Humanized Chimeric Antigen Receptor (CAR) T cells

T cells to unique antigens, the T-cell activation in a major complex chimeric as of the antigen recognition moiety to a single-chain variable fragment domain (scFv) and it the ζ chain of the TCR/CD3 modular recombinant, the intracellular CD3 ζ to T-cell activation following antigen engagement. it the origin of the chimeric antigen receptors (CARs) used clinically to of this very that CD3 with CD3 or growth factor receptor-bound protein optimal structural reconfigurations

from antibody libraries originating from an immunized host (non-human) [4][5][6][7]. There are inherent limitations to this approach as adoptive transfer can trigger an immune response characterized by the production of neutralizing antibodies against the foreign scFvs [5,6]. This limits "durable efficacy" as the administered cells are targeted for destruction and eliminated from the circulation [4][5][6]. Recent advances have addressed this with the aim of increasing long-term persistence and immune-surveillance following T-cell transfer.
scFv humanization is increasingly recognized as an important design feature to optimize CAR-T cell longevity following infusion ( Figure 1) [4,6]. An scFv is composed of four framework regions and three complementarity-determining regions (CDRs), which are responsible for antigen recognition [8]. CDR grafting describes a process where amino acids in the scFv framework of a murine-based CAR are substituted with those of its human counterpart [4,9]. This method is one of the most widely used approaches for the humanization of antibody fragments [9]. Given the dedicated effort to maintain high residue identification during this process, the humanized antibody fragment is expected to have similar characteristics with respect to affinity, sensitivity, and specificity as those of its native counterpart [9]. Another strategy to overcome the immunogenicity issue of animal-derived targeting moieties is to incorporate fully human antibody fragments into CAR constructs [6,10,11]; however, a limited number have been developed thus far. On this basis, scFv humanization remains the preferred option, and their therapeutic promise is currently being tested in preclinical as well as clinical settings (summarized in Table 1) within the CAR arena [12][13][14][15][16][17][18].
Herein, we mention some CAR-T cell products that have humanized scFvs as their targeting domains and highlight humanized monoclonal antibodies (mAbs) that achieved success in the clinics.
In 2006, Kershaw and co-investigators conducted a Phase I clinical trial to investigate the safety of folate receptor-redirected CAR-T cells in patients with metastatic ovarian cancer [26]. As reported, the administered CAR-T cells failed to react with folate receptorexpressing tumor cells in 3 out of 6 subjects (50%) which was attributed to the development of inhibitory factors in their sera [26]. Moreover, in 2011, Lamers and colleagues generated CAR-T cells against carbonic anhydrase IX (CAIX) and investigated their ability to control tumor burden in metastatic renal cell carcinoma patients [27]. Persistence issues were observed following infusion which resulted from immune reactions against the CDRs and framework regions of the CAR targeting domain. This compromised CAR-T cell-mediated antitumor responses [27]. Of note, CAR gene delivery was achieved by retroviral infection in this trial. Immune reactions against the γ-retroviral vector-encoded epitopes were also observed in 2 of the patients further exemplifying immunogenicity issues surrounding CAR transgenes and vectors used for gene transfer [27]. Finally, in a clinical trial (NCT01865617) which investigated the effectiveness of CD19-redirected CAR-T cells against B-cell acute lymphoblastic leukemia (B-ALL), Turtle et al. noted a CD8 + T-cell-mediated immune response against adoptively transferred cells expressing the synthetic receptor. This limited persistence of the administered CAR-T cells and increased the risk of disease relapse [28].  (Table 1) [29]. 18 patients were enrolled in this study from which 14 did not have previous CAR-T cell therapy [29]. Among patients without previous CAR-T cell treatment, 13 (92.9%) achieved complete remission (CR) with incomplete count recovery (CRi) on day 30 [29]. Of note, CRi is defined as <5% bone marrow blasts, absence of extramedullary disease, and no recovery of peripheral blood counts independent of transfusion. Moreover, 17 patients (94.4%) experienced cytokine release syndrome (CRS) and 1 (5.5%) developed reversible neurotoxicity [29]. Of 4 patients with previous CAR-T cell therapy, 1 died on day 14 due to intracranial hemorrhage [29]. Moreover, 2 patients died after undergoing salvage therapy (one on day 145 and the other on day 169) [29]. The remaining patient was reported to be MRD-negative until day 168 [29]. These findings show that CD19-redirected CAR-T cells equipped with humanized scFvs can effectively mediate disease remission in R/R B-ALL patients even in those who have had multiple previous conventional CAR-T cell treatment [29].
In 2020, Heng and co-workers reported the results of another clinical trial (NCT02349698) investigating CAR-Ts with a humanized scFv against CD19 for the treatment of R/R B-ALL patients (Table 1) [30]. Ten patients with R/R B-ALL were enrolled in this study, all of which (100%) achieved CR, 8 patients (80%) remained CR (report published in 2020) and 6 patients (60%) had CR for more than one year and a half [30]. The researchers also reported CRS and neurotoxicity in 4 patients which was mitigated using tocilizumab, glucocorticoid, and plasma exchange [30]. They concluded that CAR-T cells equipped with humanized targeting domains demonstrate prolonged persistence leading to low rates of disease relapse [30].
In a recent clinical trial (NCT02374333), Myers et al., evaluated the antitumor response, persistence, and toxicity of CD19-redirected CAR-T cells with humanized scFvs as the targeting domain in children and young adults with B-ALL (72 patients) and Blymphoblastic lymphoma (2 patients) ( Table 1) [31]. Among these patients, 33 had previous CAR-T cell treatment with a CAR construct containing a murine scFv (FMC63) [31]. 62 patients (84%) experienced CRS and neurotoxicity was observed in 29 patients (39%) [31]. The overall response rate one month after CAR-T cell administration was 98% among the patients with no CAR-T cell treatment history and 64% among the patients with prior CAR-T cell treatment [31]. The researchers also indicated that the relapse-free survival rate at 24 months was 58% and 74% among patients with and without previous CAR-T cell treatment, respectively [31]. Collectively, these findings show that CAR-T cells with humanized targeting domains are capable of mediating durable disease remission with prolonged persistence in children and young adults with R/R B-ALL, even in patients that underwent unsuccessful treatments with CAR-T cells [31].
Several parameters are widely acclaimed to influence the effectiveness of CAR-T cell therapies including CAR-T cell quality, differentiation status, metabolic profile, and importantly CAR design [32][33][34][35][36][37]. Given the limited persistence and immunogenicity issues surrounding CAR-T cell products designed with murine-based scFvs, efforts to develop humanized versions without impairing affinity, specificity, and sensitivity might further enhance the therapeutic promise of CARs redirected against tumor antigens (Table 2). Exemplifying their therapeutic promise, several iterations of humanized CD19-specific CAR T cells are being tested in clinical trials.