December 8, 2021

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Use cell and genome modification to generate CAR T and CAR NK cells

Use cell and genome modification techniques to generate improved “off-the-shelf” CAR T and CAR NK cells



 

Use cell and genome modification to generate CAR T and CAR NK cells.  Use cell and genome modification to generate CAR T and CAR NK cells. CARs are synthetic receptors that contain an extracellular antibody-like region designed to target a specific antigen called a single-chain variable fragment (scFv), which is a hinge region that can have different lengths, the choice of which may depend on proximity The recognition epitope, transmembrane domain, one or more costimulatory domains and signal domains on the surface of target cells induce cytotoxicity upon antigen binding (Figure 1).

Use cell and genome modification to generate CAR T and CAR NK cells

Figure 1. Modification of T or NK cells with reverse transcription and lentiviral vectors encoding CAR.

 

The choice of costimulatory domain and signal domain is mainly based on the components of T cell receptor (TCR), which includes CD28 and/or 4-1BB costimulatory domain and CD3ζ signal domain.


The clinically approved second-generation CAR contains a CD3ζ signal domain combined with CD28 (Yescarta®) or 4-1BB (Kymriah®) costimulatory domain. Although CAR T cells containing CD28-CD3ζ show faster and stronger signal transduction and are conducive to the development of effector cell phenotypes, CAR T cells containing 4-1BB-CD3ζ have a longer-lasting memory cell phenotype ( 12). A direct comparison of the CD28 and 4-1BB costimulatory domains in the anti-CD19-CAR shows that 4-1BB contributes to the greater CAR T cell persistence and greater survival in B-cell non-Hodgkin’s lymphoma (B-NHL) patients. Favorable toxicity profile (13).


The efficacy of CAR T cells and more recently CAR NK cells has been shown in liquid tumors, most prominently in CD19+ lymphoid cancers. There are currently several clinical studies exploring the conversion of these promising results into solid tumors. However, in order to further improve the CAR T cell approach, some important clinical challenges must be addressed. For example, a major adverse event that often occurs during CAR T cell therapy is cytokine release syndrome (CRS), in which the levels of inflammatory cytokines such as interleukin (IL)-6 are observed to be greatly increased. The severity of CRS is related to the level of IL-6 in patients, and the anti-IL-6 receptor antibody tocilizumab can be used to reverse the symptoms of CRS without interfering with the anti-tumor activity of CAR T cells (14). Compared with 4-1BB-CD3ζ CAR T cells, B-NHL patients treated with CD28-CD3ζ CAR T cells have more severe CRS, which may be due to the high immune response induced by CD28 stimulation (13).


Since it is not always possible to recognize neoantigens expressed only on tumor cells targeted by CAR-modified cells, healthy cells may also be eliminated through targeted-non-tumor activity. Although in some cases this can be controlled clinically, for example, a CAR targeting CD19 causes the loss of healthy B cells, but for other targets, adverse events due to targeted-non-tumor activity may be more serious, such as Harmful destruction of lung tissue after administration of anti-ERBB2(HER2)-CAR T cells aimed at treating metastatic ERBB2+ cancer (15). The severity of targeted off-target tumor activity may be adjusted by the CAR T cell dose used, because another study that tested HER2-CAR T cells in patients with sarcoma showed that it is comparable to the 1010 (or 6.25×1010, Compared with CAR T cells based on the average female body surface area of ​​1.6 m2, it is safer to give 1×108 CAR T/m2 cells (16).


Disease recurrence due to lack of CAR T cell persistence has also been reported. The loss of anti-CD19 CAR T cells was found to be due to CD8+ immunity of CAR T cells in some patients, which may be due to the use of murine scFv in clinical CAR constructs (17). In order to reduce the potential immunogenic effects of CAR scFv sequences derived from mouse monoclonal antibodies and thereby increase the persistence of CAR T cells, these should be humanized (18) (NCT02659943).


In order to improve the safety of CAR T cell therapy, a vector used to deliver CAR can be designed to co-express suicide genes so that CAR T cells can be removed in the event of uncontrollable serious adverse events. Examples of clinically available suicide gene strategies include the HSV-tk suicide gene (19), which sensitizes cells to ganciclovir-induced cytotoxicity or the inducible caspase 9 (iCasp9) gene cassette, resulting in expression Rapid caspase-mediated apoptosis of cells (for example, CAR T cells) is after the application of synthetic dimerization inducers, such as AP1903 or AP20187 (20, 21).


T and NK cells designed to express CAR still eliminate target cells through the same cytotoxic mechanism as unmodified T and NK cells, that is, through the release of perforin and granzyme, and death receptor interactions (22, 23). However, the cytotoxic activity is specifically amplified by the binding of scFv to the respective tumor-associated antigen. In addition, the concept of CAR-T cell therapy is also applicable to other disease indications, including autoimmune diseases, in which CAR is introduced into regulatory T cells (Tregs) with anti-inflammatory activity (24).


In the context of developing new CAR therapies to treat cancer, one of the key decisions that needs to be made is which tumor-associated antigens the scFv is designed to target. This will largely determine the specificity and degree of tumor targeting, as well as the side effects outside the tumor. Another important consideration is the design of the remaining domains of CAR, such as which transmembrane domains, costimulatory domains, and signal domains should be included. This decision may also be influenced by the cell types used as “live” drugs (for example, T cells, NK cells, other immune cells) and the time window during which these cell therapies should be active. Interestingly, CAR designs based on T cell receptors also work in NK cells (26-28). However, this does not exclude the possibility of designing immune cell type-specific CARs for optimal use in selected cell types (Figure 1). For example, modification of NK cells with chimeric receptors consisting of NK cell activation receptors NKG2D, DNAX activating protein 10 (DAP10) and CD3ζ resulted in increased cytotoxic activity against cancer cell lines and improved the activity of a mouse model of osteosarcoma (29, 30).


In the following sections, we will introduce the important concepts of how to generate “off-the-shelf” CAR cell therapy, such as the source of immune cells to be modified, strategies to overcome tumor immune escape mechanisms, and genome engineering methods that can be used for improvement. CAR T and CAR NK cell functions will be considered.

 

 


Use cell and genome modification to generate CAR T and CAR NK cells

T cell source: autologous, allogeneic, induced pluripotent stem cell derived and expanded progenitor cell derived

At present, the most common source of CAR T cells in clinical applications is patient-derived autologous T cells, which are then genetically modified to express the selected CAR, amplified and reinjected into the patient. Lentiviral or retroviral vectors are usually used in clinical trials to deliver CARs to the T cell genome (Figure 1) (32, 33), but it has also been shown that non-viral integration technologies (such as sleeping beauty transposons) can effectively generate CARs T cells (34).

Although it is tempting to use autologous cells because it avoids the challenge of immune incompatibility, such as complications such as GVHD, the source of autologous cells also has disadvantages. For example, immune cell populations may be adversely affected in patients undergoing extensive pretreatment, so the quality and quantity of cells used for in vitro modification and expansion may not be optimal.

In addition, cancer patients who are infected or rapidly progressing may not survive the weeks required to produce autologous CAR T cells, because these cells must be collected by apheresis, transported to the facility site for genetic modification, amplification and preparation, and then re Transport back to the hospital, where the patient will receive an injection of CAR T cells.

The advantages of allogeneic CAR T cells include lower genetic modification and the risk of reinfusion of leukemia cells (35), and the ability to prepare and store allogeneic cells for future use, thereby shortening the waiting time for infusion into the patient’s body . Therefore, “off-the-shelf” allogeneic cell sources can provide greater flexibility in treatment options. If multiple patients can be treated from a single CAR T cell product, and it is expected to allow more widespread use of these clinical procedures, it may be Reduce overall costs (36).

Therefore, the effective and reliable method of producing “off-the-shelf” T cells is still a highly sought after goal in the field of cellular immunotherapy.

 

 

 


Use cell and genome modification to generate CAR T and CAR NK cells

NK cell source

Another method is to use the natural cytotoxic activity of NK cells to generate allogeneic “ready-made” CAR NK cells to target cancer cells. One of the advantages of NK cells is that they will not induce GVHD even in a mismatched environment (43). As more and more CAR NK cell data accumulate and more and more CAR NK cells are included in clinical trials (Table 1), the relative risk of GVHD after the application of NK cells will become clearer.

Use cell and genome modification to generate CAR T and CAR NK cells

Table 1. Selected clinical trials testing potential “off-the-shelf” CAR cell therapies.

Different NK sources have been used to generate CAR NK cells for preclinical and clinical testing, including cell lines such as NK-92 cells (45), cord blood-derived NK cells (43, 46) and peripheral blood-derived NK cells (28). It is worth noting that a recent landmark phase 1 and phase 2 study showed the feasibility of umbilical cord blood-derived CAR NK cells in the treatment of relapsed or refractory CD19+ B-cell carcinoma (43). Eight of the 11 patients (73%) responded quickly (within 30 days after CAR NK cell infusion), including 7 patients in complete remission. Of particular interest is that the only major adverse events related to lymphocyte clearance strategies (ie, neutropenia, lymphopenia), and no cytokine release syndrome, neurological events or GVHD were observed, Even if there are 2-5 HLA allele mismatches (43). CARNK cells persist for at least 12 months after infusion, which may be at least partly due to the inclusion of the IL-15 expression cassette in the CAR construct, a cytokine known to enhance the survival and proliferation of NK cells (43). The same group previously demonstrated that one cord blood unit can be used to produce more than 100 doses of CAR NK, further emphasizing that allogeneic CAR NK cells are potential “off the shelf” drugs (47).

 

 


Use cell and genome modification to generate CAR T and CAR NK cells


iPSC and other cell sources

Other cell sources for producing “off the shelf” CAR cells include stem and progenitor cell populations, such as induced pluripotent stem cells (iPSC) and precursor T cells. iPSC has almost unlimited proliferation potential and can differentiate into various cell types, including T and NK cells. Therefore, iPSC provides a renewable source of potentially standardized cells for immunotherapy, and can be easily genetically modified to produce immune cells with improved properties (54).

By transducing iPSCs derived from peripheral blood lymphocytes with a lentiviral vector encoding the second-generation anti-CD19-CAR, the feasibility of producing CAR T cells from iPSCs was demonstrated (55). After hematopoietic regulation and expansion, the authors used T lymphoid tissue co-culture protocol to generate anti-CD19-CAR-T-iPSC-T. The author directly compared iPSC-derived CAR T cells with TCR-αβ and TCR-γδ peripheral blood lymphocytes from the same donor and transduced with the same CAR, and proved that iPSC-derived CAR T cells showed similar anti-cancer activity CAR TCR-γδ cells use CD19+ Raji human Burkitt lymphoma cell line in immunodeficient mouse xenograft tumor models (55).

Adaptation of CAR design to take advantage of the signal transduction pathways naturally used for cell activity may lead to improved activity of CAR NK or other CAR cell types. For example, the design of “NK-CAR” containing NKG2D transmembrane domain, 2B4 costimulatory domain and CD3ζ signal domain is used to modify iPSC cells and then differentiate them into NK-CAR-iPSC-NK cells (iPSC is equipped with NK- NK cells derived from CAR). Compared with T-CAR-iPSC-NK cells in ovarian cancer xenograft models, NK-CAR-iPSC-NK cells showed excellent anti-tumor activity, and compared with those observed with CAR T cells expressing typical CAR. similar. T cells (CD28-CD3ζ) (56). The advantages of iPSC-derived CAR T/CAR NK cells include their huge proliferation and expansion capabilities and the relative ease of genome modification, which provides the possibility to create a cell bank with different CAR constructs as a standardized “off-the-shelf” immunotherapy.

Therefore, great progress has been made in identifying alternative “off-the-shelf” therapeutic CAR cells. Since any manipulation of the genome, such as the insertion of a therapeutic CAR vector (Figure 1), has inherent risks, these risks must be carefully assessed. Although the possible genotoxicity risk (for example, the transformation of healthy cells into cancer cells) is low in terminally differentiated somatic cells (for example, T and NK cells), such in stem cells (for example, HSC, iPSC) or progenitor cell populations Modifications that can differentiate into T or NK cells may have a higher risk, which should be evaluated and mitigated as needed. There are also differences in the regulatory requirements for the clinical use of primary lymphocytes and cell lines.

 

 


Car T cell genome modification technology and application

In addition to gene transfer technology used to improve CAR T cell function, genome modification strategies have been used to advance “off the shelf” cell therapies. Zinc finger nuclease (ZFN), transcription activator-like nuclease (TALEN), and clustered regularly spaced short palindrome repeats (CRISPR)-Cas9 (CRISPR-associated protein 9) systems are currently the most commonly used genome editing technologies.

 

ZFN “off-the-shelf” CAR products


In order to produce “off-the-shelf” allogeneic CAR T cell products, it is necessary to destroy GVHD and other adverse reactions caused by endogenous TCR activation in donors and recipients with HLA mismatch. Possible gene editing strategies to avoid this situation include eliminating endogenous TCR expression to generate universal donor T cells (66) and targeting the CAR into the TCR alpha constant (TRAC) locus (67). Electroporation using the adeno-associated virus (AAV) serotype 6 vector (AAV6) to deliver homologous donor templates and ZFN mRNA for efficient genome editing of CD8 and CD4 T cells via HDR (68).

Electrotransfer is also used to deliver designer ZFN to delete TCR α or β chains in CD19-CART cells, and the TCR-CAR+ population maintains CD19 specificity and does not respond to TCR stimulation (69).

 

TALEN “off-the-shelf” CAR products

Universal CAR19 T (UCART19) cells are generated using TALEN to target the constant region (TRAC) of the TCRα chain and the CD52 gene, making UCART19 cells resistant to Alemtuzumab (Campath®), which is a type used to eliminate B- Cell chronic lymphocytic leukemia (66). UCART cells caused rapid molecular remission (28 days) in two infants with refractory high-risk B-ALL. One patient had Grade 2 skin GVHD, and the second patient may have mild skin GVHD, which resolved quickly with topical steroids (66). TALEN is also used to disrupt the TCRαβ locus to generate universal allogeneic CAR T cells against the tumor-associated antigen CS1 (UCARTCS1A), and is currently being tested in patients with relapsed and refractory multiple myeloma (NCT04142619) (Table 1). A similar method is used to generate universally applicable anti-CD22 CAR T cells (UCART22) to treat patients with relapsed and refractory CD22+ B cell B-ALL (NCT04150497).

 

CRISPR-Cas9 “off-the-shelf” cell product and development technology

CRISPR-Cas9 RNPs and AAV6 were used to specifically deliver an engineered 2.3kb long TCR construct TCR25D6, which when presented on HLA-B7, recognized peptides derived from myeloperoxidase as a medullary tumor formation The patient’s tumor-associated antigen enters the TRAC locus (70). The combination of CRISPR-Cas9-mediated TCRα/β and B2M knockout delivers anti-CD19 CAR lentivirus to allogeneic T cells, resulting in universal CAR T cells (UCART019), which are used in relapsed or refractory CD19+ leukemia and Clinical test (NCT03166878) was performed in patients with lymphoma. CRISPR-Cas9-mediated TCRα/β and B2M knockout to generate “ready-made” allogeneic CAR T cells are also targeted at CD19+ leukemia and lymphoma patients (NCT04035434), multiple myeloma patients (NCT04244656) and kidney cells Cancer patients were evaluated in clinical trials (NCT04438083) (Table 1).

Multiplex CRISPR-Cas9 allows simultaneous editing of multiple genomic sites. Recently, a phase I trial (NCT03399448) (71) demonstrated the feasibility and safety of using multiple CRISPR-Cas9 to design autologous T cells with enhanced anti-cancer activity. CRISPR guide RNA is electroporated into T cells to delete the endogenous TCRα and TCRβ chains and the PDCD1 gene encoding programmed cell death protein 1 (PD-1). Perform endogenous TCR destruction to enhance the expression of cancer-specific TCR NY-ESO-1 introduced by lentiviral transduction. In addition, PD-1 knockout improves the activity of engineered T cells by avoiding checkpoint suppression of tumor-related cells. This may be an important strategy, because destroying PD-1 on T cells may help avoid the immune-related side effects observed when anti-PD-1 monoclonal antibodies are administered systemically, while still improving the anti-tumor effect of CAR T cells. active.

 

Advances in improving the specificity and safety of CRISPR technology

Although the CRISPR-Cas9-based RNA-guided (sgRNA) programmable nuclease is a very versatile and useful tool, and mainly generates accurate and precise DNA DSB, off-target effects may also occur. In addition, chromosomal translocation is a rare unwanted side effect, especially in the case of multiplexing (72). In order to reduce the risk of these unwanted events, further engineering is being developed, such as the CRISPR-Cas9 system with fewer off-target effects and newer gene editing methods, as described below. These advancements will lead to more efficient and safer production of genome-modified “off-the-shelf” CAR T and CAR NK cell products (Figure 2).

Use cell and genome modification to generate CAR T and CAR NK cells

Figure 2. CRISPR-Cas9-mediated gene editing of CAR T cells. TCR can be knocked out to reduce the possibility of graft-versus-host disease (GVHD). HLA can be knocked out to increase the persistence of genetically modified cells. Knockout of targetable receptors for other drugs (such as antibodies) can be accomplished to allow selective survival of genetically modified cells (such as CD52).

 

CRISPR-Cas9 has two nuclease domains, and introducing an inactivating mutation into one of the domains produces a so-called nickase, which cuts only one strand of the target DNA (73). As a further improvement, “dead” Cas9 variants with inactivating mutations in both nuclease domains were created. These variants can be fused with DNA modifying enzymes, such as Apobec-like nucleobase deaminase. These “base editors” enable clear base changes without cutting DNA, further reducing the possibility of side effects (74). Recently, catalytically damaged Cas9 is fused with an engineered reverse transcriptase (derived from murine leukemia virus) to achieve “initial editing” as a new technology to write new genetic information into specific DNA sites (75) . Therefore, major editing has further expanded the capabilities of gene editing and created new options for immunotherapy.

Other methods have been developed to minimize the potentially harmful activities of genome editing described above. For example, because effective genome modification does not require long-term CRISPR-Cas9 expression, transient RNA-protein (RNP) complexes can be delivered to target cell populations to replace viral vectors or DNA constructs. Alternatively, non-integrating lentiviral vectors can be designed to achieve the transient delivery of CRISPR-Cas9 editing and possibly target specific cell populations (76). The high-fidelity CRISPR-Cas9 nuclease variant is designed to have less interaction with non-specific DNA sequences, but has also been developed to maintain target DNA activity (77). As mentioned above, the new Cas9 fusion protein is designed to create base editors, namely cytosine base editor (CBE) and adenosine base editor (ABE), capable of editing individual bases (78, 79).

 

 


 

Mechanisms to improve the anti-cancer activity of immune cells

In addition to enhancing the recognition of tumor cells by immune cells through CAR expression, additional modifications to CAR cells may be needed to effectively overcome the drug resistance mechanism of tumor cells. One mechanism used by tumor cells to evade immune cell-mediated cytotoxicity is to use immune checkpoint signals, which are used to inhibit the immune damage of “self” cells in a healthy state.

Immune checkpoints are a key component of autoimmune tolerance and can avoid autoimmune diseases such as rheumatoid arthritis (81, 82), type I diabetes (83) and multiple sclerosis (84). Checkpoint receptors on immune cells recognize ligands expressed on the cells being monitored, and ligands activate these immune checkpoint receptors to cause immune cell inactivation.

This mechanism is used by tumor cells, which may overexpress these ligands or induce other cells in the tumor microenvironment (TME) [e.g., tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), regulatory Sex T cells (Treg)]) express checkpoint ligands to form an immunosuppressive shield throughout the TME, thereby helping tumor cells to evade immune surveillance (85-87).

The immunosuppressive factors secreted by TME cells, such as transforming growth factor-β (TGF-β), can directly inhibit the cytotoxic activity of CAR T cells, and even directly inhibit the differentiation of effector T cells into regulatory T cells (88-90).

Immune checkpoint molecules include cytotoxic T lymphocyte-associated antigen 4 (CTLA4), PD-1 (PDCD1, CD279), lymphocyte activation gene 3 (LAG-3) and T cell membrane protein 3 (TIM3, HAVCR2) (91-94 ). The interaction of immune checkpoints with their cognate ligands leads to the suppression of immune cell function. Therefore, tumor cells may express CD80/86 to inhibit T cell activity by binding to CTLA4, or express PD-1 ligand PD-L1 (CD274) or PD-L2 (PDCD1LG2, PD-2 ligand). Therefore, LAG-3 binds to MHC class II or fibrinogen-like protein 1 (FGL1), or TIM3 to galectin 9, carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), high mobility group box protein 1 (HMGB1) ) Or non-protein ligand phosphatidylserine showed negative regulation of the cytotoxic activity of immune cells (95-97).

Several antibodies have been developed to inhibit the activity of immune checkpoint molecules, and the clinical anticancer activity of some of these checkpoint inhibitors has been demonstrated. Currently, most studies have investigated checkpoint inhibition of CTLA4 and PD-1 activity (98, 99). However, the strategy of inhibiting LAG-3 may be more promising, because antibodies targeting LAG-3 have been shown to enhance the activation of cytotoxic T cells and may inhibit the immunosuppressive activity induced by Tregs because of the discovery of LAG-3 in Tregs + Subpopulation levels are elevated in the tumor site and peripheral blood mononuclear cells of patients with melanoma or colorectal cancer (100).

The combination of anti-LAG-3 antibody IMP321 and paclitaxel can improve the immune response and greater anti-tumor activity in patients with metastatic breast cancer (101). Currently, more than 240 clinical studies are evaluating the efficacy of checkpoint inhibitors in several different treatment modalities for cancer patients (link to corresponding studies on clinical trials.gov). Since CAR-modified immune cells may be functionally inactivated or depleted due to immune checkpoints and other tumor escape mechanisms, checkpoint suppression can help promote the persistence and anti-tumor activity of CAR T and CAR NK cells. Currently, a clinical trial (NCT03545815) for patients with mesothelin-positive solid tumors is testing the efficacy of mesothelin-targeted CAR T cells and the accompanying CRISPR-Cas9-mediated TCRαβ and PD-1 knockout. Such studies will help clarify the feasibility of incorporating checkpoint suppression in “off-the-shelf” CAR T cell settings.

 

In addition to the application of immune checkpoint inhibitors, CAR-mediated cytokine secretion at the tumor site using T cells redirected for universal cell killing (TRUCKs) has also demonstrated anti-tumor activity (102-105). This strategy involves the modification of T cells with a constitutively expressed CAR and a cytokine expression cassette controlled by an inducible promoter. The TRUCK concept uses the NFAT signaling pathway to activate the CAR CD3ζ signaling domain after tumor antigen recognition to produce pro-inflammatory cytokines. This leads to the modification of TME through cytokine secretion and recruitment of additional anti-tumor immune cells to increase anti-cancer activity (see Figure 3). In the original design, TRUCK was produced by the transfer of two independent vectors mediated by retroviral vectors, one for CAR and the second for inducible cytokine expression cassettes. Recent work has shown that it is feasible to deliver the necessary genetic cargo on a single lentiviral vector (106), thus promoting the potential use of this technology in “off the shelf” immunotherapy.

Use cell and genome modification to generate CAR T and CAR NK cells

Figure 3. T cells redirected for universal cytokine killing (TRUCK) are used to reshape the tumor microenvironment. After antigen binding, CAR activates the CD3z(eta) signal, thereby activating the NFAT-driven promoter that controls the expression of anti-tumor cytokine cassettes (such as IL12 or IL18). Cytokines are then secreted from CAR T or CAR NK cells into the tumor microenvironment, where they recruit additional immune cells to enhance anti-tumor activity.

 

 

 


 

Use targeted viral vector nanoparticles to directly apply CAR-based principles in vivo

Due to the high requirements for the entire origin of CAR-carrying cell products, future-oriented “off-the-shelf” application methods are also considering the direct application of GMP-grade viral vector preparations to directly apply CAR-based principles to targeted immune cells, such as T And NK cells. This method completely bypasses immune rejection barriers, avoids time-consuming in vitro operations and cell culture, and can directly reach individual target effector cells. It is worth noting that in preclinical studies, when used systemically or locally, the receptor targeting carrier particles have the same selectivity for their target cell types as their antigen antibodies. In this regard, receptor targeting using viral vector nanoparticles opens up possibilities for new concepts in immunotherapy and cell-type-specific delivery of CAR in the in vivo environment (107). For non-viral delivery of mRNA in lipid nanoparticles, a similar delivery principle is shown (108).

As a prerequisite for this method, the natural tropism of the viral vectors used (such as gammaretro-/lentivirus and AAV vectors) needs to be shielded so that the viral vector no longer binds to its natural target receptor. In the second step, the definition and specific target selectivity must be added by introducing new selective target cell binding principles, such as scFv antibody, peptide or DARPin (designed ankyrin repeat protein) (107, 109, 110).

 

Retroviral vector for “off-the-shelf” CAR delivery

In the case of enveloped reverse transcription and lentiviral vectors, the pseudotyped envelope is replaced with a newly designated target-specific “targeted” measles (111) or Nipah virus-derived envelope (112). Proved proof of concept of targeted delivery for various target cells (including various hematopoietic cells and endothelial cells). Of particular importance to CAR technology is that surface-engineered lentiviral vectors have been successfully applied to mediate selective gene transfer to various subtypes of lymphocytes, including T cells (110, 113). Impressively, this leads to The in vivo generation of human CD19-CAR T cells has signs of B cell depletion and cytokine release syndrome in a humanized mouse model (114).

 

AAV carrier for “off-the-shelf” CAR delivery

For AAV vectors derived from non-enveloped viruses, capsids are the target of engineering. The protruding capsid structures are protrusions that contain natural receptor binding motifs and pores for loading vector DNA. Gene targeting methods are currently the preferred strategy for modifying carrier tropism and are used to insert receptor binding peptides [reviewed in (115)], immunoglobulin binding domains (116) or nanobodies (117) at the tips of the protrusions. Alternatively, the N-terminus of the non-essential capsid protein VP2 can be used as an insertion site.

This is particularly useful for (I) incorporation of large peptides, (II) targeting of parts that depend on its 3D structure function, or (III) incorporation of whole proteins (118–122). Each fusion protein is exposed on the surface of the capsid through the pore structure. The tropism can be expanded or redirected, depending on the specificity of the inserted targeting moiety and whether the natural tropism has been ablated, for example by site-directed mutagenesis.

By fusing DARPins with antibody-like specificity and VP2 into the AAV2 capsid, it is proved that off-target and non-targeted delivery is possible after intravenous administration of viral vector particles. The capsid is its main receptor heparan sulfate The binding of proteoglycans is unknown (109). These AAV particles can separate somatic target cells and non-target cells in mixed cell cultures and in the effective area in vivo. For example, the precise delivery of suicide genes into tumor tissues and the specific targeting of CD4+ lymphocytes in vivo have proved this ( 109, 122).

These systems will further enrich the “off-the-shelf” application portfolio of cancer immunotherapy.

 

There are several factors that influence the effectiveness and successful transformation of adoptive cell therapy (such as CAR T and CAR NK cells) in the treatment of cancer. As mentioned above, the choice of cell source is a key decision. Most CAR-based therapies use autologous T cells, which have been successfully used in a number of clinical studies. So far, the success in hematological malignancies (especially lymphatic compartments) is more widespread than in solid tumors. Compared with allogeneic CAR T cells, autologous CAR T cells have advantages such as no GVHD risk and lower risk of rejection. However, autologous CAR T cells may have some immunodeficiencies, and patients must wait several weeks before using autologous CAR T cells.


The development of strategies to overcome tumor-induced immunosuppression has been extensively studied, and the use of immune checkpoint inhibitors or genetically engineered CAR T and CAR NK cells with low response to checkpoint signals are the two main methods to solve this challenge. The emergence or selection of tumor cells that do not express the target antigen, the concept of “antigen loss”, will also have a negative impact on the anti-tumor activity of CAR T and CAR NK cells.

 

 


In short, several possibilities for producing “off-the-shelf” anti-cancer immunotherapies are currently being explored. For example, the control of TCR expression through genome knockout or RNAi down-regulation proves the feasibility of producing “off-the-shelf” allogeneic CAR T cell products. However, other allogeneic cell sources, such as NK cells and macrophages, also seem to be suitable as “off-the-shelf” anticancer CAR cells. In addition to providing cell therapy, the possibility of using a designed viral vector to target immune cells in vivo with CAR is another effective “off-the-shelf” strategy to generate CAR T and CAR NK cells. With the rapid progress of research in these areas, we look forward to the development of effective “off the shelf” therapies that will be widely available to many cancer patients around the world.

 

 

 

 

 

 

 

(source:internet, reference only)


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