CD229 CAR T cells eliminate multiple myeloma and tumor propagating cells without fratricide

Multiple myeloma (MM) is a plasma cell malignancy and most patients eventually succumb to the disease. Chimeric antigen receptor (CAR) T cells targeting B-Cell Maturation Antigen (BCMA) on MM cells have shown high-response rates, but limited durability. CD229/LY9 is a cell surface receptor present on B and T lymphocytes that is universally and strongly expressed on MM plasma cells. Here, we develop CD229 CAR T cells that are highly active in vitro and in vivo against MM plasma cells, memory B cells, and MM-propagating cells. We do not observe fratricide during CD229 CAR T cell production, as CD229 is downregulated in T cells during activation. In addition, while CD229 CAR T cells target normal CD229high T cells, they spare functional CD229neg/low T cells. These findings indicate that CD229 CAR T cells may be an effective treatment for patients with MM.

Minor points: -Intro mentions 30,000 new MM cases per year. I believe this is the # of new cases in the US alone, so this should be clarified. -Intro mentions that "BCMA-negative relapses occur often" after anti-BCMA CARs. This is unsupported by the cited studies. I don't think Raje et al reported on BCMA expression on MRD or at relapse. Cohen et al showed diminishment of BCMA expression on residual disease after initial CAR expansion, but BMCA expression later rebounded in most cases, at least by flow cytometry. It might be more accurate to say that modulation of BCMA has been observed after anti-BCMA CAR therapy, but significance is still uncertain. Authors are right to point out, however, that BCMA expression is heterogeneous, to the point that many patients were excluded from trials due to low/absent BCMA expression.
-Authors should state the source of the anti-BCMA scFv used in their comparative experiments. -Last sentence in discussion: I suggest avoiding mention of "curative potential" in a preclinical paper, given the humbling clinical experience with every therapeutic advance in MM over the last many decades.

Reviewer #2 (Remarks to the Author): Expert in CAR-T cells
In the submitted the authors describe the generation of CD229-CAR T cells for the cell therapy of multiple myeloma (MM). They explore the effector function of CD229-CAR T cells in vitro and in vivo and demonstrate potent anti MM activity. The manuscript is well written, and the authors' conclusion are supported by the presented data. However, I have several major and minor concerns the authors should address.
1. Animal experiments: it seems that each animal experiment was only performed once; please provide data for additional animals to substantiate the promising results shown in Figure 4.
2. CAR expression: I might have missed it, but I could not find that the authors demonstrate CAR expression of transduced T cells -please provide data for all CAR constructs used in this study.
3. CEFT Elispot assay: the experimental set up is unclear, important controls are missing and the results in its present form are impossible to interpret; recommend removing from manuscript.
Minor concerns: 1. Humanized mouse model: calling a model in which human PBMCs are injected into NSG mice after 2 doses of busulfan, injecting CAR T cells on day 2 and harvesting cells on day 6, a humanized mouse model is misleading unless the authors can show engraftment of human CD34+ progenitor cells. With no engraftment, recommend calling this experiment an 'in vivo cytotoxicity assay with human PBMCs'. Figure 4.

Comment 2.1: "Animal experiments: it seems that each animal experiment was only performed once; please provide data for additional animals to substantiate the promising results shown in
Reply: We have now repeated both xenograft experiments as described in our manuscript and observed comparable efficacy of CD229 CAR T cells against multiple myeloma cell lines U266 (Fig. 1A) and RPMI8226 (Fig. 1B) by in vivo imaging. For the more rapidly growing RPMI8226 cell line, we also provide survival data (Fig. 1C). If requested, we can also provide survival data for the U266 experiment, which, however, will require a much longer follow-up period.

Comment 2.2: "CAR expression: I might have missed it, but I could not find that the authors demonstrate CAR expression of transduced T cells -please provide data for all CAR constructs used in this study."
Reply: We have now added CAR expression data as Suppl. Fig. 2C.

Comment 2.3: "CEFT Elispot assay: the experimental set up is unclear, important controls are missing and the results in its present form are impossible to interpret; recommend removing from manuscript."
Reply: We have removed these data from the manuscript. Alternatively, if requested, we could also include additional controls or provide further clarification.

Comment 2.4: "1. Humanized mouse model: calling a model in which human PBMCs are injected into NSG mice after 2 doses of busulfan, injecting CAR T cells on day 2 and harvesting cells on day 6, a humanized mouse model is misleading unless the authors can show engraftment of human CD34+ progenitor cells. With no engraftment, recommend calling this experiment an 'in vivo cytotoxicity assay with human PBMCs'.
Reply: We have changed the description of this experiment throughout the manuscript as requested. NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice were irradiated with 200cGy and injected with (A) 3x10 6 U266 or (B) 1x10 6 RPMI8226 expressing luciferase. After 1 week, mice were injected with 3x10 6 CAR T cells and bioluminescence was determined weekly using in vivo imaging. Statistical significance of differences in survival determined for mice engrafted with RPMI8226 cells (C) were determined by log-rank test.