IL-7 and CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor

Background BCMA CAR-T cells have demonstrated substantial clinical activity against relapsed/refractory multiple myeloma (RRMM). In different clinical trials, the overall response rate (ORR) varied from 50% to 100%. Complete remission (CR) rate varied from 20% to 80%. Here we developed a BCMA CAR-T cell product manufactured via lentiviral vector-mediated transduction of activated T cells to express a second-generation CAR with 4-1BB costimulatory domain and evaluated the efficacy and safety, moreover, dynamics of immune cell subsets using single-cell mass cytometry during treatment were analyzed. Methods Our trial (ChiCTR1800017404) is a phase 1, single-arm, open-label single center study to evaluate the safety and efficacy of autologous BCMA CAR-T treatment for RRMM. Patients were subjected to a lymphodepleting regimen with Flu and Cy prior to CAR-T infusion. BCMA CAR-T cells were administered as a single infusion at a median dose of 3.5 (1 to 6) ×106/kg. MM response assessment was conducted according to the International Uniform Response Criteria. Cytokine-release syndrome (CRS) was graded as Lee DW et al described (Blood.2014;124(2):188-195). Phenotypic analysis of peripheral blood mononuclear cells (PBMCs), frozen BCMA CAR-T aliquots, phenotype and in vivo kinetics of immune cell subsets after CAR-T infusion were performed by single-cell mass cytometry. Results As of the data cut-off date (August 1st, 2019), 33 patients, median age 62.5 (49 to 75) years old were infused with BCMA CAR-T cells. The median observation period is 8.0 (0.7 to 18) months. ORR was 100% (The patient who died of infection at 20 days after CAR-T infusion were excluded). All the 32 patients achieved MRD negative in bone marrow by flow cytometry in 2 weeks after CAR-T infusion. Partial response (4 PR, 12.1%), VGPR (7 VGPR, 21.2%), and complete response (21 CR, 63.6%) within 12 weeks post CAR-T infusion were achieved. Durable responses from 4 weeks towards the data cut-off date were found in 28/33 patients (84.8%) (Figure 1a). All patients had detectable CAR-T expansion by flow cytometry from Day 3 post CAR-T cell infusion. The peak CAR-T cell expansion in CD3+ lymphocytes of peripheral blood (PB) varied from 35% to 95% with a median percentage of 82.9%. CRS was reported in all the 33 patients, including 4 with Grade 1, 13 with Grade 2 and 16 with Grade 3. During follow-up, 1-year progression-free survival (PFS) was 70.7% (Figure 1b) and overall survival (OS) was 71.7% (Figure 1c). Multivariate analysis of patients with PR and patients with CR+VGPR revealed that factors including extramedullary infiltration, age>60 years old, high-risk cytogenetics, late stage and CAR-T cell dose were not associated with clinical response (P>0.05). Single-cell mass cytometry revealed that the frequency of total T cells, CD8+ T cells, NK cells and CD3+CD56+ NKT cells in PB was not associated with BCM CAR-T expansion or clinical response. CD8+ Granzyme B+ Ki-67+ CAR-T cells expanded prominently in CRS period. As serum cytokines increased during CRS, non-CAR-T immune cell subsets including PD1+ NK cells, CD8+ Ki-67+ ICOS+ T cells expanded dominantly implying that non-CAR-T cells were also activated after CAR-T treatment. After CRS, stem cell like memory CAR-T cells (CD45RO+ CCR7- CD28- CD95+) remain the main subtype of CAR-T cells (Figure 1d). Conclusions Our data showed BCMA CAR-T treatment is safe with prominent efficacy which can overcome the traditional high-risk factors. We also observed high expansion level and long-term persistence of BCMA CAR-T cells contribute to potent anti-myeloma activity. Stem cell like memory CAR-T cells might be associated with long-term persistence of BCMA CAR-T cells. These initial data provide strong evidence to support the further development of this anti-myeloma cellular immunotherapy. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Engineered triple inhibitory receptor resistance improves anti-tumor CAR-T cell performance via CD56

Nature Communications ◽

10.1038/s41467-019-11893-4 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 11

Author(s):

Fan Zou ◽

Lijuan Lu ◽

Jun Liu ◽

Baijin Xia ◽

Wanying Zhang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Tumor Infiltrating Lymphocytes ◽

Interferon Γ ◽

Inhibitory Receptors ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

T Cell Survival

Abstract The inhibitory receptors PD-1, Tim-3, and Lag-3 are highly expressed on tumor-infiltrating lymphocytes and compromise their antitumor activity. For efficient cancer immunotherapy, it is important to prevent chimeric antigen receptor T (CAR-T)-cell exhaustion. Here we downregulate these three checkpoint receptors simultaneously on CAR-T cells and that show the resulting PTL-CAR-T cells undergo epigenetic modifications and better control tumor growth. Furthermore, we unexpectedly find increased tumor infiltration by PTL-CAR-T cells and their clustering between the living and necrotic tumor tissue. Mechanistically, PTL-CAR-T cells upregulate CD56 (NCAM), which is essential for their effector function. The homophilic interaction between intercellular CD56 molecules correlates with enhanced infiltration of CAR-T cells, increased secretion of interferon-γ, and the prolonged survival of CAR-T cells. Ectopically expressed CD56 promotes CAR-T cell survival and antitumor response. Our findings demonstrate that genetic blockade of three checkpoint inhibitory receptors and the resulting high expression of CD56 on CAR-T cells enhances the inhibition of tumor growth.

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Human Hyaluronidase PH20 Potentiates the Antitumor Activities of Mesothelin-Specific CAR-T Cells Against Gastric Cancer

Frontiers in Immunology ◽

10.3389/fimmu.2021.660488 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ruocong Zhao ◽

Yuanbin Cui ◽

Yongfang Zheng ◽

Shanglin Li ◽

Jiang Lv ◽

...

Keyword(s):

Gastric Cancer ◽

T Cells ◽

T Cell ◽

Solid Tumors ◽

Cell Infiltration ◽

Car T Cells ◽

Car T Cell ◽

T Cell Infiltration ◽

Car T

T cell infiltration into tumors is essential for successful immunotherapy against solid tumors. Herein, we found that the expression of hyaluronic acid synthases (HAS) was negatively correlated with patient survival in multiple types of solid tumors including gastric cancer. HA impeded in vitro anti-tumor activities of anti-mesothelin (MSLN) chimeric antigen receptor T cells (CAR-T cells) against gastric cancer cells by restricting CAR-T cell mobility in vitro. We then constructed a secreted form of the human hyaluronidase PH20 (termed sPH20-IgG2) by replacing the PH20 signal peptide with a tPA signal peptide and attached with IgG2 Fc fragments. We found that overexpression of sPH20-IgG2 promoted CAR-T cell transmigration through an HA-containing matrix but did not affect the cytotoxicity or cytokine secretion of the CAR-T cells. In BGC823 and MKN28 gastric cancer cell xenografts, sPH20-IgG2 promoted anti-mesothelin CAR-T cell infiltration into tumors. Furthermore, mice infused with sPH20-IgG2 overexpressing anti-MSLN CAR-T cells had smaller tumors than mice injected with anti-MSLN CAR-T cells. Thus, we demonstrated that sPH20-IgG2 can enhance the antitumor activity of CAR-T cells against solid tumors by promoting CAR-T cell infiltration.

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G-CSF/Plerixafor Dual-Mobilized Donor Derived CD33CAR T-Cells As Potent and Effective AML Therapy in Pre-Clinical Models

Blood ◽

10.1182/blood-2021-153868 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1716-1716

Author(s):

Giacomo Canesin ◽

Hillary Hoyt ◽

Reid Williams ◽

Mariana Silva ◽

Melissa Chng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Immune Cell ◽

Ex Vivo ◽

Publicly Traded ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract There are currently no known acute myeloid leukemia (AML) specific antigens. Genetic ablation of CD33 using CRISPR/Cas9 engineering of the hematopoietic stem cell (HSC) transplant (VOR33) represents a synthetic biology approach to generating a leukemia-specific antigen in the transplant recipient. VOR33 enables anti-CD33 CAR-T cell killing of AML cells while sparing normal myeloid lineage development and function, thereby potentially avoiding myelosuppression and increasing the therapeutic index of anti-CD33 CAR-T therapy. Mobilized leukapheresis product represents an attractive starting material for the generation of both a CD33 null HSC transplant and a complementary CD33CAR T-cell product. In this study, we sought to determine the impact of dual mobilization with Granulocyte-Colony Stimulating Factor (G-CSF) and plerixafor (mozobil) on immune cell composition, T cell phenotype, and the functionality of these T cells to control AML tumor growth upon chimeric antigen receptor (CAR) transduction. Mobilized (mob) peripheral blood mononuclear cells (PBMCs) were collected from healthy donors injected with G-CSF (10µg/kg/day, 5 consecutive days) and plerixafor (240µg/kg, on day 4 and 5). Non-mobilized (non-mob) PBMCs, collected from the same donors, were used as controls. Cells were analyzed by flow cytometry for immunophenotyping and T cell characterization including differentiation and bone marrow homing markers, as well as responses to T cell activation with anti-CD3 (OKT3) and IL-2. Non-/mob PBMC populations were also analyzed by single-cell next generation sequencing (CITEseq) using 127 immune cell phenotypic markers in combination with extensive transcriptome and T cell receptor repertoire analysis. In addition, lentiviral transduction of anti-CD33 CAR constructs enabled functional comparisons of mob- and non-mob-CAR T-cells in AML cell co-cultures as well as AML mouse models. Ex vivo immunophenotyping of PBMC from a total of over 30 healthy donor samples showed that mobilization decreases the overall percentage of CD3 + T cells but increases that of naïve T cells (CD45RA +/CCR7 +), at the expense of T effector-memory (CD45RA -/CCR7 -) and central-memory (CD45RA -/CCR7 +) populations. Bone marrow homing factors (e.g.: CXCR4) were increased in mob compared to non-mob T cell samples. As expected, higher percentages of monocytes (CD14 +) were detected in mob compared to non-mob donor samples, but this difference disappeared after culture under T cell activation conditions. T cell activation also led to similar increases in CD25, CD69 and CD137 expression, and a decrease in CD62L expression. Single cell sequencing analyses confirmed mobilization-induced increases in naïve T cells as well as changes in monocytes/macrophages, CD4 + T cells and NK cells percentages. Notably, functional in vitro cytotoxic assays demonstrated that mob-CD33-CAR T-cells are as effective as non-mob-CD33-CAR T-cells in killing CD33 + AML cells, with reduced 'bystander' activation of non-transduced T cells. Furthermore, results from in vivo AML mouse models indicate that mob-CD33-CAR T-cells are equally effective in clearing CD33 + tumors as non-mob-CD33-CAR T-cells. Our analysis showed phenotypical ex vivo differences between mob and non-mob PBMCs, which disappeared upon activation, indicating similar responses to T cell-specific stimulation. These findings are corroborated by similar in vitro cytotoxicity profiles of non-/mob-CAR T-cells. Non-transduced T cells in the mob-CAR T-cell population showed limited 'bystander' activation, indicating a potentially favorable clinical toxicity profile. Additional in vivo assessment of mob-CAR T-cell function shows effective tumor clearance, which supports further efforts towards their clinical use in combination with engineered HSCs for the treatment of AML patients. Disclosures Canesin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hoyt: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Williams: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Silva: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chng: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Cummins: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ung: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Qiu: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Shin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hu: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ge: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Scherer: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chakraborty: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Kassim: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company.

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Chimeric Antigen Receptor-Engineered T-Cells - A New Way and Era for Lymphoma Treatment

Recent Patents on Anti-Cancer Drug Discovery ◽

10.2174/1574892814666191022164641 ◽

2020 ◽

Vol 14 (4) ◽

pp. 312-323

Author(s):

Romeo G. Mihăilă

Keyword(s):

T Cells ◽

T Cell ◽

Response Rate ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Car T Cells ◽

Future Developments ◽

Car T Cell ◽

Large B Cell Lymphoma ◽

Car T

Background: Patients with refractory or relapsed diffuse large B-cell lymphoma have a poor prognosis with the current standard of care. Objective: Chimeric Antigen Receptor T-cells (CAR T-cells) are functionally reprogrammed lymphocytes, which are able to recognize and kill tumor cells. The aim of this study is to make progress in this area. Method: A mini-review was achieved using the articles published in Web of Science and PubMed in the last year and the new patents were made in this field. Results: The responses to CAR T-cell products axicabtagene ciloleucel and tisagenlecleucel are promising; the objective response rate can reach up to 83%, and the complete response rate ranges between 40 and 58%. About half of the patients may have serious side effects, such as cytokine release syndrome and neurotoxicity. Current and future developments include the improvement of CAR T-cell expansion and polyfunctionality, the combined use of CAR T-cells with a fusion protein between interferon and an anti-CD20 monoclonal antibody, with checkpoint inhibitors or small molecule sensitizers that have apoptotic-regulatory effects. Furthermore, the use of IL-12-expressing CAR T-cells, an improved technology for the production of CAR T-cells based on targeted nucleases, the widespread use of allogeneic CAR T-cells or universal CAR T-cells obtained from genetically engineered healthy donor T-cells are future developments actively considered. Conclusion: CAR T-cell therapy significantly improved the outcome of patients with relapsed or refractory diffuse large B-cell lymphoma. The advances in CAR T-cells production technology will improve the results and enable the expansion of this new immunotherapy.

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Structure of the Signal Transduction Domain in Second-Generation CAR Regulates the Input Efficiency of CAR Signals

International Journal of Molecular Sciences ◽

10.3390/ijms22052476 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2476

Author(s):

Kento Fujiwara ◽

Masaki Kitaura ◽

Ayaka Tsunei ◽

Hotaka Kusabuka ◽

Erika Ogaki ◽

...

Keyword(s):

Signal Transduction ◽

T Cells ◽

T Cell ◽

Second Generation ◽

Independent Manner ◽

Cell Functions ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

The Impact

T cells that are genetically engineered to express chimeric antigen receptor (CAR) have a strong potential to eliminate tumor cells, yet the CAR-T cells may also induce severe side effects due to an excessive immune response. Although optimization of the CAR structure is expected to improve the efficacy and toxicity of CAR-T cells, the relationship between CAR structure and CAR-T cell functions remains unclear. Here, we constructed second-generation CARs incorporating a signal transduction domain (STD) derived from CD3ζ and a 2nd STD derived from CD28, CD278, CD27, CD134, or CD137, and investigated the impact of the STD structure and signaling on CAR-T cell functions. Cytokine secretion of CAR-T cells was enhanced by 2nd STD signaling. T cells expressing CAR with CD278-STD or CD137-STD proliferated in an antigen-independent manner by their STD tonic signaling. CAR-T cells incorporating CD28-STD or CD278-STD between TMD and CD3ζ-STD showed higher cytotoxicity than first-generation CAR or second-generation CARs with other 2nd STDs. The potent cytotoxicity of these CAR-T cells was not affected by inhibiting the 2nd STD signals, but was eliminated by placing the STDs after the CD3ζ-STD. Our data highlighted that CAR activity was affected by STD structure as well as by 2nd STD signaling.

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Anti-Mesothelin CAR T cell therapy for malignant mesothelioma

Biomarker Research ◽

10.1186/s40364-021-00264-1 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Laura Castelletti ◽

Dannel Yeo ◽

Nico van Zandwijk ◽

John E. J. Rasko

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Malignant Mesothelioma ◽

Oncolytic Viruses ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

AbstractMalignant mesothelioma (MM) is a treatment-resistant tumor originating in the mesothelial lining of the pleura or the abdominal cavity with very limited treatment options. More effective therapeutic approaches are urgently needed to improve the poor prognosis of MM patients. Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a novel potential treatment for this incurable solid tumor. The tumor-associated antigen mesothelin (MSLN) is an attractive target for cell therapy in MM, as this antigen is expressed at high levels in the diseased pleura or peritoneum in the majority of MM patients and not (or very modestly) present in healthy tissues. Clinical trials using anti-MSLN CAR T cells in MM have shown that this potential therapeutic is relatively safe. However, efficacy remains modest, likely due to the MM tumor microenvironment (TME), which creates strong immunosuppressive conditions and thus reduces anti-MSLN CAR T cell tumor infiltration, efficacy and persistence. Various approaches to overcome these challenges are reviewed here. They include local (intratumoral) delivery of anti-MSLN CAR T cells, improved CAR design and co-stimulation, and measures to avoid T cell exhaustion. Combination therapies with checkpoint inhibitors as well as oncolytic viruses are also discussed. Preclinical studies have confirmed that increased efficacy of anti-MSLN CAR T cells is within reach and offer hope that this form of cellular immunotherapy may soon improve the prognosis of MM patients.

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Enhanced CD4+ and CD8+ T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses

Scientific Reports ◽

10.1038/s41598-021-96327-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alexander J. Dwyer ◽

Jacob M. Ritz ◽

Jason S. Mitchell ◽

Tijana Martinov ◽

Mohannad Alkhatib ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Convex Hull ◽

Pancreatic Islets ◽

Immune Cell ◽

Nod Mice ◽

Cell Infiltration ◽

Immune Cell Infiltration ◽

Analytical Strategy ◽

Islet Mass

AbstractThe notion that T cell insulitis increases as type 1 diabetes (T1D) develops is unsurprising, however, the quantitative analysis of CD4+ and CD8+ T cells within the islet mass is complex and limited with standard approaches. Optical microscopy is an important and widely used method to evaluate immune cell infiltration into pancreatic islets of Langerhans for the study of disease progression or therapeutic efficacy in murine T1D. However, the accuracy of this approach is often limited by subjective and potentially biased qualitative assessment of immune cell subsets. In addition, attempts at quantitative measurements require significant time for manual analysis and often involve sophisticated and expensive imaging software. In this study, we developed and illustrate here a streamlined analytical strategy for the rapid, automated and unbiased investigation of islet area and immune cell infiltration within (insulitis) and around (peri-insulitis) pancreatic islets. To this end, we demonstrate swift and accurate detection of islet borders by modeling cross-sectional islet areas with convex polygons (convex hulls) surrounding islet-associated insulin-producing β cell and glucagon-producing α cell fluorescent signals. To accomplish this, we used a macro produced with the freeware software ImageJ equipped with the Fiji Is Just ImageJ (FIJI) image processing package. Our image analysis procedure allows for direct quantification and statistical determination of islet area and infiltration in a reproducible manner, with location-specific data that more accurately reflect islet areas as insulitis proceeds throughout T1D. Using this approach, we quantified the islet area infiltrated with CD4+ and CD8+ T cells allowing statistical comparison between different age groups of non-obese diabetic (NOD) mice progressing towards T1D. We found significantly more CD4+ and CD8+ T cells infiltrating the convex hull-defined islet mass of 13-week-old non-diabetic and 17-week-old diabetic NOD mice compared to 4-week-old NOD mice. We also determined a significant and measurable loss of islet mass in mice that developed T1D. This approach will be helpful for the location-dependent quantitative calculation of islet mass and cellular infiltration during T1D pathogenesis and can be combined with other markers of inflammation or activation in future studies.

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CARTmath—A Mathematical Model of CAR-T Immunotherapy in Preclinical Studies of Hematological Cancers

Cancers ◽

10.3390/cancers13122941 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2941

Author(s):

Luciana R. C. Barros ◽

Emanuelle A. Paixão ◽

Andrea M. P. Valli ◽

Gustavo T. Naozuka ◽

Artur C. Fassoni ◽

...

Keyword(s):

Mathematical Model ◽

T Cells ◽

T Cell ◽

Mouse Models ◽

In Silico ◽

Car T Cells ◽

Car T Cell ◽

Cell Immunotherapy ◽

Car T ◽

T Cell Immunotherapy

Immunotherapy has gained great momentum with chimeric antigen receptor T cell (CAR-T) therapy, in which patient’s T lymphocytes are genetically manipulated to recognize tumor-specific antigens, increasing tumor elimination efficiency. In recent years, CAR-T cell immunotherapy for hematological malignancies achieved a great response rate in patients and is a very promising therapy for several other malignancies. Each new CAR design requires a preclinical proof-of-concept experiment using immunodeficient mouse models. The absence of a functional immune system in these mice makes them simple and suitable for use as mathematical models. In this work, we develop a three-population mathematical model to describe tumor response to CAR-T cell immunotherapy in immunodeficient mouse models, encompassing interactions between a non-solid tumor and CAR-T cells (effector and long-term memory). We account for several phenomena, such as tumor-induced immunosuppression, memory pool formation, and conversion of memory into effector CAR-T cells in the presence of new tumor cells. Individual donor and tumor specificities are considered uncertainties in the model parameters. Our model is able to reproduce several CAR-T cell immunotherapy scenarios, with different CAR receptors and tumor targets reported in the literature. We found that therapy effectiveness mostly depends on specific parameters such as the differentiation of effector to memory CAR-T cells, CAR-T cytotoxic capacity, tumor growth rate, and tumor-induced immunosuppression. In summary, our model can contribute to reducing and optimizing the number of in vivo experiments with in silico tests to select specific scenarios that could be tested in experimental research. Such an in silico laboratory is an easy-to-run open-source simulator, built on a Shiny R-based platform called CARTmath. It contains the results of this manuscript as examples and documentation. The developed model together with the CARTmath platform have potential use in assessing different CAR-T cell immunotherapy protocols and its associated efficacy, becoming an accessory for in silico trials.

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Adoptive Immunotherapy beyond CAR T-Cells

Cancers ◽

10.3390/cancers13040743 ◽

2021 ◽

Vol 13 (4) ◽

pp. 743

Author(s):

Aleksei Titov ◽

Ekaterina Zmievskaya ◽

Irina Ganeeva ◽

Aygul Valiullina ◽

Alexey Petukhov ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Solid Tumors ◽

Adoptive Immunotherapy ◽

Γδ T Cells ◽

Car T Cells ◽

Car T Cell ◽

Cell Immunotherapy ◽

Car T

Adoptive cell immunotherapy (ACT) is a vibrant field of cancer treatment that began progressive development in the 1980s. One of the most prominent and promising examples is chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of B-cell hematologic malignancies. Despite success in the treatment of B-cell lymphomas and leukemia, CAR T-cell therapy remains mostly ineffective for solid tumors. This is due to several reasons, such as the heterogeneity of the cellular composition in solid tumors, the need for directed migration and penetration of CAR T-cells against the pressure gradient in the tumor stroma, and the immunosuppressive microenvironment. To substantially improve the clinical efficacy of ACT against solid tumors, researchers might need to look closer into recent developments in the other branches of adoptive immunotherapy, both traditional and innovative. In this review, we describe the variety of adoptive cell therapies beyond CAR T-cell technology, i.e., exploitation of alternative cell sources with a high therapeutic potential against solid tumors (e.g., CAR M-cells) or aiming to be universal allogeneic (e.g., CAR NK-cells, γδ T-cells), tumor-infiltrating lymphocytes (TILs), and transgenic T-cell receptor (TCR) T-cell immunotherapies. In addition, we discuss the strategies for selection and validation of neoantigens to achieve efficiency and safety. We provide an overview of non-conventional TCRs and CARs, and address the problem of mispairing between the cognate and transgenic TCRs. Finally, we summarize existing and emerging approaches for manufacturing of the therapeutic cell products in traditional, semi-automated and fully automated Point-of-Care (PoC) systems.

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