Friday, October 28, 2016

Halloween and the Academy

The Academy has become the extreme distortion of our society. The NY Times writes:

And when it comes to offensive behavior, one rarely hears students critique or police pop music that is riddled with abusive-sounding insults, mostly about women. Profanity, in general, doesn’t seem to be something students worry about. Walk through a crowded cafeteria and curse words pop up gratuitously among students as if vocabulary has failed them. When I say, “Hey, ease up on the language, O.K.?” they look at me with incomprehension, not realizing they’ve said anything offensive. College officials won’t tell students to avoid this kind of horrible stuff, though. Establishing behavioral codes that curtail hookup culture and profanity — those old-fashioned proprieties — are sexist and puritanical, we are told.

Boy am I old! As an Engineer we never had time to cavort at Halloween. I never saw a costume. In 1962 for example we were more concerned about a nuclear strike on New York that about offending some "protected" group. 

If one has so much free time as Liberal Arts students seem to have, then this is not an education, it is expensive baby sitting. For $70,000 per year you get to ship your kid off to some place that teaches them some useless stuff so that when they "graduate" the cannot get a job and move in back home. 

Worse they have debt which the parent co-signed for and the parent's  retirement goes out the window.

Thursday, October 27, 2016

More on the Academy and VCs

MIT sent out a letter to its Community making a statement as to what it is trying to do with it purported VC fund. It states:

Today, innovators in fields like energy, manufacturing, robotics, biotech and medical devices often find it extremely difficult to secure the sustained funding, space, equipment, expertise and networks to fully develop their technologies; this struggle itself can needlessly prolong the development process, stretching it to a decade or more. All too often, "tough-tech" entrepreneurs never find sufficient support, which discourages others from trying, a dynamic that leaves many promising ideas stranded in the lab.  MIT's mission statement directs us not only to "advance knowledge" and "educate students," but also to bring "knowledge to bear on the world's great challenges." If we hope to deliver serious technological solutions to urgent global challenges — like clean water, climate change, sustainable energy, cancer, Alzheimer's, infectious disease, and more — we need to make sure the innovators working on those problems see a realistic pathway to the marketplace. The Engine can provide that pathway by prioritizing breakthrough ideas over early profit, helping to shorten the time it takes these ventures to become "VC-ready," providing local space and comprehensive support in the meantime, and creating an enthusiastic community of inventors and supporters focused on delivering new science-based innovation to make a better world.

The essence of Venture Capital is to seek out people with interesting ideas. However it is "people" first. A leader and a team capable of dealing with the uncertainties of an early stage business.

Yes, at a place like MIT, one whose mission is education and research, there is often a gap between the results of research and the reality of a business. I have seen that for fifty years. On the business side we want dedication, complete and absolute dedication, to the execution of the business. Research is allowed if and only if it is essential to supporting the imminent goals of the business. VC firms are making investments. We are not like DARPA or NSF. I have run research groups in the corporate world, yet even there I demanded a nexus to executable reality.

Now the statement above in my opinion shows gross ignorance of the VC world. Yes there often is a gap between the research product and the marketable entity. Oftentimes the gap is the people. Researchers are all too often not those who can execute. They go on to the next unknown not to delivery of a financially viable product.

Also a University should not, must not, be in the business of selecting winners and losers regarding business options. They know and understand research, not business. So what is "VC ready". To me it means that a team is in place, people who can execute. Those people are dedicated and devoted to the task. They do not want to continue research but want to spend the totality of their existence to execution of a business.

I am still working in this space after fifty years. Ideas from the edge of a University to commercial application. A day to me is eternity. A day to an academic is another Faculty meeting. Grad students oftentimes do not want to go into the real world. Post docs are often worse. They have accepted a position paying a paltry salary to continue research in hopes of some faculty position somewhere in the future. They are often disappointed. Making them entrepreneurs in my experience is both a disservice to them and to any investor.

Medical research is very risky, very costly, and takes many years. Just try and get a new drug through the FDA. The success rates in this area are de minimis. So why encourage researchers to think they can succeed when so many other real business have not.

One must ask why this step was even taken at MIT, an institution surrounded by VCs. It truly is not for want of opportunities or money. It could in my opinion be a poorly envisioned money pit, but worse in my opinion is may take competent researchers and make them think they are entrepreneurs while keep warm in the confines of MIT.

ACA and Reality

We have been following Health Care Policy for eight years now. It is easier to do cancer genomics than it is to unravel the uncertainties and complexities of Government Health Care Policy.

Along comes the MIT "Professor", one of the authors, who thinks we are all stupid, if memory serves me correctly, in NEJM and states:

As the country focuses on the 2016 election, we offer several key messages from our findings. State implementation continues to strongly affect the success — or shortcomings — of the ACA. This reality is most obvious in decisions about whether to expand Medicaid under the law, since the lack of expansion in 19 states has left roughly 3 million adults without coverage. But state policies also affect middle-income families’ ability to sign up for exchange coverage, which has been impaired in some states by legislative barriers to enrollment and lack of outreach. In essence, some state policymakers who rail against the ACA as a failed policy have created a self-fulfilling prophecy by taking steps to prevent people from signing up and benefiting from new coverage. Such actions may have contributed to the large gap between exchange enrollment rates in states participating in the federal exchange and those in states with their own exchanges. Though undermining coverage expansion may be politically expedient in some places, it is indefensible from a public health perspective. With one presidential candidate pledging to build on the ACA and the other pledging to repeal it, and with state-level battles over the law ongoing, much is at stake in this year’s election. Overall, our results reveal several ACA provisions working effectively to expand health insurance coverage to millions of Americans. Whether the law continues to expand coverage in the future most likely hinges on the outcome of the November election.

The ACA has become a bureaucratic mess. From ICD 10, the EHR, Quality Metrics, ACOs, and the like we now have a system which has massive overhead, dramatic burdens, reduced care etc. One of the major problems is that the Millennials do not get the mandate policies thus making private, or any other plan, excessively costly. We argued eight years ago that it must be universal, it must cover all, like auto insurance. Otherwise there will be arbitrage. Humans, even the Millennials, are economic machines optimizing their own returns. So why pay for something if you don't need it.

Then again in this article in NEJM we have the author who feels we folks are just fools, I think that is what he implied, and we can be manipulated, again I believe he implied that as well. Thus in my opinion why would one ever listen again? Poor MIT, what is happening? Leadership perhaps?

The Academy and Venture Investments

In a recent piece by xconomy they write:

MIT is investing $25 million in a potentially $150 million venture capital fund and opening a 26,000-square-foot startup space on the edge of its campus....But MIT hadn’t gone that route. A few months before she retired in June, former MIT Technology Licensing Office director Lita Nelsen told Xconomy she felt the Institute simply hasn’t needed to form a VC fund on campus because the school has no trouble getting “decent companies” funded. She raised concerns about conflicts of interest, clearly defining the mission of such a fund, and setting realistic expectations. Nelsen also presented a hypothetical scenario in which MIT spinouts that didn’t receive an investment from a university venture fund might have trouble raising money later from outside VC firms. “There’s a negative-select bias there for what we don’t invest in,” she said at the time. “So, better to [create] a level playing field for anybody who wants to play.”

MIT makes the announcement as follows:

The Engine is designed to meet an underserved need. In Kendall Square and Greater Boston, many breakthrough innovations cannot effectively leave the lab because companies pursuing capital- and time-intensive technologies have difficulty finding stable support and access to the resources they need.

They continue:

To fuel The Engine, MIT will seek to attract hundreds of millions of dollars of support and to make available, for entrepreneurs, hundreds of thousands of square feet of space in Kendall Square and nearby communities. The Engine will also introduce startups to their entrepreneurial peers and to established companies, in innovation clusters across the region and around the world: It seeks to power a network of innovation networks.

Frankly the above statement is in my opinion inaccurate on its face. There is a wealth of investment opportunities in the area, as in and around Stanford. It is a well running Darwinian machine. So why change? 

Frankly I could not agree more with Lita. She had spend decades managing the MIT IP portfolio. As a result she had first hand experience in how VCs operate. Having done over 35 start ups myself, the investments are "ruthless". Namely the investment works or does not work. If it does then monetize it and if not burn it.

Fundamentally doing a start up is a "burn your boats" scenario. You leave the comfort of MIT, go to some cheap place, tell your story again and again, manage a team, raise money by selling a viable dream. You do not stay at MIT in some incubator.

I firmly believe that this will become a disaster. The amount of $125 million could be be spent better on MIT's mission, education and research, not high tech investment. It is worse than a telephone company trying to get into the entertainment business. It is a clash of cultures. Lita presented all the correct responses. However it appears that the ever increasing administrative hierarchy wants more and more stuff to control. Investments in and operation of a VC is NOT the way to go.

My first start up involvement was in 1969. An EG&G back company, it made one mistake and it was dean in a heart beat. How do you do this if your people are comfortable in a MIT owned and operated facility? Or is this the Millennial generations approach to high tech, never leave home and have perpetual care and up keep?

Don't Have a Horse in the Race But...

This is a messy political year and I am not saying anything new. I don't have any horse in this race, and the race is truly a horse race...however one sees it. But I am surprised, say even shocked, to see Nature have some PhD candidate form U Penn, that bastion of socialist values comment on the American public.

This writer for example opines:

My own research shows that voters with the largest discrepancies in their affective evaluations of the two candidates, which I refer to as emotional investment in the election, experienced the largest changes in perceptions of electoral integrity following the 2012 presidential election. Among supporters of a losing candidate, the stronger their affective preference for the candidate, the greater are their doubts about the fairness of the process. Regardless of the outcome in 2016, the supporters of the loser are all but guaranteed to have a historically extreme dislike for the winner. Unfortunately, we should expect confidence in the election result to suffer accordingly.

I suspect this young person has never been at a Bingo parlor or a racetrack! Democracy is messy, it always has been, even in Greece, just ask Socrates...

But why, one wonders, does such an esteemed journal, such as Nature, dig up some neophyte to comment on American democracy? Why not just ask people at random waiting for the Times Square Shuttle...after all, they are people!

Wednesday, October 26, 2016

What Took So Long?

In a report by Tech Dirt they note:

Back in August a report emerged claiming that Google Fiber executives were having some second thoughts about this whole "building a nationwide fiber network from the ground up" thing. More specifically, the report suggested that some executives were disappointed with the slow pace of digging fiber trenches, and were becoming bullish on the idea of using next-gen wireless to supplement fiber after acquiring fixed wireless provider Webpass. As such, the report said the company was pondering some staff reductions, some executive changes, and a bit of a pivot. Fast forward to this week when Access CEO Craig Barrett posted a cheery but ambiguous blog post not only formally announcing most of these changes, but his own resignation as CEO. According to Barrett, Google will continue to serve and expand Google Fiber's existing markets (Austin, Atlanta, Charlotte, Kansas City, Nashville, Provo, Salt Lake City, and The Triangle in North Carolina), and will also build out previously-announced but not yet started efforts in Huntsville, Alabama; San Antonio, Texas; Louisville, Kentucky; and Irvine, California. 

I love the new Valley words; pivot, disrupt, etc.  We have argued for a decade that fiber is too costly and rant with delays and that wireless is the way to go. Yes a decade, ten years, 10% of a century, 1% of a millennium. So much for those bright minds at Google. Perhaps the fact of life has finally hit them in the head.

Now for wireless. You need a license. Lots of them actually. So where do they get them?  Acquisitions, but costly one.

CAR T Cells and Cancer

We have recently published a Technical Report summarizing CAR T Cells and their application to cancers. We provide a summary here. The immune system is a powerful tool that can be used in a variety of ways. One problem is that it seems that every day we discover another subtlety regarding how this functions. It is a tool, and a very powerful tool, that can be used as a scalpel or a butchering ax. With the advent of CARs, specifically designed killer T cells, CTLs or cytotoxic T cells, one can attack cancer cells, however at times this tool can explode in our hands. This paper is an attempt to examine the CAR T Cells as a tool which can be engineered. The problem we face however is that in engineering the tool we oftentimes do not have a full grasp of its effects.

Our intent herein is not to provide a detailed up to date review of CARs but to provide a summary introduction to the potential they provide. This area is still very much a work in progress and as such is subject to ongoing change.

Steven Rosenberg has been studying how best to use the immune system to fight cancer. His 1992 was a prescient piece that laid out the future opportunities. From then until now, some 25 years, we know a great deal about the immune system which was lacking then and furthermore we have a wealth of tools to manipulate the cells involved.

From Kahilil et al we have an introduction to CARs which provide continuity from the work on monoclonal antibodies, MABs:

In the past decade, advances in the use of monoclonal antibodies (mAbs) and adoptive cellular therapy to treat cancer by modulating the immune response have led to unprecedented responses in patients with advanced-stage tumors that would otherwise have been fatal. To date, three immune-checkpoint-blocking mAbs have been approved in the USA for the treatment of patients with several types of cancer, and more patients will benefit from immunomodulatory mAb therapy in the months and years ahead.

Concurrently, the adoptive transfer of genetically modified lymphocytes to treat patients with hematological malignancies has yielded dramatic results, and we anticipate that this approach will rapidly become the standard of care for an increasing number of patients. In this Review, we highlight the latest advances in immunotherapy and discuss the role that it will have in the future of cancer treatment, including settings for which testing combination strategies and 'armored' CAR T cells are recommended.

From Batlevi et al we have a discussion on the flow from MABs to CARs with a nexus to checkpoint inhibitors, namely PD-1 inhibitors:

The success of the anti-CD20 monoclonal antibody rituximab in the treatment of lymphoid malignancies provided proof-of-principle for exploiting the immune system therapeutically. Since the FDA approval of rituximab in 1997, several novel strategies that harness the ability of T cells to target cancer cells have emerged.

Reflecting on the promising clinical efficacy of these novel immunotherapy approaches, the FDA has recently granted 'breakthrough' designation to three novel treatments with distinct mechanisms.

First, chimeric antigen receptor (CAR)-T-cell therapy is promising for the treatment of adult and pediatric relapsed and/or refractory acute lymphoblastic leukemia (ALL).

Second, blinatumomab, a bispecific T-cell engager (BiTE®) antibody, is now approved for the treatment of adults with Philadelphia-chromosome-negative relapsed and/or refractory B-precursor ALL.

Finally, the monoclonal antibody nivolumab, which targets the PD-1 immune-checkpoint receptor with high affinity, is used for the treatment of Hodgkin lymphoma following treatment failure with autologous-stem-cell transplantation and brentuximab vedotin.

Herein, we review the background and development of these three distinct immunotherapy platforms, address the scientific advances in understanding the mechanism of action of each therapy, and assess the current clinical knowledge of their efficacy and safety. We also discuss future strategies to improve these immunotherapies through enhanced engineering, biomarker selection, and mechanism-based combination regimens.

One of the observations when dealing with cancer and the immune system is that once when on tries a specific approach one often finds new mechanisms which can either be used or must be thwarted.

From Jackson et al there is a discussion of the work of CARs using CD-19 targets:

The engineered expression of chimeric antigen receptors (CARs) on the surface of T cells enables the redirection of T-cell specificity. Early clinical trials using CAR T cells for the treatment of patients with cancer showed modest results, but the impressive outcomes of several trials of CD19-targeted CAR T cells in the treatment of patients with B-cell malignancies have generated an increased enthusiasm for this approach. Important lessons have been derived from clinical trials of CD19-specific CAR T cells, and ongoing clinical trials are testing CAR designs directed at novel targets involved in hematological and solid malignancies.

In this Review, we discuss these trials and present strategies that can increase the antitumor efficacy and safety of CAR T-cell therapy. Given the fast-moving nature of this field, we only discuss studies with direct translational application currently or soon-to-be tested in the clinical setting.

CAR T cells are chimeric antigen receptors on T cells. Chimeric because one designs them specifically for the target cells and essentially crated a multiheaded receptor that matches the antigen presented by the tumor cell.

We provide a simple example below for a third-generation CAR:

The function of this designed T cell it to allow a normal CTL, killer T cell, attach to a cancer cell with a recognizable antigen, and then to do what CTLs do well, allow the attacked cell to go into apoptosis, and just disappear, its constituents being used elsewhere.

We demonstrate this process graphically above. We now review some of the key functions of T cells. The two types of T cells of interest are T helper and T killer or cytotoxic T cells, CTL. The CTL is the prime target of interest for it is the cell which can attach to a tumor cell and effect apoptosis of the tumor cell by its normal operations. The T helper supports the CTL by expressing IL-2 which allows for proliferation of that specific CTL type.

The CTL has surface receptors as shown below. Two are extending well beyond the cell wall and the remaining four are below the cell wall and provide for intra cellular activation. The complex acts in unison attaching to targeted cells. Now the essence of CARs is to modify this receptor so as to effect targeting of tumor cells and their exposed antigens.

This CTL binding process is shown below. Simply the process is as follows:

1. An antigen presenting cell, APC, in this case a tumor cell, presents an antigen using the MHC I molecule. Also, the tumor cell may have another surface protein that results in the presentation of a tumor specific surface molecule like CD-19 in the case of hematological malignancies. The process starts with the ability to identify this molecule.

2. Then the CTL has a matching or cognate receptor which aligns with the MHC I and Ag combination and it attaches itself, and via CD-8 strongly binds to the cell, also using CD-3.

3. Upon binding the CTL can release cytokines or equivalents that result in the apoptosis of the cell.

We show the apoptosis below. Here the bound CTL recognizes the cancer cell and then releases apoptosis inciting proteins.

Thus, for any cancer cell we should be able to use this process, if we first know the Ag that is presented and second if we can create a receptor on a T killer cell, CTL, that recognizes that ligand and in turn can activate the apoptotic process.

In the simplest terms this is how we might proceed.

1. Extract a tumor cell.

2. Ascertain the surface molecules and determine which one is unique to that type of cell and NOT common in other cells. You don't want the CTLs attacking everything.

3. Create a binding receptor for that ligand.

4. Extract the patient's CTL and insert by some reverse transcription manner, or CRISPR type approach the genes for that designed receptor.

5. Grow these modified cells in vitro using IL-2 or the like.

6. Insert these back in the patient.

This is the "back of the envelope" approach to CAR therapy. Of course, there are many obstacles and the approach uses tools which may have to be gathered from afar. But as those who have developed CARs have shown it is doable.

Now what we have described above is not that simple. There are what are called a variety of "Checkpoint Inhibitors" that are an integral part of the control mechanisms of the immune system so that it does not go wild and destroy itself.

Let us begin with a brief review of PD-1 pathways. We have previously discussed the CTLA-4 blockage and the current approaches used to inactivate that element of T cell suppression. We summarize that again in the figure below.

 Now CTLA-4 is not the only inhibitor of T cell action. PD-1 also can be activated and thus suppress T cell activity. This means that is we can find a way to inactivate or inhibit PD-1 then we have another way to seek possible activation of the T cells. In fact, perhaps we can do both and secure a super active T cell base. That is in essence the Wolchok approach. We depict this in the figure below.

The paper by Okazaki and Honjo in 2007 also details many of the critical elements regarding the PD-1 and its ligands. It details many of the recognized disease states as well. As they state:

Since the discovery of PD-1 in 1992, the biological function of PD-1 remained mystery for many years. Generation of Pdcd1mice and the discovery of its ligands turned around the situation and the function of PD-1 was unveiled thick and fast in these 5 years. Consequently, it became clear that PD-1 plays critical roles in the regulation of autoimmunity, tumor immunity, infectious immunity, transplantation immunity, allergy and immune privilege. The development of autoimmune diseases by Pdcd1 mice especially enchanted clinicians and promoted clinical research as well.

Currently, many groups are trying to generate not only PD-1 antagonists for the treatment of cancer and infectious diseases but also PD-1 agonists for the treatment of autoimmune diseases, allergy and transplant rejection. Among these, humanized antibody against human PD-1 was approved by Food and Drug Administration of the United States as an investigational new drug in August 1, 2006. Clinical trials will test its clinical efficacy on cancer and infectious diseases.

Now we can examine the features of PD-1. As Freeman states:

T cell activation requires a TCR mediated signal, but the strength, course, and duration are directed by costimulatory molecules and cytokines from the antigen-presenting cell (APC). An unexpected finding was that some molecular pairs attenuate the strength of the TCR signal, a process termed co-inhibition.

The threshold for the initiation of an immune response is set very high, with a requirement for both antigen recognition and costimulatory signals from innate immune recognition of ‘‘danger’’ signals. Paradoxically, T cell activation also induces expression of co-inhibitory receptors such as programmed death-1 (PD-1).

Cytokines produced after T cell activation such as INF- and IL-4 up-regulate PD-1 ligands, establishing a feedback loop that attenuates immune responses and limits the extent of immune-mediated tissue damage unless overridden by strong costimulatory signals. PD-1 is a CD28 family member expressed on activated T cells, B cells, and myeloid cells. In proximity to the TCR signaling complex, PD-1 delivers a co-inhibitory signal upon binding to either of its two ligands, PD-L1 or PD-L2.

Engagement of ligand results in tyrosine phosphorylation of the PD-1 cytoplasmic domain and recruitment of phosphatases, particularly SHP2

Additional insight can also be provided by examining the regulatory T cells as well. As Francisco et al state:

Regulatory T cells (Tregs) and the PD-1: PD-ligand (PD-L) pathway is both critical to terminating immune responses. Elimination of either can result in the breakdown of tolerance and the development of autoimmunity. The PD-1: PD-L pathway can thwart self-reactive T cells and protect against autoimmunity in many ways. In this review, we highlight how PD-1 and its ligands defend against potentially pathogenic self-reactive effector T cells by simultaneously harnessing two mechanisms of peripheral tolerance: (i) the promotion of Treg development and function and (ii) the direct inhibition of potentially pathogenic self-reactive T cells that have escaped into the periphery.

Treg cells induced by the PD-1 pathway may also assist in maintaining immune homeostasis, keeping the threshold for T-cell activation high enough to safeguard against autoimmunity. PD-L1 expression on non-hematopoietic cells as well as hematopoietic cells endows PD-L1 with the capacity to promote Treg development and enhance Treg function in lymphoid organs and tissues that are targets of autoimmune attack. At sites where transforming growth factor-β is present (e.g. sites of immune privilege or inflammation), PD-L1 may promote the de novo generation of Tregs.

CAR cells are essentially engineered T cells, specifically cytotoxic T lymphocytes, CTL, engineered to target specific cells such as those in various hematopoietic cell lines. such as leukemias and lymphomas. There is no fundamental reason that they cannot be used for solid tumors but there are certain operational barriers which must be overcome.

As Kershaw et al note:

There are two main types of antigen receptors used in genetic redirection.

The first utilizes the native alpha and beta chains of a TCR specific for tumor antigen.

The second is termed a chimeric antigen receptor (CAR), which is composed of an extracellular domain derived from tumor-specific antibody, linked to an intracellular signaling domain. Genes encoding these receptors are inserted into patient's T cells using viral vectors to generate tumor reactive T cells….

The specificity of CARs is derived from tumor-specific antibodies, which are relatively simple to generate through immunization of mice. Recombinant techniques can be used to humanize antibodies, or mice expressing human immunoglobulin genes can be used to generate fully human antibodies. Single-chain variable fragments of antibodies are used in the extracellular domain of CARs, which are joined through hinge and transmembrane regions to intracellular signaling domains.

As Miller and Sadelain note:

The advent of gene transfer technologies, in particular those enabling the transduction of human T lymphocytes using gibbon ape leukemia virus envelope-pseudotyped g-retroviral vectors, created new opportunities for immune intervention based on T cell engineering. Patients’ T cells, easily accessible in peripheral blood, can be genetically instructed to target tumors by transduction of receptors for antigen, utilizing either the physiological TCR or synthetic receptors now known as CARs.

Both approaches have shown clinical successes, particularly in melanoma, targeting NYESO1, and in acute lymphoblastic leukemia, CARs are artificial, composite receptors for antigen that integrate principles of B cell and T cell antigen recognition. They are particularly attractive in that they elude human leucocyte antigen (HLA) restriction and are thus applicable to all patients irrespective of their HLA haplotypes, unlike TCRs. CARs may also overcome HLA downregulation by tumors, which deprives T cells of a ligand for their endogenous TCR.

The critical function of CARs is, however, not to merely target the T cells to a tumor antigen, but to enhance T cell function. Thus, effective CARs further integrate principles of T cell costimulation and provide a broad spectrum of functional enhancements acquired by directly soliciting selected costimulatory pathways

Juillerat et al note:

Adoptive immunotherapy using engineered T-cells has emerged as a powerful approach to treat cancer. The potential of this approach relies on the ability to redirect the specificity of T cells through genetic engineering and transfer of chimeric antigen receptors (CARs) or engineered TCRs1. Numerous clinical studies have demonstrated the potential of adoptive transfer of CAR T cells for cancer therapy but also raised the risks associated with the cytokine-release syndrome (CRS) and the “on-target off-tumor” effect.

To date, few strategies have been developed to pharmacologically control CAR engineered T-cells and may rely on suicide mechanisms. Such suicide strategies leading to a complete eradication of the engineered T-cells will result in the premature end of the treatment. Consequently, implementing non-lethal control of engineered CAR T-cells represents an important advancement to improve the CAR T-cell technology and its safety.

 Small molecule based approaches that rely on dimerizing partner proteins have already been used to study, inter alia, the mechanism of T-cell receptor triggering15. Very recently, Lim and colleagues have adapted this approach to control engineered T-cells through the use of a multichain receptor.

Here, we describe a strategy to create a switchable engineered CAR T-cells. Our approach is based on engineering a system that is directly integrated in the hinge domain that separate the scFv from the cell membrane. In addition, we chose to implement this strategy in a novel CAR architecture that relies on the FceRI receptor scaffold.

The particularity of this design resides in the possibility to split or combine different key functions of a CAR such as activation and costimulation within different chains of a receptor complex, mimicking the complexity of the TCR native architecture. In this report, we showed that the hinge engineering approaches allowed to turn a T-cell endowed with an engineered CAR from an off-state to an on-state.

By controlling the scFv presentation at the cell surface upon addition of the small molecule, our system allowed to further induce the cytolytic properties of the engineered T-cell. Overall, this non-lethal system offers the advantage of a “transient CAR T-cell” for safety while letting open the possibility of multiple specific cytotoxicity cycles using a small molecule drug.

Principles of T Cell Engineering and CAR Design

(A) Integration of B cell and T cell antigen recognition principles in the design of CARs. The heavy and light chain chains, which are components of the B cell receptor and Igs, are fused to the T-cell-activating z chain of the TCR-associated CD3 complex to generate non-MHC restricted, activating receptors capable of redirecting T cell antigen recognition and cytotoxicity.

(B and C) Integration of T cell activation and costimulation principles in dual signaling CARs designed to enhance T cell function and persistence in addition to retargeting T cell specificity. In

(B), the physiological abTCR associated with the CD3 signaling complex is flanked by the CD28 costimulatory receptor.

(C) shows a prototypic second-generation CAR, which comprises three canonical components: an scFv for antigen recognition, the cytoplasmic domain of the CD3z chain for T cell activation, and a costimulatory domain to enhance T cell function and persistence. Unlike the abTCR/CD3 complex, which comprises g, d, ε, and z signaling chains and is modulated by a multitude of costimulatory receptors, CARs possess in a single molecule the ability to trigger and modulate antigen-specific T cell functions.

There are currently three generations of CAR T cell design. We examine each here. As Cartellieri et al note:

In an attempt to extend the recognition specificity of T lymphocytes beyond their classical MHC-peptide complexes, a gene-therapeutic strategy has been developed that allows redirecting T cells to defined tumor cell surface antigens. This strategy uses both the cellular and humoral arm of the immune response by assembling an antigen-binding moiety, most commonly a single chain variable fragment (scFv) derived from a monoclonal antibody, together with an activating immune receptor.

Once this artificial immune receptor is expressed at the surface of a modified T lymphocyte, upon binding of the scFv to its antigen an activating signal is transmitted into the lymphocyte, which in turn triggers its effector functions against the target cell (Figure 2). In the first attempts to reconfigure T cells with antibody specificity the variable parts of the TCR α and β chains were replaced with scFv fragments derived from monoclonal antibodies. These hybrid T-cell receptors were functionally expressed and recognized the corresponding antigens in a non-MHC-restricted manner. As a consequence of the finding, that CD3ζ chain signaling on its own is sufficient for T-cell activation, the first “true” chimeric single-chain receptors were created by fusing a scFv directly to the CD3ζ chain. At that time this concept was called the “T body approach”. Nowadays these types of artificial lymphocyte signaling receptors are commonly referred to as chimeric immune receptors (CIRs) or chimeric antigen receptors (CARs).

The use of CARs to redirect T cells specifically against TAA-expressing tumor cells has a number of theoretical advantages over classical T-cell-based immunotherapies. In contrast to the long-lasting procedure of in vitro selection, characterization, and expansion of T-cell clones with native specificity for MHC tumor peptide complexes, genetic modification of polyclonal T-cell populations allows to generate TAA-specific T cells in one to two weeks. Engraftment with CARs enables T cells to MHC-independent antigen recognition; thus, major immune escape mechanisms of tumors such as downregulation of MHC molecules are efficiently bypassed.

Furthermore, proliferation and survival of modified T cells can be improved by the implementation of a multitude of signaling domains from different immune receptors in a single CAR

Following Cartellieri et al we note regarding all three generations that:

Evolution of CAR signaling capacities.

First generation CARs transmitted activating signals only via ITAM-bearing signaling chains like CD3ζ or FcεRIγ, licensing the engrafted T cells to eliminate tumor cells.

Second generation CARs contain an additional costimulatory domain (CM I), predominantly the CD28 domain. Signaling through these costimulatory domain leads to enhanced proliferation, cytokine secretion, and renders engrafted T cells resistant to immunosuppression and induction of AICD.

(Third Generation) Recent developments fused the intracellular part of a second costimulatory molecule (CM II) in addition to CD28 and ITAM-bearing signaling chains, thus generating tripartite signaling CARs. T cells engrafted with third generation CARs seem to have superior qualities regarding effector functions and in vivo persistence.

The first generation shown below is the simplest.

The second generation is as per below with the added element.

The third generation has added flexibility as shown below and described above.

Now the insertion of the genes to create the previously described receptors uses a reverse transcription process. It is akin to what we see in HIV reverse transcription and specifically uses lentiviruses as the delivery mechanism.

As Naldini notes regarding lentiviruses:

Major hurdles for hematopoietic-stem-cell (HSC) gene therapy include achieving efficient ex vivo gene transfer into long-term repopulating HSCs, preventing activation of oncogenes by the nearby integration of a vector and controlling transgene expression to avoid ectopic or constitutive expression that leads to toxicity.

As compared to early generation gammaretroviral vectors (γ-RVs), HIV-derived lentiviral vectors result in more efficient gene transfer and stable, robust transgene expression in HSCs and their multilineage progeny. Extensive preclinical work indicated important features in vector biology and design that affect genotoxicity and highlighted strategies to alleviate it. The self-inactivating long terminal repeats (LTRs) and integration-site preferences of lentiviral vectors were shown to substantially alleviate insertional genotoxicity.

When tested in γ-RVs, the self-inactivating LTR design was shown to improve the safety of this platform as well. Retrospective analysis of several earlier trials suggests that disease background, transgene function, ex vivo culture and the efficiency of host repopulation can all influence the likelihood that insertional genotoxicity will manifest in a trial.

These data helped to shape the ideas that not all integrating vectors have the same effects and that genome-wide integration of improved vector designs, although still a mutagenic event, can be tolerated in the absence of aggravating circumstances. Self-inactivating lentiviral vectors are also being used to engineer T cells with chimeric antigen receptors (CARs) or T-cell antigen receptors for use in adoptive immunotherapy for the treatment of cancer. The advantages of this new platform in comparison to earlier-generation γ-RVs, which perform satisfactorily in this cell target, are yet to be fully established. Lentiviral vectors are thought to give rise to more robust and stable transgene expression in T cells in vivo, and could facilitate more efficient and versatile ex vivo gene transfer while supporting coordinated expression of multiple transgenes.

These advantages will become more relevant as the gene-therapy field implements refined strategies, such as improved T-cell manipulation to preserve T memory stem cells, or more demanding cell-engineering tasks, such as the co-expression of multiple CARs (to improve specificity) or a conditional safety switch/suicide gene (to improve safety).

We now review the process below. We have initially presented a logical approach, then we explained how it could be accomplished and now we return and demonstrate how this could be accomplished. We explain in detail in the Appendix a multiplicity of such protocols in use today.

Now the mechanism above may lose some elements of control and switch mechanisms to turn it on or off have been considered.

From Wu et al a specific mechanism is presented with its advantages and possible concerns. They state:

Cell-based therapies have emerged as a promising treatment modality for diseases such as cancer and autoimmunity. T cells engineered with synthetic receptors known as chimeric antigen receptors (CARs) have proven effective in eliminating chemotherapy resistant forms of B cell cancers. Such CAR T cells recognize antigens on the surface of tumor cells and eliminate them. However, CAR T cells also have adverse effects, including life threatening inflammatory side effects associated with their potent immune activity.

Risks for severe toxicity present a key challenge to the effective administration of such cell-based therapies on a routine basis.

The ON-switch CAR exemplifies a simple and effective strategy to integrate cell-autonomous decision-making (e.g., detection of disease signals) with exogenous, reversible user control. The rearrangement and splitting of key modular components provides a simple strategy for achieving integrated multi-input regulation. This work also highlights the importance of developing optimized bio-inert, orthogonal control agents such as small molecules and light, together with their cellular cognate response components, in order to advance precision-controlled cellular therapeutics.

We graphically demonstrate this mechanism below.

The authors continue:

Titratable control of engineered therapeutic T cells through an ON-switch chimeric antigen receptor. A conventional CAR design activates T cells upon target cell engagement but can yield severe toxicity due to excessive immune response.

The ON-switch CAR design, which has a split architecture, requires a priming small molecule, in addition to the cognate antigen, to trigger therapeutic functions. The magnitude of responses such as target cell killing can be titrated by varying the dosage of small molecule to mitigate toxicity. scFv, single-chain variable fragment; ITAM, immune receptor tyrosine-based activation motif.

CAR T cell therapy has had successes and failures. It seems to be appropriate for hematological cancers and some related ones where immunodeficiency is an element. However, it often has some several unintended consequences. The immune system is a very powerful system in the body. Setting CTLs loose to do what they do best can be at times very overpowering. In addition, the use of these systems without a means to throttle them back can present a danger to a wide selection of patients. We examine some of these issues as follows.

As Brudno1 and Kochenderfer have noted:

CAR T cells could damage tissues that express the antigen recognized by the CAR. This mechanism of toxicity can be minimized but not eliminated by an exhaustive search for expression of a targeted antigen on normal tissues during preclinical development of a CAR.

Examples of this mechanism of toxicity have been reported in the literature. In one study, 3 patients with metastatic renal cell carcinoma who received infusions of autologous T cells transduced with aCAR targeting carboxyanhydrase- IX experienced grade increases in alanine aminotransferase, aspartate aminotransferase, or total bilirubin.

Liver biopsies of affected patients revealed a cholangitis with a T-cell infiltration surrounding the bile ducts, and bile duct epithelial cells were unexpectedly found to express carboxy-anhydrase-IX.

A patient with metastatic colorectal cancer who received an infusion of autologous CAR T cells directed against the antigenERBB2 (Her-2/neu) experienced acute respiratory distress and pulmonary edema requiring mechanical ventilation. The patient subsequently died.

As Pegram et al note:

CD19-targeted chimeric antigen receptor (CAR) T cells are currently being tested in the clinic with very promising outcomes. However, limitations to CAR T cell therapy exist. These include lack of efficacy against some tumors, specific targeting of tumor cells without affecting normal tissue and retaining activity within the suppressive tumor microenvironment. Whilst promising clinical trials are in progress, preclinical development is focused on optimizing CAR design, to generate “armored CAR T cells” which are protected from the inhibitory tumor microenvironment. Studies investigating the expression of cytokine transgenes, combination therapy with small molecule inhibitors or monoclonal antibodies are aimed at improving the anti-tumor efficacy of CAR T cell therapy. Other strategies aimed at improving CAR T cell therapy include utilizing dual CARs and chemokine receptors to more specifically target tumor cells. This review will describe the current clinical data and some novel “armored CAR T cell” approaches for improving anti-tumor efficacy therapy.
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