June 26, 2022

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What hurdles are there for in vivo CAR-T cell therapy to go to the clinic?

What hurdles are there for in vivo CAR-T cell therapy to go to the clinic?



 

What hurdles are there for in vivo CAR-T cell therapy to go to the clinic?

 

Chimeric antigen receptor T-cell (CAR-T) therapy is a special kind of cancer immunotherapy: it has unprecedented efficacy, but the development process is complex and expensive.

So far, the therapy has not been a treatment option that would benefit all patients. This has prompted researchers around the world to work together to improve CAR-T therapy.

 

Of all the proposed improvements, generating CAR-T cells directly in patients is arguably the most challenging and clinically valuable strategy.

This could transform CAR therapy from a cell-based autologous drug product to a universal off-the-shelf therapy.

 

What hurdles are there for in vivo CAR-T cell therapy to go to the clinic?

 

Recently, the journal Molecular Therapy published a review paper introducing the current cutting-edge in vivo CAR treatment options.

 

 

This article will describe the properties of different vector platforms, such as their immunogenicity, potency, and CAR delivery modes, etc.

Finally, the authors also outline the work needed to advance in vivo CAR therapy from proof-of-concept to clinical application .

 

Following studies of nanocarriers (NCs) for CD3 targeting of mouse T cells and CD8-lentiviruses for human T cells, several studies have further explored in preclinical mouse models using various carrier platforms In vivo CAR generation strategy.

The heterogeneity of model systems, routes of application, vector doses, cellular targets, time points, and readouts in reported studies hinders direct quantitative comparisons between platforms.

Nonetheless, these studies reveal important essential differences between carrier platforms that may profoundly affect the future development trajectories of each platform technology.

 

 


Targeting and Biodistribution


With the exception of one report using AAV vectors, almost all studies on in vivo CAR-T cell generation have relied on targeting vectors.

Overall, these studies can be divided into two categories: one targeting markers on T cell subsets; the other targeting pan-T cell markers.

Of the latter, CD3 is a very common choice. Unlike CD3, CD5 is not required for T cell effector function, but it is also expressed on some B cells.

Since the carrier particles are attached to the target receptor, it is possible that the CAR gene could be delivered to non-T cells when the expression of the target receptor is downregulated.

Future research needs to further explore this.

 

CD3 has been the target of non-viral and lentiviral (LV) vectors. Studies evaluating the biodistribution of mouse CD3 ε-chains showed that nanocarriers (NCs) were predominantly bound to T cells (75% of particles) shortly after injection into C57BL6 mice , while 25% of NCs were in peripheral blood. Adhesion to CD3-negative cells (mainly B cells, neutrophils and monocytes) .

Furthermore, this targeting reduced particle load in the mouse liver by a factor of three and improved the distribution of carrier particles in the spleen, lymph nodes and bone marrow. The biodistribution of gene expression closely matched the particle distribution.

MCD3-NC exhibited similar reporter activity in liver as control NC, but 3-8-fold higher activity in lymphoid tissue.

While most reporter activity was present in T cells in these tissues, substantial activity was also detected in non-T cells such as B cells, dendritic cells, and macrophages.

These off-target activities account for approximately 20%-30% of on-target gene transfer. In order to deepen this understanding, researchers need to further deepen the research on tumor cell transduction.

 

Two types of LVs (SINV and NiV) have also been used to target human CD3.

The researchers used activated CD3 antibody-derived single-chain antibodies to target LV pseudotypes containing the NiV glycoprotein to achieve selectivity for T cells while facilitating gene editing of T cells.

These CD3-LVs induce T cell activation and proliferation and can efficiently transduce primary T lymphocytes in the absence of cytokines or stimulatory CD3/CD28 antibodies.

In the absence of any CAR signaling that restricts CD3 + CD45 + cells, the scholars delivered the CD19-CAR into CD34 + hematopoietic stem cell (huCD34 + NSG) humanized NSG mice, resulting in a functional CAR-T cell.

On this basis, Huckaby et al. injected a bispecific tandem Fab-targeted SINV -pseudotyped LV containing a CD3-binding moiety (CD3-SINV-LV) into NSG mice containing human PBMCs.

Final analysis showed that approximately 70-90% of CAR-positive cells were CD3 positive.

 

When looking at the biodistribution of carrier particles targeting human pan-T cell markers, most humanized mouse models only provide on-target cells in human cells.

In contrast, when CD4 or CD8 serve as target receptors, T cell subsets offer the opportunity to monitor target cell selectivity between closely related human target cells and non-target cells, as CD4 + and CD8 + T lymphocytes Cells share the vast majority of cell surface proteins.

 

Some scholars have used NiV-pseudotyped CD8-LV to achieve subpopulation-specific in vivo CAR cell generation.

The researchers injected the vector intravenously into huCD34 + NSG mice and detected CAR signaling only in CD8 + cells in the blood, spleen, and bone marrow.

Similarly, mRNA delivery of hCD8-NCs in T cell-humanized NSG mice resulted in CD8 + CAR-T cells. Notably, in a follow-up study of CD8-LV, NK and T cells, the scholars found that cells other than CD8 T cells also expressed CAR.

Targeted transduction of NK cells by CD8-LV may facilitate tumor clearance, as CAR NK cells may also have potent antitumor effects.

 

CAR-T cells generated from MV-pseudotyped LV targeting human CD4 by displaying DARPin (CD4-LV) have surprising activity.

The administration of CD4-LV not only enables the specific expression of the CAR on CD4 + cells, but also enables these CAR-T cells to develop faster than cells derived from CD8-LV, while they also completely eliminate the targeted tumor cell.

Although CD4 + T cells have been described previously to have good cytotoxic activity, in the field of CAR-T cells, the 1:1 combination of CD4 + and CD8 + CAR-T cells is still recognized as an effective method for tumor clearance.

 

Recent studies have shown that it is mainly CD4 + CAR-T cells that persist in patients for ten years .

In addition, also through in vivo methods, CAR-T cells can be generated in specific subsets by combining vectors targeting CD4 and CD8 in any ratio.

 

 


Dosage and expression kinetics


The expression and effector kinetics observed in preclinical in vivo CAR cell generation experiments, and the doses at which they were achieved, reflect the different modes of action of vector platforms that lead to permanent or transient gene transfer.

Although no direct comparison has been made , it appears that nanoparticles and AAV are higher than LV in terms of the number of carrier particles required per animal. This may be related to LV-mediated permanent gene transfer and its high target cell specificity.

However, other parameters such as kinetics and efficiency of cell entry and intracellular delivery may also play a role.

 

In addition, the pharmacokinetic parameters of in vitro and in vivo CAR-T cell therapy differ greatly.

In traditional methods, large numbers of CAR-T cells are activated immediately after administration.

In contrast , in vivo delivery of the vector produces CAR-T cells that are orders of magnitude fewer and then expand in vivo over time.

 

LVs and transposase-activated NCs can stably integrate transgenes into their host chromatin.

Therefore, a single injection of the vector is sufficient to induce the generation of persistent CAR-T cells.

For Paramyxa pseudotyped LV, the per-mice injection of particles ranging from 4x 10 10 to 2.5×10 11 was effective; for SINV-LV, the per-mice dose was 5x 10 10 .

 

CAR-T pharmacokinetics are dependent on the activation state of human immune components: in mouse models reconstituted with activated human PBMCs, CAR-T cell (or target cell killing) becomes evident within one to two weeks after vector injection , thus acting earlier than in huCD34-NSG mice.

Furthermore, peak CAR-T levels were higher in PBMC-NSG than in huCD34-NSG mice, reaching 40% of the targeted T-cell subtype.

Notably, CAR-T cells were detected in some CD34 + huNSG mice during a complete monitoring period of up to eighteen weeks after vehicle administration.

It has also been reported that permanent CAR expression can be achieved using NC-transferred CAR plasmids and the highly active PiggyBac translocase, albeit at significantly higher particle doses than previously reported LV.

In this experiment, a total dose of 1.5 x 10 12 NC particles injected over 5 days resulted in up to 20% of T cells in mice being converted to CAR positive 12 days after injection, 7 out of 10 mice tested were successful Leukemia cells were eliminated.

While this seemed promising, this strategy was later abandoned due to low transfer efficiency and concerns about transposase toxicity, and scholars instead focused on transient delivery of mRNA-NCs.

 

In transient gene transfer, ie when non-integrating vectors are used, transgene expression is eventually lost.

This is caused by degradation and inactivation of the transgene and its dilution as the cells proliferate. Then, despite antigen-induced proliferation, re-dosing may be required to maintain adequate CAR-T cell levels.

Indeed, multiple doses of mRNA-transferred PBAE-NCs are necessary to maintain antitumor CAR activity. To treat hematological and solid tumors in T-cell-humanized NSG mice, the researchers administered six doses of 50 μg over 6 weeks for a total of 300 μg of mRNA.

In a syngeneic mouse model of acute lymphoblastic leukemia, the researchers administered 12 doses of 15 μg over 4 weeks for a total of 180 μg of mRNA. A human equivalent dose is equivalent to 2.08 g/mL of blood, or about 10 mg of mRNA.

In contrast, complete Covid-19 vaccination of adult humans with mRNA-based vaccines was achieved with 90-300 μg of mRNA in LNPs.

Notably, both solid and liquid tumors recurred within weeks of treatment cessation, suggesting that sustained CAR activity is necessary for durable tumor suppression.

The rapid kinetics of mRNA transfer may be associated with these relapses, as CAR levels peaked after two days and were almost completely lost 7 days after nanoparticle infusion into NSG mice.

 

When long-term CAR cell viability is not required, lower doses appear to be sufficient: results after three weeks of nanoparticle injection suggest that a single injection of 10 μg of FAP-CAR-mRNA in LNP experiments reduces fibrotic tissue burden and Significant improvement in cardiac function in a syngeneic model of fibrosis .

In this case, the researchers prefer to use short-term CAR-T cell activity to reduce the effect of CAR-T cells on healthy fibroblasts.

 

CAR-T cells can be generated in a humanized NCG T cell leukemia mouse model by delivering DNA rather than mRNA by a single injection dose of 1-2x 10 11 non-targeted AAV.

Surprisingly, CAR-T cells were still detectable in mice 35 days after AAV injection.

This unexpected persistent transgenic signal may be related to the high transduction efficiency of intraperitoneal injection and the spontaneous integration of the CAR coding sequence into the T cell genome.

In vitro, the transgenic signal from DART-AAV targeting mCD8 was almost completely lost in activated mouse lymphocytes within one week of vector addition.

 

 

 


Security and Control


An imminent safety concern for the study of vectors capable of permanently transferring CAR genes is the genotoxicity of transcriptional dysregulation due to misplaced insertion of the transferred gene into host chromatin .

In vitro transduction of first-generation autologous hematopoietic stem cells ( HSG) in children with X-linked severe combined immunodeficiency , non-self-inactivating γ- Retroviral vectors induce leukemia in children. LMO2 causes uncontrolled clonal expansion.

The occurrence of this event and subsequent studies have led to a better understanding of retrovirus-mediated oncogene insertion formation and the use of self-inactivating retro and lentiviral vectors.

Notably, T lymphocytes are relatively less likely to undergo retrovirus-mediated oncogene transformation than HSCs.

 

Overall, there have been no events of insertional tumorigenesis in the clinical application of LV-mediated T cell transduction.

However, there are also some examples of benign expansion. In contrast to LV, transposon-mediated gene transfer caused “severe combined immunodeficiency” last year, and two patients developed T-cell lymphoid T cells after receiving in vitro CAR-T cells generated using the piggyBac transposon enzyme. tumor.

Although the mechanism of tumorigenesis is still under investigation, this event will inevitably force the development of safer translocation strategies.

 

In light of these security incidents, academics recommend close monitoring of long-term research in the field.

A focus on risk factors, such as vector copy number in CAR-T cells, will ensure that the benefits of CAR delivery outweigh the potential risk of genotoxicity.

 

In addition to concerns about the risk of genotoxicity, for permanent transfer of CAR genes, one may also need pharmacokinetic tools to better control CAR activity, especially in patients with severe cytokine release syndrome (CRS) or immune effects organ-associated neurotoxicity syndrome (ICANS) .

The two syndromes often co-occur and are characterized by high levels of proinflammatory cytokines. Interestingly, CRS-like symptoms were also observed when CD8-LV was used to generate CAR-T cells in CD34 + huNSG mice.

This was associated with the infiltration of CD8 + cells in the lung, brain, liver and spleen B-cell regions and the elevation of cytokines such as IL-6, IFNγ, GM-CSF and TNFα.

 

Clinically, CRS and ICANS patients are treated with tocilizumab , a monoclonal antibody, to reduce systemic inflammation without compromising the therapeutic function of the CAR.

More precise control can be achieved with the tyrosine kinase inhibitor dasatinib, which has been used in preclinical experiments to intermittently shut down CAR-T cells in mouse models of CRS. It reduces the inflammatory burden and improves mouse survival.

 

Another approach that has already been applied in humans utilizes inert UniCARs equipped with soluble antigen-specific targeting modules (TMs) . In this strategy, control of the CAR is achieved by selecting TMs with longer or shorter half-lives. Once all TM is degraded, the activity of CAR-T cells is lost.

 

In addition to short-term control strategies, researchers can also combine inducible caspase 9 (IC9) suicide genes to CAR molecules to achieve long-term control of CARs. Using this approach, doctors can stop CAR cell function in time after a patient’s tumor has been removed.

Through small molecule administration, IC9 protein dimerization is induced, resulting in apoptosis of CAR-T cells. For patients with milder disease, the existence of this mechanism may make CAR cells derived from permanent transfer vectors more attractive.

Importantly, all of these control strategies can be combined with in vivo CAR-T cell generation, thereby facilitating the clinical application of this approach.

 

Uncontrolled CAR cell activity is not a problem when transient transfer vectors are used. Quite the contrary, it is unclear whether CAR activity can be maintained long enough to achieve durable tumor clearance when transient transfer vectors are used.

Available short-term experimental safety data do not indicate severe acute toxicity, however, increased levels of cytokines following in vivo administration of synthetic and viral vectors, as well as NC-induced activation of complement components and increased mitochondrial oxidative stress, suggest that inflammatory responses may be involved.

Affects repeated administration of vehicle.

Long-term experiments in syngeneic models enable assessment of the risk of induction of inflammatory and other host immune responses following vector administration or repeated administration.

 

 


Host immunity


The immune response of the host to the delivery vehicle can affect the safety and efficacy of the drug.

Antibody-mediated immune responses to carrier particles can reduce the number of effective target particles, induce inflammation, and affect the efficacy of initial and repeated dosing.

LV and AAV vectors of viral origin are particularly important because of their apparent immunogenicity.

 

In fact, a single systemic injection in rodents has been shown to induce the formation of neutralizing antibodies against virions and the VSV-LV envelope protein.

Although there is currently no vaccine against AAV, a significant proportion of people carry neutralizing antibodies against AAV.

Although the immunogenicity of LNPs carrying modified mRNAs appears to be acceptable, the introduction of protein components for receptor targeting may increase the risk of antibody formation after repeated administration .

Plasma exchange to remove serum IgG is a time-consuming process that requires complex instrumentation, and an alternative is the use of streptococcal IgG-degrading enzymes such as IdeS. Systemic treatment with IdeS not only allowed liver transduction of rhesus monkeys already carrying anti-AAV8 antibodies, but also effectively achieved multiple administrations.

 

Phagocytosis is another factor in the clearance of carrier particles. In mice treated with CD3-targeted NCs, approximately 20% of both macrophages and monocytes were NC-positive, indicating that these cells have phagocytic activity toward the vector.

Notably, phagocytosis did not appear to be affected by targeting, as it was able to affect particle distribution of CD3-NCs and isoform NCs equally.

In liver transduction experiments using LV and adenoviral vectors, the researchers observed a non-linear dose response.

When the threshold dose was reached, most of the administered particles were sequestered by liver-resident Kupffer macrophages. Incorporation of the phagocytosis inhibitor CD47 into carrier particles reduced LV phagocytosis.

Uptake of this particle by macrophages in cell culture is reduced, and this vector results in higher hFIX transfer efficiency in non-human primates. Similar effects were observed in LV-targeted packaging cells with CD4 and CD8.

The presence of macrophages reduces the average transduction efficiency of conventional CD4 and CD8-LV on human T cells by approximately 25-50%, whereas vectors that inhibit phagocytosis can greatly enhance the ability of gene transfer into T cells.

Furthermore, in CD34+ HSC humanized NSG-SGM3 mice reconstituted at physiological levels of human bone marrow cells, the phagocytosis-inhibiting vector achieved more efficient in vivo CAR-T cell generation and more pronounced target cell clearance.

 

 


How to the clinic?


Using cutting-edge carrier platform technology, CAR-T cells can generate and eliminate tumor cells in vivo.

Without a doubt, this is a milestone in CAR therapy and gene therapy. However, more research is still needed to explore the short-, medium-, and long-term safety and efficacy issues involved in permanent or temporary transfer approaches .

Recent or earlier reported gene therapy deaths urge us that the effects of vectors on the host need to be examined more thoroughly in long-term preclinical studies before moving forward with first-in-human trials.

 

In addition to ensuring patient safety, rigorous characterization of each vector-host interaction will help us understand how innate and acquired host immunity interfere with vector administration, leading to drug toxicity failure and vector particle clearance, and how best The host immune system is well regulated to deal with this disturbance.

This is critical because when targeting lymphocytes, we cannot perform extensive immunosuppression or lymphatic clearance.

Therefore, we must identify regulatory protocols that minimize inflammation associated with vector administration and immune-mediated loss of particle dose, while maintaining lymphocyte viability and activity.

A syngeneic model with fully immune function would be helpful in this research.

The mouse T cell targeting vectors required for these experiments can be derived from all four vector platforms, and it has been reported that LV and AAV targeting mouse CD8 via DARPin have been reported.

 

Another key issue is vector manufacturing, as a sufficient quantity of high-quality vector stocks is critical for further development.

The design of the targeting vector production process should comply with Good Manufacturing Practice (GMP) requirements.

The development of GMP compliant processes for the production of targeted LVs, AAVs and LNPs can draw on previous experience with the production of non-targeted GMP grade vectors for approved drugs.

While the global rollout of Covid-19 vaccines has demonstrated considerable scalability of synthetic particle production, it remains to be determined whether cell-generated target receptor conjugates can be integrated into the production process without compromising yield.

 

Furthermore, in the context of in vivo CAR therapy, advances in addressing host immunity and vector manufacturing issues (among other as yet unidentified issues) may benefit the gene therapy field as a whole.

These breakthroughs will help access in vivo treatments for blood, cardiac, infectious and developmental diseases for many patients in need. Through the development of targeted vector technology and the continuous rigorous work and tenacity of scholars, the field of gene therapy may soon enter the “in vivo era”.

 

 

References :
Michels, Alexander, Naphang Ho, and Christian J. Buchholz. “Precision Medicine: In Vivo CAR Therapy as a Showcase for Receptor-Targeted Vector Platforms.” Molecular Therapy (2022).

What hurdles are there for in vivo CAR-T cell therapy to go to the clinic?

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