KHYG-1 NK cells expressing CD38-specific chimeric antigen receptors in Myeloma New Horizons: Validation of the concept of "off-the-shelf" therapy for multiple myeloma

In June 2021, "HemaSphere" published a study on the proof of concept of KHYG-1 NK cells expressing CD38-specific chimeric antigen receptors for multiple myeloma (MM) "off-the-shelf" therapy
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To explore the preclinical anti-MM efficacy and potential toxicity of CD38CAR NK cells by expressing affinity-optimized CD38CARs in KHYG-1 cells, let us find out what the specific results are
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Research background MM is a malignant disease in which clonal plasma cells proliferate abnormally.
Chimeric antigen receptor T cells (CAR-T cells) are effective in treating hematological malignancies
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The lack of universal donors and the long-term production limit the wide application of CAR-T cell therapy.
However, CAR-based treatment methods continue to achieve clinical success.
The rapid increase of candidate antigens for MM and other hematological malignancies has increased the convenience of research and development.
The need for a wider range of treatment strategies facilitates patients who no longer rely on individualized therapies and can directly use "off-the-shelf" CAR-based cell therapies
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Natural killer cells (NK) from a third host may be a source of "off the shelf" CAR therapy
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Unlike allogeneic T cells, NK cells do not produce graft-versus-host response (GvHD).
In addition to reducing the risk of heterologous reactions, NK cells also have other advantages, such as easy expansion, induction of cytokine storm, and independent antigen killing risk Lower
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NK cells have different characteristics from T cells and a shorter life cycle, which makes CAR-NK cells have lower cytokine release syndrome and other chronic toxicity than CAR-T cells
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It is not clear whether NK cells are suitable as CAR carriers for each target antigen, especially CD38
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CD38 has moderate expression levels in different tissues (including non-malignant hematopoietic cells)
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In order to solve the potential efficacy and preclinical safety of CD38CAR NK cells, this study explored the preclinical anti-MM efficacy and potential toxicity of CD38CAR NK by expressing CD38CARs with optimized affinity in KHYG-1 NK cells
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  Research methods 1.
Primary cells: select a single cell from the bone marrow of patients with newly diagnosed or ineffective daratumumab, and cryopreserved in liquid nitrogen until use
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Patient characteristics are shown in Table 1
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2.
Cell line: Culture the target cell line (CD38+ cell line UM9 and THP-1; and CD38-cell line U266) in complete medium, and culture KHYG-1 cells in complete medium supplemented with IL-2
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According to experimental requirements, KHYG-1 cells were incubated with different concentrations of IL-15 or irradiated with 10Gy during culture or before the experiment
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3.
Retroviral transduction: construct CD38CAR and transduce it with MOCK-CAR into KHYG-1 cells.
Three days after transduction, analyze CAR expression by flow cytometry
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4.
Flow cytometry: Use several antibodies in different culture plates to identify the phenotype of CARKHYG-1 cells and target cells: NGFR, CD2, CD3, CD8, CD16, CD14, CD19, CD38, CD56, CD138
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5.
Based on flow cytometry cytotoxicity experiment: stain CAR KHYG-1 or target cells to distinguish effector cells from target cells
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The cells were incubated with the specified effect-to-target ratio (E:T) and stained with appropriate markers.
If necessary, MM cells were further distinguished from other cells, and the cells were analyzed by flow cytometry
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6.
The in vivo efficacy of CD38CAR KHYG-1 cells: MM tumors derived from UM9 cells were inoculated on the backs of RAG2-/-γc-/- mice, and unmodified or irradiated CAR-KHYG-1 cells were combined at the specified dose At the time point, the mice were injected intravenously and IL-15 was injected at the same time
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The growth of the tumor was monitored by BLI analysis and manual tumor size measurement every week.
The bone marrow, spleen, blood, and stent were taken from the mouse after death, and the single cell suspension was counted, stained and measured by flow cytometry
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  Research results CD38CAR KHYG-1 cell anti-tumor effect in vitro Two kinds of affinity-optimized CD38 CARs (CD38A4 with medium affinity and CD38B1 with low affinity to CD38) were transferred into KHYG-1 cells, and the transduced KHYG- 1 The positive expression rate of CAR on the cell surface is >85% and can be amplified efficiently
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Both CD38A4CAR and CD38B1CAR transduced KHYG-1 cells can effectively kill the CD38+ malignant cell line UM9 (MM) in a CD38-dependent manner, but the lysis level of MOCK-transduced KHYG-1 cells is very low
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  The lysis effect of CD38B1CAR KHYG-1 cells on THP-1 cells is not inferior to that of CD38A4CAR KHYG-1 cells (Figure 1A)
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After KHYG-1 cells were transduced with MOCK and CD38A4CAR, the CD38 negative control MM cell line U266 was lysed in the same amount.
The results showed that KHYG-1 cells have inherent killing ability to this cell line
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The lysis of U266 by CD38CAR KHYG-1 cells was not higher than that of MOCK-CAR KHYG-1, again indicating that the lysis of CD38+UM9 and THP-1 cells is dependent on CD38 (Figure 1A)
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The anti-MM effect and targeted tumor suppressor activity of CD38CAR KHYG-1 cells in primary newly diagnosed patient bone marrow mononuclear cells (BMMNCs) are based on flow cytometry cytotoxicity test, even in the case of low E:T ratio, CD38A4CAR KHYG-1 cells can effectively mediate CD38-dependent lysis on primary CD38highCD138+MM cells (n = 13)
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The low-affinity CD38B1CAR KHYG-1 cell-mediated MM cell lysis was significantly lower (Figure 1B)
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In the analysis of non-malignant cells, the two CD38CAR KHYG-1 cells had no obvious CAR-mediated lysis on T cells, NK cells and monocytes (Figure 1B)
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Only at higher E:T ratios, CD38A4CAR KHYG-1 showed CAR-dependent lysis of some (about 20%) B cells
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These low lysis levels are only slightly higher than the cell blank lysis level induced by MOCK and significantly lower than the lysis level of MM cells
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These results confirm that NK cells can be used as a suitable CAR carrier for affinity-optimized CD38CARs
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Since the CD38A4 variant has significant anti-MM activity, especially the anti-MM activity of primary MM cells is significantly better than that of CD38B1-transduced cells, CD38A4 variants are mainly used in the following experiments
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The cytotoxic activity of CD38CAR KHYG-1 cells on CD38+ regulatory T cells and hematopoietic progenitor cells.
In the bone marrow samples isolated from 4 patients with MM, CD38A4CAR-KHYG-1 cells effectively killed CD138+CD38+MM cells, while also inhibiting CD34 +CD38+ progenitor cells did not show CD38CAR-mediated lysis, but showed an increase in the level of CD38CAR-mediated lysis of CD38+ regulatory T cells (Tregs) (Figure 1C), indicating that CD38A4CAR-KHYG-1 cells inhibited CD38+Treg cells.
CD38-Treg cells are stronger and may have immunomodulatory properties by depleting CD38+ Treg
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The irradiated affinity optimizes the anti-MM activity of CD38CAR KHYG-1 cells.
The constant proliferation of KHYG-1 cells enables them to be effectively expanded in vitro.
This feature is beneficial to the concept of “off the shelf” therapy
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To verify this phenomenon, MOCK and CD38CAR transduced cells were continuously expanded in the presence of different cytokines (Figure 2A)
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CD38CAR KHYG-1 cells are easy to expand in long-term culture, and the expansion rate is slightly higher than MOCK-transduced KHYG-1 cells.
After adding IL-15 (10mg/mL) or IL-2 (100 ~ 900 U/mL) The cell expansion rate is similar, the combined effect of cytokines does not significantly increase the cell expansion rate, and even slight toxicity occurs under the culture of the combination of IL-15 and the highest dose of IL-2 (Figure 2A)
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When detecting CD38CAR-dependent cytotoxic activity, even long-term culture (after passage >140) CD38CAR KHYG-1 cells can effectively mediate CAR-dependent lysis of the MM cell line UM9 (Figure 2B)
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Although the constant proliferation of KYGH-1 cells facilitates in vitro expansion, it is necessary to stop replication before being used in vivo, for example, by irradiation
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The short-lived, non-proliferating KHYG-1 cells may be a better model of primary NK cells
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The proliferation activity and functional characteristics of CD38CAR KHYG-1 cells after irradiation were tested (Figure 2C)
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10Gy irradiation can almost kill all MOCK or CD38CAR-transduced KHYG-1 cells that are viable under the microscope within one week.
The cell survival rate is about 50% within 3 days, and the cells cannot be detected even for up to 2 months.
Recovery (Figure 2C)
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In order to evaluate the effect of irradiation on cytotoxicity, we tested the lysis of THP-1 cells by CD38CAR KHYG-1 cells that were still alive 5 days after irradiation
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Although the number of CD38CAR KHYG-1 cells decreased rapidly, all living cells could mediate effective cytotoxicity when performing cytotoxicity tests, indicating that irradiation did not limit the cytotoxicity of still viable cells (Figure 2C)
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The anti-MM effect of the affinity-optimized CD38CAR KHYG-1 cells in vivo was first compared with the irradiated and unirradiated CD38CAR KHYG-1 cell viability after 6 repeated injections of 10 × 106 CAR+ cells (Figure 3A)
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Regardless of whether the cells are irradiated or not, CD38CAR KHYG-1 cells significantly delayed tumor growth, while MOCK-transduced KHYG-1 cells did not show any anti-tumor effects (Figure 3B-D)
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The autopsy analysis results showed that no CD38CAR KHYG-1 cells were found in the bone marrow, blood, spleen or stent regardless of irradiation
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In multiple tissues of mice, there is no evidence that CD38 downregulation of the MM tumor cell line UM9 leads to tumor escape
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If the treatment is repeated, the capacity of unirradiated or irradiated CAR-NK cells is similar
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By increasing the number of injections to 10 and reducing the number of live cells per injection to 4 million, the effect of long-term repeated injections of irradiated CD38CAR KHYG-1 cells was evaluated
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It was again observed that compared with the MOCK-transduced cell therapy and untreated mice, the tumor growth was significantly delayed after CD38CAR KHYG-1 cell therapy, and the anti-tumor effect disappeared after the injection was stopped
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In CD38CAR KHYG-1 treated mice, the overall survival rate also slightly improved
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Similarly, autopsy analysis results showed that no viable CAR-KHYG-1 cells were detected in the blood, spleen, bone marrow or stent
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The anti-MM efficacy of optimized CD38CAR KHYG-1 cells on primary MM cells derived from daratumomab refractory patients was tested for the susceptibility of CD138+ MM cells in BMMNCs of daratumomab refractory patients (n = 6 ) (Figure 4A)
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When the E:T ratio is 1:1, it can be clearly observed that the cell dose-dependent MM cell lysis reaches 70% on average, which is similar to the lysis level of daratumumab in unexposed patients
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The killing efficiency and the percentage of CD38 positive cells showed a strong linear correlation
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In non-linear regression analysis, there is a strong non-linear correlation between MM cell lysis level and CD38 expression level (MFI) (Figure 4B and C)
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  Conclusion The results of this study indicate that for MM patients (including patients refractory to CD38 targeting antibodies), engineered affinity-optimized CD38CARs NK cells are a potential therapeutic method
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References: 1.
Stikvoort A, van der Schans J, Sarkar S, Poels R, Ruiter R, Naik J, Yuan H, de Bruijn JD, van de Donk NWCJ, Zweegman S, Themeli M, Groen R, O'Dwyer M , Mutis T.
CD38-specific Chimeric Antigen Receptor Expressing Natural Killer KHYG-1 Cells: A Proof of Concept for an "Off the Shelf" Therapy for Multiple Myeloma.
Hemasphere.
2021 Jun 12;5(7):e596.
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