Osteosarcoma (OS) remains a frustrating malignancy in children and young adults, with a dismal prognosis for metastatic and recurrent disease. Immunotherapy in OS is less promising than in some other specifically targeted cancer types due to intratumoral heterogeneity and uncertainty in potential target proteins leading to substantial off-target expression.

(Data source: Chen C, et al. Cancer Lett. 2021)

Nature , June 2023, Nature Communication s published a research paper titled "ALPL-1 is a target for chimeric antigen receptor therapy in osteosarcoma." Based on two anti-osteosarcoma cell antibodies isolated more than 30 years ago, the study screened and determined that the antibodies recognized the target ALPL-1 specifically on osteosarcoma cells (especially lung metastasis). CAR molecules were constructed based on the antibody sequences, and it was demonstrated that T cells expressing these CAR molecules could specifically recognize and kill osteosarcoma cells. Preclinical animal model validation also showed that the CAR-T cells effectively controlled tumor progression in different human xenograft models (including in situ and metastatic environments), thus confirming that ALPL-1 is a very attractive target for CAR-based therapy in osteosarcoma.

(Article publication information)

Anti-osteosarcoma cell antibody discovery:

Over 30 years ago, monoclonal antibodies against sarcoma-associated cell membrane antigens were generated by immunizing BALB/c mice with cells derived from human osteosarcoma. Seven of the antibodies generated bound to osteosarcoma cells but not to autologous skin fibroblasts. Monoclonal antibodies from two of these seven clones (TP-1 and TP-3) also did not cross-react with non-sarcoma tumor cells or peripheral blood lymphocytes. Immunohistochemical studies on frozen tissue sections demonstrated that TP-1 (IgG-2a) and TP-3 (IgG-2b) antibodies had characteristic and identical specificity profiles.

(Data source: Bruland O, et al. Int J Cancer. 1986)

Confirmation that TP-1 and TP-3 recognize their targets:

The researchers used the Retrogenix platform to screen a collection of nearly 5,000 different cDNAs encoding membrane proteins. For both TP antibodies, the same target was identified in two consecutive rounds and confirmed in the final panel. The target was identified as isoform 1 (NP000469.3) of the ALPL gene (NM_000478.6). Interestingly, isoform 3 (NP 001120973.2) was also included in the screen, differing from isoform 1 by only 55 amino acids at the N-terminus and not recognized by the TP antibodies.

(Retrogenix platform screening candidate target proteins)

The specificity of the antibody against ALPL-1 was further confirmed by overexpression and knockout experiments (Figures 1a, b, c below). In addition, the presence of ALPL-1 was studied in relevant healthy tissues (bone and lung) compared with osteosarcoma lung metastasis samples, and a significant increase in diseased tissue was detected (Figures 1e, f, g below).

(Characterization of TP -1 and TP -3 antibodies)

Construction of CAR-T targeting osteosarcoma:

Using previously published hybridoma sequences, scFvs were designed and subcloned into second-generation CAR backbones (Figure 2a). The TP-1- and TP-3-derived CARs were named OSCAR-1 and OSCAR-3, respectively. OSCARs were first validated to retain the specificity of the TP antibody for ALPL-1 (killing activity was detected only in the presence of ALPL-1, Figure 2c). Using ALPL knockout osteosarcoma OHS cells (Figure 2b), it was observed that target ablation protected OSCAR-directed killing (Figure 2c). This was further confirmed when additional osteosarcoma cell lines stained positive with the TP antibody, demonstrating sensitivity to OSCAR-T cells (Figure 2d). These data confirm the specificity of OSCAR for cells expressing ALPL-1 on their surface.

(Characterization of CAR-T targeting osteosarcoma)

OSCAR-T efficacy assessment:

We then analyzed whether OSCAR-expressing T cells could generate sufficiently robust stimulation to trigger cytokine release. Following overnight incubation with osteosarcoma cell lines, OSCAR-3 appeared more effective at triggering the release of certain cytokines (Figure 2e). OSCAR-expressing T cells maintained robust short-term effector function until day 21, mediating effective elimination of osteosarcoma cells (Figure 2f).

(OSCAR-T cell assay)

In human xenograft models, both OSCAR-1 and OSCAR-3 effectively controlled tumor growth in the model.

(OSCAR-T Animal Model Test)

OSCAR-T safety assessment:

The researchers examined the reactivity of OSCART cells to selected primary tissues or healthy tissue cell lines: mesenchymal stem cells (MSCs) of bone cells, human renal epithelial cells (HREpC), human hepatocytes (HH), human alveolar epithelial cells (HPAEpiC), human lung endothelial cells (Hulec-5a) and human fetal lung fibroblasts (MRC-5) in co-culture experiments: consistent with the lower in vitro activity of OSCAR-1, these cells had a slight reactivity to osteoblasts, and only MSCs differentiated for 18 days were able to stimulate OSCAR-3 T cells to a similar extent as control osteosarcoma cells (Figure 1a below); while other tissue cells could not induce stimulation of OSCART (Figure 1b below).

(OSCAR-T cell assay)

Summary:

ALPL-1 has never been designated as a target for antibody-based therapy, likely because it is present in diverse healthy tissues and reports of its cellular localization, namely the plasma membrane and cytosol, remain controversial. This study confirmed ALPL-1 as an osteosarcoma-specific target (exclusively present in osteosarcoma samples, barely detectable in healthy tissues, and expressed at trace levels in osteoblasts). A chimeric antigen receptor (CAR) T cell therapy based on this target was developed, initially showing promising therapeutic potential. The study will further explore the role of ALPL-1 in osteosarcoma and osteoblast activity, and advance to first-in-human clinical trials.

The Wuhan Mabnus Bio R&D team has over a decade of experience in recombinant protein and antibody development, focusing on recombinant protein expression in eukaryotic mammalian cells and development of recombinant rabbit monoclonal antibodies in single B cells. We provide simpler and more efficient recombinant protein and antibody products and development services. We currently offer effective recombinant expression targeting ALPL-1 protein and TP-1 and TP-3 antibody sequence information.