Advances in Treatment of Hemophilia and Targeting dsRNA in Tumors to Overcome Immunotherapy Resistance

Transcript

• 00:00The hearts go out to his wife.
• 00:03Doctor Kellie Martin is two
• 00:05children tests and Jacob and just
• 00:08take a moment just to silence
• 00:10just to recognize Tony's legacy.
• 00:21Well, thank you, so let's now turn
• 00:24to our first of two great speakers.
• 00:28We were very fortunate this year
• 00:31to recruit Doctor Jeffrey Ishizuka.
• 00:34Jeff is an assistant professor of medicine.
• 00:38And Jeff's work.
• 00:39Previously at Harvard was was
• 00:42focused on the biology of T cells.
• 00:44Discovering knew better understanding
• 00:46of that biology and and and
• 00:49ultimately leveraging that science
• 00:50to what is likely to be the next
• 00:54generation of amino therapies.
• 00:55And we're really very fortunate to have
• 00:58Jeff as one of our physician scientists in
• 01:01the center of molecular Italian Colosseum,
• 01:04member of the Melanoma program,
• 01:07and physician scientists in general at.
• 01:09At Yale and Smile also,
• 01:11Jeff really excited to hear about your work.
• 01:14Turn it over to you.
• 01:17Thank
• 01:17you so much Charlie, really appreciate
• 01:20it and let me just project my slides.
• 01:23How there we go?
• 01:25Yes, thank you so much and thank you
• 01:28for the opportunity to speak today.
• 01:30Today I'm going to be talking to you
• 01:32about some of the work we've done.
• 01:34Targeting double stranded RNA in
• 01:37order to overcome immunotherapy
• 01:38resistance and also update on
• 01:40other ongoing projects in the lab.
• 01:42This is my disclosure slide.
• 01:45I wanted to begin with the overall survival
• 01:48curves from the Checkmate 067 trial,
• 01:51which is likely familiar to this audience.
• 01:54These curves represent survival in
• 01:56advanced Melanoma by patients treated
• 01:58with immune checkpoint blockade.
• 01:59In this case,
• 02:00with antibodies targeting PD 1C,
• 02:02TL A4 or the combination.
• 02:05I wanted to start here because
• 02:06Melanoma has been something of
• 02:08a touchstone for the use of
• 02:10checkpoint blockade in solid tumors.
• 02:11First indication approved and remains
• 02:13one of the indications in which immune
• 02:15checkpoint blockade is most effective
• 02:16in these data are outstanding,
• 02:18particularly when compared with the pre
• 02:20immunotherapy standard of Care Dakar Busine,
• 02:22which had an overall survival
• 02:23of five to 10% at five years.
• 02:26However,
• 02:26even in this disease,
• 02:28large proportion of patients
• 02:29don't experience durable benefit.
• 02:31The situation which is which is
• 02:33actually more challenging in other
• 02:35diseases where responses are less good.
• 02:38And this is really the focus of
• 02:39our work to improve responses
• 02:41in this disease and in others.
• 02:46Certainly, however, if you check,
• 02:48my blockade is rapidly reshaping the
• 02:49landscape of cancer care across indications.
• 02:51I was preparing for this talk and I
• 02:53had to go through and update this slide
• 02:56because indications have nearly doubled
• 02:58since its original publication by
• 02:59Tony Ribas and Jed will Chuck in 2018.
• 03:01Although many of us have followed
• 03:03this emerging data very closely,
• 03:05I have to admit that it gave me pause to
• 03:08consider the pace of change in this field.
• 03:11The advancement of PD one access approvals
• 03:14continues through lymphomas and solid
• 03:17tumors of desperate tissue origins.
• 03:19Combination approaches have also
• 03:20proliferated, including approaches,
• 03:22approvals in music, leoma,
• 03:24breast cancer, and others.
• 03:26Successful combinations include
• 03:27combinations of checkpoint inhibitors
• 03:29with other checkpoint inhibitors,
• 03:31chemotherapies and touristing
• 03:32kinese inhibitors,
• 03:33and notably many here at Yale,
• 03:36have played critical roles in this dance.
• 03:42Still, for all the advances,
• 03:43there have been a lot of failures and
• 03:46there remain a lot of ongoing challenges.
• 03:48For most, many patients don't respond,
• 03:50indeed, considered across all indications,
• 03:52most patients don't respond in a few
• 03:54of the response rates listed are
• 03:56really based on earlier trials that
• 03:58likely overestimated response rates.
• 04:00Many of them also include
• 04:02biomarker cutpoints,
• 04:03PDL 1 positive ITI and this sort of thing.
• 04:07And in my mind there are really a couple
• 04:09of big areas in which we can improve.
• 04:111st for all of the new indications,
• 04:14few combinations involving novel
• 04:17targets have been approved.
• 04:192nd, we have a limited mechanistic
• 04:22understanding of how these agents work.
• 04:25Accordingly,
• 04:25the biomarkers that we used to deploy
• 04:27them lack sensitivity and specificity,
• 04:29and there's not a great way to rationally
• 04:32prioritize combinations with anti PD one.
• 04:36So it's worth considering for a moment what
• 04:37we've learned about response and resistance,
• 04:39not so much in the interest of
• 04:41an extensive overview for which
• 04:42we wouldn't have time today,
• 04:43but in terms of the pathways that have
• 04:46given the strongest clinical signals to date.
• 04:48The data shown here are from the study
• 04:51by Merck of over 300 different patients
• 04:53across 22 different tumor tissue types.
• 04:56These figures show responses.
• 04:59Non response defined as CR
• 05:01or PR versus no CR PR.
• 05:04When graphed with tumor mutational burden
• 05:06on the Y axis and a gene expression profile
• 05:09representing tumor microenvironment,
• 05:11inflammation kind of T cell
• 05:12inflammation on the X axis.
• 05:14The genes in this profile are listed in the
• 05:17upper right here and notably include PDL,
• 05:20one among them as well as several MHC related
• 05:23genes and kind of T cell related genes.
• 05:28Tumor mutational burden, as you know,
• 05:30is often used as a surrogate for
• 05:32too many antigens and the gene
• 05:34expression profile really points
• 05:35to information of the tumor,
• 05:37micro environment and the authors make
• 05:39two points that are important here.
• 05:41First, that these are two of the
• 05:43strongest predictors they could find.
• 05:45Reviewing one of the largest
• 05:46and most comprehensive datasets
• 05:48that existed at the time.
• 05:49Really, it's telling us in second
• 05:51that they appear to predict response
• 05:52independently of one another.
• 05:54That is to say that although the
• 05:56best responses are in that kind of.
• 05:58Upper right quadrant that you actually get
• 06:01a good number of responses in a T cell.
• 06:05Inflamed only micro environment,
• 06:06or in TMB only TB high only tumors.
• 06:12For the sake of time today,
• 06:13I won't spend a lot of time
• 06:15on TMB or antigen load,
• 06:17so it's obviously an important consideration.
• 06:18Instead, I'm just going to talk about
• 06:20tumor microenvironment information,
• 06:21which is really the focus of our lab.
• 06:23Aside from the work by the Merck
• 06:25Group A number of lines of evidence
• 06:27have established inadequate tumor
• 06:28microenvironment information.
• 06:29As one of the most prominent mechanisms
• 06:31of resistance to me, no therapy.
• 06:33Most dramatically,
• 06:34this occurs in immune desert type tumors,
• 06:37which entirely lack T cell infiltrate,
• 06:39as depicted here. However,
• 06:41it can also occur in a different phenotype.
• 06:44The so-called immune excluded
• 06:45tumors which have anti tumor immune
• 06:47cells at the site of the tumor,
• 06:49although they are excluded
• 06:50from the tumor core,
• 06:51either by physical barriers
• 06:53or by immune signaling.
• 06:55Finally,
• 06:55we believe that there is the T cell
• 06:57inflamed type of tumor that have
• 06:59diffuse infiltration of T cells
• 07:01that tend to be PD L1 positive,
• 07:03and these are the ones that we
• 07:06believe respond best to immunotherapy.
• 07:09To date,
• 07:09there's been progress in identifying
• 07:11therapeutic strategies to enhance this
• 07:13tumor microenvironment information,
• 07:14many of which involve either real
• 07:16or simulated infection of the tumor
• 07:18to trigger anti tumor immunity,
• 07:20and I think about them in kind
• 07:22of two big buckets.
• 07:24The first is the provision of
• 07:26exogenous sources that mimic
• 07:28nucleic acid ligands to tumors.
• 07:30This includes sting agonist,
• 07:31MDA 5 or rig I agonist,
• 07:33double stranded RNA sensing
• 07:35pathways and uncle lytic viruses.
• 07:37The other is the induction of endogenous
• 07:40sources of nucleic acid ligands,
• 07:43primarily endogenous retroviruses,
• 07:44although others have been published recently,
• 07:47alualu repeats in humans.
• 07:51And examples of this include a
• 07:53deciding in CDK 46 inhibitors.
• 07:57So my interest in turning these cold
• 08:00microenvironments hot and kind of
• 08:02providing these logins to tuners really
• 08:04developed out of work in the Canings
• 08:07lab was finishing my postdoctoral work
• 08:09there and through the type of experiment
• 08:11that I'm showing here on the left,
• 08:14you have kind of a transplantable tumor
• 08:16cell line, something like a B16 Melanoma,
• 08:19and the way the experiment works is to,
• 08:22in fact, that cell line with a
• 08:24library of CRISPR CAS 9 guides
• 08:26that knockout thousands of.
• 08:28Immunologically relevant genes.
• 08:29In the genome and then to kind of
• 08:33select those guides until you have
• 08:35a pool of knockout tumor cell lines
• 08:38that is then implanted into mice
• 08:40under increasing immune selective
• 08:42pressure from extremely immunodeficient
• 08:44mice that lack T cells to mice with
• 08:46an intact immune cell system.
• 08:482 mice treated with immunotherapy.
• 08:50In this case,
• 08:51the irradiated GM CSF secreting
• 08:53whole tumor cell vaccine GBX,
• 08:55plus anti PD one kind of strong
• 08:58immunotherapy treatment regiment.
• 08:59Would grow these tumors for about
• 09:012 weeks and then remove them.
• 09:04Harvested tumors and sequence the sequence.
• 09:06The barcodes sequence the guides using
• 09:09them as barcodes and quantitating.
• 09:11Enrichment and depletion of each
• 09:13guy and the way we interpreted
• 09:15this experiment was to compare high
• 09:17to lower mean selective pressure.
• 09:20So immunotherapy treated to
• 09:22immunodeficient mice, for example,
• 09:23and to interpret it that guides
• 09:25that were depleted.
• 09:27Comparing height alone,
• 09:28selective pressure represented Jews that,
• 09:30when deleted, convert sensitivity.
• 09:32To the mean system,
• 09:34and therefore potential targets
• 09:36for combination therapy.
• 09:38In contrast,
• 09:39guides that were enriched under
• 09:41strongly selective pressure suggested
• 09:42to US jeans that were lost made
• 09:44tumors resistant to new therapy.
• 09:48And a lot of the targets that we
• 09:50found this way actually ended up in
• 09:52the kind of realm of double stranded
• 09:54RNA sensing or antiviral triggering,
• 09:56and this is really the area that
• 09:58I focused on throughout my time.
• 10:01And this guy is thinking because a lot of
• 10:03what we know about viral infection comes
• 10:06from the study of exonerees viruses.
• 10:08But of course the genome is comprised
• 10:11largely of repetitive elements that have
• 10:14the potential to form double stranded RNA.
• 10:17These could be small interspersed
• 10:19nuclear elements and obvious retrovirus.
• 10:21Endogenous retroviruses are long
• 10:23interspersed nuclear elements or or others.
• 10:26And so we considered that that we've
• 10:29Co evolved with these elements.
• 10:31With these kind of viral remnants
• 10:33in many cases and ourselves have
• 10:35developed systems to regulate double
• 10:37stranded RNA sensing to distinguish
• 10:39between double stranded RNA.
• 10:41That's a result of normal cellular
• 10:43activity and exogenous viral threats.
• 10:45And so we thought that by targeting some
• 10:48of the genes that control this regulation,
• 10:51we might sensitize tumor cells
• 10:52to tumor therapy.
• 10:53Trigger this kind of anti virus state.
• 10:58And the top hits that we discovered
• 11:01through this process in the antiviral
• 11:03sensing arena was this paid.
• 11:05R18 R is an adenosine deaminase
• 11:07that acts on double stranded RNA.
• 11:10It has a long cytoplasmic P-150 isoform.
• 11:12That's interferon inducible and a short.
• 11:15Constitu Tively Express P110I support him.
• 11:19The main known function of edar is to
• 11:21catalyze the conversion of adenosine
• 11:22to in a scene and double stranded RNA.
• 11:25And it's thought that in so doing it
• 11:27prevents double stranded RNA sensing in
• 11:30the triggering of antiviral immunity.
• 11:32Kind of autoimmunity.
• 11:33Accordingly,
• 11:33there is an autoimmune syndrome called
• 11:36Acardi Goutieres syndrome that is
• 11:38associated with biallelic mutations
• 11:40of a Darwin on the catalytic domain.
• 11:43It can be quite severe effects
• 11:45children and mimics viral infection.
• 11:47However, Interestingly,
• 11:47the parents of affected patients
• 11:49who have monolith mutations in the
• 11:52catalytic domain have evidence of
• 11:54increased signatures of interferon
• 11:55gene expression in the blood,
• 11:57but have no detectable disease phenotype,
• 11:59suggesting that there's a gene dose effect.
• 12:04So to begin to validate our
• 12:06one as a potential drug target
• 12:08for combination immunotherapy.
• 12:09We created dedicated knockout tumor cell
• 12:12lines again using the B16 Melanoma model.
• 12:15This transplantable tumor model and
• 12:16we implanted these into mice under
• 12:19increasing selective pressure.
• 12:20It means selective pressure starting
• 12:23with the extremely immunodeficient
• 12:24nods give gamma mice that entirely
• 12:27lack adaptive immunity and have
• 12:29only impaired innate immunity.
• 12:31In these mice,
• 12:32looking at the 8 Arnold tumors,
• 12:34either P-150 knockouts in Orange,
• 12:36P-150 P, 110 knockouts in red
• 12:38compared to controls and Gray,
• 12:39and looking at tumor volume on the top,
• 12:42or survival in the bottom,
• 12:44you can see a sort of minimal
• 12:46decrease in the growth of the
• 12:49Darnell tumors compared to controls.
• 12:51And a minimal increase in survival.
• 12:55In contrast,
• 12:55when planted these tumors into wild
• 12:57type mice with an intact immune system,
• 12:59you see a significant decrease in
• 13:01the growth of tumors in a significant
• 13:03survival advantage for the mice.
• 13:05Finally,
• 13:05when we implemented these tumors into
• 13:08mice and treated with anti PD one,
• 13:10we saw a near 100% cure rate for
• 13:12mice treated that were a Darnall and
• 13:15almost no cures in the control chambers.
• 13:18So to start to understand
• 13:20the mechanism of this,
• 13:21we looked at the tumor micro environment
• 13:23of untreated a Darnall and control
• 13:25tumors 14 days after implantation,
• 13:27and we did this using immuno histo
• 13:29chemistry and as you can see on the
• 13:32left in control tumors you have
• 13:34the immune desert type phenotype.
• 13:36Almost no CD8T cells infiltrating.
• 13:38In contrast,
• 13:39in a Darnall tumors we saw this
• 13:41T cell inflamed phenotype with
• 13:42diffuse infiltration of CD8T cells.
• 13:44Quantitative here on the right.
• 13:49To understand this more deeply,
• 13:50we next perform flow cytometry.
• 13:52Again with tumors 14 days after
• 13:54implantation in the untreated setting,
• 13:56and as you might predict,
• 13:58we saw an increase in CD 45
• 14:01positive immune cells and a Darnell
• 14:04tumors compared with controls.
• 14:06And then looking within the CD 45 compartment
• 14:08we saw increases in CD 3 positive T cells,
• 14:12CD 4 positive T cells,
• 14:13CD 8 positive T cells,
• 14:15gamma Delta T cells and NK cells.
• 14:19In contrast, when we looked at
• 14:22immunosuppressive populations,
• 14:23including mdse and tumor
• 14:25associated neutrophils,
• 14:26we saw significant increases in control
• 14:30tumors relative to a Darnall tumors.
• 14:34Finally, to probe the micro
• 14:35environment yet more deeply,
• 14:37we perform single cell RNA sequencing.
• 14:39These are the populations we
• 14:41recovered with myeloid populations
• 14:43in the upper right and T cell
• 14:46populations in the bottom left.
• 14:48As you can see,
• 14:50using these density plots that
• 14:51we adapted for this purpose,
• 14:53you get a strong signal from suppressive
• 14:56myeloid populations and to like
• 14:58macrophages and mdse in control tumors.
• 15:00But a weaker signal from inflammatory
• 15:02monocytes and CD8T cells.
• 15:03In contrast,
• 15:04in the 8 Arnold tumors you
• 15:06have hardly any signal from the
• 15:08suppressive minded populations
• 15:09and and enrichment of single from
• 15:11inflammatory monocytes and CD8T cells.
• 15:16To understand what's driving this
• 15:18change in the micro environment,
• 15:20we wanted to study the double
• 15:22stranded RNA sensing pathways that
• 15:24we thought could be associated
• 15:25with the phenotypes we'd observed.
• 15:28Specifically, we wanted to understand the
• 15:30role of protein kinase are an MD5 rig,
• 15:33I and nouns which are both associated
• 15:35with his internal sensors of nucleic
• 15:38acids in double stranded RNA,
• 15:40specifically protein kinase power
• 15:41is associated with translation
• 15:43arrest in a pop ptosis.
• 15:44Upon binding double stranded RNA.
• 15:47Where is MD5 regarding mass induced type
• 15:50one interferon in the antiviral state?
• 15:53To test the role of each of these sensors,
• 15:56we generated a series of double
• 15:58and triple knockout tumor cell
• 16:00lines and probe some of the in
• 16:02vitro phenotypes that we previously
• 16:04previously studied in a Darnell tumors.
• 16:06Specifically,
• 16:07we looked 1st at growth inhibition.
• 16:09So when you stimulate control
• 16:11tumors with interferon in vitro,
• 16:13there's a slight defect in growth that's
• 16:17magnified when you knockout eight R1.
• 16:20Looking at our double knockouts,
• 16:22we saw no effect of knocking out rig.
• 16:25I MDA 5 or Mens but saw that knocking
• 16:28out peak PQR reduced the phenotype to
• 16:31the levels observed in control tumors,
• 16:34suggesting a PQR was alone.
• 16:37Responsible for the in vitro
• 16:39growth defect that we'd observed.
• 16:41We next looked at interferon beta production.
• 16:44And this was again an in vitro Aliza and
• 16:47tumor cells stimulated with interferon.
• 16:50As you can see,
• 16:52control tumors produce no
• 16:54detectable interferon,
• 16:54whereas a Darnall tumors
• 16:57produces significant quantity.
• 16:58This is maintained from the loss
• 17:00of Rig I suggesting that guy is
• 17:02not involved in the phenotype.
• 17:04However,
• 17:04following the loss of MDA,
• 17:06Five Man's or PK are you see a
• 17:08significant reduction suggesting
• 17:09that all three of these sensors,
• 17:11or these two sensors in this adapter
• 17:13have a role to play in phenotype.
• 17:17We next wanted to understand which of
• 17:20these double stranded RNA sensing pathways
• 17:21was required for the in vivo phenotype of
• 17:24sensitization to whom checkpoint blockade.
• 17:26So we took our double and triple knockout
• 17:28tumor cell lines and implanted them into
• 17:30mice, treating the mice with PD one.
• 17:33Antibodies targeting PD one,
• 17:34and as you can see in our control experiment,
• 17:37control tumors continue to grow out
• 17:39as they did previously for us in the
• 17:41eternal summers respond well to,
• 17:43you know, therapy.
• 17:45This phenotype persisted following
• 17:47loss of PQR, suggesting that PQR is
• 17:50alone not required for the phenotype.
• 17:53Similarly.
• 17:53It persisted following loss of MD5,
• 17:57suggesting MDA 5 alone does not
• 18:00explain the phenotype.
• 18:01However.
• 18:02Following the deletion of both PK
• 18:04are in MDA 5 together with eight
• 18:06or one we no longer observe any
• 18:08difference between the growth of
• 18:10eight R1 knowledge control tumors
• 18:12treated with immunotherapy.
• 18:14Together,
• 18:14these results suggested to us that
• 18:16growth inhibition by PQR or antiviral
• 18:19sensing by MDA 5 amounts sufficient
• 18:21mediate sensitivity to no therapy
• 18:23but that at least one is required.
• 18:28We next wanted to understand which
• 18:30double stranded RNA sensing pathway
• 18:32was required for the enhanced community
• 18:34filtration for the inflammation in the
• 18:36tumor microenvironment that we'd observed.
• 18:39And so we again used our double and
• 18:41triple knockout tumor cell lines.
• 18:43In this time return to our habit of
• 18:45looking at the tumor microenvironment,
• 18:47dissecting the tumors out,
• 18:49separating out the cells,
• 18:50and quantitating them.
• 18:52To look which sensor was was required.
• 18:57In our control tumors,
• 18:58you see a relatively low infiltration
• 19:01of immune cells that significantly
• 19:03increased following loss of eight R1.
• 19:05And Interestingly,
• 19:06this phenotype is,
• 19:07if anything exaggerated following
• 19:09loss of protein kinase are however
• 19:12it's attenuated following loss of
• 19:13MBA 5 and oblated following the
• 19:16loss of the two senses together.
• 19:18A similar pattern followed when
• 19:19we looked at the proportion of
• 19:22the 45 positive immune cells that
• 19:24was comprised of CD8T cells,
• 19:25again,
• 19:26increases in eight are null that
• 19:28persisted following loss of PQR
• 19:30was attenuated following loss
• 19:32of MD5 with loss following the
• 19:34loss of both sensors together.
• 19:36When we look at a immunosuppressive mdse,
• 19:39we saw the opposite pattern
• 19:41increases in control that persisted
• 19:43or work were even increased.
• 19:45Further following loss of PQR and no
• 19:48loss of the phenotype following loss
• 19:50of MDA 5 for the two sensors together.
• 19:57This suggested to us that MBA five
• 19:58may be playing the predominant role.
• 20:00And inducing tumor microenvironment
• 20:02inflammation may darnel tumors.
• 20:04To confirm this,
• 20:06we looked at the production of interferon
• 20:08beta interferon gamma in the tumor
• 20:11microenvironment of the eternal jiggers.
• 20:13And we saw a similar pattern again
• 20:15increases in a terminal tumors that
• 20:18persisted following loss of PQR but was
• 20:20lost after law after loss of MD5 or the
• 20:23two sensors together in the same pattern.
• 20:25Again looking at tumor
• 20:28lysate interferon gamma.
• 20:29So haven't seen having seen
• 20:31this powerful dual mechanism for
• 20:33sensitizing tumors to immunotherapy.
• 20:35We asked whether loss of eight R1 was
• 20:38sufficient to overcome commonly acquired
• 20:40mechanisms of resistance to amino therapy,
• 20:43including genetic aberrations
• 20:45that have been identified as
• 20:47enriched when comparing discordant,
• 20:48responsive,
• 20:49pretreatment,
• 20:49and resistant posttreatment lesions.
• 20:51Matched with the same patient.
• 20:54Known mechanisms that fit this
• 20:57description include the loss of MHC one
• 21:00through mutations of HLA or beta 2M,
• 21:02loss of targeting,
• 21:03children expressing through Mino,
• 21:05editing mutations and interferon sensing
• 21:07pathways including interferon gamma receptor,
• 21:09the Jackson,
• 21:10the stats.
• 21:13And we focused first on the loss of MHC one,
• 21:16as mediated by loss of data to microblogging
• 21:19which has been repeatedly identified as
• 21:21important in challenging form of resistance.
• 21:24To create this model we
• 21:26again use CRISPR CAS 9.
• 21:28This time deleting beta 2 micro
• 21:30globulin and eight are together.
• 21:32Along with creating match
• 21:34control tumor cell lines.
• 21:37To validate our model of resistance,
• 21:39we compared control in beta two of null
• 21:41tumors in the untreated that is dashed
• 21:44line state versus the treated state.
• 21:47That's the solid lines using again,
• 21:49this strong immunotherapy treatment
• 21:51regimen of GBX and PD one.
• 21:54And we did this because the normal
• 21:56control chambers responded very poorly
• 21:58to PD one and we wanted to make sure
• 22:00that we could see a response in control
• 22:03tumors and then validate that it was
• 22:05lost in the beta two unknown tumors.
• 22:08And sure enough,
• 22:09that's what we did see you can see the
• 22:11control tumors respond albiate transiently.
• 22:13Alternately,
• 22:13do grow out to this strong unit
• 22:15therapy treatment regiment,
• 22:16but made it to heaven.
• 22:18All tumors hardly respond at all.
• 22:22We next looked at a Darnall tumors.
• 22:25This is our positive control
• 22:26experiment using strong again
• 22:27with therapy treatment regimen.
• 22:29We got a great response to treatment.
• 22:31The untreated tumors grow out,
• 22:33albeit more slowly than controls.
• 22:36Strikingly, however,
• 22:37this sensitivity persisted following
• 22:39loss of beta two microglobulin,
• 22:41suggesting that loss of a Darwin
• 22:43in tumors is sufficient to overcome
• 22:46this mechanism of resistance.
• 22:48This result was a bit surprising actually.
• 22:50At first, as it suggests that CD8T
• 22:52cell recognition with MHC one in
• 22:54tumors is not in all cases required
• 22:56for the response to amino therapy.
• 22:58It also raises the question as to
• 23:01whether it could be possible to
• 23:03target tumors that entirely lack
• 23:04high quality CDH cell antigens.
• 23:06A lot of ongoing work in the
• 23:08lab is focused on dissecting the
• 23:09mechanism of this finding,
• 23:11and one of the first
• 23:12things we wanted to know.
• 23:14Is whether antigenic vaccine GBX,
• 23:16which was unsuccessful in
• 23:17translating to human use,
• 23:19was required for this response.
• 23:23This is actually pretty new data
• 23:24or afraid with PD one alone,
• 23:26and found that indeed you still get
• 23:28great responses in a Darwin all tumors.
• 23:32Even without the gmax.
• 23:34To start to understand this
• 23:36mechanism further, we again looked
• 23:38in the tumor microenvironment,
• 23:39this time focusing on our beta 2M
• 23:41null compared to control tumors.
• 23:43And so, as you would expect,
• 23:46increased immune infiltration
• 23:47CD 45 positive cells.
• 23:50But now focused on some of these
• 23:52MHC one non MHC one restricted
• 23:55cytotoxic populations and these
• 23:57include granzyme B positive CD
• 24:004 positive T cells and NK cells.
• 24:02With the hypothesis that perhaps
• 24:04these cells which don't require MHC
• 24:07one for recognition of tumor cells.
• 24:09May be involved in the phenotype
• 24:13we've observed.
• 24:14We've also begun to dissect the
• 24:16cytokinin kyma kind drivers,
• 24:17by which these populations may
• 24:19be recruited and activated.
• 24:21These graphs are from side to kinda be
• 24:24Teresa Beta to null and a Darnall tumors.
• 24:28The two prominent chemo kinds
• 24:29were identified so far.
• 24:31CX CL 10 in CCL 5.
• 24:34Which are both significantly
• 24:35increased in our beta to emulate
• 24:37our one all tumors compared with
• 24:39beta to a control control tumors.
• 24:44Notably Ehrenring here at Yale has
• 24:46described a similar phenotype of
• 24:47being able to overcome the loss
• 24:49of MHC one using a modified I'll
• 24:5118 side kind that he designed.
• 24:52So this remains another possibility
• 24:54that we haven't yet explored.
• 24:56However, we think this type of study
• 24:59is important 'cause articulating the
• 25:00general principles by which loss of MHC
• 25:02one can be overcome could lead to new
• 25:05treatment approaches to target tumor
• 25:06specific immune evasion mechanisms.
• 25:11In summary, I hope I've convinced
• 25:13you have several points.
• 25:14First aid are one loss over improves the
• 25:18response to me to therapy. Specifically,
• 25:21it can overcome the lack of evidence.
• 25:23Plain tumor, micro environment and the
• 25:26loss of antigen presentation by image C1.
• 25:29Additionally, this phenotype is
• 25:31driven both by tumor microenvironment,
• 25:33inflammation mediated by MDA
• 25:355 and sensitization.
• 25:36Interferon driven by PK are.
• 25:40Finally, and I think this may be important.
• 25:43Tumor cells contain sufficient innate
• 25:45lightning into drive therapeutic information.
• 25:47If they are in need.
• 25:49Nucleic acid sensing
• 25:50checkpoints are disabled.
• 25:52And what we think this implies is that
• 25:54there may be other similar innate
• 25:56immune checkpoints that limit the
• 25:57sensing of double stranded RNA or other
• 26:00nucleic acid ligands that we could
• 26:01think about as therapeutic targets.
• 26:05And really, those questions inform the
• 26:07rest of the work that the lab is doing.
• 26:10I've mentioned already a focus on
• 26:12double stranded RNA and eight R1.
• 26:14We're also applying functional genomics
• 26:15to try to identify other novel targets.
• 26:18Really, with the insight that we
• 26:19have to focus on turning on some of
• 26:22these pathways of double stranded RNA
• 26:24sensing or micro violent information.
• 26:27And then we're involved in human translation,
• 26:29doing kind of in depth tumor
• 26:31microenvironment investigation across
• 26:32several different tumor indications.
• 26:34We're always looking for new
• 26:37collaborators there.
• 26:38And all of this comes under the rubric
• 26:41of therapeutically targeting the
• 26:43information in the tumor microenvironment.
• 26:45In just the last couple of minutes here,
• 26:47I want to quickly mention some of
• 26:49the ongoing projects in the lab that
• 26:51I haven't talked about this far.
• 26:52First, I mentioned just the project.
• 26:57Describing how to Riker environment
• 26:59inflammation can overcome the
• 27:01loss of MHC one.
• 27:02This is being led by Jessica Way,
• 27:05but she's Additionally leading a project.
• 27:09Looking at human tumors and trying
• 27:10to turn these pathways on in ex
• 27:13vivo samples as well as doing deep
• 27:14dissection of the micro environment.
• 27:16Where we go is working on novel
• 27:19strategies to detect double stranded
• 27:20RNA and to mimic the sensors of double
• 27:23stranded RNA that we believe will be
• 27:26compatible with functional genomic
• 27:28screening in the identification of
• 27:30novel cancer immunotherapy targets.
• 27:32And finally,
• 27:32even Kim who is in the lab focused on the
• 27:36comparison of discordant response lesions.
• 27:39So responsive and resistant lesions.
• 27:42From the same patient trying to
• 27:44understand novel mechanisms of
• 27:46resistance to new therapies so
• 27:47that we can focus on overcoming.
• 27:50With that I want to thank everybody in
• 27:52our lab as well as our collaborators
• 27:55and mentors here at, you know,
• 27:57have been fantastic.
• 27:58I also wanted knowledge at Nikki
• 28:00Ning my form. Enter drumming.
• 28:01So much of the work that I presented early
• 28:04derives from from studies with them,
• 28:06and of course our funding here
• 28:08at the Cancer Center and the
• 28:10International Research Alliance.
• 28:11With that, I will wrap up.
• 28:13Thank you so much for the chance to present,
• 28:16and I'm happy to take questions.
• 28:19Jeff, thank you. That's just
• 28:21terrific work and really exciting.
• 28:23And we we have folks can submit questions.
• 28:27We have one question.
• 28:28Mike Hurwitz asked.
• 28:30So given the response in eight R1
• 28:33knockouts in the absence of MHC class one,
• 28:36do you think that's function of
• 28:38CD4T cells or NK cells, or both?
• 28:42Or some other mechanism? Yeah,
• 28:44I think that's a great question and we
• 28:46definitely would love to know that answer.
• 28:50Best hypothesis Now is that partially
• 28:52based on some of the work that Ehrenring
• 28:56is presented in Marcus Bosenberg.
• 28:58NK cells could be an important player there.
• 29:01Certainly there increased and we
• 29:02started to see some cytokines in Kemah
• 29:04kinds that may activate them further,
• 29:06but you know, we don't even know for
• 29:08sure that CD8T cells aren't important.
• 29:10That's an experiment we're doing now.
• 29:12We just know they're not recognizing the
• 29:14tumor, but could they be activated through
• 29:16cross presentation or another means is
• 29:18another question that we're investigating.
• 29:21And then you know, in related work.
• 29:24Obviously Akiko, Saki,
• 29:25and Anna Pile of working independently on
• 29:28Rig Rig I are iguana, which which it is.
• 29:31But which obviously is not necessarily
• 29:34related to the function vadar one,
• 29:36and you know how?
• 29:38How do you see those two with those
• 29:41two sort of bodies of work relating?
• 29:44Yeah, so this is
• 29:46a great question Charlie and actually
• 29:48Akiko is one of my mentors here and.
• 29:52Collaborators and we've talked about this.
• 29:55We're actually in the process of testing.
• 29:58Are a guy at. Egotist with the innate
• 30:03arnolin control tumor cell lines and
• 30:05you know the colloquial way we we
• 30:07thought about this is kind of as a
• 30:09maximum inflammation bomb because what
• 30:11we've shown is that any interferon
• 30:13producing stimulus can trigger this
• 30:158 Arnold amplification of sensing,
• 30:16and so our hypothesis would be that if
• 30:18you initiate signaling through a guy,
• 30:20even if there a guy is not involved
• 30:23in the pathways we've described here,
• 30:25you basically create a massive
• 30:26amplification of interferon,
• 30:27buy by further knocking out eight
• 30:29R1 so that remains to be seen,
• 30:32but that's what I would hypothesize.
• 30:34Yeah, that's interesting.
• 30:35It sounds like a great
• 30:37opportunity to look at that.
• 30:38Well, I I want to keep us on time,
• 30:41so Jeff, thank you.
• 30:42I know there are other questions
• 30:44coming in and people should certainly
• 30:46reach out to you directly, Jeff.
• 30:48But thank you for a superb presentation
• 30:51and let me now turn to our second speaker,
• 30:54doctor Robert Bone and Bob Bone is a
• 30:56professor of medicine in hematology,
• 30:58and recently the past year joins us as the
• 31:01director of the Benign Hematology program,
• 31:03as well as the medical director of
• 31:06the Hemophilia Treatment Center.
• 31:08Prior to joining Yale,
• 31:09Bob was founding faculty member
• 31:11and leader at the Frank Netter
• 31:14School of Medicine at Quinnipiac,
• 31:16as well as a professor of medicine at
• 31:19the University of Connecticut School of
• 31:21Medicine and Bob throughout his career,
• 31:24really has been a leader in in in the
• 31:27clinical care and sort of advancing
• 31:30work in hemostasis thrombosis as well
• 31:32as benign hematologic conditions.
• 31:35And we're really,
• 31:36very fortunate Bob to.
• 31:38That Bob,
• 31:38now leading this section and sharing
• 31:40with his work with us.
• 31:42So Bob thank you.
• 31:44Thank you, Charlie for that introduction
• 31:47and for the opportunity to speak today.
• 31:50Let me just share my screen here.
• 31:53So good afternoon everybody.
• 31:56And what I would like to do in the
• 31:58next 25 minutes or so is discuss with
• 32:01you some of the advances that have a
• 32:04curd in the treatment of hemophilia
• 32:07and what I hope to show you is that
• 32:09over the past five years there have
• 32:11really been significant and substantial
• 32:13advances which came in the background
• 32:16of really several decades of really
• 32:18only modest advances in therapy.
• 32:21So just as a brief review here,
• 32:24these are excellent disorders,
• 32:26mostly affecting men,
• 32:27but can also affect women who might
• 32:30have low factor levels due to
• 32:33unequal X chromosome inactivation,
• 32:35hemophilia A&B or deficiencies
• 32:37in factor 8 or 9 respectively.
• 32:39They are clinically identical disorders
• 32:42and the severity of the disease
• 32:45is really relies primarily on the
• 32:48residual factor that is remaining
• 32:50in the blood with those with severe.
• 32:53And moderate disease having less
• 32:55than 5% of factor 8 or factor 9
• 32:57and those with mild disease having
• 33:00a higher value and morbidity and
• 33:02mortality is due to spontaneous
• 33:04and trauma induced bleeding,
• 33:06including bleeding into joints which
• 33:09can cause a hemophilic arthropathy
• 33:12which we can be quite quite disabling.
• 33:15And just the history of hemophilia
• 33:17treatment in the last century
• 33:19is seen briefly on this slide,
• 33:21and at the end of World War Two
• 33:24blood or plasma transfusions were
• 33:26used to treat patients.
• 33:28This these were largely ineffective,
• 33:30is only small amounts of factor 8 or
• 33:33factor 9 could be transfused in the 1960s.
• 33:36Cryoprecipitate was discovered
• 33:37as a source of Factor 8,
• 33:40and that quickly gave way to the
• 33:42use of factor concentrates either
• 33:44factor 8 or factor 9.
• 33:46Purified from the plasma of
• 33:4910s of thousands of donors.
• 33:51And of course,
• 33:52while this advanced care,
• 33:54it also exposed individuals to a
• 33:56number of viral viral particles and
• 33:59hepatitis C and HIV became a very
• 34:02significant problem in this population.
• 34:04And then in the early 90s
• 34:07recombinant factors 8:00 and 9:00,
• 34:09or produced and for the developed world,
• 34:12where economically this was allowable
• 34:14of the treatment of hemophilia
• 34:17with recombinant factors 8 and 9.
• 34:19Became really the standard of
• 34:23care up until very recently.
• 34:26There are now about 145 federally
• 34:29funded hemophilia treatment centers
• 34:31in this country and of course jeliz is
• 34:34one of those is one of those centers.
• 34:37And the therapeutic.
• 34:38The approach in clinical issues
• 34:40are outlined here.
• 34:42Patients with hemophilia can either be
• 34:44treated in what's known as on-demand
• 34:46or episodic factor replacement,
• 34:48which is the treatment with Ivy
• 34:51Factor 8 or factor 9 to treat a
• 34:54bleed or prophylactic therapy.
• 34:56An inhibitor development,
• 34:57that is an Allo antibody directed
• 35:00against Factor 8 or less commonly,
• 35:02factor 9 is a significant problem
• 35:05for patients and may occur in 30 or
• 35:0840% of individuals with hemophilia A
• 35:10and makes treatment very difficult
• 35:13and the goals of therapy are really
• 35:15here to prevent
• 35:16any bleeding. If possible,
• 35:18prevent joint disease and optimize a
• 35:21quality of life for these individuals.
• 35:24And the infusion of factor 8 or
• 35:25factor 9 by patients is traditionally
• 35:27given at home intravenously.
• 35:29Patients from a very young age learn to start
• 35:32an Ivy and infuse factor 8 or factor 9,
• 35:35but because of the short
• 35:37half-life of these drugs,
• 35:38about 12 hours for factor 8 and
• 35:4018 to 24 hours for factor 9,
• 35:42they need to be administered two to
• 35:45three to sometimes four times per
• 35:47week to keep the factor levels in a
• 35:49range that will prevent bleeding.
• 35:51So this is an onerous thing
• 35:53for patients to do.
• 35:54And any advances here would be
• 35:58greatly appreciated by them.
• 36:00So here's the obligatory coagulations
• 36:02slide that I would like to show
• 36:06to to reinforce and emphasize
• 36:08the role that Factor 8 and factor
• 36:119 having blood coagulation.
• 36:14So what we're seeing here is the
• 36:17tissue factor initiated pathway and
• 36:20activation of factor 10 by tissue
• 36:23factor 7A or activation by factor
• 36:269 to 9 A by tissue factor 7A.
• 36:29And 9A is also able to activate 9:50
• 36:33A O2 pathways to get down to this
• 36:36all important enzyme factor 10A,
• 36:39and in this latter reaction factor
• 36:418 serves as a cofactor for the
• 36:44enzyme factor 9A.
• 36:45To act on its substrate factor 10
• 36:48and increases the rate of reaction
• 36:51hundreds of 1000 fold when factor 8
• 36:54is able to align the substrate and
• 36:56enzyme on a phospholipid surface in
• 36:59the correct in. In the correct fashion.
• 37:03One other thing to mention about
• 37:05Factor 8 before we get into some of
• 37:08the details of the advances is that
• 37:11factor 8 travels if you will in the
• 37:13blood bound to von Willebrand factor.
• 37:15Von Willebrand factor is seen here in
• 37:18this linear structure at the bottom,
• 37:20factor 8 is the yellow diagram above,
• 37:23and the binding of factor 8 von
• 37:25Willibrand factor enhances the
• 37:27half life of factor 8 from about
• 37:292 hours to about 12 hours.
• 37:31So this is a very important interaction.
• 37:34And just to point out here,
• 37:36'cause this will become important
• 37:38later is that the binding site
• 37:40on von Willebrand factor is these
• 37:42two protein domains,
• 37:43designated D prime and D3,
• 37:45and another important point is there
• 37:47appears to be a large portion of the
• 37:50factor 8 molecules termed the B domain,
• 37:52which is not required for factor 8 function,
• 37:55so you could remove that domain
• 37:58and in fact factor 8 has a similar
• 38:01activity than it does with that domain.
• 38:04So the advances in care of hemophilia
• 38:07really over the past five to six
• 38:10years come into three different areas.
• 38:12One is extended,
• 38:13half-life factor concentrates,
• 38:14allowing for patients to infuse
• 38:16less frequently.
• 38:17The development of non factor 8
• 38:19or 9 therapeutics,
• 38:20and then gene therapy and we'll
• 38:24go through these individually
• 38:26in the next 15 minutes or so.
• 38:28So the extended Half-life products
• 38:30have been produced by manipulating
• 38:32the recombinant factor eight
• 38:34or nine in a number of
• 38:35different ways, many of which are familiar
• 38:38to you by either adding polyethylene
• 38:40glycol or conjugating the factor to the
• 38:43FC portion of immunoglobulin or albumen,
• 38:45to improve half-life, or,
• 38:47in the case of factor 8,
• 38:49to remove that B domain, which causes
• 38:52a slight increase in the half life.
• 38:55And there are now a number of products
• 38:58that have been approved for use at
• 39:00our extended Half-life products,
• 39:02and I'll draw your attention
• 39:04to the last three on this list.
• 39:07These are factor 9 products which have
• 39:09been manipulated by these methods,
• 39:11seen here and the half life of these
• 39:14products has been extended from 18 to
• 39:1724 hours to upwards of 90 or 100 hours.
• 39:20So this is allowed patients with factor 9
• 39:23deficiency or hemophilia B to be treated.
• 39:26Once a week,
• 39:27once every 10 days and in some circumstances,
• 39:31even once every two weeks.
• 39:32So a significant advance for
• 39:34people needing to give intravenous
• 39:36therapy themselves at home.
• 39:38The advances in hemophilia A with factor 8.
• 39:41However,
• 39:41a much more modest with this
• 39:43type of manipulation,
• 39:45and it turns out that the the
• 39:47degradation in the catabolism and
• 39:49clearance from the circulation of
• 39:51factor 8 is much more linked to the
• 39:54clearance of von Willebrand factor,
• 39:56the protein that it's bound to.
• 39:59So making modifications in the FAQ.
• 40:00After 8 molecule has really had
• 40:03minimal effect up until recently
• 40:06on Factor 8 Half-life.
• 40:08So an interesting construct has been devised,
• 40:11and it's shown on the top panel here
• 40:13and in this construct the D prime and
• 40:16D3 regions of von Willebrand factor,
• 40:19the binding region to factor 8,
• 40:22is linked to an FC portion of an
• 40:25immunoglobulin and linked to the
• 40:27B domain less factor 8 molecule,
• 40:29which also has linked on at
• 40:32this hydrophilic polypeptide,
• 40:33which also can extend the half life.
• 40:36So this product has been called bib 001.
• 40:39And was treated with.
• 40:41Was used to treat a handful of
• 40:43patients in a safety study,
• 40:45and those results were were
• 40:47reported in the New England Journal
• 40:49of Medicine earlier this year,
• 40:51and patients were either treated
• 40:53at two different doses of this new
• 40:56product and the factor a clearance
• 40:58from the circulation was compared
• 41:00to the typical factor 8 clearance
• 41:02seen in the lighter blue bars here
• 41:05and what you can see I think,
• 41:07is that the half life of this newer product.
• 41:11Is now about two days increased,
• 41:13about five or six fold the half life
• 41:15of the standard factor 8 product.
• 41:18So this this product is now in
• 41:20large scale clinical trials and I
• 41:22think in the next year or two we
• 41:24should have some more information,
• 41:26and this may be an advanced
• 41:29for for some of our patients.
• 41:32So let me shift for a minute for
• 41:34the to the non factor product for
• 41:36the treatment of hemophilia and I
• 41:38think their significant advance
• 41:39has been made here and there are
• 41:41three drugs that will talk about
• 41:43will really focus primarily on this
• 41:45first drug which is called EMAS
• 41:48ISM AB. A nemesis Omab is a
• 41:51bispecific monoclonal antibody.
• 41:53That binds the factor 9 and factor 10,
• 41:56so it simulates the activity of Factor 8.
• 42:00Remember that factor 8 is able
• 42:02to colocalize factor 9 and factor
• 42:0510 on a phospholipid surface.
• 42:07This antibody is able to bind factor
• 42:109A and factor 10 in the circulation an
• 42:14again simulate the activity of Factor 8.
• 42:18So this drug is not exactly like Factor 8.
• 42:21There are.
• 42:22There are certain differences here.
• 42:24It binds to factor 8 and nine
• 42:27in the circulation,
• 42:28not just on the phospholipid membrane.
• 42:30It has different infinities
• 42:32for the substrate and enzyme,
• 42:34and whether or not that becomes an
• 42:37issue for this drug will only know
• 42:40as more experience is is accumulated.
• 42:43But nonetheless,
• 42:43this drug is really shown dramatic activity,
• 42:46so this this is a study that was
• 42:49published a few years ago in the
• 42:52New England Journal of Medicine.
• 42:54Here we had patients who have hemophilia
• 42:57A with inhibitors to factor 8,
• 42:59so a challenging group of patients to
• 43:01treat were treated either with their
• 43:04typical regimen of recombinant factor
• 43:067A or factor 8, bypassing activity,
• 43:08or with Emma system AB given by
• 43:11subcutaneous injection once a week
• 43:13and the annual bleeding rate.
• 43:15Is been been described on this slide
• 43:17here and you could see if we just look
• 43:21at these blue histograms for a minute here.
• 43:23The annualized bleeding rate in the
• 43:26EMA system app Prophylaxis Group
• 43:29was about five or six and it was
• 43:32almost 30 in the standard of care.
• 43:35Treatment of patients with
• 43:37hemophilia A and inhibitors.
• 43:39So a really significant
• 43:41advantage for these individuals.
• 43:43And then a second study was published
• 43:45with looked at patients with hemophilia
• 43:48A without inhibitors and these.
• 43:50This was a randomized trial.
• 43:52Patients were treated with one
• 43:54of two doses of Emma's is a map
• 43:56either given weekly or every other
• 43:58week by subcutaneous injection,
• 44:01compared with no prophylaxis.
• 44:02About 100 patients in the trial,
• 44:04and again the annual annualized
• 44:06bleeding rate went from about
• 44:0940 to about one or two.