Next Phase Newsletter

Juan Carlos Lopez

Andy Marshall

Just over a week ago, AbbVie paid $2.1 billion for Capstan Therapeutics’ in vivo anti-CD19 chimeric antigen receptor (CAR)-T cell therapy (CPTX2309) for B cell-mediated autoimmune disorders, which is currently in phase 1 testing. In the past few days, EsoBiotec (acquired by AstraZeneca earlier in the year) also published its first clinical data on a lentiviral-delivered anti-B-cell maturation antigen (BCMA) CAR-T approach (ESO-T01) for multiple myeloma, detailing responses in four patients, two of whom showed complete remission. With a host of other companies working on in vivo delivery into endogenous T cells—including Interius BioTherapeutics, Umoja Biopharma, and Orna Therapeutics, the field of in vivo delivered CAR-T cells appears poised at a tipping point.

Since transforming the face of cancer treatment in 2017, autologous CAR-T cell therapy has been dogged by logistical issues that have limited commercial rollout and increased costs—the need for leukapheresis, laborious cell harvesting, heterogeneous cell expansion, lengthy turnaround times, and inconsistency of batches—with access limited to just a few clinical centers. Extensive waiting lists can mean many patients die before even being treated, which has driven the search for ex vivo approaches that shorten manufacturing times using fully closed systems and/or miniaturization. Given these challenges, delivery of a CAR-encoding mRNA to a T cell in vivo could be a game-changing technology: No need for viral vectors; no leukapheresis/chemo; no ex vivo manipulation, no requirement for multiple patient hospital visits; no convoluted training of personnel; and no risk of second primary T-cell cancers due to insertional mutagenesis. This last issue has loomed over the field, with all CAR-T therapies carrying black box warnings, although at the end of June the FDA removed all requirements for Risk Evaluation and Mitigation Strategies (REMS).

​Writing in Science, the founding team of Capstan Therapeutics, headed by Carl June and Bruce Levine at the University of Pennsylvania and Haig Aghajanian of Capstan, report proof of concept data that functional CAR T cells with antitumor activity can be produced in animal models without any ex vivo manipulation. A key breakthrough in their effort was the development of lipid nanoparticles (LNPs) specifically designed to target T cells and to overcome the propensity of LNPs to accumulate in the liver. To avoid this problem, the authors screened a set of ionizable lipids to identify L829, a lipid that incorporates a tertiary amine headgroup that reduces non-specific interactions with the hepatic system due to its pH-dependent protonation and neutral charge. Ester cleavage sites in the lipid also promote rapid breakdown in, and clearance from, hepatocytes. A final step was to decorate L829 LNPs with a mAb targeting CD5, a T-cell specific marker. The resulting LNP showed limited liver uptake in rodents and non-human primates compared with control LNPs.

To test the potential of L829-containing LNPs to generate functional CAR-T cells, the team engineered them to incorporate 1) mRNA encoding a CAR that binds CD19 on B cells and 2) an antibody targeting CD8⁺ T cells. These CD8-L829-CD19 targeted (t)LNPs successfully delivered the mRNA in vitro to CD8⁺ T cells from healthy subjects and from people with B cell-mediated autoimmune diseases. In vivo, these CAR T cells had anti-tumor activity in a humanized mouse model of B cell acute lymphoblastic leukemia.

In cynomolgus monkeys that received repeated doses of CD8-L829 tLNPs containing anti-CD20 CAR mRNA (instead of anti-CD19, which is not cross-reactive between human and monkey), sustained B-cell depletion was observed that lasted for one month. Importantly, reconstituted B cells were predominantly naïve, implying an immune reset — a key therapeutic goal in autoimmunity.

The Capstan in vivo mRNA-encoded CAR T platform eliminates the need for ex vivo manipulation and lymphodepleting conditioning. It avoids the risks often associated with the use of viral vectors that integrate into the genome. It also is transient, allowing dosages to be optimized and quickly stopped if patients suffer adverse events associated with neurotoxicity or cytokine-release syndrome. It will be interesting to see whether the approach is scalable and whether it can open up conditions where long-term CAR-T cell persistence might not be necessary, such as autoimmune disease.

Going forward, an important question will be to determine the potential immunogenicity of the tLNP formulation (especially as the mRNA treatment may be given multiple times), and whether tLNPs cause elevations of human liver enzymes like alanine transaminase or aspartate aminotransferase. Liver toxicity of a novel liposome formulation already caused a clinical hold for Verve Therapeutics’ base editing therapy last year. Future work will also need to define optimal dosing, durability, and long-term safety of this approach. But the work of June, Aghajanian and their colleagues is a compelling advance promising a new era of widely available adoptive T-cell therapies for B-cell driven hematological cancers and autoimmune conditions. A single dose of any of the seven currently approved commercial ex vivo CAR-T therapies costs ~$500,000. A vial of an in vivo treatment is likely to cost an order of magnitude less.

Tags: alzheimers, art, biology, books, cancer, food, genetics, health, immune-system, nutrition, painting, poetry, science, writing

Juan Carlos Lopez

Andy Marshall

​Drug development efforts targeting the constitutive 26S proteosome have led to the development of several important multiple myeloma (MM) and mantle cell lymphoma treatments, including the first landmark FDA approval of Millennium Pharmaceuticals’ (now Takeda) dipeptide boric acid Velcade (bortezomib) in 2003 and second-generation molecules, such as Amgen/Ono Pharmaceutical’s irreversible inhibitor Kyprolis (carfilzomib) and Takeda’s orally available inhibitor Ninlaro (ixazomib). Second-generation versions of these ‘pan-proteosome’ drugs have longer duration of effect, reduced peripheral neuropathy and increased safety in renally impaired patients, but may cause gastrointestinal and cardiac toxicity. This toxicological profile has shifted attention to developing inhibitors selective for an alternative form of the core 20S proteosome—the immunoproteasome, which processes peptides for presentation to CD8⁺ T cells in the MHC-I complex and is constitutively expressed only in hematopoietic cells, induced in immune cells stimulated in the presence of IFN-γ, and upregulated in certain cancers like MM.

Currently, Kezar Life Sciences’ is furthest along in development; in April, it completed a phase 2a trial in autoimmune hepatitis of zetomipzomib (KZ-616), a small-molecule that inhibits both the immunoproteasome core particle component beta subunit 8 (PSMB8; LMP7/β5i) and PSMB9 (LMP2/β1i). Merck kGaA (Darmstadt, Germany) is also pushing forward with a phase 1 clinical program of M3258, a small-molecule inhibitor specific for PSMB8 and intended for use in MM (Principia Biopharma’s selective PSMB8 inhibitor was swallowed up by Sanofi in 2020 when the pharma acquired the San Francisco-based biotech’s Bruton’s tyrosine kinase inhibitor program). Elsewhere, Leiden University startup iProtics recently received a €200K grant from the Dutch Biotech Booster to develop selective immunoproteosome inhibitors, while Auburn University spinout Inhiprot (West Lebanon, NH) received SBIR funding to develop a dual PSMB6/PSMB9 inhibitor for MM. Now, a new study reveals immunoproteosome targeting may also have benefits in neuroinflammatory diseases like multiple sclerosis.

The work, published in Cell and led by Catherine Meyer-Schwesinger and Manuel Friese, from University Medical Center Hamburg-Eppendorf, identifies a neuron-intrinsic mechanism of neurodegeneration in multiple sclerosis (MS) driven by the immunoproteasome.

Under healthy conditions, neurons utilize the constitutive proteasome subunit PSMB5 to regulate proteostasis and degrade 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a potent stimulator of glycolysis. This degradation is key because neurons rely more on the pentose phosphate pathway than on glycolysis to produce antioxidants like NADPH and glutathione for protection against oxidative stress.

However, Meyer-Schwesinger, Friese and their colleagues show that, during neuroinflammation, chronic exposure to interferon-γ leads to the induction of the immunoproteasome in neurons, triggering the replacement of constitutive proteosome PSMB5 (β5c) with PSMB8 (β5i). This subunit swap in neurons reduces proteasomal activity, resulting in accumulation of PFKFB3, which in turn enhances glycolysis, diminishes the activity of the pentose phosphate pathway, and impairs redox homeostasis — conditions that sensitize neurons to oxidative injury and ferroptosis.

The team showed that this mechanism was operational in both experimental autoimmune encephalomyelitis (EAE; a mouse model of MS) and brain tissue from MS patients. Moreover, neuron-specific knock-out of Psmb8 or pharmacological inhibition using the small-molecule PSMB8 inhibitor ONX-0914 (originally developed at Onyx Pharmaceuticals/Proteolix) protected neurons in vivo from inflammation-induced damage. Similarly, blocking PFKFB3 with the small-molecule inhibitor PFK-158 or through conditional knockout in neurons reduced disease severity in EAE, prevented neuronal and synaptic loss, and reduced markers of oxidative stress and lipid peroxidation.

It is important to highlight that, unlike cancer or immune cells, neurons do not upregulate PSMB8 in response to a series of MS-related cytokines. So, the neuron-specific effect reported in this study might only become active upon chronic neuroinflammation (i.e. chronic exposure to interferon-γ). Understanding this mechanism might reveal new targets related to the immunoproteosome in the treatment of MS.

This brings us to challenges for translational efforts seeking to develop immunoproteosome inhibitors against MS. Several important neuronal processes, such as synaptic transmission and calcium signaling, are tightly linked to proteasome function; thus, pan-proteosome inhibitors like Velcade could be detrimental to the CNS. The saving grace of approved proteosome inhibitors is that current chemotypes (boronate-based peptides or epoxyketone-based binders) do not cross the blood brain barrier, at least in healthy individuals. Thus, any MS program might need to use intrathecal injection for compounds derived from existing chemical series or engage a medicinal-chemistry effort to design molecules that can breach the BBB and retain potency.

The gambit for immunoproteosome-selective drugs is that they avoid inhibiting constitutive 26S proteosome activity in most tissues (and non-inflammed CNS), which is what makes Velcade and its derivatives so difficult for patients to tolerate; an immunoproteosome inhibitor should therefore have a more favorable safety profile. But so far, immunoproteosome-targeting drugs have had their own share of toxicity problems in the clinic.

Last October, Kezar abandoned its program for zetomipzomib in lupus nephritis after the FDA placed a clinical hold on the trial after 4 patient deaths. The agency placed a second partial hold on the company’s autoimmune hepatitis trial in 24 patients last November due to concerns about steroid control and injection site reactions in 4 patients who were waiting to roll over into the open-label extension arm. Concerns about compromised immune surveillance of acute or latent viral infections due to hobbled antigen processing and presentation would also need to be explored.

In sum, the new work provides strong evidence that the immunoproteosome plays a key role not only in inflammation or infiltration of immune cells, but also in a metabolic switch in neurons which is a key driver of vulnerability in MS. It will be interesting to see whether either targeting immunoproteosome component PSMB8 or taking a completely different tack, blocking PFKFB3, will prove more practical as a neuroprotective strategy in MS.

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Tags: alzheimers, art, biology, books, cancer, food, genetics, health, immune-system, nutrition, painting, poetry, science, writing