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"Dysregulated Transmembrane Ion Gradients Highlight Cancer Invasion and Proliferation" and "Over the River and Through the Woods and into the Brain we Go: lupus autoantibodies offer a new pathway to treating brain tumors"

February 24, 2022

Presentations by: Fahmeed Hyder, PhD and James Hansen, MD, MS

ID
7475

Transcript

  • 00:00We have a number of people on
  • 00:02the zoom already and I'm sure
  • 00:04there will be more that the join.
  • 00:07I'm very pleased today to have two speakers.
  • 00:13Doctors, Hyder and Hanson.
  • 00:15They will they will go in that
  • 00:19order and so first is Pharmd Hyder,
  • 00:22who is a professor of radiology
  • 00:24and biomedical imaging and
  • 00:26biomedical engineering.
  • 00:28He received his PhD in
  • 00:30biophysical chemistry from Yale,
  • 00:32where he was also an associate
  • 00:35research scientist and postdoctoral
  • 00:36associate with Douglas Rothman.
  • 00:38He's been in the faculty since 1999,
  • 00:41seems hard to believe.
  • 00:43You look very young and currently
  • 00:46holds dual professor appointments
  • 00:49in diagnostic radiology
  • 00:51and biomedical engineering.
  • 00:53Doctor Hatter is the director of
  • 00:55the High field horizontal smallbore
  • 00:57systems at Yale's MRI Research Center.
  • 01:00He uses MRI.
  • 01:01He uses magnetic resonance methods
  • 01:03to map Physiology and chemistry
  • 01:05that underlie brain function
  • 01:07for early disease detection,
  • 01:09but also for targeted drug
  • 01:11delivery and monetary treatments.
  • 01:12It's also the founder and director
  • 01:15of Yale's quantitative neuroscience
  • 01:17with magnetic resonance course enter
  • 01:19the only NIH supported programmatic
  • 01:21effort at Yale on neuroimaging.
  • 01:23With Mr technologies.
  • 01:24His work has produced over 100
  • 01:27peer reviewed publications and
  • 01:29he's written and edited books
  • 01:32on functional brain imaging.
  • 01:34I have to say something that I'm
  • 01:37becoming increasingly interested
  • 01:38as I age and he has received early
  • 01:41career awards from various scientific
  • 01:43societies and funding scientific
  • 01:46agencies for me to pleasure.
  • 01:48To have you.
  • 01:49Thanks for being here and we
  • 01:50look forward to hearing from
  • 01:51you. Thank you, thank you very much
  • 01:54for that generous introduction.
  • 01:56I hope I can live up to that introduction.
  • 02:00It's a pleasure to be here.
  • 02:02So hopefully you can see the slide.
  • 02:05Yep, perfect.
  • 02:09So my topic today is about disregulated
  • 02:12transmembrane ion gradients.
  • 02:14I like cancer invasion of liberation.
  • 02:17I believe the latter 4 words are probably
  • 02:20more akin to a lot of the audience members,
  • 02:22but I'm going to make the connection or
  • 02:25try to at least before the the initial
  • 02:28part of this title, which is about.
  • 02:32So a little bit of.
  • 02:34Preface I guess I'm interested
  • 02:36in bringing the tablets in why?
  • 02:39Because they tell us about the path weights.
  • 02:42Which you know end field cells use
  • 02:45nutrients to fill their function,
  • 02:47but also growth.
  • 02:49I'm interested in how cells work.
  • 02:52What their made nutrients
  • 02:54are in different situations,
  • 02:55different states how they
  • 02:57get these nutrients.
  • 02:58That's actually a very important
  • 03:00indicator of disease onset.
  • 03:02A lot of fences,
  • 03:04but also how they use these
  • 03:06nutrients to generate energy,
  • 03:07which is crucial for the
  • 03:08brain but also to regrow.
  • 03:11And disease and damage and so on.
  • 03:14And all of these processes happen
  • 03:17naturally for normal situations,
  • 03:18but they also begin to malfunction.
  • 03:22So these questions that I pose for myself,
  • 03:27as well as my group, my movies and vibrators.
  • 03:30These questions are fundamental for
  • 03:33functional imaging of the brain because
  • 03:35the energy demand for normal brain
  • 03:37work is extremely high and that still
  • 03:40is a very unique property of how.
  • 03:43This organ varies from a lot of
  • 03:46other buildings in the body,
  • 03:47but these questions are also relevant
  • 03:49in cancer and molecular energy
  • 03:51of brain disorders in general,
  • 03:53but especially concert,
  • 03:55because this fundamentally is
  • 03:56a disease of both,
  • 03:58and both requires lots of fuel.
  • 04:01Whenever you need to regulate
  • 04:03to compete for fuel that is a
  • 04:06medical question that it's tough.
  • 04:08So that's my preference,
  • 04:09but my main objective today is to
  • 04:12talk to you about the importance
  • 04:15of transparent votes.
  • 04:16If they're crucially linked
  • 04:18to cellular metabolism,
  • 04:20and this property differs substantially
  • 04:23between normal cells and cancer cells.
  • 04:27Abnormal transmembrane protein and sodium.
  • 04:30Specifically,
  • 04:30I'll talk about protein and sodium gradient.
  • 04:34Are there consequences of health
  • 04:37physiological alterations
  • 04:39occurring with cellular level?
  • 04:41And the two primary things I'll
  • 04:43focus on is the city and salinity
  • 04:46of the interstitial fluid.
  • 04:48These two quantities,
  • 04:50reflected as a proton and sodium ions,
  • 04:54respectively.
  • 04:54They actually regulate central cellular
  • 04:57functions in health as well as improved,
  • 05:00especially cancer under
  • 05:01their bark cancer tomorrow.
  • 05:03Basicness,
  • 05:04which is one of the hallmarks
  • 05:06and even resistance to therapy.
  • 05:08This is actually enhanced
  • 05:10by acidic interstitial.
  • 05:14Which is actually a consequence
  • 05:16of popularity by policies.
  • 05:18I will talk about this in a few minutes.
  • 05:21But there's also recent
  • 05:23discoveries from our work,
  • 05:24and I work with and related groups
  • 05:28that show that enhance proliferation.
  • 05:31Which is also a hallmark of cancer
  • 05:35is impacted by interstitial so,
  • 05:39so we both these city and the salinity
  • 05:42of legislation that is crucial.
  • 05:45A little bit of background,
  • 05:46I think that's the background
  • 05:48is probably redundant,
  • 05:49but for the sake of being bad
  • 05:51protein and sodium lines are
  • 05:53vital for numerous processes for
  • 05:55maintaining blood pressure to fire.
  • 05:58Only statically we maintain
  • 06:00a very large sodium gradient.
  • 06:03Almost two orders of magnitude,
  • 06:05almost an order of magnitude.
  • 06:06Sorry. Lending to a very strong
  • 06:09trance member Radiant.
  • 06:10Well, certainly, but similarly.
  • 06:11For for time buildings,
  • 06:13which is usually measured in terms of pH,
  • 06:15which if you know it's
  • 06:16just a log rhythmic scale.
  • 06:18So even though the pH between interstitial
  • 06:22and intracellular intracellular
  • 06:24compartments are 7.4 and 7.2 in PS units,
  • 06:29it's small.
  • 06:30But in terms of actual concentration,
  • 06:33it's again very large.
  • 06:35There are various mechanisms with
  • 06:37which are located the cell membrane,
  • 06:39how regularly.
  • 06:40A proton and certain levels to
  • 06:43avoid these mishaps that are
  • 06:45that sells try very hard before.
  • 06:49First,
  • 06:49Megan mechanisms that regulate
  • 06:51transforming what time gradients
  • 06:53are shown here in the parking form.
  • 06:56Let me just go around the cell here first.
  • 06:59Is this mechanism here carbonic
  • 07:02anhydrase mine specifically?
  • 07:04But these kinds of.
  • 07:09Instruments are used to basically take out
  • 07:12department dioxide and water generated by
  • 07:16oxidation and take them out and it
  • 07:19basically takes them out and comes
  • 07:21to bicarbonate and protons and which
  • 07:25signifies the intracellular space.
  • 07:27Then there is these channels at BCS and NC.
  • 07:30Is which abbreviations are shown here,
  • 07:33but the second time I'll just
  • 07:35go through the abbreviations.
  • 07:36These bring in both bicarbonate
  • 07:39and sodium and this one.
  • 07:41Runs in bicarbonate itself, right islands.
  • 07:44Both of these contributed to a contribute to.
  • 07:48Changing of the intracellular field.
  • 07:52Maximo carboxylate transporters
  • 07:54are those kinds and city one and
  • 07:58NC 3-4 working opposite days.
  • 08:00They bring in assets for NCT one
  • 08:04and take out assets for actually 4
  • 08:08altering hydrogen ion concentration.
  • 08:12It's all specifically.
  • 08:15Important one entity,
  • 08:17one sodium proton exchanger,
  • 08:19takes like for every proton taken out,
  • 08:22brings in every brings in a sodium and
  • 08:25that can alter the full time as well.
  • 08:30These are ATP and ATP dependent,
  • 08:34vascular full-time ATP ace,
  • 08:36where you're bringing in or taking
  • 08:38out protons to a city public service
  • 08:41place as well as these acid.
  • 08:46Sensing line channels as well as these
  • 08:50epithelial specific certain channels.
  • 08:53They also are known to affect more
  • 08:56time but as well as sodium because a
  • 08:58lot of things different channels as
  • 09:00sodium gradients and therefore beans
  • 09:03won't be repeated because these are
  • 09:05the channels that I just talked about.
  • 09:07But there are other mechanism.
  • 09:09This is a channel that I just
  • 09:10showed in the previous slide.
  • 09:11See energy as well as the
  • 09:14sort of important change.
  • 09:16Are all of these have a role in altering
  • 09:19the sodium level in the exercise of space?
  • 09:23This particular channel, the NSX,
  • 09:25the sodium counseling exchanger,
  • 09:27it's exchanges sodium for
  • 09:29calcium between the compartments.
  • 09:32It's interesting, it was recently discovered,
  • 09:34but in cancer specifically,
  • 09:36this is actually need to.
  • 09:39In addition,
  • 09:40there is this additional new channels
  • 09:43mentioned here that are specific to
  • 09:46sodium regulation across the department.
  • 09:48The most important one of this is
  • 09:50probably a well known as the support.
  • 09:52The sodium proton switches exchanging
  • 09:56of sodium and potent potassium at the
  • 10:00cost of ATP to basically maintain a balance.
  • 10:06To keep resting potential.
  • 10:10The same so that the cell can
  • 10:12actually keep on doing that.
  • 10:14It's designed to do so.
  • 10:15These are the mechanisms that are in play.
  • 10:19Glucose metabolism is key to all
  • 10:22of these processes and the healthy
  • 10:24miracle as blood drains in both
  • 10:27glucose and oxygen in the form of
  • 10:30oxyhemoglobin glucose is its transport
  • 10:33is expedited by these transporters,
  • 10:35but oxygen goes through just
  • 10:37passive diffusion into the cell.
  • 10:41Just breakdown appointments through
  • 10:42the bike clinic steps and then further
  • 10:45breakdown in in in the prep cycle
  • 10:48of generating large amounts of ATP.
  • 10:51All of these processes,
  • 10:52generally small amounts of protons
  • 10:54but also carbon dioxide water which
  • 10:57I just mentioned.
  • 10:58Carbonic anhydrase has a has a role in in,
  • 11:02in, in taking out these these these products.
  • 11:05So this is a.
  • 11:06Complete oxidation of glucose if you will,
  • 11:09but even under normal conditions
  • 11:12there is some.
  • 11:16Less oxidation than expected from theory,
  • 11:20where some of the glucose is actually
  • 11:23shunted towards biosynthetic processes,
  • 11:25but some of the invoices actually.
  • 11:29Excluded in terms of lactate by the public.
  • 11:33Feeds on CTV's that you
  • 11:36have mentioned earlier.
  • 11:37A lot of the protons generated are removed.
  • 11:40As I said from these different
  • 11:43mechanisms which I mentioned me
  • 11:45cheese also about the heart races.
  • 11:47But if cancer and the unhealthy durable the.
  • 11:51A lot of dysfunction happens in
  • 11:54terms of amount of bike policies,
  • 11:56which is the amount of
  • 11:59oxidation that happens.
  • 12:00And therefore some of these
  • 12:02there will be glycolytic.
  • 12:04Steps are augmented to the point.
  • 12:06Where these machinery's that I speak
  • 12:09just mentioned with carbonic and
  • 12:12hybrid sister upregulated in NCT 4?
  • 12:15Is that up regulated to handle
  • 12:17exclusion of lactate more efficiently,
  • 12:21but also in terms of the NET,
  • 12:25which is also up regulated to alter the
  • 12:28whole time in the access center space.
  • 12:31We vacuumed.
  • 12:35Along with some of the same mechanism,
  • 12:37the same machinery or additional machinery,
  • 12:40you can also experience a lot of
  • 12:43imbalances in a certain environment,
  • 12:45but key player as I just mentioned
  • 12:47is the mutation of the NSX,
  • 12:49which happens which is most
  • 12:51recently discovered in cancer,
  • 12:53but also the fact that there
  • 12:56is actually downregulation of
  • 13:00oxidation within a cancer cells.
  • 13:04Reduce potassium sodium is also an important
  • 13:11role of causing these sodium imbalances as.
  • 13:15And it's culture.
  • 13:17So this is the unhealthy miracle and our.
  • 13:21Our role is to understand and observe
  • 13:24these the impact of the altered proton
  • 13:27as well as transgender, Ingrid.
  • 13:29So tumors are highly like little
  • 13:31even when off, well oxygenated,
  • 13:34and this aerobic glycolysis phenotype.
  • 13:38Enables enabled by metabolism.
  • 13:40Different nutrients generates
  • 13:41lots of their civic products
  • 13:44that are extremely deficient.
  • 13:46For example,
  • 13:46even carbon dioxide and water generated
  • 13:49from oxidative metabolism are,
  • 13:50you know,
  • 13:51they contribute to certification
  • 13:53of the interstitial space by these
  • 13:55these anionic exchangers like.
  • 14:00Invite.
  • 14:03Interstitial acidosis actually
  • 14:04helps with tumor cell invasion.
  • 14:07They they actually did break
  • 14:09the interstitial matrix,
  • 14:11and they also promote angiogenesis
  • 14:14while suppressing entry. Sense.
  • 14:18So this is an example from Bob
  • 14:22Giles work where it is shown that
  • 14:26whenever there is use acidification,
  • 14:28this is done in a culture system
  • 14:32where there's reduce acidification,
  • 14:35there is enhanced invasion of
  • 14:38cells in terms of timber growth.
  • 14:41This is an example where
  • 14:42you study shown in situ,
  • 14:44but in people evidence for this
  • 14:46from our lab and other labs,
  • 14:47so security is absolute.
  • 14:51Impact with the altered transplant in
  • 14:53sodium gradient normal cells maintain.
  • 14:55Hyperpolarized membrane potential.
  • 14:58But before I see member interventional
  • 15:01and non excitable cells is linked to
  • 15:06their proliferation of point to this in
  • 15:10in the next slide a little bit more.
  • 15:12In fact, even in mean cells consensus
  • 15:15in the interstitial in the loop.
  • 15:17Therefore,
  • 15:18salinity of the interstitial and
  • 15:20intracellular compartments may be crucial.
  • 15:23As has been pointed out by recent.
  • 15:26Work that may be important for
  • 15:29early diagnosis of cancer,
  • 15:30but also maybe contracting
  • 15:32in cancer treatments.
  • 15:34So this point, this light points this out.
  • 15:36This is the scale of membrane potential.
  • 15:38On the bottom are these
  • 15:41non proliferating cells?
  • 15:42These are real cells,
  • 15:44neuronal cells.
  • 15:44They have a very hyperpolarized
  • 15:48membrane potential equation which
  • 15:50takes into account sodium and
  • 15:52the poor at concentrations up.
  • 15:54Here are the perfect.
  • 15:55Or looks writing cells which are
  • 15:57non tumor cells but the membrane
  • 15:59potential measure of tumor cells
  • 16:01in the rieti of different kinds.
  • 16:03You see they right up here in the
  • 16:07depolarized level. So in essence.
  • 16:10What a lot about solutions from
  • 16:13various groups have shown is that.
  • 16:18The week transmembrane sodium
  • 16:20gradient maintaining a depolarized
  • 16:22membrane potential is actually
  • 16:24necessary for these cells to survive
  • 16:26in their altered environment that
  • 16:28they create for their survival.
  • 16:31Update at the.
  • 16:32Cost of normal cells that they're replacing.
  • 16:36Whereas you know these normal cells,
  • 16:39especially in the brain,
  • 16:40maintain strong,
  • 16:41transmembrane certain buildings
  • 16:42and they may do so by maintaining
  • 16:45their hyperpolarized.
  • 16:49So these are the hallmarks of cancer,
  • 16:52our goal, and my goal in the next few
  • 16:55minutes is to show that you can employ
  • 16:57sodium and proton imaging methods
  • 17:00to actually observe 2 properties,
  • 17:04invasion and proliferation of cancer cells
  • 17:07by using proton and imaging perspective.
  • 17:11We talked a little bit about that.
  • 17:13I'm sure this method is quite
  • 17:15familiar with most.
  • 17:21Slash house light.
  • 17:38Turns out that.
  • 17:45In like one step. Slide disappeared, yeah.
  • 17:49I realized that it just didn't like 1 slide.
  • 17:55Do you wanna stop sharing and start
  • 17:56again or is that what you're doing?
  • 17:58Yeah, I'm trying to do that right now, OK?
  • 18:01OK, so I'll skip that slide.
  • 18:10And see if I can get from that.
  • 18:15So the slide that I unfortunately had that.
  • 18:21You know it bounced me out of PowerPoint.
  • 18:24Basically this describes a key late.
  • 18:27That is the design structure of most contrast
  • 18:30agents that is used in the preferable
  • 18:32design is where you take a gadolinium.
  • 18:35Mine is very car magnetic and you
  • 18:38use that to reduce its toxicity
  • 18:42Yuzuki late with donating.
  • 18:44Components like oxygen atoms that would
  • 18:47provide the stability for conjugation,
  • 18:50reducing discovery toxicity
  • 18:52of the metal iron.
  • 18:54But discriminating iron is some of the
  • 18:57key of the types of imaging observations
  • 18:59that I'm going to show you about.
  • 19:02So this is essentially a proton
  • 19:04spectrum of a key late like this.
  • 19:06This is a it's called appear TP molecule.
  • 19:09It's basically a molecule that
  • 19:12contains multiple phosphinate groups,
  • 19:14much like phosphonates that exist
  • 19:16in molecules like ATP and ADP.
  • 19:18So this is a very
  • 19:20common diamagnetic range of signals that
  • 19:22you would see emanating for different.
  • 19:25Protons, the non exchangeable protons that
  • 19:28exist in these carbons as well as the
  • 19:31carbons existing in these pendant arms.
  • 19:36By the way, I point out that these
  • 19:37phosphates on this on a molecule like this.
  • 19:40These protons exchange with protons
  • 19:42of water and that kind of exchange
  • 19:45is actually a a a pH mediated
  • 19:48and and and therefore they are.
  • 19:52These molecules are essentially
  • 19:54create sensor much like ATP and
  • 19:58ADP is using phosphorus and.
  • 20:00So if you now collate this,
  • 20:03take this molecule and and complexes
  • 20:05with the paramagnetic metal.
  • 20:07In this case,
  • 20:07I'm not using gadolinium,
  • 20:08but a fully am I and you essentially
  • 20:12caused this large expansion of the
  • 20:14same chemical shift that you see here
  • 20:16within a few partner million and that's
  • 20:19expanded by hundreds across people.
  • 20:21So these signals have no potential
  • 20:24overlap with other types of signals
  • 20:27that you may observe in vivo.
  • 20:29And this is the water signal.
  • 20:31And so this hyperfine shifted
  • 20:33signal has very unusual properties.
  • 20:36Basically, all it means is that
  • 20:38you can observe them quickly.
  • 20:39You can observe them under very
  • 20:43precarious in vivo situations that we
  • 20:46may experience the relaxation times are
  • 20:49so short because of the paramagnetic
  • 20:51environment that that these photons
  • 20:53are in create a very sensitive scenario.
  • 20:57That's most, importantly,
  • 20:58that the chemical shift of these signals,
  • 21:00not the amplitude,
  • 21:01of these signals.
  • 21:02But how these signals are
  • 21:05located and how they shift?
  • 21:08Is the readout that we observe again,
  • 21:10so this is a molecule which is the sensor.
  • 21:13So if you change the
  • 21:15environment of the sensor.
  • 21:17The structure of the molecule will change
  • 21:19and as the structure of the molecule changes.
  • 21:22What you then read out that structural
  • 21:25changes from the chemical change.
  • 21:28And so this is encapsulated here.
  • 21:30This is a molecule that we
  • 21:32are using as our sensor.
  • 21:33These are the types of signals that we
  • 21:37observe using this as an example 86 proton.
  • 21:40It has a pH sensitivity,
  • 21:42but obviously any molecule reports
  • 21:43environment by changes like these
  • 21:45molecules have or molecules like this
  • 21:47have very very short relaxation times.
  • 21:49So you can observe them quickly
  • 21:51and therefore imaging these have
  • 21:53been possible and the readout from
  • 21:56them in terms of chemical shift is.
  • 21:58Not confounded by other overlapping signals.
  • 22:02So this is an in vitro example of this
  • 22:05kind of work from my colleague Dan
  • 22:08Employment and then this method is.
  • 22:11Paul by sensor imaging,
  • 22:13redundant deviation shifts, birds for short.
  • 22:15Essentially showing you that in these
  • 22:18two phantoms which have very different pH,
  • 22:21you can read out the pH with high confidence
  • 22:23even if you change the temperature.
  • 22:25So you can essentially
  • 22:27give these two things out.
  • 22:29Simultaneously,
  • 22:30the key thing is is that this is
  • 22:33not like most imaging contrast.
  • 22:36This contrast is not based on
  • 22:39the amount of agent that you have
  • 22:42in in the given compartment,
  • 22:44but really their chemical shift.
  • 22:45So it's it's a readout that's
  • 22:47independent of concentration,
  • 22:48and so using this method we've
  • 22:50been able to apply it to look at
  • 22:53the cancer environment,
  • 22:54specifically the the pH in the extracellular
  • 22:57space, and a variety of different.
  • 22:59Scenarios these these are examples
  • 23:02from breaking specifically shown for
  • 23:05two different types of tumors here,
  • 23:06but you can note and appreciate the acidity.
  • 23:09Acidity within the tumor poor as shown here.
  • 23:13Core being identified by the line
  • 23:18that shows up in the MRI contrast.
  • 23:21But you can also appreciate this
  • 23:23extended area of a certification
  • 23:24for this tumor and not that tumor,
  • 23:27and that has to do with the
  • 23:29how evasive this tumor is,
  • 23:31and using cellular markers,
  • 23:33you can corroborate that funding,
  • 23:35so this goes back to the
  • 23:37point that our pH readout,
  • 23:39so this is a case of a a
  • 23:41very non invasive tumor,
  • 23:42versus this one is tumors invade and this.
  • 23:47Invasion if you will.
  • 23:49I we believe is essentially preconditioning
  • 23:53for the expansion of this tumor within time.
  • 23:56For these two specific cases,
  • 23:58here in the 2nd and R2, but not the help.
  • 24:03So talked about a little bit about what
  • 24:06we do with the Proton imaging part to
  • 24:08read up pH and that uses the proton nucleus,
  • 24:12right?
  • 24:12This is a very highly sensitive
  • 24:15nucleus to observe because with the
  • 24:17hygiene gentlemen medical issue.
  • 24:19But what I'm going to talk about next
  • 24:21is the sodium imaging part which has
  • 24:25a lower gyromagnetic ratio but aided
  • 24:27by the fact that we are looking at.
  • 24:30You know our product nucleus we are.
  • 24:33So we can benefit a little bit from the
  • 24:36certain relaxation times of this nucleus.
  • 24:39So that's kind of where we are.
  • 24:42Our basic rationale for this type of
  • 24:45experiment is that the problem that
  • 24:47we face is that the sodium signal,
  • 24:49whether looking at the the interstitial
  • 24:53or the intercellular pool,
  • 24:55signals they overlap.
  • 24:57They're not chemically different,
  • 24:59and therefore the total signal
  • 25:00you observe is is a representative
  • 25:03of these two types, etc.
  • 25:05Our expectation is that somehow we can
  • 25:08separate the two and that separation.
  • 25:11Will allow us to look at things
  • 25:13like the transmembrane great.
  • 25:16So the idea stemmed from work
  • 25:17that existed in the field,
  • 25:19but the advanced it in the last few
  • 25:22years is that if we have a polyanionic.
  • 25:26Our magnetic agent,
  • 25:27much like pet agents that I've talked about.
  • 25:30And we take a sodium ion because
  • 25:33of its negative charge.
  • 25:34It's attracted to it and there
  • 25:37is superbound bounding of some of
  • 25:41these ions because of the negative.
  • 25:44Target of this agents and we have
  • 25:46some exchange of free sodium with
  • 25:49this sort of bound sodium,
  • 25:51and if that exchange happens.
  • 25:55Fast enough that we can reflect the
  • 25:58shifting of the sodium signal from
  • 26:01a from this process to reflect the
  • 26:04true 2 compartments of service.
  • 26:06So in essence,
  • 26:07what we do and this is our rationale take.
  • 26:11Before we add an agent like this,
  • 26:13this is the total signal we will look here.
  • 26:16And when we inject or introduce
  • 26:19an agent like this because it's so
  • 26:22negative and it can attract positive
  • 26:24recharge sodium ions towards it,
  • 26:26we can separate these two sodium
  • 26:30compartments.
  • 26:31And this is a theory which we've
  • 26:33contributed to, and a key.
  • 26:34Two key factors that we point out in in
  • 26:38this theoretical and practical demonstration
  • 26:40of pages like this is the fact that.
  • 26:43There is a certain bound fraction of sodium
  • 26:46towards these agents that's important,
  • 26:48but also their exchange and these two
  • 26:51mechanisms have NKX actually contribute to
  • 26:54how much the sodium signal will be shifted.
  • 26:58So the shift ability factor,
  • 27:00but also how these signals could be broadened
  • 27:03because of their kind of like nature.
  • 27:06So, uh, another key thing that we point
  • 27:08out is that these shifted signals
  • 27:11brought in signals of of of sodium
  • 27:13that we will observe is dependent
  • 27:16on the sodium concentration on the
  • 27:19on the agent concentration.
  • 27:20And here we are limited by the fact
  • 27:23that we need sufficient amount of
  • 27:25this agent present to process certain
  • 27:28for the broadening and therefore
  • 27:30this is something that will act as
  • 27:33a benefit for certain sensor.
  • 27:36But it was not necessary for
  • 27:38them for the poor concepts.
  • 27:40So our expected idea for these
  • 27:42experimentation is that before we add
  • 27:45the agent, we're seeing one single,
  • 27:46and after we add this agent,
  • 27:48we're gonna separate the signal.
  • 27:50Now you see three and I point out
  • 27:53three because you also have sodium.
  • 27:55Plenty of sodium also in the
  • 27:57blood department.
  • 27:58But because the the blood department is much
  • 28:01smaller than the interstitial compartment,
  • 28:03these representative amplitudes
  • 28:05of these beats are depicted.
  • 28:09So in vivo inside a tumor voxel.
  • 28:12This is what we see.
  • 28:13This is before the agent.
  • 28:15It's been separated here.
  • 28:17And outside of the tumor,
  • 28:19we see something that's similar but
  • 28:21to the shifting and the broadening
  • 28:23is to some lesser extent.
  • 28:25So this is kind of what we
  • 28:27typically do nowadays,
  • 28:28and this is work by Mohammad Khan who
  • 28:30did his thesis work on this project.
  • 28:32Is is this is the conventional
  • 28:34location of the tumor.
  • 28:35We inject the agent to
  • 28:37identify the blood compartment,
  • 28:38interstitial compartment,
  • 28:40interstitial intracellular compartment
  • 28:42in terms of sodium.
  • 28:44We integrate these signals to
  • 28:46get maps like this,
  • 28:47and using a combination of these two,
  • 28:49we can get what's called
  • 28:51an endothelial gradient.
  • 28:52This is asserting gradient representing
  • 28:54the and the feeling department.
  • 28:56And this is the critical one
  • 28:57that we will talk about,
  • 28:59which is the transmembrane gradient
  • 29:01which is obtained from the intracellular
  • 29:04and the interstitial signals also.
  • 29:06And we we can now map this into ID
  • 29:09to get the transmembrane gradient
  • 29:11how it appears within the tumor.
  • 29:14I point out there's these blobs of high
  • 29:17intense signal represents the ventricles.
  • 29:20Ventricles hasn't has a
  • 29:22lot of sodium in them,
  • 29:24and that has variety of them applications.
  • 29:28Or other disease disorders as well,
  • 29:31but the key thing is that we can
  • 29:33obtain a clear readout of the
  • 29:36transmembrane gradient shown for two
  • 29:38different three different tumor types,
  • 29:40and so this is a pretty uniform and
  • 29:43vigorous observation that we can now make.
  • 29:46We even began to employ these
  • 29:48methods for variety of
  • 29:50different treatments.
  • 29:53And and so this is a very unique entry.
  • 29:57Genic antiangiogenic treatment and
  • 29:59what is shown here is a comparison
  • 30:01of what's your afternoon, does it?
  • 30:04It it blunts it.
  • 30:06It sort of impedes that's in the growth
  • 30:08which is most significant within two weeks.
  • 30:11And this kind of effect can be
  • 30:13read out by the teenage as well
  • 30:16as the transmembrane gradient.
  • 30:17As you can see the sorafenib against
  • 30:20renormalize the pH with treatment and.
  • 30:24As well as normal, you know.
  • 30:28Strengthening the sodium transmembrane
  • 30:30gradient upon treatment even
  • 30:32the Chamber making it similar
  • 30:34to the normal tissue as well,
  • 30:36so I hope that's been able to show
  • 30:39within the last few minutes is
  • 30:41that we now have a quote which can
  • 30:44sense both quote photon and sodium.
  • 30:46A lot of credit to both.
  • 30:49I'm sharing design quotes of
  • 30:52this particular quote,
  • 30:54and quotes like this,
  • 30:55which with proton imaging can be a very
  • 30:58powerful proton sensor for pH imaging,
  • 31:01but also a sodium sensor in terms
  • 31:04of its its signal shifted.
  • 31:07It it can be also used simultaneously.
  • 31:11So I hope I've what I've
  • 31:12shown is that all cells,
  • 31:13not just excitable neurons and muscle,
  • 31:16generate and receive by electrical
  • 31:19signals that are encoded.
  • 31:21Within changes in the transmembrane
  • 31:24potential and the iron fluxes that
  • 31:26occur at the cell membrane and
  • 31:28these things happen regularly,
  • 31:30you know on timescales of milliseconds,
  • 31:32seconds to even days.
  • 31:34But these are inextricably
  • 31:37regulated by catabolism,
  • 31:39and that is the connection that it is is.
  • 31:44This is needed to find the proper readouts
  • 31:48of various treatments that we see.
  • 31:51And I, I think this is another way of
  • 31:54saying this is that we need advancing
  • 31:56before imaging methods to assess the
  • 31:58non genetic by physical aspects of
  • 32:00tumor microenvironment that regulates
  • 32:02balance between normal growth
  • 32:04but also the disorganization that
  • 32:06happened with most solid solid cancer.
  • 32:11But importantly,
  • 32:12simultaneous imaging invasion and
  • 32:15qualification can hold a promise for
  • 32:18early cancer diagnosis and tracking
  • 32:21therapies. From chemotherapy to two.
  • 32:25There's always grateful for
  • 32:27tonight support and this work is
  • 32:29unique collaboration among many
  • 32:31colleagues within our group.
  • 32:35Khan and John Walsh,
  • 32:38PhD and MD,
  • 32:39PhD students are involved in a
  • 32:40lot of this work,
  • 32:41but also by colleagues in
  • 32:43terms of post starts.
  • 32:44You're still present Doctor
  • 32:46Kumar Mishra and and the high
  • 32:48leverage but also my colleagues.
  • 32:52Within radiology and
  • 32:54surgery that department so.
  • 32:57And I thank you for your.
  • 33:01Your attention.
  • 33:03Thanks, thanks very much it.
  • 33:05I think that was great.
  • 33:06You mentioned chemotherapy and
  • 33:09immunotherapy, and you know,
  • 33:10our next talk is from a radiation
  • 33:12oncologist and one wonders whether this
  • 33:15might also help identify tumors and
  • 33:17tract tumors that are being irradiated.
  • 33:20Since, as he well knows,
  • 33:23it is that his doctor Hanson.
  • 33:26It is sometimes confusing
  • 33:27after someone's been treated,
  • 33:28whether there's active tumor or or not.
  • 33:31So without further ado.
  • 33:33Guy James Hansen is an associate
  • 33:36professor of therapeutic radiology
  • 33:38and chief of the Gamma Knife
  • 33:40program in therapeutic radiology.
  • 33:42He received his medical degree
  • 33:44from UCLA School of Medicine and
  • 33:47Masters degree in biochemistry and
  • 33:49Molecular Biology at UCLA as well.
  • 33:51Doctor Hanson,
  • 33:52Clinical area of expertise is gamma
  • 33:54knife stereotactic radiosurgery for the
  • 33:56premium of CNS tumors had neck tumors,
  • 33:58lung cancer as well as lymphoma,
  • 34:00skin,
  • 34:01GI and GUI will bet that he spends more
  • 34:03time on CNS disease than other things.
  • 34:06But he can tell us his research focus
  • 34:08is studying the interplay between
  • 34:10autoimmunity and malignancy and
  • 34:12attempting to harness and optimize select
  • 34:14autoantibodies to use against cancer.
  • 34:16James, thanks for.
  • 34:18Joining us.
  • 34:19Thank you and thank you
  • 34:21for that introduction.
  • 34:21And yes, if if you can explain
  • 34:23my clinical career to me,
  • 34:25that would be great.
  • 34:26I would love to. But it's hot.
  • 34:30Alright, so let's see,
  • 34:31can you see my slides and hear me?
  • 34:34Yeah, it's in that presentation
  • 34:35now that's great. Yeah
  • 34:36alright. Here we go.
  • 34:38So I'm gonna be talking today about
  • 34:40my particular interest in using
  • 34:43lupus antibodies against cancer,
  • 34:45and in this case, how we can maybe use lupus
  • 34:48antibodies against brain tumors and so.
  • 34:51Already, I probably have lost half
  • 34:53of youth that are just saying,
  • 34:54well, that's impossible.
  • 34:56Antibodies can't do that.
  • 34:57But just give me 20 minutes or so,
  • 34:59give me a chance.
  • 35:00I I think that there's something
  • 35:01here that's worth talking about.
  • 35:03Do you have some disclosures?
  • 35:04I'm a consultant.
  • 35:05I have grants from inventor
  • 35:07on a patent licensed by this
  • 35:09company called Patrys Limited,
  • 35:11who has licensed the DEOXY mab technology.
  • 35:14So with that said, let's jump head first in.
  • 35:17I don't think there's any
  • 35:19secret that antibodies have
  • 35:21revolutionized our approaches.
  • 35:22In modern day two treatment of cancer.
  • 35:25But I think it's important to recognize
  • 35:27that all the antibodies that we really
  • 35:29rely on in the clinic right now are
  • 35:31targeted towards extracellular targets.
  • 35:33So things like surface receptors
  • 35:36or circulating growth factors.
  • 35:38But doggone it seems like there
  • 35:40are so many intracellular antigens
  • 35:41that if we could just get an
  • 35:43antibody in there to engage,
  • 35:45we could add an entire new
  • 35:47dimension to immunotherapy.
  • 35:48But the dogma has always
  • 35:50been that that's impossible.
  • 35:52Antibodies don't penetrate live cells.
  • 35:56Now this is where critics will often
  • 35:57interrupt me and say that's not true.
  • 35:59We have antibodies that are already in
  • 36:01clinical use that are internalized.
  • 36:03What about cats?
  • 36:04I love the TDM ones and I I would
  • 36:07say I say DNA.
  • 36:08That's not what I'm talking about.
  • 36:10I'm talking about an antibody that can get
  • 36:12in and engage its actual native antigen.
  • 36:15In these ADC constructs,
  • 36:17these antibodies are still looking
  • 36:18for surface receptors like her
  • 36:20two in this example and then the
  • 36:22antibody gets eaten up and destroyed
  • 36:24in the endosome and lysosome which
  • 36:26is great for this mechanism because
  • 36:28that's what we want,
  • 36:28we want that drug to be released,
  • 36:30but that doesn't utilize the exquisite
  • 36:33binding specificity of an antibody
  • 36:35against intracellular targets.
  • 36:36So I think we need an antibody
  • 36:38that can get into cells and not get
  • 36:40stuck in those Endo celebs, but.
  • 36:42How are we going to do that well?
  • 36:45I guess we could try to invent one.
  • 36:46We could stick things like cell
  • 36:49penetrating peptides onto antibodies
  • 36:50like that at peptides and such,
  • 36:52and that's been tried,
  • 36:54but those tend to still get stuck
  • 36:56in the endosomes.
  • 36:57I guess we could try gene therapy, but boy,
  • 36:59that's got all kinds of challenges.
  • 37:01I'm going to leave that to other people.
  • 37:04Maybe if we can just find a naturally
  • 37:06occurring antibody that penetrates
  • 37:07cells and use that as a platform to
  • 37:09teach us how to invent these antibodies,
  • 37:11that seems the most appealing to me.
  • 37:14But anybody have any idea where we're
  • 37:17going to find an antibody like that well?
  • 37:20How about in lupus?
  • 37:22Systemic lupus erythematous?
  • 37:24It is the prototype autoimmune disease.
  • 37:27Patients suffer widespread tissue
  • 37:29destruction and inflammation
  • 37:30as their immune
  • 37:32systems recognize their
  • 37:33own cells and tissues.
  • 37:35And one of the laboratory hallmarks of
  • 37:37lupus is the presence of circulating
  • 37:39autoantibodies that are reactive
  • 37:41against the patient's own DNA.
  • 37:44Now those antibodies are a big mystery.
  • 37:46We still don't know exactly how they
  • 37:48contribute to lupus pathophysiology,
  • 37:50but remarkably it is now finally
  • 37:53accepted that a small percentage
  • 37:55of them actually have the ability
  • 37:57to cross through membranes and
  • 37:59penetrate into live cell nuclei.
  • 38:02Well, hey, so there we have it right.
  • 38:03We have a source of naturally
  • 38:05occurring cell penetrating antibodies.
  • 38:06We can use those for all kinds of therapies,
  • 38:08right?
  • 38:08Well, hold on now everybody
  • 38:11we're talking about Lupus right?
  • 38:13At last I checked Lupus is still
  • 38:15in the textbooks as a disease.
  • 38:17And indeed,
  • 38:18a lot of these cell penetrating lupus
  • 38:20autoantibodies are just broadly cytotoxic.
  • 38:22And there there wouldn't be any
  • 38:24benefit to giving them to a patient.
  • 38:26But thankfully,
  • 38:26that's not true for all of them.
  • 38:28There's an antibody called 310,
  • 38:31which is the hero of this story.
  • 38:33It was discovered in the early
  • 38:341990s at UCLA by Richard Weisbart
  • 38:36who's pictured there at the left,
  • 38:38along with his technician Grace Chan and
  • 38:40his great colleague Robert Nishimura.
  • 38:42Their great,
  • 38:43great friends and colleagues.
  • 38:45What makes 310 so remarkable is that
  • 38:48it was isolated from a lupus mouse.
  • 38:50It penetrates extremely effectively
  • 38:52specifically into the nucleus of live cells,
  • 38:55and so it does not go through endosomes,
  • 38:57and it does not kill or is not
  • 38:59toxic in any way to normal cells.
  • 39:02So now we've got our chance.
  • 39:04Now we have an opportunity to use a
  • 39:07platform antibody that penetrates cells.
  • 39:09And in fact,
  • 39:10as we dive a little deeper into
  • 39:13how this antibody really works,
  • 39:15it turns out this antibody is really
  • 39:17well situated for targeting things
  • 39:19like tumors or sites of tissue damage.
  • 39:22And that is because part of its
  • 39:25mechanism of penetration is dependent
  • 39:27on presence of extracellular
  • 39:28DNA or nucleosides in the area.
  • 39:31So what we're showing on the left
  • 39:32here is something we call the
  • 39:34three E 10 bullseye effect of the.
  • 39:35The dark stain represents where
  • 39:37the antibody is, and as you see,
  • 39:39that dark stain is getting lighter
  • 39:40and lighter as you get further
  • 39:42out from the center,
  • 39:43and that's because there's a
  • 39:44dead cell there in the middle.
  • 39:46And it's releasing DNA into its
  • 39:47surroundings and helping the antibody
  • 39:49penetrate the live cells that are
  • 39:51closest to it, but less effective.
  • 39:52As we get further out.
  • 39:54And in the middle we showed we can
  • 39:56reproduce this in the laboratory
  • 39:57just by adding DNA to the antibody.
  • 39:59At the bottom we allow the antibody to
  • 40:01penetrate 100% of the cells in the culture,
  • 40:03so the DNA has to be there.
  • 40:06And then if you take this antibody
  • 40:08to mice and you give it to mice that
  • 40:10don't have any tumors or damage,
  • 40:12you don't see the antibody really
  • 40:14going anywhere.
  • 40:15But if you give it to a mouse with a
  • 40:17tumor that is necrotic and is releasing DNA,
  • 40:20you do see the antibody
  • 40:21localising to that tumor.
  • 40:22That's what's shown in the top right.
  • 40:24The brown stain are the nuclei
  • 40:26penetrated by this antibody.
  • 40:28So what on Earth is going on here?
  • 40:30Why is this antibody using
  • 40:32DNA to penetrate cells?
  • 40:33How is it doing that?
  • 40:34Well, that's a little bit of a longer
  • 40:36story than we have time for today,
  • 40:37but just to jump to the punch line,
  • 40:40it is using a specific nucleoside
  • 40:42salvage pathway to get into live
  • 40:45cells that are salvaging DNA and
  • 40:47nucleosides from their surroundings,
  • 40:48and that transporter is called ENT two.
  • 40:51It is my best friend transporter
  • 40:53in the whole world stands for
  • 40:56equilibrated Nucleoside transporter 2.
  • 40:59So in order for 3:10 to cross membranes
  • 41:01to penetrate into cells and nuclei,
  • 41:03you have to have two things.
  • 41:05The cell has to express ENT two and
  • 41:08there has to be DNA or nucleosides
  • 41:10around that cell so that the antibody
  • 41:13can bind to the nucleosides and then
  • 41:15follow them through ENT 2 into the cell.
  • 41:18And that's why the antibody likes
  • 41:20to preferentially accumulate
  • 41:21in vivo insights of damage,
  • 41:23like tumors where DNA is being released
  • 41:26and ENT two is salvaging salvaging
  • 41:28the DNA and along with it the 3:10.
  • 41:31Networks for injury sites as well,
  • 41:33and that's what we've seen
  • 41:35in multiple studies.
  • 41:37For example,
  • 41:37moving from left to right,
  • 41:39if you take the three E 10 antibody and
  • 41:42you link it to a heat shock protein.
  • 41:45And then give it to mice or rats.
  • 41:47Actually that have had strokes.
  • 41:48You find the antibody gets the heat
  • 41:50shock protein into the ischemic brain and
  • 41:53improves neurologic function and recovery.
  • 41:55In the middle here we're showing
  • 41:58heart attacks in rabbits.
  • 41:59The 3:10 antibody finds the site
  • 42:01of the heart attack and delivers
  • 42:03the heat shock protein.
  • 42:04It works remarkably well.
  • 42:05And on the right we're looking at tumors.
  • 42:08You take the antibody and you fuse it to P53.
  • 42:10That protein from long time ago
  • 42:12and it absolutely localizes the
  • 42:13tumors and shuts them down.
  • 42:15So 310 has great potential as a delivery
  • 42:18vehicle for tumors or sites of damage,
  • 42:21and there's now more companies
  • 42:24looking at this than I can even count.
  • 42:27But that's not all the antibody does.
  • 42:29It turns out that it does more.
  • 42:31It's not just a delivery agent.
  • 42:34When the antibody penetrates into the
  • 42:36nucleus of a cell, it will bind DNA.
  • 42:38But if it has its choice,
  • 42:40it will preferentially bind
  • 42:42a DNA that's broken.
  • 42:43And then it's going to mess around with
  • 42:45DNA repair so it blocks base excision,
  • 42:46repair,
  • 42:47and rad 51 mediated molega's recombination.
  • 42:50And that means it can make cancer
  • 42:52cells more sensitive to DNA
  • 42:54damaging therapy like certain
  • 42:56chemotherapies and radiation.
  • 42:57But even more significantly,
  • 42:59if the cancer cell or the tumor has a
  • 43:03pre-existing defect in DNA repair due
  • 43:05to a mutation such as Bracco or P-10 loss.
  • 43:08The antibody doesn't need radiation.
  • 43:10It doesn't need chemotherapy.
  • 43:11It will kill that cancer cell
  • 43:13by itself by causing persistence
  • 43:15of DNA double strand breaks,
  • 43:17but that doesn't happen in normal
  • 43:19cells that have intact DNA repair,
  • 43:21so we have a selective toxicity.
  • 43:244HR deficient tumor cells with this antibody.
  • 43:28So we reported that quite a while ago,
  • 43:32and then we asked the question,
  • 43:33is it just 310?
  • 43:34Is this just something magic about
  • 43:363:10 or is this true for other
  • 43:37cell penetrating antibodies?
  • 43:39And it turns out indeed
  • 43:40it's not just three ten.
  • 43:42There are antibodies that
  • 43:44penetrate cells and cut DNA and
  • 43:46kill the HR deficient cells.
  • 43:47So there's a pattern emerging here
  • 43:50where lupus antibodies some lupus
  • 43:52anti DNA antibodies seem to be
  • 43:53toxic to HR deficient cancer cells,
  • 43:56and so then that led us to start
  • 43:58asking some questions about.
  • 44:00What does this mean for lupus
  • 44:02and and cancer risk in lupus?
  • 44:03And I'm not a rheumatologist.
  • 44:06I did not know,
  • 44:07but we started reading and looking.
  • 44:08And overall,
  • 44:10cancer risk is increased in lupus,
  • 44:13but it's driven mostly by
  • 44:15haematological legacy.
  • 44:16So if you back up and you say, well,
  • 44:18let's go tumor type by tumor type,
  • 44:20you get a surprising finding in
  • 44:23that breast cancer occurs at a
  • 44:25lower than expected rate in lupus.
  • 44:27And this has been widely recognized
  • 44:28for years,
  • 44:29but no one can figure out exactly why.
  • 44:30There's no clear associating factor.
  • 44:34But if you look a little deeper,
  • 44:35it looks like it's the triple
  • 44:37negative breast cancer that is
  • 44:38specifically suppressed in lupus.
  • 44:40And we do know that triple negative
  • 44:42breast cancer is associated with the
  • 44:44braknis phenotypes and the HR deficiency.
  • 44:47So then we start to think,
  • 44:48well, if that's true, like why?
  • 44:50Why aren't lupus patients making these
  • 44:52triple negative breast cancers as much?
  • 44:54Well,
  • 44:54we know about these DNA damaging
  • 44:56autoantibodies that are killing
  • 44:57these HR deficient cells.
  • 44:58Is it possible that lupus anti
  • 45:00DNA antibodies actually are
  • 45:01protective against breast cancer?
  • 45:03And if so,
  • 45:04maybe we can re engineer them
  • 45:05and use them to treat triple
  • 45:07negative breast cancer and so?
  • 45:09We're excited about this.
  • 45:10We sent it in as an opinion letter.
  • 45:12I thought I was extremely clever.
  • 45:14I was very impressed with
  • 45:15myself when I called it.
  • 45:17The lupus butterfly effect,
  • 45:18because lupus is the symbol of that.
  • 45:21Sorry, the butterfly is a symbol of lupus.
  • 45:24The butterfly effect is a symbol of chaos.
  • 45:26There's chaos and immunology.
  • 45:28Chaos of using lupus
  • 45:30antibodies against cancer.
  • 45:31I thought it was great title and then
  • 45:32rear said now you haven't proven it.
  • 45:34You gotta call the theory so it was
  • 45:36published as the lupus butterfly theory,
  • 45:38but I will forever want to
  • 45:39remember it as the lupus butterfly
  • 45:40effect theory hypothesis.
  • 45:41Postulate whatever you want to call it.
  • 45:45But we do need to prove it,
  • 45:46so we published that in 2016
  • 45:48and we started asking around.
  • 45:50Anybody can help us with
  • 45:52epidemiology 'cause we have no idea
  • 45:53what we're doing in that regard.
  • 45:55And it turns out Lupus is
  • 45:57rare enough that it's a.
  • 45:58It's a challenge to actually prove
  • 46:00the association between lupus
  • 46:02antibodies and breast cancer risk.
  • 46:04But not an insurmountable one because
  • 46:06John Hopkins was able to do it and
  • 46:08they published it just last year.
  • 46:09So this. Kind of blew my mind
  • 46:11and I was very exciting to see
  • 46:13this the Hopkins Lupus cohort.
  • 46:15They were able to treat.
  • 46:17Sorry they were able to analyze
  • 46:192000 plus lupus patients that
  • 46:21entered their cohort without a
  • 46:23cancer diagnosis and then evaluate
  • 46:24their risk of breast cancer.
  • 46:28Over the years and they were able
  • 46:30to associate that with the patients,
  • 46:32laboratory studies and anti
  • 46:34DNA antibody profiles.
  • 46:35And we do see finally proof that
  • 46:38there is an association between
  • 46:41anti double stranded DNA antibody
  • 46:43positivity and that reduction in
  • 46:45breast cancer risk patients that make
  • 46:47those anti D antibodies have 45%
  • 46:49reduction in the breast cancer risk.
  • 46:52And if you dig even deeper and you
  • 46:54stratify the patients based on the amount
  • 46:56of those antibodies they are making,
  • 46:58the low producers did not have any
  • 47:01reduction in breast cancer risk.
  • 47:02But the high producers had
  • 47:04a 59% reduction in risk,
  • 47:06so I've I'm taking this as
  • 47:08backing up my theory.
  • 47:09I think the lupus butterfly theory can be
  • 47:11called the lupus butterfly effect now,
  • 47:12so I might ask for a revision
  • 47:14to that article.
  • 47:15Although it's been five years here.
  • 47:17But regardless,
  • 47:18it's it seems like we're learning
  • 47:20something about lupus antibodies
  • 47:21and at least breast cancer,
  • 47:23and it gives me more confidence
  • 47:25in what we're doing and trying
  • 47:26to reengineer lupus antibodies
  • 47:28to treat triple negative breast
  • 47:30cancer and other tumors.
  • 47:32So what are we doing in that regard?
  • 47:33Well? Again.
  • 47:35I remember and I recognize 310,
  • 47:39although it's technically
  • 47:40safe in normal cells.
  • 47:41It's still a lupus anti DNA antibody.
  • 47:44And you don't want to give
  • 47:46anybody lupus like symptoms.
  • 47:47So we gotta rethink this a little
  • 47:49bit before we start taking
  • 47:51this to clinical trials.
  • 47:52The last thing you want is a lupus FC
  • 47:54region being administered to patients,
  • 47:56because the FC is going to activate
  • 47:59compliment and ABC and all kinds of madness.
  • 48:01But the good news is, 310 does not
  • 48:03care whether or not it has the FC tail.
  • 48:06It'll penetrate cells with or without the FC.
  • 48:08It'll bind DNA and inhibit DNA
  • 48:10repair with or without the FC.
  • 48:12So first thing we've done, cut it off.
  • 48:14There is no more FC tail,
  • 48:16the danger is gone.
  • 48:17And that's how we make what we call
  • 48:19a single chain variable fragment,
  • 48:21SCFE.
  • 48:21It's only the variable sequences
  • 48:23of the light and heavy chains.
  • 48:26And that's been optimized to
  • 48:27increase the affinity for DNA.
  • 48:29And then we stuck a couple of those
  • 48:31together to make a dye single chain
  • 48:33fragments to bump up the avidity for DNA.
  • 48:35And that works really well against HR
  • 48:37deficient cancer cells and tumors.
  • 48:38And that's the product that was
  • 48:40finally licensed by biotech company
  • 48:42Patriss as we talked about who
  • 48:44then has helped with funds to
  • 48:45allow us to humanize and demonize
  • 48:47and further optimize the CDR's.
  • 48:49To develop the antibody,
  • 48:51I would like to now introduce
  • 48:53named Deoxy Mab one or DX1 deoxy
  • 48:55mab 'cause DNA mab for antibody.
  • 48:58Yeah,
  • 48:58you get it.
  • 49:00DX1 is well if ENT two is my
  • 49:03favorite transporter DX one is
  • 49:05probably my favorite antibody,
  • 49:06although there is some other ones that
  • 49:09are kind of catching my attention too.
  • 49:11It works great,
  • 49:11penetrates so clearly into the
  • 49:13nucleus we see triple negative
  • 49:14breast cancer cells on the left and
  • 49:16breast cancer brain Mets else on the right.
  • 49:18It's killing the cells,
  • 49:20it's sensitizing to radiation,
  • 49:22but it's still leaving normal cells alone.
  • 49:24Just what we want and we are
  • 49:26excited to move this towards
  • 49:27clinical trial testing.
  • 49:28But wait a second did I?
  • 49:30Am I saying something about
  • 49:32brain Mets cells but?
  • 49:36Am I arguing that we could maybe
  • 49:37be using this to treat brain Mets?
  • 49:38This is an antibody, right?
  • 49:39I mean this, the story is
  • 49:40already kind of far fetched, but.
  • 49:42Brain Mets well, The thing is one of
  • 49:44the key biologic markers that predict
  • 49:46sensitivity to our antibody is loss
  • 49:49of P-10 even more so than bracket for.
  • 49:51And I know that all the DNA repair
  • 49:53experts are probably jumping
  • 49:54up and down right now saying,
  • 49:55well the P-10 HR link is not
  • 49:59completely proven and fine, but.
  • 50:01Cells at a P-10 deficient are
  • 50:04killed by this antibody and brain.
  • 50:06Mints often exhibit P-10 loss even when
  • 50:09the primary tumor is P 10 positive
  • 50:11and that's either evolution towards
  • 50:14metastasis or secretion of P-10
  • 50:17suppressive micro RNAs by astrocytes.
  • 50:20It's also worth noting P-10 is lost
  • 50:21an awful lot in primary GBM, so.
  • 50:23Maybe we've got treatment for GBM as well.
  • 50:28But I hear what you're saying.
  • 50:30Brain Mets brain tumors with an antibody.
  • 50:34I I don't understand what the
  • 50:35why the skepticism.
  • 50:36I mean,
  • 50:37all we need is DNA and nucleosides
  • 50:39and the E NT 2 transporter right?
  • 50:41It seems like the antibody should be able
  • 50:43to get into the brain tumors just fine it.
  • 50:45Oh oh right.
  • 50:49The blood brain barrier.
  • 50:53I guess that does pose something of an issue.
  • 50:57Maybe I should have thought this through
  • 50:59before I signed up for this talk.
  • 51:00I guess it's too late to back out now though.
  • 51:03If we look really closely at the
  • 51:06blood brain barrier and we look in
  • 51:08at the brain endothelial cells.
  • 51:10Whoa, there's my buddy.
  • 51:13E and T2. The luminal surface
  • 51:15of the brain endothelial cell.
  • 51:17And it actually regulates nucleoside flux
  • 51:19into and out of the central nervous system.
  • 51:22So wait a second.
  • 51:23This is all starting to come full circle now.
  • 51:26I think I'm arguing that our
  • 51:28antibody DX1 can get over the river,
  • 51:30the bloodstream, and through the woods.
  • 51:33The blood brain barrier and
  • 51:34into the brain tumor.
  • 51:35We will go where the antibody will
  • 51:38then bind DNA and inhibit DNA repair
  • 51:41and kill those brain Mets and also.
  • 51:44The possibly last owner.
  • 51:48And we are seeing that this indeed is
  • 51:51working out very well season. Sorry.
  • 51:55We are finding that DX1 does cross transwell
  • 51:58models of the blood brain barrier and it
  • 52:00does so in an EMT 2 dependent manner.
  • 52:02If you shut down EMT 2 the antibody
  • 52:04can't get across in the middle panel.
  • 52:06Here we're showing mice with
  • 52:08orthotopic brain tumors.
  • 52:09GBM in this case.
  • 52:11And we gave the antibodies.
  • 52:13We gave the mice antibodies labeled so
  • 52:14that we can see it on imaging and the
  • 52:17antibody gets into the brain tumor great.
  • 52:19But not if we treat the mice
  • 52:20with an inhibitor of ENT two,
  • 52:22so you shut down ENT 2,
  • 52:23the antibody can't get into the brain tumor,
  • 52:25so it's working like we think.
  • 52:27And it seems to actually matter.
  • 52:29It makes a difference.
  • 52:30The panel on the right.
  • 52:31These are mice treated with the antibody
  • 52:34with P-10 deficient patient derived GBM.
  • 52:37DX1 by itself.
  • 52:38No radiation.
  • 52:39No chemotherapy significantly suppresses
  • 52:41the tumor growth and extends survival.
  • 52:49There we go. Brain Mets are a little
  • 52:52harder to study, and GBM in mice.
  • 52:54Just implant the tumor intracranially
  • 52:57and watch and it goes, but that's not
  • 52:59really fair for a brain met model.
  • 53:01So the way we study brain Mets has been
  • 53:03taught to us by our good friend and
  • 53:06colleague Jangling Zhao and mastered by
  • 53:08research associate my lab benedet caffari.
  • 53:10These are not easy experiments.
  • 53:14Breast cancer cells are injected
  • 53:15into the hearts of the mice to allow
  • 53:18them exposure to the circulation,
  • 53:19and we use a brain seeking some type of
  • 53:21the cancer cells to go to the brains.
  • 53:24And you can then track those based
  • 53:26on their signal on serial imaging.
  • 53:28So if we give the mice brain mats and
  • 53:30we treat them with tail vein injections
  • 53:32with control, or the antibody,
  • 53:34just the antibody, no radiation,
  • 53:36no chemotherapy.
  • 53:37I think that picture up in the
  • 53:39top right says it all.
  • 53:41It suppresses the brain
  • 53:42Mets quite phenomenally,
  • 53:44and it does also extend survival,
  • 53:47so this is looking really, really good.
  • 53:49But now I start to hear the words
  • 53:51again in the back of my head.
  • 53:52I don't know if anybody else can hear them.
  • 53:54Do we really need a new
  • 53:56way to treat breast cancer?
  • 53:57Brain Mets, I think isn't that
  • 53:58what the gamma knife is for?
  • 53:59It's it's easy, right?
  • 54:00You see a brain,
  • 54:01but you just send the patient to the
  • 54:02gamma knife and I would say, Oh no.
  • 54:05It's not so easy.
  • 54:06First of all,
  • 54:07not everybody is a candidate for
  • 54:08the gamma knife and gamma knife
  • 54:10does work really quite well,
  • 54:11but there are risks.
  • 54:12Radiation, necrosis,
  • 54:13as Doctor Chang will attest
  • 54:15to is is a problem,
  • 54:17and there's a lot of a lot of it that occurs.
  • 54:19And when disease comes back after the game,
  • 54:21and if it's even harder to take care
  • 54:23of and then there are the patients for
  • 54:25which gamma knife is unfortunately not an
  • 54:27option that require whole brain radiation.
  • 54:29And even with our fancy
  • 54:30spinning of the beams,
  • 54:31hippocampal sparing in the manting,
  • 54:33it carries risks of neurotoxicity so.
  • 54:37I, I don't think there's any question.
  • 54:39If there was a way to reduce the
  • 54:40need for radiation or at least lower
  • 54:42the dose of radiation required,
  • 54:43that would make a big benefit for
  • 54:45patients with breast cancer and brain
  • 54:48metastases and other tumors as well.
  • 54:50So we have already started looking.
  • 54:54Into this and we have a DoD grant
  • 54:57to help us conduct this work.
  • 54:59I think DX one is perfectly poised to
  • 55:02lower the needed dose of radiation,
  • 55:04and I think radiation is perfectly
  • 55:06poised to help the X one get into
  • 55:08the brain nuts because remember.
  • 55:10DX1 is looking for and using DNA to get
  • 55:13into the tumors so dose of radiation
  • 55:15to increase tumor death and release
  • 55:17DNA should recruit more DX1 to the brain.
  • 55:20Mets and the bar graphs are
  • 55:21shown on the bottom there.
  • 55:22That's so that's exactly what we see.
  • 55:25We see the most.
  • 55:25DX one get in the brain.
  • 55:26Mets in the mice that get treated
  • 55:29concurrently with the radiation.
  • 55:30And take it to the obvious extreme.
  • 55:33Those mice that get DX1 with the
  • 55:35radiation also have the best response
  • 55:37we see the less number of metastases,
  • 55:39so it's all working the way we expect.
  • 55:41I understand it's all preclinical,
  • 55:43it's in mice, but sometimes things
  • 55:45start to come together and it's
  • 55:47it's looking very promising to me.
  • 55:49Obviously very biased and.
  • 55:51Very grateful to Benedet and Marta
  • 55:53and Caroline and who for all their
  • 55:55work with this it's it's been tough
  • 55:57but we've hung through here and it's
  • 55:59it's going really quite well now.
  • 56:02And again, can't do any of this
  • 56:04without help from your friends.
  • 56:06I had to cross out the word little and say
  • 56:08a lot because jangle from neurosurgery has.
  • 56:11Been great friend and
  • 56:12colleague to me for years.
  • 56:13Now he is an expert in all things related
  • 56:15to the brain and brain tumors and ways
  • 56:18to treat tumors in the brain and he's
  • 56:20helped us figure out these models.
  • 56:21He's very interested in the dxy antibody.
  • 56:23We work together all the time,
  • 56:25but he also does some other things.
  • 56:26Believe it or not,
  • 56:27a lot of other things.
  • 56:29And just recently had a great
  • 56:31paper in nature cell biology where
  • 56:33he reported on a new effect of an
  • 56:36LRRC 31 protein that significantly
  • 56:38sensitizes breast cancer brain
  • 56:40metastases to radiation therapy.
  • 56:42So it's almost like the universe is.
  • 56:44Telling us something because that
  • 56:48LRRC 31 protein is great.
  • 56:51They can't cross the blood brain barrier.
  • 56:54I think I just spent the last 20 minutes
  • 56:56talking about an antibody that can
  • 56:58carry things across the blood brain barrier.
  • 57:00So we got the Hanson lab and the
  • 57:02shower lab working together and we
  • 57:04can put them together and make a
  • 57:06DX1 LRRC 31 fusion protein that I'm
  • 57:09hopeful is going to be next in line
  • 57:11to cross over the river through the
  • 57:14woods and into the brain and increase
  • 57:16sensitivity to radiation therapy.
  • 57:18So hopefully that was of some
  • 57:21interest to people listening here.
  • 57:23I have a lot of people to thank.
  • 57:25Obviously jeongbang Zhao.
  • 57:27Great colleague.
  • 57:29Of.
  • 57:29Joe Contessa and Marta Bero in his lab
  • 57:32have been incredibly helpful to us.
  • 57:34My own lab.
  • 57:35I'm extremely grateful to your efforts.
  • 57:37I'm I'm usually at the gamma knife nowadays,
  • 57:38so you probably are wondering
  • 57:40where I've been here I am.
  • 57:42If you're looking for me,
  • 57:42it still looks like me,
  • 57:43right?
  • 57:44And the Yale Gamma knife team
  • 57:46everybody is is great help.
  • 57:48I'm appreciative to everybody but
  • 57:50last anyone that knows me knows
  • 57:52that I'm a fan of the Marvel movies
  • 57:54and the best part about the Marvel
  • 57:56movies is always there's one more
  • 57:57cut scene at the very end, right?
  • 57:59And I think if you.
  • 58:00Listen very carefully and you
  • 58:02look off into the horizon.
  • 58:04You will see that there is a team
  • 58:07coming assembled led by Pi Megan King.
  • 58:10Getting the best breast cancer,
  • 58:11brains researchers together to
  • 58:13expand and improve DNA targeted
  • 58:15therapies towards better breast
  • 58:16cancer treatment with Pat Larusso,
  • 58:18Megan Kingaby Patel, Ryan Jensen,
  • 58:20myself and Zhangzhou,
  • 58:21and we're very thrilled to have
  • 58:23been notified.
  • 58:24Just recently that we've received
  • 58:25the YCC Team Challenge Award
  • 58:27to conduct this work.
  • 58:29With that I will take a breath
  • 58:32and stop talking and answer any
  • 58:34questions if there are.
  • 58:36Well, I think we're a little short on time,
  • 58:40so it's it's exactly 1 now.
  • 58:43James that was great.
  • 58:46I'm certainly extraordinarily supportive
  • 58:47of anyone who wants to study breast cancer.
  • 58:51Brain tests is an area that I've
  • 58:53thought about a lot over the years,
  • 58:55and I would agree with you 100% that it's
  • 58:59an area that's perhaps the most in need,
  • 59:04and potentially the most
  • 59:05in need in breast cancer.
  • 59:06Because we we we may well be able
  • 59:08to eradicate disease elsewhere in
  • 59:10virtually everyone in the next decade.
  • 59:13But the brain is the hardest place,
  • 59:14it seems so.
  • 59:16With that I wanna thank both
  • 59:19both of you for great talks.
  • 59:21It's been a great grand rounds and James.
  • 59:23Congratulations on the on
  • 59:25the challenge award.
  • 59:28Thank you so much, alright?
  • 59:31I made thanks again bye bye.
  • 59:35And thanks to our audience.