xenotransplantation. So, going back to that, I

7 just think that\--one of my personal comments here

8 is that, at some point, we need to also consider

9 how we are characterizing the quality and the

10 integrity of the payload through all these changes

11 because everything we have talked about, up to now,

12 has not really dealt with that.

13 DR. NOGUCHI: I would actually disagree.

14 I think there has been a lot of very good

15 discussion on that, and you note that Dr. Wilson

16 and Takefman are diligently noting these things.

17 It is actually central to some of the evaluation

18 because it does appear as though the payload may

19 actually push the virus to recombine and do

20 different mutations, deletions and so forth which

21 clearly is an activity we need to be monitoring

22 even from just the safety aspect.

23 So I think we have actually gotten very

24 good advice on that.

25 DR. CORNETTA: This is Ken Cornetta. Can

> 238 1 I make a comment?

2 DR. SALOMON: Yes; go ahead.

3 DR. CORNETTA: I guess just maybe to pick

4 up a little bit of what you were saying about the

5 T-cell function after transduction and the

6 stimulation process. That bothered me, too, as I

7 was reading through. While a lot of cancer

8 patients have gotten T-cells that have been

9 manipulated and given back, our experience,

10 although limited, has been that those cells don\'t

11 function very well, at least after allogeneic

12 transplantation.

13 So, in the process, their ability to do

14 what the T-cell initially was designed to do seems

15 to be lacking. So one of the real advantages I saw

16 to lentiviruses was that you might be able to avoid

17 this in vitro stimulation. It bothered me a little

18 bit that there seemed to be a fair amount of

19 stimulation that would occur in these cells and

20 that, again, the concern that their ability to

21 function, once they got back into the patient,

22 would be not as we hoped.

23 DR. SALOMON: Do you want to comment on

24 that?

25 DR. LEVINE: Yes. We have several lines

> 239 1 of evidence, and I could spend an hour talking

2 about them, that, by stimulating by CD3 and 28, we

3 reverse defects of the T-cells as they are removed

4 from both cancer patients and HIV patients.

5 We have recently completed at phase I

6 study in lymphoma where we have looked at

7 intracellular cytokine response following TMA and

8 antimycin stimulation at Day 0 and at Day 12 of in

9 vitro culture and showed that we can reverse what

10 is a substantially diminished response at Day 0

11 that is increased at Day 12.

12 In the HIV setting, we have looked at

13 response to allogeneic mixed lymphocyte reaction

14 and show that we can increase in the study

15 population that we did several years ago\--we

16 increased that allo-MLR response.

17 With respect to a CCR5 population that is

18 different from the population with this study, we

19 looked at CCR5 in vivo expressed on CD4 patients,

20 both before and after infusions, dose-escalating

21 infusions of 3, 10 and 30 times 109 and could show,

22 specifically on the CD4 cells, that we have reduced

23 the CCR5 levels.

24 Also, in vitro, we have looked at cytokine

25 production in the HIV patients both before and

> 240 1 after stimulation and it is very much higher. So I

2 think there is a wide spectrum of T-cell functions

3 that are improved following CD3-28 stimulation.

4 I think the point also is that it may be

5 nonphysiologic but that might be better. So, by

6 not stimulating CHLA4, by stimulating CD28

7 specifically, you are upregulating BCL2 protecting

8 against apoptosis. So there really is a wide

9 spectrum of things that are improved following CD28

10 stimulation.

11 DR. SAUSVILLE: I guess I would offer

12 that, certainly if this is successful to the extent

13 that we work through some of these issues, or the

14 sponsor works through these issues and gets into

15 the clinic, one can imagine many different flavors

16 of mix and match. That would be the subject for

17 future clinical investigations.

18 I certainly would agree that, being vested

19 in this particular way of doing it, this is one way

20 to do. And I would leave it at that.

21 DR. SALOMON: Fine. Again, as I said, we

22 had agreed to disagree a bit ago and nothing has

23 changed.

24 Any other comments or can we come to a

25 close?

> 241 1 DR. GAYLOR: I haven\'t said anything for

2 the last two days and feel compelled to earn my way

3 here somewhat. But, as a statistician, I need data

4 to work with. There is obviously a paucity of data

5 here for understandable reasons. It is a brand-new

6 area. So my role, I think, has changed from being

7 one that could have any scientific input to really

8 how does the man on the street feel about this,

9 somebody that not really been terribly involved in

10 this.

11 I feel very comfortable with the

12 discussions I have heard. There has been a lot of

13 thought. A lot of questions have been raised.

14 There has been a lot of good discussion and I

15 guess, again, it is a theoretical comfort because I

16 don\'t have a lot of data to look at.

17 But I think the committee and the people

18 involved, the research that has been done, makes me

19 feel like everything is being done that could be

20 done at this point.

21 DR. SALOMON: Again, anyone else have any

22 final comments that they would like to make?

23 DR. NOGUCHI: On behalf of the FDA, I hope

24 I could take this liberty to really thank the

25 committee, VIRxSYS especially, for being so bold as

> 242 1 to come here and face the stings and arrows, I

2 guess, as best we can put it.

3 The committee, especially, for this round,

4 has been exceptional both in its civility as well

5 as rigor in pursuing obvious and not-so-obvious

6 questions. I would especially like to thank our

7 chair for keeping us on keel and getting us through

8 this very difficult set of questions.

9 I think that, on our side, we can say

10 that, with this advice, we are confident we will be

11 able to make the appropriate decisions to move the

12 entire field forward and we thank you for that.

13 DR. SALOMON: Thank you, Phil. Then, as

14 chair, let me speak for everyone. I think that

15 VIRxSYS, you guys did a really good job. I have

16 always said, going back a couple of years now, that

17 this committee functions the best when a sponsor

18 can step up and provide us a real protocol to look

19 at. That is when we can really deal with the kinds

20 of specifics that allow the fields to move forward.

21 You guys have done that and I respect

22 that. I also thank the sponsors who presented

23 yesterday for doing the exact same thing in a

24 situation that they even have more to lose, if you

25 will, because we were taking them on on some of

> 243 1 their things that they hadn\'t even brought quite as

2 far as you guys have. Again, I thank them.

3 I think everyone from the committee for

4 two to three days, depending on which group you are

5 in, hanging in there with us. To Rosanna Harvey

6 and Gail Depolito and the rest of the FDA staff who

7 worked so hard to put all this together, to get us

8 here, to take us to dinner, to move us around in

9 hotel rooms, and to the audiovisual staff and

10 everyone else involved.

11 Thank you very much. Everyone travel

12 safe.

13 \[Whereupon, at 1:50 p.m., the meeting was

14 adjourned.\]

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